Professional Documents
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Dunadiella
Dunadiella
Lilitskaya
Photomovement
of Dunaliella Teod.
VIEWEG+TEUBNER RESEARCH
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ISBN 978-3-8348-0974-2
In Memory of Professor Nadiya Massjuk
1931-2009
One of the authors of this monograph, Professor Nadiya Massjuk, Dr. Sci. Biol. and Leading
Researcher of M. Kholodny Institute of Botany of the National Academy of Sciences of
Ukraine, passed away on 13 March, 2009.
Her scientific interests were related to algology, particularly biodiversity, flora, syste-
matics, ecology, geography, origin, evolution, phylogeny, the role of algae in the world of
living organisms, and applied algology.
She was interested in the biology of algal photomovement from the point of view of
diversity, phylogeny of phytoflagellates, classification, biotechnology of cultivation of caro-
tene-containing algae, and carotenoid production.
Dr. Massjuk was an author of the classic monograph “Morphology, Systematic, Ecol-
ogy, Geographical Distribution of Genus Dunaliella Teod and Perspectives of its Practical
Applications” (Kiev, Naukova dumka, 1973) in which the results provide essential basic in-
formation on the genus Dunaliella, the main principles of systematics of the genus and eluci-
dation of its species, subspecies, versions and forms.
Dr. Massjuk’s published work (over 260 books and articles) and her impact on her
friends and collegues has left an invaluable and lasting legacy to the scientific community.
V
This monograph represents 30 years of scientific cooperation on the study of the basic biology
of photomovement in algae between the National University of Life and Environmental Sci-
ences of Ukraine (Prof. Yuriy Posudin) and the M.G. Kholodny Institute of Botany of Na-
tional Academy of Sciences of Ukraine (Prof. Nadiya Massjuk and Dr. Galyna Lilitskaya). It
reviews the historical development and current state of the art in the biology of photomove-
ment in algae. Problems in terminology and a logical basis for classification of photomove-
ment in microorganisms are discussed. The research has focused on two species of Dunaliella
Teod., D. salina Teod. and D. viridis Teod., as the principal organisms investigated.
The results of experimental investigations on the critical factors controlling and
modulating photomovement are described and include the effects of various abiotic factors,
critical aspects of photomovement such as photoreception (i.e., location and structure of pho-
toreceptor systems, composition of photoreceptor pigments, mechanisms of photoreception
and photoorientation), sensory transduction of absorbed light into signals that govern the ac-
tivity of the motor apparatus, and flagellar activity.
Various aspects involved in the utilization of these species as models for studying
photomovement, such as testing aquatic media and the effects of surface-active substances,
salts of heavy metals, and pesticides on algal photomovement parameters are described.
Vector methods for testing are proposed for assessing the action of various chemicals.
Likewise, the potential of using the two species as organisms for transgenic alteration, such as
enhanced production of E-carotene, ascorbic and dehydroascorbic acids, glycerin and other
valuable organic compounds are described.
The results of photomovement investigations are assessed relative to the evolution-
ary biology of algae and their phylogenetics, systematics, taxonomy, ecology and geography.
Critical aspects of photomovement biology that remain to be investigationed in flagellates are
discussed.
The monograph is intended for algologists, protistologists, hydrobiologists, bio-
physicists, physiologists, ecologists and biotechnologists, teachers, post-graduate students and
students of related biological specialities.
The authors express their deep and sincere gratitude to Professor Francesco Lenci
and Doctor Giuliano Colombetti (Institute of Biophysics CNR, Pisa, Italy) for stimulating our
interest in the photobiology of microorganisms and introducing the authors to the fascinating
world of algal photomovement.
The authors are grateful to Professor Felix Litvin (Moscow State University, Rus-
sia) and Professor Boris Gromov (St.-Pertersbourg State University, Russia) for their contin-
ued interest in the investigation of photomovement in Dunaliella and fruitful discussions of
the results.
The authors are much indebted to Prof. D.P. Häder (Friedrich-Alexander Univer-
sity, Erlangen, Germany), Prof. A. Flores-Moya (University of Malaga, Malaga, Spain), Prof.
H. Kawai (Kobe University, Kobe, Japan), Prof. C. Wiencke (Alfred Wegener Institute for
Polar and Marine Research, Bremerhaven, Germany) and Prof. D. Hanelt (Hamburg Univer-
sity, Hamburg, Germany) for providing the opportunity to conduct research on the photobiol-
ogy and photomovement of algae in their laboratories.
The authors would like to express their grateful thanks to Prof. Ami Ben-Amotz
(National Institute of Oceanography, Israel) for illustrative materials and Prof. Shogo Naka-
mura (Toyama University, Japan) for an electron micrograph of Dunaliella.
VII
Special gratitude to Dr. Igor Zaloilo for developing the computer versions of fig-
ures in the book.
The authors would like to express their very grateful thanks to Professor Stanley J.
Kays and Betty Schroeder (The University of Georgia, USA) for technical assistance.
Yuriy Posudin
Nadiya Massjuk
Galyna Lilitskaya
VIII
Introduction ................................................................................................................................ 1
Chapter 1
Photomovement of Algae Historical Overview of Research and Current State of the Art .... 5
Chapter 2
Terminology and the Fundamentals of Classification of Light-Induced Behaviour in
Freely Motile Microorganisms ................................................................................................. 13
2.1. State of the Art.............................................................................................................. 13
2.2. Parametrical Classification Principles for Photomovement in Organisms.................. 18
2.3. Summary....................................................................................................................... 22
Chapter 3
Investigations with Species of Dunaliella Teod. ..................................................................... 23
3.1. History of the Discovery and Description of the Genus Dunaliella ............................ 23
3.2. Characteristics of the Test Species ............................................................................... 27
3.3. Cultivation of the Species ..................................................................................... 29
Chapter 4
Investigation of Photomovement in Dunaliella ....................................................................... 31
4.1. Methods of Investigating the Photomovement Parameters in Dunaliella .................... 31
4.1.1. Experimental Installation ........................................................................................ 31
4.1.2. Measuring the movement velocity of the cells ....................................................... 32
4.1.3. Measuring the phototopotaxis ................................................................................. 32
4.1.4. Fourier Transform of Angular Distribution of the Cells ......................................... 34
4.2. Results of Measurement of Photomovement Parameters in Dunaliella ....................... 35
4.2.1. Photokinesis and Photokinetic Reactions ............................................................... 35
4.2.2. Phototopotaxis ........................................................................................................ 38
4.2.3. Results of Fourier Transform of Angular Distribution of the Cells ....................... 44
4.3. Summary....................................................................................................................... 46
Chapter 5
Effect of Abiotic Factors on Photomovement Parameters f Dunaliella................................. 49
5.1. Effect of Temperature................................................................................................... 49
5.2. Effect of Electrical Fields ............................................................................................. 50
5.3. Effect of pH .................................................................................................................. 52
5.4. Simultaneous Effect of Several External Factors ......................................................... 55
5.5. Effect of Ultraviolet Radiation ..................................................................................... 58
5.6. Effect of Ionizing Radiation ......................................................................................... 66
5.7. Summary....................................................................................................................... 69
Chapter 6
Structure of the Photoreceptor System ..................................................................................... 71
6.1. Problems associated with Photoreception of Algae ..................................................... 71
IX
6.2. Structure of Photoreceptor Systems in Green Algae .................................................... 71
6.3. Structure of the Photoreceptor System of Dunaliella................................................... 7
6.3.1. Stigma ..................................................................................................................... 7
6.3.2. Structure of the Photoreceptor ................................................................................ 77
6.3.3. Application of Two-Beam Irradiation to Dunaliella ells ..................................... 79
6.4. Summary....................................................................................................................... 81
Chapter 7
Identification of Photoreceptor Pigments ................................................................................. 83
7.1. Characteristics of Photoreceptor Pigments ................................................................... 83
7.2. Identification of Photoreceptor Pigments in Euglena gracilis ..................................... 83
7.2.1. Euglena gracilis Photoreceptor Pigments ............................................................... 83
7.2.2. Pigment Isolation .................................................................................................... 84
7.2.3. Microspectrophotometry and Microfluorometry of Pigments ................................ 84
7.2.4. Determination of the Action Spectra for Photobiological Reactions ...................... 86
7.2.5. Biochemical methods .............................................................................................. 87
7.2.6. Effect of Exogenous Chemicals on Photomovement .............................................. 88
7.2.7. Introduction of Alternative Photoreceptor Pigments .............................................. 88
7.3. Identification of Photoreceptor Pigments in Green Algae ........................................... 89
7.4. Identification of the Photoreceptor Pigments in Dunaliella ......................................... 93
7.4.1. Analysis of the Phototopotaxis Action Spectra in Dunaliella ................................ 93
7.4.2. Application of Lateral Ultraviolet Irradiation ......................................................... 93
7.5. Summary....................................................................................................................... 96
Chapter 8
Mechanisms of Photoreception and Photoorientation in Dunaliella ....................................... 97
8.1. Photoreception and Photoorientation Mechanisms in Algae........................................ 97
8.2. Diffractional Mechanisms of Photoreception and Photoorientation in Dunaliella ... 100
8.3. Role of Proteins in Photoregulation Mechanisms in Flagellates ................................ 103
8.4. Summary..................................................................................................................... 104
Chapter 9
Sensory Transduction ............................................................................................................. 105
9.1. Methods for Investigation of Sensory Transduction .................................................. 105
9.2. Sensory Transduction in Euglena gracilis ................................................................. 105
9.3. Sensory Transduction in Green Algae ........................................................................ 107
9.4. Sensory Transduction in Dunaliella ........................................................................... 109
9.4.1. Methods of Investigation....................................................................................... 109
9.4.2. Effect of Calcium Ions .......................................................................................... 10
9.4.3. Effect of Ionophore A23187 ................................................................................. 111
9.4.4. Effect of Ouabain .................................................................................................. 112
9.4.5. Effect of Cobalt Ions ............................................................................................. 112
9.4.6. Effect of Cinnarizine and Isoptin ........................................................................ 113
9.4.7. Effect of Sodium Azide ......................................................................................... 115
9.5. Summary..................................................................................................................... 117
X
Chapter 10
Flagella Apparatus.................................................................................................................. 119
10.1. Structure ................................................................................................................... 119
10.2. Peculiarities of Flagellar Beating ............................................................................. 119
10.2.1. Flagella Beating in Euglena gracilis .................................................................. 119
10.2.2. Flagella Beating in Green Algae ........................................................................ 120
10.2.3. Flagella Beating in Dunaliella............................................................................ 121
10.3. Analysis of Flagellar Beating ................................................................................... 122
10.3.1. High-Speed Microcinematography .................................................................... 122
10.3.2. Laser Light Scattering ........................................................................................ 122
10.3.3. Method of Microphotometry .............................................................................. 123
10.4. Summary................................................................................................................... 124
Chapter 11
Applied Aspects of Aquatic Biomonitoring Using the Photomovement of Dunaliella ......... 125
11.1. Algae of Genus Dunaliella as Test-Objects ............................................................. 125
11.2. Photomovement Parameters of Dunaliella as Test-Functions ................................. 129
11.3. Effect of Surface-Active Substances on Photomovement of Dunaliella ................. 130
11.3.1. Characteristics of Surface-Active Substances …………………………... 130
11.3.2. Effect of Various Types of Surface-Active Substances, their Combinations and
Duration of Action on the Velocity of Movement in Dunaliella…………….. 131
11.4. Investigation of the Effect of Heavy Metals on Photomovement in Dunaliella
Using Laser Doppler Spectroscopy.......................................................................... 134
11.5. Vector Method of Biomonitoring & ............................................................................. 137
11.5.1. Dependence of Vector R on the Type and Concentration of Surface-Active
&
Substances .......................................................................................................... 139
11.5.2. Dependence of Vector R &on the type and Concentration of Heavy Metals ....... 141
11.5.3. Dependence of Vector R on the Type and Concentration of Pesticides……… 148
11.5.4. Advantages of the Vector Method for Biomonitoring………………………… 150
11.6. Summary.................................................................................................................. 150
Chapter 12
Dunaliella Biotechnology ...................................................................................................... 151
12.1. Carotenoids, E-carotene Biosynthesis and Stereoisomers ........................................ 151
12.2. Use of Dunaliella salina for the Commercial Production of E-carotene .................. 153
12.3. Summary................................................................................................................... 158
Chapter 13
General Results and Perspectives of Further Investigations .................................................. 159
13.1. Problems of Terminology ......................................................................................... 159
13.2. Phenomenology of Photomovement......................................................................... 160
13.3. Photoreactions .......................................................................................................... 160
13.4. Photokinesis .............................................................................................................. 160
13.5. Phototopotaxis .......................................................................................................... 161
13.6. Motility ..................................................................................................................... 162
13.7. Photoreceptor System ............................................................................................... 16
13.8. Mechanisms of Photoreception ................................................................................ 16
13.9. Sensory Transduction of the Light Signal ................................................................ 16
XI
13.10. Importance of Data on Algal Photomovement for Related Fields of Science ....... 16
13.11. Applied Importance of Data on the Photomovement of Algae .............................. 16
Author’s Index....................................................................................................................... 2
Chemicals ............................................................................................................................... 22
About the Authors .................................................................................................................. 22
XII
XIII
Fig. 5.1 Dependence of mean linear velocity X of D. salina and D. viridis
on the temperature t and kinetic reactions R (t) in both species
on the change of temperature 't [Posudin et al., 1988]………………… 50
Fig. 5.2 Effect of an external electric field of 20 V/m applied to the algal
suspension on the angular distribution of the cells and intensity of photo-
topotaxis of D. salina [Posudin et al., 1991]………………………… 51
Fig. 5.3 Dependence of the linear velocity X of movement, phototopotaxis F , and
relative quantity of immobile Nim/N0 cells of Dunaliella salina on the pH
of the medium at the end of the first day of cultivation.......………......... 53
Fig. 5.4 Dependence of the linear velocity of movement and phototopotaxis F
of the cells of two species of Dunaliella on the intensity (I) of preliminary
exposure to nonfiltered ultraviolet radiation (wavelength range 250–350
nm, duration of irradiation 5 min) [Posudin et al., 2004]......………..… 61
Fig. 5.5 Dependence of the linear velocity of movement and phototopotaxis F
and relative motility Nm/N0 of the cells of two species of Dunaliella on
the duration t of preliminary exposure to nonfiltered ultraviolet radiation
(wavelength range 250–350 nm; intensity of radiation 10 W/m2 [Posudin
et al., 2004]………………………………………………………………. 62
Fig. 5.6 Dependence of the linear velocity of movement and phototopotaxis F
of the cells of two species of Dunaliella on the wavelength of ultraviolet
radiation (intensity of radiation is 2 W/m2; duration of irradiation 5 min;
c – control) [Posudin et al., 2004]…………………………………….. 64
Fig. 5.7 Dependence of the linear velocity of movement and phototopotaxis F of
the cells of two species of Dunaliella on the wavelength of ultraviolet
radiation (intensity of radiation is 2 W/m2; duration of irradiation 10 min;
c – control) [Posudin et al., 2004]………………………………………. 64
Fig. 5.8 Phototopotaxis of Dunaliella salina and D. viridis 2 hours after cessation
of a 10 min pulse of ultraviolet radiation (intensity of radiation is 2
[Posudin et al., 2004]………………………………………..…….. 65
Fig. 5.9 Dependence of the linear velocity and phototopotaxis F on the dose of
ionizing radiationafter oneday of irradiation [Posudin et al., 1992]…… 67
Fig. 5.10 Histograms which characterize relation between scattering and fluores-
cence of the cells, fluorescence of the cells, scattering of laser radiation
on the cells, and scattering at an angle of 900 for D. salina [Posudin et al.,
1992]…………………………………………………………………….… 68
Fig. 5.11 Histograms that characterize the relation among scattering and fluores-
cence of the cells, fluorescence of the cells, scattering of laser radiation
on the cells, and scattering at an angle of 900 for D. viridis [Posudin et al.,
1992]……………………………………………………………………… 68
XIV
Fig. 6.1 Interaction of light with a quarter-wave stack of alternating layers of high
and low refractive indices wavelength [adapted from Foster and Smyth,
1980]............................................................................................................. 72
Fig. 6.2 Schematic of the relative orientation of dipole moments of photoreceptor
&
molecules inside the photoreceptor Ph and direction of propagation n of
stimulating light........................................................................................... 78
Fig. 6.3 Fourier-analysis of the angular distribution of Dunaliella salina cells due
to two light flows of moderate illuminance (E1 = E2 = 500 lx) [Posudin et
al., 1991]………………………..…………………………........................ 79
Fig. 6.4 Fourier-analysis of the angular distribution of Dunaliella salina cells due
to two light flows of high illuminance (E1 = 10,000 lx and E2 = 60,000
lx) [Posudin et al., 1991]…………..............................…………..……… 80
Fig. 7.1 Absorption spectra of the photoreceptor pigments [Britton, 1986]……… 86
Fig. 7.2 Patch clamp technique for studying membrane potential………………… 92
Fig. 7.3 Absorption spectra of pigments and transmission spectra of interference
filters in the ultraviolet and visible portion of the spectrum [Posudin et al.,
1990]…………………………………………………………..………. 94
Fig.7.4 Action spectra of positive phototopotaxis in Platymonas subcordiformis,
phototopotaxis in Chlamydomonas reinhardtii, phototopotaxis in Duna-
liella spp., photoinduction of phototoptaxis potential in Haematococcus
pluvialis, phototopotaxis in Euglena gracilis…………………….. 96
Fig. 8.1 Modulation mechanism for the photoorientation of Euglena gracilis.
[Colombetti et al., 1982]…………………………………………………. 97
Fig. 8.2. Structure and location of photoreceptor system in Chroomonas Hansg….. 98
Fig. 8.3 Morphology of the ocelloid in Nematodium armatum….…………….. 99
Fig. 8.4 Schematic of the optical phenomena that occur during the interaction of
light with the structure formed by spherical or hexahonal globules that are
densely packed due to mutual compression [Posudin and Massjuk, 1996].. 101
Fig. 8.5 The dependence of a function F(p) of the light intensity diffracting on
pigmented globules of Dunaliella on the parameter p of diffraction……. 102
Fig. 8.6 The dependence of a function F(p) of the light intensity on the wave-
length O of the light falling on stigma of Dunaliella [Posudin and
Masssjuk, 1996, 1997]…………………………………………………… 103
Fig 8.7 Deformation of peptide groups under symmetrical and antisymmetrical
excitation………………………………………………………………….. 104
Fig. 9.1 Dependence of photomovement parameters F and in Dunaliella salina
and Dunaliella viridis on the concentration of CaCl26H2O in the water
[Posudin et al., 1993]……………………………………………………… 110
XV
Fig. 9.2 Temporal dependence of photomovement parameters F and υ in two spe-
cies of Dunaliella on the addition of ionophore 23187 to the medium
[Posudin et al., 1993]……………………………….……………………. 111
Fig. 9.3 Dependence of photomovement parameters F and υ in two species of
Dunaliella on the concentration of CoCl2 [Posudin et al., 1993]………… 113
Fig. 9.4 Dependence of photomovement parameters F and υ of two species of
Dunaliella on the concentration of [Posudin et al., 1993]…… 114
Fig. 9.5 Dependence of photomovement parameters F and of two species of
Dunaliella on the concentration of isoptin [Posudin et al., 1993]…….. 114
Fig. 9.6 Effect of sodium azide on the velocity (X ) of movement and positive and
negative phototopotaxis (F) in D. salina and D. viridis illuminated with
white light [Posudin et al., 1995]……………….…………………….… 116
Fig. 10.1 Flagellar beatings in Chlamydomonas……….……………………………. 120
Fig. 10.2 Principle of microphotometry for the study of flagella beating …………. 123
Fig. 11.1 Dependence of linear velocity of two species of Dunaliella on the
concentration of Surface-Active Substances during 4 hours of contact
[Parshikova et al., 1990]…………………………………………….. 133
Fig. 11.2 Dependence of phototopotaxis in two species of Dunaliella on the
concentration of Surface-Active Substances during 4 hours of contact
[Parshikova et al., 1990]………………………………….………………. 134
Fig. 11.3 Doppler correlation spectrometer [Begma et al., 1989]…………………. 135
Fig. 11.4 Dependence of energy expense W in the cells of Dunaliella on the
duration exposure to the toxicant (Cu2+) at a concentration of 10 mg/l
[Begma et al., 1989]………………………………….……………… 136
Fig. 11.5 The dependence of parameter W on the concentration of two toxicants
(Cu2+ and triton X-100) [Begma et al., 1989]…………………………… 136
&
Fig. 11.6 Value r and direction of vector R in a two-dimensional system of
coordinates (V/Vc,F/Fc) [Posudin et al., 1996]………………………….. 137
&
Fig. 11.7 Value r and direction (T1 and T2) of vector R in a three-dimensional
system of coordinates (V/Vc; F/Fc; (Nim/N0)/(Nim/N0)c) [Posudin et al.,
1996]…………………………………………………………………….. 138
&
Fig. 11.8. The dependence of value r and the direction T of vector R , in a two-
dimensial system of coordinates (V/Vc; F/Fc), on the type and
concentration of SAS. [Posudin et al., 1996a, b]………………………. 140
&
Fig.11.9 Dependence of the value r and direction T of vector R for Dunaliella vir-
idis in two-dimensional system of coordinates (V/Vc;F/Fc) on the type and
concentration of heavy metal salts [Posudin et al., 1996a,b]………… 140
XVI
&
Fig.11.10 Dependence of the value r and direction T of vector R , that is constructed
in two-dimensional system of coordinates, on the type of the salts of
heavy metals at the same concentration [Posudin et al., 1996a, b]……….. 143
Fig.11.11 The primary tendencies for changes in the value r and direction T of the
&
vector R in a two-dimensional system of coordinates (P1/Pc; P2/Pc)
[Posudin et al., 1996]…………………………………………………….... 143
&
Fig. 11.12 The dependence of the value r and direction (T1 and T2) of the vector R
in a three-dimensional system of coordinates (/c; F/Fc;
(Nim/N0)/(Nim/N0)c) [Posudin et al., 1996]…………………….................... 145
&
Fig. 11.13 The dependence of the value r and direction (T1 and T2) of a vector R on
the concentration of heavy metal salts in a three-dimensional system of
coordinates (/c; F/Fc; (Nim/N0)/(Nim/N0)c) [Posudin et al., 1996]……… 145
&
Fig. 11.14 Changes of the value r and direction T of vector R when one parameter
(/c) is decreasing and there is a simultaneous increase the second
((Nim/N0)/Nim/ N0)k) in response to an increase of concentration of copper
[Posudin et al., 1996]……..………………………………………………. 146
&
Fig. 11.15 Changes of the value r and direction T of vector R during a simultaneous
increase in parameters (/c) and ((Nim/N0)/Nim/N0)k) in response to an
increase in the concentration of copper [Posudin et al., 1996]…………. 146
&
Fig. 11.16 The dependence of the value r and direction T of vector R in a two-
dimensional system of coordinates using simultaneous monitoring of two
parameters (/c) and (F/Fc) on the type and concentration of pesticides
[Posudin et al., 1996]………………………………………………….. 148
Fig. 12.1 Accumulation of algae and carotene in biomass of Dunaliella salina
when produced in mass culture [Massjuk, 1973]………….…………... 15
XVII
Photograph 3.1 An image of Dunaliella sp. from an electron microscope.
Courtesy of Prof. Shogo Nakamura (Toyama University,
Japan)…………..................................................................... 29
Photograph 4.1 An experimental videomicrograph developed by the
Biophysics Department at National University of Life and
Environmental Sciences of Ukraine for investigating the
photomovemt of Dunaliella [Posudin et al., 1992,
1996a]…….................................................……………… 32
Photograph 12.1 Experimental carotene production station at the Saksky
chemical plant, Crimea, 1965-1969 [Massjuk,
1973]………………………………………………………. 155
Photograph 12.2 A general view of the reactor used for biomass production
of Dunaliella near the city Eilat (courtesy of Prof. A. Ben-
Amotz)................................................................................... 156
Photograph 12.3 Details of the reactor used for biomass production of Du-
naliella near the city Eilat (courtesy of Prof. A. Ben-
Amotz)………..............................................................……. 157
.
XVIII
XIX
&
Table 11.7 Dependence of the value r and direction (1 and 2) of vector R in a
three-dimensional system of coordinates on the type and concentration
of heavy metal salts[Posudin et al., 1996a, b]………………………… 144
&
Table 11.8 The dependence of value r and the direction (1, 2, 3) of vector! in
a four-dimensional system of coordinates on the type and concentration
of heavy metals using the simultaneous measurement of four photo-
movement parameters in Dunaliella viridis [Posudin et al., 1996]…… 147
Table 11.9 Effect of the type and concentration of pesticides on photomovement
parameters in Dunaliella viridis [Posudin et al., 1996a, b]………….... 149
XX
"The only generalization that can be madefor
photomovement is its diversity"
W. Haupt, 1983
Introduction
In a broad context the term photomovement encompasses any movement or its alteration in-
duced by light. Photomovement is the result of the photoregulation ofmovement - which in-
cludes an entire complex of elementary processes caused by a light stimulus such as photore-
ception, primary reactions of the photoreceptor pigments, and the sensory transduction of the
light stimulus into a physiological signal that governs the activity of the motor apparatus and
results in the photoorientation of the organism.
The study of photomovement and the photoregulation of movement in micro-
organisms is of considerable interest due to the importance of these phenomena and that they
are closely tied to fundamental biological processes such as photosynthesis, photoreception,
energy transformation, membrane-coupled and membrane-mediated phenomena. The investi-
gation of photomovement and its photoregulation are also closely tied to the elucidation of the
basic principles of intracellular developmental processes, as well as ontogenesis, embryo-
genesis, and morphogenesis. A better understanding of light mediated responses impacts our
understanding of light's role in the ecology and bioceno10gy of these organisms since light is
an important factor in their spatial and temporal distribution. While photomovement has an
independent function, it also conveys information on the complexity of related environmental
factors (e.g., temperature, pH, biogenesis of compounds, oxygen content, the presence of
other microorganisms [Kritsky, 1982; Sineschekov and Litvin, 1982]).
The investigation of photomovement mechanisms is also of interest from the stand-
point of bionics, evolutionary biology, morphology, phylogeny, and systematics. It is known,
for example, that the structure of the motor apparatus and photoreceptor is an important sys-
tematic character at higher taxonomic levels (divisions and classes) in phycology [Sedova,
1977; Topachevsky and Massjuk, 1984; van den Hoek et aI., 1995; Graham and Wilcox,
2000; Massiuk and Kostikov, 2002]. Thus, it is possible to assume the specificity of the
mechanisms of photoperception and photoregulation of photomovement among members of
different divisions or classes of algae. Finally, the study of photomovement has the potential
for stimulating the practical application of this technology in areas such as biomonitoring of
the environment, biotechnology, and the use of these organisms for the synthesis of useful
natural products.
There have been a number reviews on light induced movement of microorganisms
[Halldal, 1958, 1961; Haupt, 1959, 1983; Bendix, 1960; Hand and Davenport, 1970; Nultsch,
1975; Wolken, 1977; Lenci and Colombetti, 1978; Miyoshi, 1979; Nultsch and Hader, 1979,
1988; Diehn, 1979, 1980; Feinleib, 1980; Colombetti and Lenci, 1982; Lenci, 1982; Poffand
Hong, 1982; Sineshchekov and Litvin, 1982, 1988; Hader, 1987a, 1987b, 1987c; 1994;
1996a; Lenci et al., 1984; Colombetti and Petracchi, 1989; Doughty, 1991; Nultsch and Ruef-
fer, 1994; Donk and Hessen, 1996; Hader and Lebert, 2000; Lebert and Hader, 2000; Sinesh-
chekov and Govorunova, 2001a; Hegemann and Deininger, 2001; Hegemann et aI., 2001;
Williams and Braslavsky, 2001; Sgarbossa et aI., 2002; Checcucci et al., 2004]. In addition,
there have been a number of scientific conferences and schools that communicate recent ad-
vances in our fundamental understanding of the subject (e.g., "Biophysics of Photoreceptors
and Photobehaviour of Microorganisms" (Pisa, 1975), "Photoreception and Sensory Trans-
2
ronmental factors such as ultraviolet and visible radiation, temperature, pH, and electrical
fields on the photomovement parameters in these species.
The potential of algal biotechnology is likewise addressed. Both species represent pos-
sible organisms for the commercial production of !3-carotene (provitamin A), ascorbic and
dehydroascorbic acids, glycerol, feed for fish production, and other products. Assessment of
changes in photomovement by these organisms can also potentially be used as biosensors for
assessing the composition of aquatic media.
A comparative analysis of both general and specific differences in photomovement
among these flagellated algae species and representatives of different orders (classes) of algae
is also reported.
The main objective of this monograph is to critique the current understanding of pho-
tomovement in the unicellular green algae species D. salina and D. viridis.
2. Describe theoretical problems in terminology and the logic of the existing method for the
classification of photomovement in these microorganisms;
4. Critique the experimental methods utilized for the measurement of photomovement of these
species and the effects of abiotic factors on photomovement;
6. Describe the processes of sensory transduction of absorbed light into signals that govern
the activity of the motor apparatus of the two species;
7. Assess the possible application of D. salina and D. viridis as models for testing the quality
of aquatic media and estimating the effects of surface-active substances, salts of heavy metals,
and pesticides on photomovement in algae;
8. Assess the potential of the two species of Dunaliella for transgenic alteration to enhance the
synthesis of J3-carotene, ascorbic and dehydroascorbic acids, glycerol and other valuable organic
compounds;
10. Critique critical areas for future research on the biology of photomovement in flagellates.
3
Chapter 1
Photomovement of Algae - Historical Overview of
Research and Current State of the Art
Interest in understanding the mystery surrounding the movement of living organisms dates
from ancient times. The first published work in the field [De Motu Animalium ("On the
Motion of Animals")] was by Aristotle (384-322 B.C.) who was interested in similarities in
motion among animals. Leonardo da Vinci (1452-1519), a distinguished painter, architect
and engineer, also studied the mechanics of movement in organisms (biomechanics). His
Codex on the Flight of Birds was a precise study of the mechanics of flight and air movement.
The same problems captured the interest of Giovanni Alfonso Borelli (1608-1679), a famous
Italian mathematician, astronomer and compatriot of Galileo GalileL He authored the first
book on biomechanics [De Motu Animalium I and De Motu Animalium II ("On the Motion
of Animals"), 1679] that was dedicated to muscular movement and body dynamics. He also
studied bird flight and the swimming of fish [Thurston, 1999].
The nature and mechanisms of movement of living organisms preoccupied the
attention of many famous scientists - I.M. Sechenov (1829-1905), I.P. Pavlov (1849-1936),
P.F. Lestgaft (1837-1930), AA Ukhtomsky (1875-1942), N.A. Bernstein (1896-1966) and
others.
There has been a progressive increase in interest in motile behaviour of microorgan-
isms since 1674 when Antonie van Leeuwenhoek [Mosolov and Belkin, 1980] first observed,
using a microscope he developed, the movement of Euglena and Volvox [cited by: Wolken,
1975]. An article by Ludolph Christian Treviranus (1779-1864), a German botanist, was the
first work dedicated to the investigation of algae. Zoospores of Drapamaldia glomerata
(Vaucher) CA Agardh and U/othrix subtilis Kutzing accumulated near the illuminated edge of
the vessel or at the opposite side [Treviranus, 1917].
Christian Gottfried Ehrenberg (1795-1876), a German scientist, studied over a 30
years period thousands of new species, including flagellates such as Euglena, ciliates such as
Paramecium aurelia MUller and Paramecium caudatum Ehr., a group of unicellular protists
called diatoms, and many species of radiolaria. Of particular interest was his manuscript pub-
lished in 1838 describing the red eye (eyespot) or stigma of Euglena, an organelle that plays
an important role in the photomovement of the algae.
Charles Darwin wrote in 1872 "How a nerve comes to be sensitive to light, hardly
concerns us more than how life itselforiginated; but I may remark that, as some ofthe lowest
organisms, in which nerves cannot be detected, are capable ofperceiving light, it does not
seem impossible that certain sensitive elements in their sarcode should become aggregated
and develop into nerves, endowed with this special sensibility".
Experiments by F. Cohn (1865a) demonstrated that zoospores of some algae, just as
the cells of Euglena, exhibited phototaxis in response to blue-green but not red light. This was
the first indication of spectral sensitivity in microorganism photomovement.
AS. Famintzin (1843-1918) published "Action of light on algae and some other or-
ganisms close to them" (St.-Petersbourg, 1866) and was conferred the title of Doctor of Bot-
any. The author distinguished two types oflocomotion in protozoa; those that have cilia (zoo-
spores) and pseudopodia (amoeboid organisms). Cilia are present in flagellates such as: Vol-
vox, Gonium, Stephanosphaera, Euglena, and Chlamydomonas.
Famintzin (1887a, c. 19) further characterized this phenomenon: "It was observed long ago
that lateral illumination of the vessel with liquid. where zoospores are swimming, provoked
the accumulation of them along the edge of the vessel forming a green strip" (translation of
Yu. Posudin). Famintsin concluded that light induced the movement of zoospores and that the
light-induced movement of algae depended upon the light intensity, temperature, and the
composition of aquatic medium [Famintzin 1867a, b, 1887a]. A number of articles describing
photomovement in desmids and blue-green algae were published in the 1880s which repre-
sented the general understanding of the biology of photoorientation mechanisms during that
time period.
Eduard Adolf Strasburger (1844-1912), a famous Polish-German Professor of Botany,
conf"rrmed that various microorganisms use different mechanisms of photoorientation. He
believed that zoospores of Haematococcus respond to light gradients, while the motile repro-
ductive cells of Botrydium respond to the direction of the light. Strasburger was the first to
use the term ''phototaxis'' to distinguish between the light-induced transfer of mobile (photo-
taxes) and fixed (phototropisms) organisms and to distinguish between positive and negative
phototaxes. He also was the first to use coloured glass filters to study spectral peculiarities of
zoospore photomovement and likewise discovered the ability of colourless microorganisms to
respond to light [Strasburger, 1878].
Theodor Wilhelm Enge1mann (1843-1909), a German botanist, physiologist, and
microbiologist, published in 1882 the effects of different wavelengths (or colors of light) on
photosynthetic activity and showed that the conversion of light energy to chemical energy
took place in the chloroplast [Drews, 2005; Enge1mann, 1882a,b]. Enge1mann also made a
number of valuable contributions to the investigation of photomovement in algae (l882a,b).
Using the technique "projected microspectrum", he demonstrated the dependence of the
photoreaction in microorganisms on the wavelength of the light stimulus. Despite the limited
qualitative precision of these early experiments, it was possible to estimate the action spectra
6
of photoreactions in microorganisms. Engelmann determined the spectral sensitivity of Eu-
glena viridis (O.F. Mueller) Ehrenberg to be around 470 to 490 nm. He described the shock
reactions of Euglena in response to the application of a narrow beam of light and found that
the front of the cell (where the stigma is located) was more sensitivit than other areas
[Engelmann, 1882a,b].
A series of articles were published toward the end of 1890s that were dedicated to the
effect of external physical factors on photomovement in algae, for example, electric fields
[Vervorn, 1889], temperature [Wildeman, 1893] and X-rays [Shaudinn, 1899] (cited by: Jalm,
Bovee, 1968). These were followed by a number of studies at the beginning of 20th century
that focused on photomovement terminology [Rothert, 1901; Nagel, 1901; Pfeffer, 1904].
Loeb and Maxwell (1910) discovered the ability of cells to aggregate when exposed to the
blue portion of the electromagnetic spectrum. During this period, Euglena gracilis G.A. Klebs
became the classic model for photomovement investigations. A number of investigators fo-
cused on this organism in their study of the mechanisms of photoreception and photoorienta-
tion. Stigma morphology was addressed as was the colour of the cup-shaped p1astids [France,
1893] present in hexagonally-packed layers [Wolken, 1956] or aggregates of pigmented gran-
ules (globules) [Hall, Jalm, 1929; Gojdics, 1934] (all authors are cited by: Jalm, Bovee, 1968).
Research dedicated to the study of the mechanisms of photoorientation in Euglena was
also published during this period [Jennings, 1906; Mast, 1911]. The ability of Euglena to
move toward the direction of the light and photophobic reactions of algae ("Schreck-
bewegung" [Engelmann, 1882a,b] or "avoiding reaction" [Jennings, 1906]) were described.
The sensitivity of Euglena to the light in the blue portion of the spectrum was also estab-
lished. Mast supposed that the photosensitive pigments in Euglena were located near the
stigma [Mast, 1911]. He stressed the necessity of using "spectrally pure" (monochromatic)
light and its precise calibration for estimating the stimulating efficiency of light at various
wavelengths (a procedure now referred to as ''registration of the action spectrum"). He also
measured the optical transmission of the stigma of Euglena and his results (1'= 0,28) are simi-
lar to those obtained by investigators half a century later (1' = 0,32) [Wolken, 1967]. The ac-
tion spectrum for phototaxis in Euglena was measured by Mast in 1927. The spectrum is
characterised by a wavelength band between 410-540 nm with a maximum at 485 nm. Caro-
tenoids were thought to be the photoreceptor pigments [Mast, 1927].
With regard to the function of the stigma in Euglena, Arnoldi concluded "Its (the eye)
jUnction has been mysterious up to now, but it has certain similarity to the eyespot of some
protozoa, which plays the role ofa rudimentary organ ofsense .. [Arnoldi, 1908] (translation
ofYu. Posudin).
Beginning with pioneer work of A. Fisher [1894, cited by: Jalm, Bovee, 1968], where
the morphology of flagellar system of Euglena was described, an increasing number of inves-
tigations on the structure and function of the locomotor apparatus in algae were published, the
results of which have only recently been expanded through the application of modem tech-
niques.
An attempt to establish a cause and effect relation between the light stimulus and res-
ponse in microorganisms was addressed in a number of investigations [Buder, 1917; Mainx,
Wolf, 1929]. A valuable contribution to the development of ideas concerning the mechanisms
of photoorientation in Euglena was introduced by T. Buder (1917) who was the first to pro-
pose that the location of the photoreceptor was in the paraflagellar body (a hypothesis that
remains correct to this day). Buder also utilized light passing through the condenser of a mi-
croscope to measure photomovement parameters. He stressed the importance of the spectral
composition of the light and introduced the term "schwaches, tief rubinrotes licht" (weak,
deep ruby-red light), used in the microscopy study of microorganism mobility. Buder identi-
fied the problems associated with the quantitative analysis of photomotile reactions, investi-
gated flagellar activity during phototaxis and photophobic reactions, proposed the probable
7
location of the photosensitive system in the cell, and described the relationship between fla-
gellar beating and the position of stigma relative to the source of light. Many of the questions
proposed by Buder have yet to be answered.
In 1936, Sergey Chakhotin (1883-1973) studied the functions of the photoorientation
apparatus in Euglena using ultraviolet microirradiation which he developed [cited by Posudin,
1995]. A Euglena cell was placed in the illuminated portion of a quartz capillary tube. As the
cell moved along the capillary, it reversed its direction of movement when reaching the "light-
dark" boundary. Ultraviolet irradiation on the stigma stimulated its excitation and contraction
of the stigma. After exposure to UV irradiation the cell moved past the "light-dark" boundary
without reversing its movement, indicating a deactivation of the photoorientation mechanism
in the cell. The use of monochromatic light demonstrated the sensitivity of stigma to blue-
green light. Chakhotin concluded that the stigma of Euglena was a primitive sensory organ
that controlled the movement of the cell. Ultraviolet irradiation appeared to ''blind'' the cell
with the frontal part of the cell being more sensitive to the irradiation than the back.
B.V. Perfiliev (1915) studied photomovement in the blue-green algae (Oscillatoria
geminata Menegh. and Synechococcus aeruginosus Naeg.) and diatoms (Pinnularia strep-
toraphe Cl. and Anomoeoneis sculpta Pfitzer), particularly in relation to spectral sensitivity.
He supposed that " ... movement is probably the common property of blue-green algae ... "
(translation of Yu. Posudin) based on the stimulation of photomovement in blue-green algae
by the red region and in diatoms by the blue region of the electromagnetic spectrum. The role
of light on the behaviour of green algae, diatoms and desmids is mentioned by AA Elenkin
(1925). He also noted the ability of chloroplasts of Mougeotia genuflexa (Roth) C.A Agardh
to move inside the cell in response to light.
In the 1930s, A. Luntz determined the action spectra of phototaxis of Eudorina, Vol-
vox, Chilomonas, and Chlamydomonas reinhardtii P.A. Dang. [Luntz, 1931 a,b]. An attempt
to precisely measure the of absolute light energy values was a distinctive feature of his inves-
tigations. The action spectra for phototaxis in green flagellates (Eudorina, Volvox and Chla-
mydomonas) had a maximum at 492 nm while the colorless cryptophyte algae Chilomonas
was sensitive at 366 nm.
The fundamental work of Per Halldal at the beginning of the 1960s [Bjorn L. et aI.,
2007] determined the action spectra for representatives of Volvocales, Dinophyceae, and
Ulva-gametes [Halldal, 1958]. The action spectra for positive phototaxis were determined for
Dunaliella viridis Teod. and Dunaliella t: euchlora Lerche, while the action spectrum for a
negative phototaxis was characterized in Dunaliella salina Teod. The projected spectrum
method was used in these investigations. The phototactically active spectral region in the
three Dunaliella species was between 400 to 540 nm with a maximum at 493±3 nm and a
small shoulder at around 435 nm. Halldal indicated that "the stigma is a photoreceptive organ
associated with the orientation oftopo-phototactic algae ... " [Hal1da1, 1958].
The 1960s and 1970s were marked by the transition from naturalistic descriptions of
photomovement to basic photochemical explanations of the phenomenon [Clayton, 1964;
Haupt, 1966; Tollin, 1969; Nultsch, 1970; Feinleib and Carry, 1967; Hand and Davenport,
1970]. A number of investigators [Wolken and Shin, 1958; Wolken, 1967, 1971, 1977;] pub-
lished detailed studies on the photomovement in Euglena gracilis, in particular the effect of
external factors on the velocity and direction of movement, measurement of the action spectra
for the photoreactions, elucidation of the nature of photoreceptor pigments, and analysis of the
function of the flagellar apparatus. Due in part to the progressive accumulation of data, E.
gracilis became the model system of choice for studying photomovement in algae. This was
followed by the first detailed review of the literature on photomovement in which problems of
terminology, experimental and methodological approaches for studying photoreception, and
sensory transduction were critiqued [Diehn, 1979].
8
The second half of the 20th century was marked by the study of photomovement in
both individual cells and populations [Feinleib, 1977; Ascoli et aI., 1978; Barghigiani et al.,
1979; Colombetti and Lenci, 1982]. The study of individual cells is more labour intensive and
complex but it makes it possible to determine all motile reactions and photomovement pa-
rameters, as well as response characteristics to specific light stimuli. The study of populations,
however, allows obtaining data on the movement of millions cells with a high level of preci-
sion, though due to the absence of photomovement data on individual cells, interpretation of
the results can be complex. The subsequent years have seen a progressively increasing interest
in alga photomovement [Williams, 2001; Iseki and Watanabe, 2004].
We have seen the formation of several highly productive schools of scientific inquiry
into the biology of photomovement in various countries of the world under the aegis of
prominent photobiologists. A German school led by the photobiologists W. Nultsch (Mar-
burg, Germany) studied photomovement using a green alga (Chlamydomonas reinhardtii P.A.
Dang.) as the model [Nultsch, 1962, 1970, 1983]. Several extensive reviews on the analysis of
experimental and methodological approaches to the investigation of algal photomovement
were published [Nultsch, 1975, 1980; Nultsch and Hlider, 1979, 1988]. Nultsch described the
considerable confusion in the scientific literature due to a plethora of terminology utilized in
the studies of photomovement in microorganisms [Nultsch, 1973, 1975].
Also in Germany, W. Haupt (Erlangen, Germany) studied motile responses to light in
organisms and cellular organelles, and the phototropic and photonastic movement of plants.
He authored the first detailed reviews on the diversity of photobiological reactions among
alga species and also focused on the problem of terminology [Haupt and Feinleib, 1979;
Haupt, 1983, 1986; Haupt and Seitz, 1984].
D.-P. Hllder (Erlangen, Germany) and a group of scientists under his leadership stud-
ied photomovement and gravitaxis in various algae. He authored a number of reviews on the
subject [Hllder, 1979, 1987a, b, 1996a; Hllder and Lebert, 2000; Lebert and Hllder, 2000].
His research group used a computerized system of videomicrography for the quantitative
analysis of photomovement parameters [Hllder, 1994b]. Hllder and M Lebert also published a
monograph "Photomovement" (2001) on the subject.
A fourth group of Gennan scientists was lead by P. Hegemann (Regensburg). They
investigated light-sensitive processes in green algae using C. reinhardtii as the model. They
utilized optical, spectroscopic and electrophysiological methods to study the nature of the
photoreceptor. Hegemann (together with co-authors) published several reviews [Hegemann,
1997; Hegemann and Harz, 1998; Hegemann and Fisher, 2001; Hegemann and Deininger,
1999,2001; Hegemann et al., 2001].
In Italy a group of investigators at the Institute of Biophysics CNR (pisa, Italy) that
included Francesco Lenci, Giuliano Colombetti, Francesco Ghetti, Paolo Gualtieri and others
have studied photomovement for a number of years [Lenci, 1982; Lenci and Colombretti,
1978; Gualtieri, 2001]. The principal research focus of the scientists has been on photocon-
trolled biological phenomena such as the ability of microorganisms to use light to transmit
information that affects their motile behaviour under environmental light conditions. The mo-
lecular processes of photoreception and sensory transduction of light into a signal that con-
trols movement of the organism have been studied using E. gracilis as a model. Microspectro-
scopic fluorometric analysis was utilized for investigating the photoreceptor pigments which
resulted in the discovery of fluorescence by an organelle located near the base of the flagel-
lum [Benedetti, Checcucci, 1975].
It is necessary to mention a brilliant methodological approach that was used to identify
the photoreceptor pigments in alga. The paraflagellar body in E. gracilis was illuminated us-
ing a tunable dye-laser focused on it using a microscope. The technique made it possible to
determine the in vivo fluorescence excitation spectrum of the paraflagellar body and compare
9
it with absorption spectra of flavins. They concluded that these pigments participate in the
photomovement of alga [Colombetti et aI., 1980,1981; Ghetti et aI., 1985].
An additional technological breakthrough has been the use of laser Doppler spectros-
copy developed by scientists at the Institute of Biophysics CNR. The method allows the linear
and rotational velocity of movement and the frequency of flagella beatings to be measured
[Ascoli, 1975; Ascoli et aI., 1978, 1978]. Current research at the Institute is directed toward
photomovement in algae and protozoa.
A Polish school, led by E. Mikolajczyk, has studied photophobic reactions in E. graci-
lis, in particular, the dependence of these reactions on the wavelength of light and conditions
under which the alga are grown. They have also studied photomovement in the colourless
euglenophyte algae Astasia longa and Peranema trichophorum [Mikolajczyk, 1984a, b,
1986].
In the United States, the photobiologist M.E. Feinleib (Tufts University, USA) studied
photomovement in Chlamydomonas reinhardtii [Feinleib, 1977, 1978, 1980, 1985]. Likewise,
Pill-Soon-Song (University of Nebraska, USA) studied the molecular mechanisms of sensory
transduction in protozoans (e.g., Stentor coeruleus Ehr.) with special emphasis on the photo-
control functions ofstentorins and blepharismins [Song, 1983, 1985].
The Japanese school of photobiologists used a unique measuring complex - an Oka-
zaki Large Spectrograph [Watanabe et aI., 1982]. The spectrograph was developed in 1980 for
the measurement of the action spectra of various photobiological reactions in the 250-1200
nm range at a high resolution (i.e., 0.8 nm/cm). Algae such as the cryptophytes Cryptomonas
sp., Cryptomonas rostrati/ormis Skuja ex T. Willen, Chroomonas nordstedtii Hansgirg,
Chroomonas coerulea (Geitler) Skuja [Watanabe and Furuya, 1982] and the green algae C.
reinhardtii [Kondo et aI., 1988] and D. salina [Wayne et al., 1991] were the primary species
studied.
A second group of photobiologists under leadership of H. Kawai (Kobe University,
Japan) studied the nature of the photoreceptor pigments in the gametes and zoospores of
golden and brown algae using microspectrofluorometry of the flagellar apparatus [Kawai,
1988, 1989, 1992; Kawai et aI., 1991, 1996; Yamano et aI., 1993].
A third Japanese research team at the University of Toyama has been led by the algo-
logist S. Nakamura. They proposed using flagellar regeneration of Dunaliella sp. to assess
seawater pollution [Horike et aI., 2002]. Together with T. Takahashi they likewise studied the
photoreceptor system (eyespot, structure and localization) in C. reinhardtii [Nakamura et al.,
2001; Suzuki et al., 2003]. At the University of Tokyo, the laboratory of R. Kamiya studied
photoreception and the mechanisms of flagellar beating during the phototactic activity in
Chlamydomonas. They were interested in the structure and function of eukaryotic flagella and
used mutants lacking flagella to address certain questions at the universities of Tokyo and
Tsukuba [Isogai et aI., 2000; Yoshimura and Kamiya, 2001; Kamiya, 2002; Okita et al., 2004;
Fujiu et aI., 2009].
A Russian school on photobiologists (e.g., F. Litvin, O. Sineshchekov, E. Govorunova
et al.) applied electrophysiological methods to study the light-induced excitation of the photo-
receptor in alga that instigates a cascade of fast electrical events in the cell membrane. The
measurement of photoreceptor currents allowed identifying the photoreceptor pigment in two
species of green algae (Haematococcus pluvialis Flotow, C. reinhardtii) that appeared to be
rhodopsin [Sineshchekov and Litvin, 1982, 1988; Sineshchekov, 1991a,b; Sineshchekovand
Govorunova, 1999, 2001a,b,c].
Research on photomovement in the Ukraine, led by Yu. Posudin, N. Massjuk, and
G. Lilitskaya began in 1980. The main research focus of the group was on problems in termi-
nology and a logical basis of classification of photomovement in microorganisms. Their ex-
perimental investigations on photomovement used two green algae species from genus Du-
naliella (Dunaliella salina Teod. and Dunaliella viridis Teod.) and the effect of abiotic factors
10
on them. They also studied the processes involved in photoreception (Le., location and struc-
ture of the photoreceptor system, nature of the photoreceptor pigments, mechanisms of photo-
reception and photoorientation), sensory transduction of light received by the photoreceptor
and its conversion into a signal controlling the movement of alga, the potential for using pho-
tomovement as a means of biotesting of aquatic media, and the potential for the use of certain
species for transgenic alteration to address biotechnological problems [Massjuk et aI., 2007].
The systematic investigation of photomovement in algae is currently in progress in
Europe (Germany, Italy, Poland, Russia, Ukraine), Asia (Japan) and the United States. The
following representatives of various divisions of cyanoprocariotic algae - Cyanophyta (Oscil-
latoria geminata, Synechococcus aeruginosus), eucariotic algae - Euglenophyta (Euglena
gracilis, Astasia longa, Peranema trichophorum), Dinophyta (Peridinium gatunense), Bacil-
lariophyta (Pinnularia streptoraphe, Anomoeoneis sculpta), Cryptophyta (Cryptomonas spp.,
Chroomonas spp.), Chlorophyta (Dunaliella salina, D. viridis, Chlamydomonas reinhardtii,
Chloromonas sp., Haematococcus pluvialis, Stephanosphaera sp., Gonium sp., Eudorina sp.,
Volvox sp.), and spores and gametes of green (Chlorophyta), golden (Chrysophyta), yellow-
green (Xanthophyta), red (RlJodophyta) and brown (Phaeophyta) algae are currently or have
been used as models in these investigations. Special attention is being directed toward further
elucidating the mechanisms of photoreception and photocontrol of movement and to the na-
ture of the photoreceptor and locomotor apparatus. In addition, continuing problems with ter-
minology and the classification of various motile reactions in microorganisms are also sub-
jects of interest.
11
Chapter 2
Terminology and the Fundamentals of Classification
of Light-Induced Behaviour in
Freely Motile Microorganisms
The problems concerning terminology and classification of the phenomena associated with
the motile properties of microorganisms have been studied by a number of researchers
[Halldal,1958; Jahn and Bovee, 1968; Nultsch, 1973, 1975, 1980; Diehn et aI., 1977; Feinleib,
1977, 1978, 1980; Lenci and Colombetti, 1978; Diehn, 1979; Hader, 1979, 1987a, 1987b,
1987c, 1996a, 1996b; Nultsch and Hader, 1979; Posudin, 1982, 1985; Sineschekov and Lit-
vin, 1982; Colombetti and Lenci, 1982; Colombetti et aI., 1982; Lenci, 1982; Haupt, 1983;
Burr, 1984; Lenci et al., 1984; Burr, 1984; Posudin et al., 1988, 1990, 1991, 1992a, 1992b,
1993, 1995, I 996a,b,c; Massjuk et al.,1988; Massjuk and Posudin,199Ia; Kawai and
Kreimer, 1992; Witman, 1994; Kreimer, 1994; Martynenko et al., 1996; Posudin and
Massjuk, 1996; Lenci et al., 1997; Hegemann and Harz, 1998; Sineschekov and Govorunova,
1999; Lebert and Hllder, 2000; Gualtieri, 2001; Hllder and Lebert, 2001; Hegemann and
Fisher, 2001; Hegemann et aI., 2001; Massjuk and Posudin, 2002]. The definition of terms
and concepts, however, remains unacceptably ambiguous. On occasion a new meaning is
attached to an old term, e.g., ''phototaxis'' or ''photokinesis'' which is interpreted differently
by various authors (e.g., compare Diehn et aI., 1977; Nultsch and Hllder, 1979). Likewise,
terms with different meanings are used as synonyms (e.g. "behavioural light response", "be-
havioural response", "light controlled cell motility", "light controlled movement', "light
response", "light-induced behavioural response", "light-induced responses offreely moving
organisms", "locomotive and motile response", "motile behaviour", "motile response to
light', "movement behaviour", "photobehaviour", "photobehaviour response", "photoinduced
behaviour", ''photomotile response", ''photomotion'', ''photomovement response", ''photomo-
vement', ''photoreaction'', ''photoregulation of movement', ''photoresponse'') - see Posudin,
1982, 1985; Sineschekov and Litvin, 1982; Wayne, 1991; Kreimer, 1994; Hegemann, 1997;
Holland et al., 1997; Matsuda, 1998; Horigushi et al., 1999; Sineschekov, Govorunova, 1999;
Lebert and Hader, 2000; Govorunova et al., 2000, 2001; Haupt, 2001; Hader and Lebert,
2001; Tahedl and Hader, 2001.
Currently there are several terminological systems that describe the motile behaviour
of organisms (e.g. [Fraenkel and Gunn, 1961; Burr, 1984]). The wide diversity in the systems
studied has been a source of terminological confusion. In addition, existing classification sys-
tems and definitions of the terms have a number of errors hindering precise communication
among scientists in photobiology.
The objective of this Chapter is to describe a logical basis for the classification ofpho-
tomovement phenomena in freely motile organisms (independent of their structure and sys-
tematic position), and to define the appropriate terminology.
14
Pfau et al., 1983; Hader and Lebert, 1998; Horiguchi et al., 1999; Lebert and Hader, 2000;
Hader and Lebert, 2001].
The plurality and diversity of these terms are undesirable. New designations are either
bulky or not exact and concrete, and they may be interpreted both in a broader meaning (e.g.
including phototropism, photoregulation) and in a more narrow one. Some authors use these
expressions as synonyms, while others distinguish photocontrol and its consequences (pho-
tomovement, photomotion) as well as photoresponses and behavioural consequences, increas-
ing the frequency of disagreements in terminology.
Taking into account the above-mentioned considerations we propose keeping the
primary interpretation of the term "phototaxis" as any light-induced movement of freely mo-
tile organisms. Photooriented movement of organisms is termed "phototopotaxis" and subdi-
vided into two basic patterns: positive (toward the light source) and negative (away from it)
[Hal1da1, 1958; Nultsch, 1975; Feinleib, 1980]. Occasionally a third pattern, transverse
phototopotaxis (perpendicular orientation relative to the direction of the light) is used [Diehn
et al., 1977; Diehn, 1979; Hader, 1979]. This includes any linear movement of organisms that
is oriented to the direction of the light except in the case of diffused light or simultaneous
illumination from all sides.
The term "photomovemenf' should have a broad meaning, i.e., any motility response
or its alteration induced by light. Thus, this term involves either phototaxis (in the broader
meaning) or phototropism. Our interpretation of this term does not contradict the definition
given by W. Nultsch and D.-P. Hlider (1979), "The term photomovement denotes any move-
ment or change of movement elicited by light ... ". As a consequence, the terms "photo-
movement" and "phototaxis" are compatible, but not identical. The former is subordinating,
while the latter is subordinate.
It is quite clear that the control of movement and photomovement in organisms are
absolutely different and are distinguished by a cause and effect relationship. Photomovement
in organisms occurs by way of controlled movement. Control of movement is realized
through the interaction of external factors (e.g., light stimulus - its intensity, spectral charac-
teristics, direction of propagation) and internal regulatory mechanisms of the organism (e.g.,
photoreception, sensory transduction, function of the motor apparatus). Spatial displacement
due to photomovement takes place as a result of such an interaction. Thus, the meanings
"photomovement" and "control of movement" are not identical and not compatible from the
point of view of formal logic.
In the latter part of the 19th century, Strasburger was the first to define the reaction of
microorganisms to a sudden change in light intensity, a response called "Schreckbewengung"
(''motion of jrighf'). Engelmann [1882a,b] observed a sharp change in the direction of
movement of some microorganisms in response to a sudden increase in light intensity. This
response was termed "phobic" (from Greek word rpOyfJ6'; - fear). Later, several terms were
proposed for indicating the shock reaction of microorganisms to changes in the intensity of
light: "apobatic phototaxis" [Rothert, 1901], "phobism" (from the Greek word "fear") [Mas-
sart, 1902, cyt. by Nultsch, 1975], "discrimination sensitivity" [Nagel, 1901] and "photo-
phobotaxis" [Pfeffer, 1904].
Within the scientific literature there are synonyms for the term "photophobic reaction"
such as "photophobic response", "light induced stop response", ''photoshock'', ''photoshock
response ", ''photoshock cell response ", ''photophobic, stop or ecclitic response ", ''photo-
phobotaxis", and "stop response" [Beckmann and Hegemann, 1991; Hegemann et al., 1991;
Govorunova et al., 1997; Holland et al., 1997; Sineschekov and Govorunova, 1999; Lebert
and Hader, 2000; Hader and Lebert, 2001].
Engelmann (1882) termed the restoration of movement in microorganisms, that were
immobile in the dark, after switching on the light stimulus ''photokinesis''(from Greek word
lClVTJm; -motion). The term orthokinesis indicates that the linear velocity of the organism is
15
altered by the stimulus while klinokinesis indicates the frequency or rate of turning of the or-
ganism induced by the stimulus. These terms continue to be used although their interpretation
has undergone significant alterations. Thus, there are at least four different interpretation for
the term "photokinesis" which indicates an essential discrepancy in the interpretation of
''phototaxis'' and ''photophobic response" [Burr, 1984].
A number of new terms (see, f.e., [Fraenkel and Gunn, 1961]) have subsequently
emerged the appearance and interpretation of which are discussed in a review by Nultsch
(1975). The author noted "these disagreements in terminology have, in the past, led to errors
and misinterpretation, so that the creation of new terms would further confuse the situation
rather than clarifY it".
This fact resulted in the creation of the Committee on Behaviour Terminology that was
convened in 1976 at the conference on Sensory Transduction in Microorganisms in Santa
Barbara, California
The recommendations of the Committee were published and signed by prominent
photobiologists from various countries (e.g., B. Diehn, M. Feinleib, W. Haupt, E. Hildebrand,
F. Lenci, W. Nultsch). The committee's recommendations follow and are presented in detail.
There are three main nomenclature sections: 1. Stimuli; 2. Responses; and 3. Behav-
ioural consequences. The first section defines a stimulus as any quantity of energy or matter
which, when interacting with the organism, can elicit a response. The nature of the stimulus is
indicated using a prefix as in photo- (stimulus is a radiant energy affecting specific receptor
molecules), thermo- (thermal energy), galvano- (ionic electric current), electro- (electrical
field), geo- or gravi- (gravitational force), mechano- (mechanical force), magneto- (magnetic
field), chemo- (molecular species acting upon specific receptor molecules). The
recommendations were in agreement with the requirements of formal logic (since nothing can
be said against them) and were considered to be generally accepted [Burr, 1984; Massjuk and
Posudin, 1991a]. The authors were cognizant of the nature of the stimulus since some
physical factors (e.g. radiation or electrical energy) are capable of generating chemical sub-
16
stances that may act as a direct stimuli. They also proposed distinguishing the dependence of
a response upon an increase ("step-up") or decrease ("step-down") in the stimulus intensity.
This section is devoted to the definition of 15as any stimulus-induced alteration in the
activity of the organism's motor apparatus which may (but not always) result in an alteration
in the movement or orientation of the organisms [Diehn et al., 1977].
The responses can be classified as:
"Kineses" - responses in which the steady-state rate of activity of the organism is
controlled by the absolute magnitude of the stimulus intensity. In positive kinesis the activity
rate is greater in the presence of the stimulus than in its absence; in negative kinesis the activ-
ity rate is lower in the presence of stimulus than in its absence.
"Phobic responses" denote the transient alteration in the activity of the organism that
is caused by a change in stimulus intensity. The authors delineate the types of response levels
in the organism: active but not due to the activity of the motor apparatus of the organism
[Diehn et al., 1977]. Therefore, the defmition that was proposed by the Committee for the
term "response" does not correspond exactly to this meaning. They proposed distinguishing
two main types of the responses: steady-state (kineses) and transient (phobic responses)
[Diehn et aI., 1977; Diehn, 1979]. Both types of responses are responsible for a change in the
linear velocity and the direction of movement. The former is controlled by the absolute
magnitude of the stimulus intensity, while the latter is controlled by a temporal stimulus
gradient. Interpretation of these terms changed when compared to the primary variant
[Engelmann, 1882]. In addition, according to Diehn et al. (1977), phobic responses that have a
long adaptation time are nearly indistinguishable from kinesis. In such cases the choice of one
or another term depends on the investigator. Therefore the response classification suggested
by the Committee (kinesis and phobic responses) lacks a common efficient basis for division
from the standpoint offormallogic.
The classification of responses according to the presence or absence of adaptation did
not suit the zoologists [Burr, 1984] whom found that it accounts for only one of the light
stimulus parameters - its intensity (absolute magnitude or temporal gradient) but ignores
other parameters such as spectral composition, polarization, etc. The fact is that the same
authors use terms such as "action spectrum of photokinesis" in their other publications
[Nultsch, 1973; Nultsch and Hader, 1979; Colombetti et al., 1982], testifies to the dependence
of movement upon the wavelength of the light stimulus.
Unfortunately, the term "behavioral consequences" is not defined [Diehn et al., 1977].
Therefore, it is not clear which criterion is used as the basis for the separation of "responses"
and "behavioral consequences". The authors considered there to be two types of "conse-
quences". The first concerns single cells when their movement is oriented relative to the
stimulus direction; the second applies to populations of cells that accumulate in the region
with the higher intensity of the stimulus or their dispersal from it. In other words, the response
of an organism affected by the stimulus must be considered at individual (microeffect) or
aggregation (macroeffect) levels.
The following prefixes were proposed for determining the dependence of movement
parameters (of all kineses and phobic responses) on the action of the stimulus: ortho-, klino-,
stropho- and morpho- [Diehn, 1979]. Therefore orthokinesis is when the linear velocity of
movement is increased (positive orthokinesis) or decreases (negative orthokinesis); klinokine-
sis - the rate of random spatial alteration in the direction of movement; strophokinesis - the
frequency or amount of turning; and morphokinesis - the rate of alteration in shape or contour
of the microorganism. Each of these kineses is induced by a light stimulus.
Thus, according to Diehn et aI. (1977) and Diehn (1979), taxis are the result of a
response or of a series of responses by the motor apparatus rather than just a single response.
We have discussed earlier that it is undesirable to use a broad meaning for the term <<taxis» in
the narrow interpretation of organism photoorientation.
17
In spite of the fact that the publication [Diehn et al., 1977] was the result of the collec-
tive thinking of the entire Committee, only one of its member [Diehn, 1979] actually followed
the terminology recommendations. The other authors [Feinleib, 1978, 1980; Lenci,
andColombetti, 1978; Nultsch and Hader, 1979; Nultsch, 1980; Lenci, 1982; Colombetti and
Lenci, 1982; Haupt, 1983; Lenci et al., 1984] accepted the classification previously suggested
by Nultsch (1975) as did other photobiologists (see, e.g. [Sineschekov and Litvin, 1982] ).
This classification was presented in detail in the review by Nultsch and Hader (1979) in which
the division of "responses" and "behavioural consequences" proposed by the Committee was
not accepted. They adopted three types of responses for freely motile organisms [Nultsch and
Hader, 1979]:
18
light stimulus (e.g., appearance or disappearance of the light, changes in its spectral com-
position, intensity or direction).
From our perspective, the term light-dependent behaviour of freely motile organisms
has a broader meaning. It includes various light-induced displacements of freely motile
microorganisms in space, independent of changes in the light stimulus over time (a meaning
that is equivalent to the term "phototaxis" in its original sense). Thus, according to our
interpretation the terms light-dependent movement behaviour of freely motile organisms
(phototaxis) and photoreactions have compatible but not identical meanings, the former is
subordinating and the latter subordinate.
Light-induced motile behaviour of biological objects is a broad physical phenomenon-
movement. Some types of movement can be described by traditional physical parameters such
as velocity (0), direction (1') and trajectory (l). These can be subdivided into other, less
general parameters for a more detailed characterization of the various types of movement. For
example, velocity may be linear or angular. Both linear and angular velocities may be
constant or may vary as a function of time. Certain classes of linear and angular velocity may
also be divided according to specific limits in velocity inherent to a particular organism or
group of organisms. Direction of propagation can be oriented or not oriented relative to the
source of light and the orientation can be positive (toward the source of light) or negative
(away from the source of light). Likewise, trajectory of movement can be rectilinear, curvilin-
ear, parabolic, zigzag, helical, or circular (around its own longitudinal or transverse axis or
around an external axis).
According to the previous assessments, the light stimulus therefore is characterized by
parameters such as intensity (l), direction (S ), spectral composition (A.), polarization (P), and
duration and frequency of light pulses. Similar to movement parameters, light parameters may
be divided into the intensity of light that is characterized by absolute magnitude (l) and its
gradient in space (dI/dx) and time (dI/dt).
According to the parametrical character of either the movement of organisms or a light
stimulus, we propose the classification of phototaxes (the dependence of the organisms
movement on a light stimulus) on the basis of the parametrical principle for both individual
freely motile organisms and populations of organisms as is presented schematically in Tables
2.1 and 2.2. Table 2.1 demonstrates the phototaxis of individual freely motile organisms (i.e.,
individual or microeffect), Table 2.2 the phototaxis of populations of organisms (i.e., group or
macroeffect).
The well-known dependences are shown in each case, e.g. 0 (I) is the dependence of
linear velocity of a single cell on the absolute magnitude of intensity of the light stimulus (or
photokinesis according to [Nultsch and Hader, 1979] or ortokinesis according to [Fraenkel
and Gunn,196l]); n(I) is the dependence of angular velocity of movement (frequency of
random rotations) on the absolute intensity of the light stimulus (klinokinesis) [Fraenk:el and
Gunn,1961]; ;(8) is the dependence of movement direction on the direction of the light
stimulus (or phototaxis according to [Diehn et al., 1977]); l(dI/dt) is the dependence of
movement on temporal changes in the light stimulus intensity (or photophobic response
according to [Diehn et al.,1977]).
19
Table 2.1. Photoresponses of individual organisms
..
individuals:
linear a
angular n
a (I)
n(I)
a (d/ldt)
n(dIldt)
a (dIldx)
n(d/ldx)
a (S)
n(s)
a (A)
n(A)
a (P)
n(p)
(frequency of
spatial changes
of trajectory of
individuals:
oscillations,
rotations,
trembling)
Direction of i' (I) i' (dIldt) i' (dIldx) i' (s) i' (A.) i' (P)
movement of
individual
i'
Trajectory of I(I) l(dIldJ) I(d/ldx) I(s) I (A.) I(P)
movement of
individual
I
Frequency of n(I) n(dIldt) n(dIldx) n(s) n(A.) n(p)
spatial changes
in trajectory of
individual
(oscillations,
trembling,
rotations)
n
We propose based upon the preceding analysis the following tenninology [Massjuk et ai.,
1988; Massjuk and Posudin, 1991a; Massjuk et ai., 1991; Massjuk and Posudin, 2002]:
Photomovement - movement or change in movement of organisms induced by light.
Phototaxis - any movement or change in character of the movement of freely motile
(not fIxed) organisms, not obligatorily oriented relative to light source.
20
Table 2.2. Photoresponses of populations and colonies of organisms
b
Intensity Gradients of intensity Direction Wavelength Polariza-
.gbt stimulus tion
Photocontro/ ofmovement - the entire complex of elementary processes that are induced by a
light stimulus, particularly photoreception, primary reactions of photoreceptor pigments, sen-
sory transduction of the light stimulus into a physiological signal(s) that governs through the
function of the motor apparatus, photoorientation, and velocity of movement of organisms.
Photokinesis - dependence of the velocity of movement of an individual organism and
their groups on any parameter of the light stimulus.
Phototopotaxis - dependence of the direction of the movement of an individual
organism and their groups on any parameter of a light stimulus.
The dependences that are included in Tables 2.1 and 2.2 may be direct or indirect,
positive or negative, though the forms of the dependence may not always be evident. Like-
wise, it is possible to further divide these terms into sub-classes. In particular, photo-
ortokinesis (the dependence of the linear velocity of movement on the parameters of the light
stimulus) andphotoklinokinesis (dependence of the quantity of rotations per unit of the time
on parameters of the light stimulus) can be distinguished. Some dependences have not yet
been given a specific tenn, e.g., the trajectory of movement, the concentration of organisms,
the shape of "ensemble", the relative number of organisms, that undergo the photoreactions
mediated by a light stimulus (see Tables 2.1, 2.2). A number of the light stimulus parameters
may also be further categorized (e.g., rhythm of the light stimulus, its constant or variable
21
character) or the possible interaction(s) of various parameters (e.g. wavelength and intensity,
change of velocity and direction of propagation).
Thus, the parametrical principles of classification of light-induced behaviour for
organisms we propose not only places in the appropriate order the available terminology and
the data of interaction of the light stimulus with the specific features of movement of living
organisms, but also forecasts and allows for characterizing peculiarities and facilitates the
development of a program of additional research needed for the completion of the
classification utilizing strict basic logic. This principle may also be applied to different types
of taxes induced by other physical factors. With regard to photoreactions, they may be
expressed through: 1. a change in the velocity (kinetic reaction); 2. a change in the direction
and trajectory of movement (vector reaction) and 3. a simultaneous change in the velocity and
direction of movement (photophobic reaction). Thus we distinguish such meanings as
photomovement and control of movement, and within the limits of photomovement -
phototaxes (in the original broad sense) and phototropisms. The parametrical principle is
proposed for the classification of the light-induced behaviour of single cells as well as for
populations of organisms. The broader meaning of ''phototaxis'' involves the term subordinate
to it - "photoreaction", that consists of three phenomelogical types: kinetic, vector and
phobic.
2.3. Summary
A critical assessment of the terminology and classification of different types of light-induced
behavior in freely motile organisms has shown considerable diversity and ambiguity in the
existing classification systems that has led to confusion. We present a new parametrical classi-
fication of light-dependent behavior for either individual motile cells (individual effect or micro-
effect, Table 2.1) or their aggregations (group effect or macroeffect, Table 2.2). The original
meaning of the term phototaxis as any light-induced movement of freely motile organisms in
space is retained. Light-dependent reactions of motile organisms (photoresponse, photoreac-
tion) indicate any immediate motion responses of the organisms to any change in the light sti-
mulus.
Mntility of biological objects represents a special case in the general physical phenome-
non of movement (mobility). Therefore, motility of organisms can be described by traditional
parameters such as velocity (0 ), direction (r ) and trajectory (l) of movement. The light sti-
mulus, in turn, is characterized by such parameters as intensity (1), direction (s), spectral com-
position (A.), and polarization (P) and the duration, frequency, and shape of light pulses. Consi-
dering the parametrical characteristics of both factors (light and movement), we believe that any
classification of the dependence of microorganism movement (phototaxis) on light should be
based on parametrical principles (Tables 2.1, 2.2). We propose photokinesis to be any depen-
dence of velocity of individual organisms or their groups on any parameters of the light stimu-
lus, and phototopotaxis as any dependence of the direction of movement of individual organ-
isms or their groups on any parameters of the light [Massjuk et al., 1988; Massjuk and Posudin,
1991a;2002].
The proposed classification can be further developed by accounting for additional para-
meters of movement and light (e.g., the rhythm of a light flux), their possible interactions (e.g.,
wavelength and intensity of the light, velocity and direction of movement), or specific features of
some parameters (e.g., the velocity of movement can be linear or angular, the light intensity can
be characterized by its absolute value (1) or its gradient in space (dI/dx) and time (dI/dt». The
suggested principles not only promote further improvement of the existing terminology but also
identify the direction of future research.
22
Chapter 3
Investigations with Species of Dunaliella Teod.
Fig.3.1. Title-page oflbe article ofE.C. Teodoresco (1905) where he described Dunaliella
Based on comprehensive studies, the genus Dunaliella was placed in the Familia Dunaliella-
ceae, Order Dunaliallales, Class Chlorophyceae, Division Chlorophyta, Regnum Viridiplan-
tae. The genus Dunaliella has seventy or more species and 30 specific and intraspecific
names, some appearing nomen nudum and are definitely synonyms. Dunaliella cordata
Pascher and Jagoda, deemed distinctly different from the other members of the Dunaliella,
was determined to be a separate genus, PapenjUssiomonas Desikachari. Currently the genus
Dunaliella includes 28 species with 33 intraspecific taxons (Table 3.1(
24
Table 3.1. Intraspeeific taxons of Duna/iel/a Teod. (continued)
Taxon Size of cells Basionyms, synonyms
(lengthxwidth, 1=)
Sub!!C!!!!S Dunaliel/a
25
The original and subsequent scientific literature on the genus Dunaliella are summarized in a
monograph by Nadia Massiuk ["Morphology, Systematic, Ecology, Geographical Distribu-
tion of the Genus Dunaliella Teod. and Perspectives of its Practical Utilization", Kiev,
Naukova Dumka, 1973 (in Russian)].
The total extent, phylogenetic relations and position of the genus in the taxonomy of
green algae was discussed, the main tendencies of intra-specific evolution indicated, and an
original system for the classification of the species within the genus proposed. Problems deal-
ing with the extent of species and principles of intra-specific systematics for algae are consid-
ered as well as determinant tables and criteria for the identification of Dunaliella species were
proposed. The potential for the commercial utilization of representatives of the genus Du-
naliella as fish forage and as a source of raw material containing carotene was described.
Classical comparative-morphological, biochemical, physiological, ecological, geographic and
biological criteria were widely used for the first time. The investigations were conducted at
the population level and used variational statistics.
N. n. M/1ClOK
MOP~)OJlO,f151,
01CTEMAH1f(A,
3KOJlO,V151,
,EO,PA¢V1'-iECKOE
PACnpOCTPAHEHV1E
POllA
DUNALIELLA TE 0 D.
VI 11EPcnEKTVlBbi Ero
nPAKTW-IECKOrO
VlCnOJlb30BAHV1~
Fig. 3.3. Title-page of the monograph by Nadia Massiuk entitled "Morphology, Systematic. Ecology, Geographi-
cal Distribution of the Genus Dunaliella Teod. and Perspectives of its Practical Utilization" (Kiev, Naukova
Dumka, 1973).
26
nalie//a: Biodiversity, Physiology, Genomics and Biotechnology" (A.Ben-Amotz, J. Polle, S.
Rao, eds.), was also recently published (2009).
Additional information on the alga can be found in reviews by M. Ginzburg ["Du-
nalie//a: a green alga adapted to salt" (1987)], M Borowitzka [''The mass culture of
Dunalie//a salina" (1990)], A. Borovkov ["Green microalga Dunalie//a salina Teod.
(Review)" (2005)], and A. Oren ["A hundred years of Dunalie//a research: 1905-2005"
(2005)].
Dunaliella:
Physiology,
Biochemistry, and
Biotechnology
MOI'rlhln Avron
UJ~C",,")
l);l'aMrn...' """ll(mllll)
1 ~\~I/IIUIIl' In''''''''"';4' 5.l:onIU
11" ".,:. bod
Ami B('ll-AmC)17,
.',l .. _t1~&.lr",~
:-;~h .,,1 I _I' ...~"" n......'o6I",p",
1....1 n..,"" l'iI[JblL -.! Um""'7:..d R ~
....,f.. lind
nH p(""
\.1> .. rl"lf I "".lian
-
TIII.~"
Fig. 3.4. Title-page of multi-author review "Dunaliella: Physiology, Biochemistry, and Biotechnology"(Mordhay
Avron and Ami Ben-Amotz, edo.) CRC Press, 1992.
27
The front of the cell has papilla where two isocontic, isomorphic and isodynamic flagella are
found. The flagella display smooth ciliary the beating of which results in both translational
and rotational (clockwise and counter clockwise) movement of the cell.
The basal body of Duna/iella spp. are interconnected by distal striated fiber. It is
thought that these connecting fibers may be involved in coordinating the flagellar beating
[Mellronian and Preisig, 1984]. A system of the flagellar roots (of the 4-2-4-2 type) is attached
to each basal body. The roots act as a cytoskeleton providing a regular framework around the
cell and conferring uniform distribution of the mechanical strain caused by flagellar beating
[Mellronian and Preisig, 1984].
According to Melkonian (1978) the flagellar roots determine the precise location of
the cellular organelles, e.g. the eyespot's (stigma) position is relative to the flagellar appara-
tus. The stigma is located in the anterior peripherical portion of the chloroplast. The stigma in
D. viridis is large, distinct, and bright-red coloured, while the stigma of D. salina is diffuse,
pale-red coloured and barely visible.
The species of Duna/iella described herein are distinguished by differences in the cell
shape and size. The cells of D. salina are 5 to 29 ~ in length and 4 to 20 IJlII wide, while D.
viridis is 3 to 18 J.1l11 in length and 2 to 15 IJlII wide [Massjuk, 1973]. The length of the fla-
gella of D. salina is approximately equal to the length of the cell, while in D. viridis flagella
are 1,3 times longer than the cell (Fig. 3.5).
I
I
I
I
J
J
a b
Fig.3.5. General schematic of two species of Dunaliel1a: a-D. salina; b - D. viridis where: 1 - stigma;
2 - flagella; 3 - chloroplast; 4 - starch; j - pyrenoid (after Posudin et aI., 1988).
28
An image of Dunaliella sp. from an electron microscope is given in Photograph 3.1.
Photograph 3.1. An image of Dunaliella sp. from an electron microscope. Courtesy of Prof. Shogo Nakamura
(Toyama University, Japan)
Species such as D. salina can accumulate certain oils in the stroma of the chloroplasts in
which carotenoids, primarily ~-carotene, are soluble giving a red coloration. Red forms of D.
salina are prevalent in the salt brines on the Crimea in southern Ukraine. However, if the salt
concentration in the brine decreases, under the low illuminance, the red cells quickly lose the
carotene and tum from red to green in color.
29
Chapter 4
Investigation of Photomovement in Dunaliella
Fig. 4.L A schematic of experimental videomicrography for studying photomovement in algae: 1 - source of
white light; 2 - collimator; 3 - glass infrared filter; 4 - liquid infrared filter; 5 - interference filter; 6 - sample in
a concave slide; 7 - halogen lamp; 8 - monochromator; 9 - source of white light; 10- condenser; 11 - polarizer;
12 - microscope; 13 - videocamera; 14 - coupling unit; 15 - monitor [Posudin et al. 1992, I 996,a].
Radiation from a white light (300 W filament lamp) source 1 passes through a collimator 2,
glass infrared filter 3, liquid infrared filter 4 (a 1 cm deep layer of distilled water), and to re-
cord the light curves, through an interference filter 5. The light is directed at an angle of 30°
to the slide plane, thus illuminating the algal suspension on a concave slide 6. Special atten-
tion was paid to avoid air bubbles under the glass cover slip in that they may cause chemotac-
tic reactions by the cells to oxygen. The spectral dependence of phototopotaxis was studied
using the halogen lamp 7, with the radiation passed through a monochromator 8. To study the
effect of the polarized light, radiation from the light source 9 was directed through a con-
denser 10 and a polarizer 11 before striking the sample. A xenon lamp (2 kW) was employed
as the source for high light intensities. Photomovement parameters (e.g., linear and rotational
velocity, number of cells moving toward or away from the light source, motility of the cells)
modulated by light stimulus parameters (e.g., intensity, polarization, spectral composition)
32
270 0
Fig. 4.2. Geometry of the interaction of light directed at an angle 000" to a slide plane containing algal cells that
results in a change in the angu\at distribution of the moving cells. M is the number of cells in i-sector; a, is the
angle that the sector forms with the projected light in the direction of the slide plane.
Several statistical methods were used for assessing the distribution of the moving cells (e.g.,
Rayleigh test, V-test method, i-criterion, method of moments) that permitted determinating
the primary direction of movement and estimating phototopotaxis quantitatively [Mardia,
1972; Batschelet, 1981; Hlider et aI., 1981]. Thus, the angular distribution of cells moving due
to the effect of light could be estimated quantitatively using vector R, whose components
were the mean values for the sum of sine and cosine of the measured angles ai, i.e R=
( ~ Lsina,;~ Lcosa, ), where N is the number of the cells measured. The vector is char-
N N
acterized by differing values for length r (in this case 0 < r < I) and angle 8that obey the fol-
lowing ratios [Hader et aI., 1981; Mardia, 1972]:
Lcosa,
arccos=------'--
8= N (4.1 )
R
If the stimulating light is absent, the angular distribution of the moving cells
should be isotropic in character; in this case r = 0, and angle 8 is not indefinable.
The statistical method utilized was based on the estimation of an estimate of vector r
that is called the Rayleigh test which provides a comparison of the value for z = Nl with a
certain tabular value of z,.. The Rayleigh test permits estimating the significance of the differ-
ence in the observed angular distribution of the cells with illumination versus isotropic. The
data obtained with the Rayleigh test were later used for a V-test that is preferable when there
is a dominant cell movement direction as indicated by the angle 80 . Using the V-test method,
33
it is possible to estimate the value of V = H Nrcos(O - 00). At V > 0, a positive phototopo-
taxis is observed, while at V < 0, a negative. The ability to determine the sign of phototopo-
taxis is the primary advantage of the V-test in comparison with the Rayleigh test.
We also used a method for analyzing the angular distribution, the %2_ criterion [Rokit-
sky, 1973; Batschelet, 1981]. In this case the entire polar diagram is divided into three (K = 3)
groups. The first group included cells moving within a sector of 60° toward the direction of
the stimulating light; the second group, also within 60°, indicates cells moving in the opposite
direction; and the third group includes the remainder of the cells. The % 2_ criterion is esti-
mated as follows:
2 ~ (0, _e,)2
% =£.., , (4.2 )
1=1 e,
where 0/ are the observable frequencies, e, are frequencies of the expected series, and K is the
number of the test groups. Under an isotropic distribution, the number of cells moving in the
above-mentioned directions would be 25, 25 and 50 %, respectively.
All the methods have the same disadvantage: it is impossible to estimate the bimodal-
ity of the angular distribution due to the effect of stimulating light. Objects, such as Du-
naliella, are characterised by the ability of different cells to move simultaneously toward and
away from the light source. The method ofmoments [Mardia et ai., 1972], in contrast, permits
determining the relative number Ji of cells moving toward and away from the light source
according to the equation:
34
quency harmonics with low amplitudes. Reversing the technique (Fourier synthesis) results in
a smoothed histogram. Fourier transform makes it possible to construct histograms devoid of
noise and thereby estimate real tendencies in the direction of movement of the microorganism
[Posudin et ai., 1991].
The microscope field of vision was divided into 48 angular (7.51) sectors on a polar
diagram and the trajectories of the moving cells were plotted into the appropriate sector on the
polyethylene film covering the monitor screen. The angle at between the trajectory of move-
ment of the cell and direction of the light stimulus was determined.
When restricted to a number of N = 7 for the Fourier transform of the angular distribu-
tion of Dunalie//a it resulted in a coarse histogram. A greater number increases the noise, sig-
nificantly distorting the shape of histogram [Posudin et ai., 1991b].
R(I).
IJ,
r.u.
Lt--f.
prnls
j 022
'l"lt"'i
50
...t.;···f-' r... ,f . a 18
0.14
40
3 () 0(,
30
007.
I I I I I I I I I I I I I
'0 100 J 50 250 550 1250 E.lx
2
0.14 02; 037 0.81 1.83 j,W/m
0.01 0.07
2
007 0.13 0.21 ()1{) 0.8 1.82 6/. \V/m
Fig. 4.3. Dependence of the linear velocity (!="s) mean of Dunaliella salina ( I ) and Dunaliella viridis
(2) on the intensity 1 (yI/m2 ) or illuminance E (Ix) of white non-polarised light (dotted deviations) and polarized
white light (solid deviations) and photokinetic reactions R(l) (relative units) of both species ( 3 ) on the change
in the intensity AI (W/m2) of the light. Axis of ordinate indicates: the linear velocity of cell movement (left);
photokinetic reaction (right). Axis of abscissa indicates the intensity 1 of light, illuminance E of the sample, and
change of the intensity AI. Vertical bars reprersent one standard error [Posudin et aI., 1988].
35
The illumination intensity that resulted in a maximum velocity for Dunaliella movement was
similar to the value reported for Euglena gracilis G.A. Klebs (illuminance 300 Ix) [Wolken
and Shin, 1958] but was lower that than at the optimum illumination for Anabaena variabilis
Kiitx. (1000 lx) [Nultsch, 1975].
Motile cells of cyanobacterium Synechocystis sp. also demonstrate photomovement in
response to a light stimulus.Under vertical irradiation, Synechocystis decreased the frequency
of the mean vectorial gliding speed depending upon the applied fluence rate, whereas the de-
viation distribution width of the speed increased. This strongly suggests the involvement of
photokinesis.The maximum photokinetic activity at 420 om and 680 om supports the continu-
ation hypothesis that the cyanobacteria's photokinesis is governed by the energy-generating
chlorophyll pigments since the action spectrum for photokinesis resembles the absorption
spectrum of chlorophyll [Chung Young-Ho et ai., 2004].
In general the cell movement velocity of the both Dunaliella species is 1 to 2 orders of
magnitude higher than that ofblue-green and red algae that do not possess a flagellate appara-
tus [Nultsch, 1980]. It is to a certain extent lower than in some species of Chlamydomonas
[Racey et ai., 1981] and E. gracilis [Jahn and Bovee, 1968; Haupt, 1959]. It is important to
note, however, that the flagellated algae display a velocity in the same order as flagellated
bacteria that have different flagella structure [Gromov, 1985] and use a different source of
energy for movement [Evtodienko, 1985] (Table 4.1).
36
Taxon Velocity of cell References
movement, jlIll!s
Chromatium okenii 46 ..
Pseudomonas aeruginosa 56 ..
Escherichia coli 16 ..
Bacillus licheniformis 21 ..
Sporosarcina urea 28 ..
We found that the cell movement velocity essentially does not change during measurement,
which lasts for several minutes per sample. At the same time, the cells respond rapidly to any
change in the intensity of the stimulating light's spectral composition.
A statistically significant variation in the linear movement velocity was found in both
species in response to irradiation with polarized versus non-polarised white light at the same
intensity (Fig. 4.3). This indicates a non-crystallite, non-dichroic photoreceptor in these spe-
cies [Posudin et aI., 1988].
The dependence of the velocity of movement of the two Dunaliel/a species on the
wavelength of the stimulating light was not established in our experiments. Likewise, there
has been contradictory data concerning the wavelength dependence of photokinesis in other
algae species. Some authors [Ascoli, 1975; Hader and Hader, 1989] explain such a depen-
dence in E. gracilis as being due to the participation of the photosynthetic pigments (chloro-
phyll band/or l3-carotene), however, there is experimental evidence that contradicts this hypo-
thesis. For example, there is a positive photokinetic reaction of Astasia longa Pringsheim
which is devoid of a photosynthetic apparatus [Mast, 1911]. Diehn (1973) reported a com-
plete immobilization of E. gracilis in the blue region of the electromagnetic spectrum. The
photokinetic reactions of Chlamydomonas have not been differentially established. Feinleib
and Carry [1967] confirmed the absence of these reactions, while [Nultsch and Throm [1975]
observed a positive photokinetic reaction in Chlamydomonas after prolonged dark adaptation.
It is not altogether clear what photoreceptors are responsible for the photokinetic reactions in
E. gracilis and Chlamydomonas, photosynthetic (as in prokaryotic organisms [Hader, 1979])
or specialized blue-light receptors. Quantitative estimation of the velocity of rotational
movement of the two Dunaliel/a species indicates that maximum velocities are 0.52 ± 0.04 S-1
for D. salina and 0.54 ± 0.04 S-1 for D. viridis at 20°C (Fig. 4.4).
The cells can rotate around either the longitudinal axis of the ellipsoid or along the
spiral trajectory. The maximum rotational velocities were obtained within the illumination
range of 150-550 lx. This is in sharp contrast to the frequency of cell body rotation of Chla-
mydomonas reinhardtii PA Dang.which is in the 1-5 Hz range [Yoshimura and Kamiya,
2001].
37
Fig. 4.4. Dependence ofthe velocity n of rotational movement of Dunaliella viridis ( -. - ) and D. sa/ina
( - 0 - ) on the illuminance E ofthe sample by white light.
4.2.2. Phototopotaxis
The primary objective of this portion of the investigation was to establish the quantitative
characteristics of phototopotaxis in the two Duna/iella species in response to the intensity and
spectral composition of the light stimulus using the statistical methods described previously.
Diagrams of the angular distribution for the Duna/iella species moving within the 12 sectors
in response to different levels of illumination are shown in Fig. 4.5a,b.
38
E = 0 Ix IxE=301x IxE=5001x E = 1500 Ix E = 40000 Ix
270~ + 0'
180
0
90~
11
Fig. 4.5. Diagrams of the angular distribution of two species of Dunaliella Teed. under different levels ofillu-
mination: a-D. salina; b - D. viridis. The direction of the stimulating light ranges 180° to 0°.
There is an absence of a light effect in an isotropic distribution of the moving cells while ani-
sotropy of the angular distribution is observed due to lateral light. Statistical analysis of the
data using various methods (Rayleigh test, V-test, I-criterion, method of moments) and the
angular distribution of moving cells are given in Table 4.2 for D. salina and in Table 4.3 for
D. viridis.
39
Table 4.2. Analysis using different statistical methods on the dependence of the angular distribution of moving
cells of Dunaliella salina on illumination intensity (NM - non-meaningful, M - meaningful differences in the
angular distribution from the anisotropic for the given level of significance p).
E,lx
Method of analy-
sis
O
I 30
I 500
N
I
1500
I
40000
Parameter z=N,J
Real value 0.85 1.59 10.73 0.75 44.56
Tabular value 2.99 4.57
(P=0.05 ) (P =0.01)
Probability level:
forP=0.05 NM NM M NM M
for P=O.OI NM NM M NM M
Method of
moments
Value of£...arame-
terp 0.50 0.63 0.76 0.60 0.71
40
Table 4.3. Analysis using different statistical methods on the dependence of the angular distribution of moving-
cells of Dunaliella viridis on illumination intensity (NM - non-meaningful, M - meaningful differ-
ences of the angular distribution from the anisotropic for the given level of significance P).
E,Ix
Method of 0 30 500 1500 40000
analysis I I I I
N
119 I 134 I 152 I 158 I 604
Parameters of Rayleigh test
vector Ii:
Parameter z=Nr'
forP=O.05 NM M M NM M
for P=O.OI NM NM M NM M
Values of parame- V-lest
ter V:
Method of
moments
Value of parame-
ter Ii 0.50 0.63 0.68 0.38 0.67
41
Our experiments allowed elucidating the dependence of angular distribution in the two spe-
cies on the intensity of the latera1light in the range 0--40,000 Ix (pH 6-7, 21 0q. Using the
Rayleigh test, it was possible to confirm that the angular distribution of the cells differed from
isotropic in response to lateral light at an illumination of E=500 Ix and 40,000 Ix for D. viri-
dis. At E=30 Ix the level of significance was P=O.OI, Table 4.3. Similar results also are ob-
tained using the V-test. The latter statistic proved to be more sensitive since it allowed analy-
sis of the anisotropic distribution of D. salina (P=0.05 at E=30 Ix, Table 4.2).
Comparison of the results between the two statistical tests (Rayleigh test and V-test)
confirmed that under laboratory conditions D. viridis begins to demonstrate its photoorienta-
tion ability at lower levels of illuminance (30 Ix) than D. salina, a response that is in accor-
dance with observations in nature [Massjuk, 1973]. The V-test's advantage is its ability to
distinguish positive (V> 0) from negative (V < 0) phototopotaxis [Mardia, 1972; Hilder, et
al., 1981]. Both species demonstrated a positive V-test for phototopotaxis at an illuminance of
30 and 500 Ix and negative phototopotaxis at 40,000 Ix (Fig. 4.6, Tables 4.2, 4.3). Isotropicity
of the angular distribution of the cells at higher (i.e., 1500 Ix) illumination may be explained
by the transition of certain cells to a negative phototopotaxis. Therefore the number of cells
moving toward and away from the light source was approximately equal.
It is necessary to note that the two species of Dunalie//a demonstrate different behav-
iours in the concentrated saline basins found in southern Ukraine where the illuminance of the
surface salt-water reaches 100000 Ix or more. D. viridis cells concentrate in the shadowy parts
near the bottom, while D. salina cells gathered near the surface where the illumination was
highest [MacIOK, 1973]. Differences in response between laboratory and natural populations
is no doubt due in part to the fact that the cells differ in color, i.e., red vs. green. The accumu-
lation of carotene in vegetative cells of D. salina under natural conditions increases consid-
erably their tolerance to high illuminance. The transition from positive to negative phototopo-
taxis was not observed in natural populations of D. salina in vivo even under an illuminance
intensity that exceeded by 100 times the threshold for the transition established under labora-
tory conditions [Massjuk,1973]. The unique behaviour of the red form of D. salina can be
explained not only by increased tolerance caused by the elevated synthesis of carotene but
also by the cells ability to accumulate a considerable amount of glycerol [Ben-Amotz et
al.,1982; Enhuber and Glinuner,1980; Wegrnann,1979; Posudin, Didyk, 2007]. Glycerol de-
creases the density of the cells, increasing their buoyancy. It also performs a protective func-
tion like carotene. The mechanisms that determine the difference in photomovement of red
cells of D. salina in natural settings needs further elucidation.
The application of i-criterion (Tables 4.2 and 4.3) confirmed the conclusions de-
rived from the Rayleigh test and V-test. The deviation of the real distribution of mobile cells
in both species from isotropic was observed under a lateral illumination of 500 Ix and 40,000
Ix. The analysis made it possible to estimate with a high level of probability (P=0,05 for D.
salina and P=O,OI for D. viridis) the relative quantity of cells moving in a given direction.
This additional information can be obtained via application of the method of moments that
permits estimation of the bimodality of angular distribution of the species due to lateral illu-
mination, i.e. relative number 11 of cells moving toward and away from the light source cor-
responds to the level of illuminance at 500 Ix and 10,000 Ix (Tables 4.2 and 4.3).
Application of these statistical methods made it possible to determine the dominant
directions during phototopotaxis, the level of the anisotropy of angular distribution of the
moving cells under lateral illuminance (Rayleigh te~t)i the sign of phototopotaxis (V-test), the
relative number of cells moving at given direction u; -criterion), and the bimodality of angu-
lar distribution of the cells (method of moments). It is necessary, therefore, to use all the sta-
42
tistical methods to adequately characterize the movement. The presence of differences in pho-
tomovement parameters between the two species and their dependence upon the external con-
ditions and the composition of aquatic medium indicates that such parameters can be used to
identify ecological peculiarities among species and as taxonomic indices [Posudin, 2007].
It is useful to compare photopotaxis of Dunaliella with other algae. The phototopo-
taxis maximum occurs at an illumination of 125 Ix in Ochromonas danica Pringsheim [Hader
et aI., 1981], 50 Ix in E. gracilis [Hader et aI., 1981], 50-200 Ix in Phormidium ambiguum
Gomont [Nultsch, 1962],200 Ix in Nitzschia communis [Nultsch, 1971], and 500-1000 Ix in
C. reinhardtii [Feinleib, 1974; Nultsch et aI., 1971]. l11umination at which the transition from
positive phototopotaxis to the negative occurs is over 125 Ix in Ochromonas [Hader et aI.,
1981],250 Ix in Euglena [Hader et aI., 1981], 103_104 Ix in Phormidium [Nultsch, 1962],
4000 Ix in Micrasterias denticulata Bn:bisson ex Ralfs [Neuscheler, 1967] and about 105 Ix in
Chlamydomonas [Nultsch et aI., 1971]. The red form of D. salina that is high in carotene and
glycerol, responds like Chlamydomonas displaying a high level of photoresistivity [Massjuk,
1973]. The diversity in photomovement characteristics among various algae species could be
due to differences in experimental conditions, faulty experimental methodology, or actual
biological differences among the taxa and strains studied. Based on the data obtained, D. viri-
dis is more sensitive to the influence of weak light than D. salina, a result that concurs with
our observations in nature [Massjuk, 1973].
At very high illumination (ca. 40,000 Ix) the cells of both Dunaliella species demon-
strate a negative phototopotaxis. The action spectrum of phototopotaxis F(A) of D. salina and
D. viridis (Fig. 4.6) falls within 400-520 nm and is characterized by two maxima - at 410-415
nm and 465-475 nm.
F,r.u.
40 - C
o
30 -
o o
20 -
o 0
10 -
00 0
I I I I I
400 420 440 460 480 500 A, run
Fig. 4.6. Phototopotaxis action spectrum for two species of Dunaliella Teod. [Posudin et al. 1991].
A similar spectral interval for the phototopotaxis action spectrum in D. salina (400-520 nm)
was observed by other authors [Wayne et aI., 1991]. They determined that the action spectrum
for phototaxis had a maximum at 450-460 nm and proposed that carotenoproteins or rhodop-
sins acted as the photoreceptor pigments. The different spectral positions of two maxima (i.e.,
near 460 nm and 520 nm) can be explained by differences in the alga strain tested and/or or
differences in salt condition within the medium [Wayne et aI., 1991].
43
In the 400-550 nm range, species of Peridinium, Gonyaulax. Platymonas, Stephanop-
tera [Halldal, 1958], Cryptomonas [Watanabe and Furuya, 1974], and Chlamydomonas
[Nultsch, 1971] have been shown to orient themselves relative to the direction of the light. In
the green alga Ochromonas danica, both green and bleached cells accumulate under blue
light, though only green cells do so under red light [Di Pasquale et aI., 1980].
0'
b
40
20
1
90' II"IIIIII!III!IIIIIII,I
n
10 20
0°
E/~E2~
N=7
°Ix d
J60~
270
180
90
270° - - - - ' r - - f - - - . L , - 90' n
10 20
180'
Fig. 4.7. Fourier-transfonn of the angular distribution of motile cells ofDunaliella Teed. in the absence ofa
light stimulus (E = 0), where: a - real histograms of angular distribution; b - amplitudes of harmonics ( A ); c -
histograms of an inverse Fourier-transfonn for the first 7 harmonics; d - the phases of harmonics; n - number of
sectors in the angular distribution [Posudin et aI., 1991].
44
If the light stimulus (E = 500 Ix) is increased, the histogram of angular distribution indicates a
prominent maxima in the real distribution of moving cells (Fig. 4.8a). It is therefore possible
to distinguish the first and second harmonics (Fig. 4.8b). The phases of the harmonics indi-
cate the cell movement in a direction of 2700 with the population more oriented toward the
source of light (Fig. 4.8c). The inverse Fourier transform makes it possible to observe the
primary movement of the cells in a smoothed histogram indicating a positive phototopotaxis
for Dunalie//a (Fig. 4.&1).
a 0°
~2700 _ 9 00
10 20
180 0
0° d
90
n
10 20
0
180
Fig.4.8. Fourier-transfonn of the angular distribution of motile cells of Dunaliella Teod. at an illuminance E of
500 Ix [posudin et aI., 1991].
Increasing the illuminance up to 40,000 Ix resulted in the appearance of at least five intense
maxima in the distribution (Fig. 4.9a). It is possible to distinguish the first four harmonics
(Fig. 4.9b) which have phases close to zero (Fig. 4.9c). The smoothed histogram for these
harmonics indicates a negative phototopotaxis for Dunaliella (Fig. 4.9d).
Fourier transform establishes the dependence of angular distribution of the cells on the
level of lateral illuminance by white light. The advantage of this method is the ability to con-
struct histograms for the angular distribution of the cells that are deprived the random factors
and to estimate the dominant tendencies in the direction of movement of the organisms in
response to the light stimulus at varying intensities.
45
1:-;0
1)'
0" F! 40.0001:-;
\-7
II
10 20
ISO"
1)'
Fig. 4.9. Fourier-transfonn of the angular distribution of motile cells of Dunaliella Tood. at an illuminance E of
40,000 Ix.
4.3. Summary
Species of Dunalie/la, as well as other flagellate algae move freely in an aquatic environ-
ment in response to light, Le. photomovement (phototaxis). The species are capable of photo-
kinetic and photovector reactions, even though photokinetic reactions in Chlamydomonas are
challenged in the literature. We did not observe photophobic reactions in Dunaliella (in con-
trast to Chlamydomonas and Euglena), though previous reports suggest the possibility of such
reactions [Wayne et al., 1991].
An average velocity of cell translational movement in Dunaliella, both of which are
hyperhalobic species, was 36 ± 2 ~s (D. viridis) and 48 ± 2 I-lrn/s (D. salina). The average
velocity of movement of the two species varied over a wide range, thus eliminating the possibili-
ty of making distinctions between the species using the parameter. The modal value for the
average velocity of movement of Dunaliella bioculata Butcher was 105 ± 5 ~s. These val-
ues exceed by 1-3 orders of magnitude those in microorganisms not possessing a flagellar appa-
ratus and are within the limits known for others flagellates, both prokaryotic and eukaryotic.
However, the average velocity of movement of both species of Dunaliella was lower
(sometimes by an order of magnitude) than those in the marine species, D. bioculata and the
freshwater species C. reinhardtii, and E. gracilis. These differences are most likely caused by
differences in viscosity of the media.
The average velocity values for rotary movement of cells in the Dunaliella species
were nearly identical Le., 0.52±0.04 rotations (revolutions) per second in D. salina and 0.54±0.04
rotations per second in D. viridis. However, these values are lower than in the freshwater species
C. reinhardtii.
46
The maximum values for the average velocity of linear and rotary photomovement in
Dunaliella cells were observed within the limits of the white-light intensity (0.22-0.81
W/m2), illumination (150-550 Ix), temperature (20-30°C), and pH (8). No statistically sig-
nificant variation in linear movement velocity was found in either species due to irradiation
with polarized light versus non-polarized white light of the same intensity (Fig. 4.3). This
suggests a non-crystallite non-dichroic photoreceptor in the species [Posudin et ai., 1988].
Phototopotaxis in the two hyperhalobic species of Dunaliella under laboratory condi-
tions was observed at an illuminance of 500 Ix (positive) and 40,000 lx (negative). The tran-
sition from positive to negative phototopotaxis occurred at 1,500 lx. These parameters are within
the limits known for other algae species. However, sensitivity thresholds to weak and strong il-
luminance and transitions from positive to negative phototopotaxis differed substantially among
alga species. This allows assessing shade-tolerance, sun-tolerance, and resistance to high light
exposure among species.
Dunaliella viridis is more sensitive to weak light (30 lx) than D. salina, a finding that
corresponds to behavioral peculiarities between the two species in nature. In laboratory cul-
tures both species were more sensitive to high illuminance than C. reinhardtii which has a tran-
sition to negative phototopotaxis at 100,000 lx.
The transition from positive to negative phototopotaxis in the Dunaliella species differs
from chlamydomonads. Unlike chlamydomonads, the change in flagellar beating from ciliary to
undulate mode was not observed in either species. The beating of only one flagellum was
observed which caused a turning of the cell and movement in the direction opposite to the di-
rection of the light source.
In Crimean hyperhaline watersheds under high illuminance (>100000 lx), natural popu-
lations of the red form displayed a complete absence of negative phototopotaxis. Thus, hyperha-
lobic species (D. salina and D. viridis) differ in their sensitivity to both high- and low-light
intensities explaining in part differences in the ecological niches occupied by the species.
The action spectrum for phototopotaxis was identical for the two Dunaliella species. It
is between 400-520 nm and has two maxima: at 410-415 nm and 465-475 nm. The photo-
topotaxis spectrum for Dunaliella differs somewhat from those of C. reinhardtii and Haemato-
coccus pluvialis Flotow that display a wide band in the 400-600 nm range and a maximum at
500 nm. This indicates differences in their photoreceptor systems and the composition of their
photoreceptor pigments.
47
Chapter 5
Effect of Abiotic Factors on Photomovement
Parameters of Dunaliella
Motile microorganisms are exposed to the influence of a number of abiotic factors such as
mechanical (mechanical shocks, hydrostatic pressure), gravitational, thermal, electromagnetic
(ultraviolet, visible, infrared, and microwave radiation), electrical and magnetic fields, and
ionizing radiation. They are also influenced by the chemical, gas and ion composition and the
pH of the aquatic media, biogenous elements and other organisms, each of which can affect
their photomovement responses [Jahn and Bovee, 1968; Marbach and Mayer, 1970; Kritsky,
1982; Sineschekov and Litvin, 1982; Colombetti et aI., 1982]. The effects of light on the pho-
tomovement parameters of two species of Dunaliella were described in the Chapter 4.
Motile microorganisms respond to various abiotic factors in their environment gravi-
tating toward conditions that enhance their survival and population growth. Thermal [Gimm-
ler et aI., 1978; Lynch, 1984; Lynch et al., 1984; Poff, 1985; Norman and Thompson, 1985;
Yang, 1988; Ramazanov et al., 1988; Ben-Amotz, 1996; Krol et aI., 1997] and chemical
[Berg, 1985] gradients, gravitational [Hllder,1987b], electrical [Mast, 1911; Hllder, 1977] and
magnetic [Esquivel and de Barros, 1986; Yamaoka et aI., 1992] fields, solar radiation
[Nultsch and Hllder,1988; Richter et al., 2007], and ionizing radiation [Saraiva, 1972] have all
been shown to modulate their behavior. Several articles have elucidated the effect of multiple
abiotic factors on the physiology and behaviour of algae [Mil'ko, 1963; TImenez and Niell,
1991; Thakur and Kumar, 1998b; Gomez and Gonzalez, 2005; Zhang et al., 2006].
The effects of abiotic factors such as temperature, electrical fields, medium pH, and
ultraviolet and ionizing radiation, as well as the influence of physical factors (i.e., optical ra-
diation, temperature, electrical fields) on the photomovement parameters of two species of
Dunaliella are discussed in this Chapter.
YY--~---<-f
50
40 I
0,20
f'J/ - ',:,1
1
0,16
30
f " 0,12
0,08
0,04
° 10 16 20 25 30 35 °
I, DC
° 4 9 14 19 LIt, DC
Fig. S.L Dependence of mean linear velocity v ofD. salina ( I ) and D. viridis ( 2 ) on the temperature t and
kinetic reactions R (t) in both species (3 ) on the change of temperature LIt [posudin et aI., 1988].
The dependence of linear cell movement velocity on temperature can be in part explained by a
decrease in the viscosity of medium with increasing temperature between 16 to 25 °e and a
progressive inhibition of the flagellar apparatus between 25 and 35 °e. It is important to note
that the temperature within this range did not affect phototopotaxis.
50
strates the inhibition of phototopotaxis in D. salina when the electrical field was switched on
and its recovery 2 minutes after the electrical field was switched off.
0°
270° 90'
Fig. 5.2. Effect of an external electric field of 20 V/crn applied to the algal suspension on the angular distribution
of the cells and intensity of phototopotaxis of D. salina: I - field is switched on; and 2 - field is switched off.
Arrows indicate the direction ofpropagation of the light stimulus (illuminance 500 Ix) [Posudin et al., 1991].
51
light by the photoreceptor molecules is accompanied by their excitation and conformational
alterations in the photoreceptor proteins. This leads to opening of calcium channels in the
plasma membrane of the stigma and corresponding increase in the flow of calcium ions into
the cell. The plasma membrane is depolarised locally resulting in a corresponding opening of
calcium channels in the flagellar membrane, increasing the flow of calcium ions into the flag-
ellar axoneme increasing flagella beating.
Our experimental results and those described in the literature support the hypothesis
that the inhibition of phototopotaxis in green algae by an external electrical field is due to one
of two phenomena - phototopotaxis and galvanotaxis, as was observed in Chlamydomonas
[Marbach and Mayer, 1971; Nu1tsch and Hlider, 1979; Dolle and Nultsch, 1988] or the par-
ticipation of light induced changes in membrane potential during photomovement was dem-
onstrated in Haematococcus [Litvin et al., 1978; Sineshchekov and Litvin, 1982]. It is evident
that photomovement in microorganisms is affected by external electrical fields and the same
relationship between photoresponse and changes of photoinduced potentials have been found
in bacteria and protozoa.
Our data on the inhibition of phototopotaxis in Dunaliella by external electrical fields
and those described for Chlamydomonas [Marbach and Mayer, 1971; Dolle and Nultsch,
1988] and Haematococcus [Litvin et al., 1978; Sineshchekov and Litvin, 1982] support the
idea of the participation of an electrical potential (in particular, an action potential that ap-
pears as a response to the light stimulus) occurring during the photoregulation of algal move-
ment. The application of an external electrical field disturbs the propagation of the potential
from the receptor to the flagellar apparatus causing an inhibition of phototopotaxis.
5.3. Effect of pH
The effect of the concentration of hydrogen (IT) ions in an aquatic medium, measured in pH
units, is a critical environmental factor influencing the viability of organisms. There is a
wealth of literature on the effect of medium pH on growth, development, reproduction, bio-
mass production of algae [Massjuk and Yurchenko, 1962; Weggmann, 1968; Weggmann and
Metzner, 1971; Malis-Arad et al., 1980; Rao, et al., 1982; Goldman et aI., 1982 a,b; Ghasi, et
aI., 1983; De Busk and Ryther, 1984; Lukas et aI., 1986; Gimmler and Weis, 1992; Lustigman
et aI., 1995; Thakur and Kumar, 1998; Thakur et al., 2000] and their distribution in nature
[Massjuk, 1973; Lopez-Archilla and Amils, 1999; Lopez-Archilla et al., 2001; Topics...,
http//www.bio.uni-potsdam.de/oeksys/fsvte.htm]. This is especially so for algae living in
highly acidic environments [Gross, 2000]). Changes in ultrastructure [Ma et al., 1999], bio-
mass and pigment synthesis [Ghazi et al.,1983; Celekli and Doenmez, 2000], photosynthetic
rate [Wegmann, 1968; Wegmann and Metzner, 1971; Gimmler and Weis, 1992], fermentation
activity [Mronuk et aI., 1980], ion transport [Balnokin et al., 1983; Lucas et al., 1986; Pick,
1992], Ca2 [Quarmby, 1996] and Na+ [Weiss and Pick, 1990] influx, phosphate uptake
[Hirsch et al., 1993], velocity of cytoplasmic movement [Masashi and Teruo, 1982], accumu-
lation of chemicals [Yamaoka et al., 1992] and heavy metals [Riisgaard, 1980; Takimura et
aI., 1989; Turker and Balcioglu, 2001; Sacan et al., 2001], changes in zeta potential [Gimmler
et aI., 1991], and cell harvesting [Horiguchi et aI., 2003] have been documented.
The relationship between the medium pH and the concentration of hydrogen ions has
been studied inside algal cells [Beardall and Entwisle, 1984; Gimmler et al., 1988; Goyal and
Gimmler, 1989; Katz et aI., 1991, 1992; Weiss and Pick, 1996; Braun and Hegemann, 1999]
within the cytosol and vacuole [Kuchitsu et aI., 1989]. Effect of trans-membrane electrical
potential [Remis et al., 1992, 1994] and membrane proton pumps in acidophile species [Sand-
ers et aI., 1981] on the regulation of intracellular pH is also under investigation. Photoinduced
pH changes in suspensions of an acid-resistant green algae were discussed by Remis et al.
(1994). Special attention has been paid to mechanisms, including those at the molecular level,
52
that provide F homeostasis within the cytosol of acidophile and hyperhalobic species of al-
gae [Gimmler et aI., 1988; Seider et aI., 1991, 1994; Gimmler et aI., 1991; Gimmler and Weis,
1992; Pick, 1992, 1999; Weiss and Pick, 1996; Ohta et al., 1997; Messerli et al., 2005;
Topics, http://www.bio.uni-potsdam.de/oeksys/fsvte.htm; Pick et al., http://www.weizmann.ac.iV
Biological_Chemistry/ scientist lPickluri---'pick.html; Pick et al., http://bioinformatics. weiz-
mann.ac.iV _Is/uri---'pick/uri---'pick.html].
While phototrophs of representatives of the genus Dunaliella can grow over much of
the wide range of pH values found in nature [Raven, 1990], little is known about the influence
of pH on photomovement parameters [Nultsch, 1977]. The pH range in which D. salina
[Massjuk and Yurchenko, 1962] and D. viridis [Baas-Becking, 1930] cells maintain their mo-
tility has been determined, though the dependence of motility, velocity of movement and pho-
totopotaxis on medium pH has not been studied. As a consequence, the dependence of various
photomovement parameters in D. salina on the pH of the medium are presented in this section
[Massiuk and Posudin, 2007].
The dependence of photomovement parameters in D. salina (velocity of linear move-
ment v in J,llOIs, phototopaxis F and relative quantity of mobile N"INo or immobile Nt"INo
cells) on pH was investigated between pH 2.95 and 9.50 created by varying the concentration
KOH or HCI. Medium pH was measured using a pH-meter at the end of the first, seventh and
twentieth day after the onset of the experiment.
The cells displayed motility between a pH of 2.95 and 9.50 at the end of the first day.
The cells were immobile, deformed and completely destroyed at pH 2.95 (Nt"INo =100 %).
The algae varied in their photomovement, exhibiting different pH-optima for the various pa-
rameters (Fig. 5.3). Optimal pH for mobility (N"INo = 100 %; NtrrlNo = 0 %) was 6.8; for pho-
totopotaxis (F = 0.7) 7.35; and for linear velocity of movement (v = 47 ± 2 J,llOIs) 8.00 (Fig.
5.3).
F,
~lntls
\,
IUU 100
so 0,8 \_ 30
60 0,6 60
",/
/
/
40
" 0,4
/// ./f:-:-·
/
00 0,:
///
/:f // : '
20
// '"
/
::::
~/ ),26
I
4,00 ,15 5.0°5,30
6.00 / 7.00 s,on\ \... . . . S.S5 9.00 \ \'9,50
4 6,29 6.80 7.35 S,:O 8.25 9.159,40 pH
Fig. 5.3. Dependence of the linear velocity v of movement (I), phototopotaxis F (2), and relative quantity of
immobile NtnlNo (3) cells of Dunaliella salina on the pH of the medium at the end of the first day of cultivation.
The left vertical axis denotes values of linear velocity V of movement (lUID's) and levels of phototopotaxis F
(r.u.); the right vertical axis denotes the relative quantity of immobile NtnlNo cells; the horizontal axis indicates
the pH.
53
The relative quantity of immobile cells N;rrlNo did not exceed 80 % of the maximum value
between pH 3.26 to 9.32 (Fig. 5.3). Likewise, the linear velocity of cell movement did not
exceed 80 % of the maximum value between pH 5.30 and 8.40 and phototopotaxis F did not
exceed 80 % of the maximum (Fig. 5.3) between pH 5.70 and 8.20. Both the linear velocity
and phototopotaxis declined rapidly at a greater or lower pH.
Thus, various photomovement parameters possess either different pH optima or sensi-
tivity pH extremes. Phototopotaxis F was the most sensitive and motility the least. The linear
velocity of movement u was intermediate in sensitivity between the two parameters.
The results support our contention that the control of various photomovement parame-
ters differ and are mediated by way of different mechanisms [Posudin et aI., 1992, 1995,
2004; Massjuk et aI., 2006]. The wide range in tolerance to medium pH (2.95 to 9.50) can be
explained through the action of intracellular mechanisms whose homeostasis is dependent
upon the concentration of if ions in the cytosol at pH 7.1 (Pick et aI.,
http://www.weizmann.ac.iV Biological_Chemistry/ scientist /Pick/uri....Pick.html). In addition, the
optimum pH varies during algal development (Table 5.1).
Table 5.1. Changes ofpH of the medium during cultivation of Dunaliella salina in 20-days experiment
[Massjuk and Posudin, 2007].
I~ 2.95 4.30 5.30 6.29 6.80 7.35 8.10 8.25 8.35 9.15 9.40 9.50
7'" - 5.10 6.00 6.70 7.20 7.70 8.10 8.20 8.30 8.25 9.10 9.20
th -
20 6.50 7.25 7.70 7.87 8.07 8.15 8.15 8.17 8.35 8.47 8.42
Difference - 2.20 1.95 1.50 1.07 0.72 0.05 --{l.1 --{l.18 --{l.8 --{l.93 -1.08
between 0
values ofpH
th
at 20 and
I~ days
As iJJustrated in Tab]e 5.1, over the course of the experiment there were changes in pH of the
medium. In acidic, neutral and weak alkaline media, the pH increased with time, however, in
alkaline and strong alkaline media the pH decreased. Maximum changes in pH occurred at pH
4.30 (+2.20) and 9.50 (-1.08). The lowest change in pH (~pH = 0.05) during the course of
the experiment was at a pH of 8.1 (Table 5.1). The pH interval between 6.50 and 8.47 is suit-
able for survival of this species; D. salina growing in nature has been observed at a similar pH
range (pH 6.5-9.5) [Massjuk, 1973].
The optimum pH for motility and phototopotaxis of D. salina does not coincide with
the optimum pH values for growth [Massjuk and Yurchenko, 1962] and catalase activity
[Myronjuk et aI., 1980] of the species. The pH optimum for linear velocity of cellular move-
ment (8.0) was observed in a medium that underwent minimal changes in pH during cultiva-
54
tion and coincides with the pH for optimum growth (8-9) [Massjuk and Yurchenko, 1962].
The optimum pH for linear movement velocity and minimum change in pH during growth
may be a useful criterion for determining optimum conditions for the commercial production
ofhyperhalophobous algae for carotenoids.
The pH of the medium changed during the course of the experiment (Table 5.1) with
the range in pH becoming narrower between pH 5.10 and 9.20 by the end of the 7tb. day.
D. salina displayed motility through out the entire initial pH range (2.95 to 9.50). With the
exception of pH 2.5, the initial pH of the medium was reduced to 6.5 to 8.47 with pH 2.50
being the only one not altered. The adjusted pH range was more favorable for growth of the
species.
The level of sensitivity of the cells to the hydrogen ion concentration and their pH op-
timum for various photomovement parameters differed indicating the possible existence of
different mechanisms governing the parameters. Photomovement parameters may be used as
an indicator aquatic media quality during the commercial production of algae for carotenoids
[Massjuk & Posudin, 2007].
55
D. salina: u = 34.8 + 0.6 L - 0.62 E - 1.66 T+ 1.3 (LxE) - 0.6(LxE)
-0.8 (LxExT); (5.1)
The most important contributor in the equations was made by a free tenD. (the uninfluenced
movement velocity ofthe cells) that was independent ofthe external factors and was equal to 34.8
for D. salina and 37.6 for D. viridis. Weight coefficients ofvariab1es L, E and T define the con-
tribution of each of the factors to the change in movement velocityu. In equation (5.1), the
greatest effect on movement velocity of D. salina was due to temperature. Increases in tempera-
ture lead to deceleration (the coefficient was -1.66). Light and an electrical field modulated the
other factors. D. salina accelerated when exposed to increased light (+0.6) while the electrical
field decelerated movement (-0.62). The interaction of the two factors was also significant (+1.3)
and resulted in acceleration. The interaction effects of temperature and light (-0.6) and the three-
way interaction of temperature, light and an electrical field (-0.8) lead to a deceleration in move-
ment. Thus, the effect of the interaction of the external factors on D. salina was statistically
significant and commensurate with the influence ofthe separate factors.
In equation (5.2), the reactions of D. viridis to the same factors differed from D. salina.
Increasing temperature (within the abovementioned limits) lead to an acceleration of cell
movement while an increase in illuminance resulted in a deceleration of cell movement. The
most significant effect on movement velocity was due to increasing temperature (coefficient
+2.0) and the interaction ofan electrical field and temperature (coefficient -2.6).
Increasing the level of illuminance resulted in an inhibiting effect on D. viridis with cell
movement decelerating (weight coefficient -0.6). The electrical field, as a separate factor, did not
have a significant affect on D. viridis cell movement velocity, but its interaction with illuminance
and temperature significantly increased movement velocity (+ 0.5). Collectively the data indicate
that under the same aquatic conditions the two species can occupy different ecological niches, a
conclusion supported by observations in nature [Massjuk, 1973].
56
Table 5.2. Dependence of linear movement velocity (V) ofceUs Duna/ie/Is sa/ina Teod. on the effect ofextemal factors:
illwninance (Xl), electrical field (X2) and temperature (X3)
Xl X2 X3 V, V2 V3 <V> SV
L e I 38.200 39.900 37.300 38.467 1.743
L e I 45.800 35.700 37.100 39.533 29.943
L E I 40.900 31.700 32.700 35.100 25.480
L E I 40.400 37.800 38.000 38.733 2.093
L e T 35.300 33.200 33.000 33.833 1.623
L e T 33.800 32.000 32.700 32.833 0.823
/ E T 33.700 32.300 31.200 32.400 1.570
L E T 33.200 33.600 33.700 33.500 0.070
Nole. Here and in Table 5.3/-100 Ix; 1-18 DC; e-OV/cm;L- 500 Ix; T-30 DC andE - 2.4 V/cm; V 1> V 2, V 3
are absolute velocity significances measured in trifold sequence; < V > - average velocity significance; S V-
standard deviation.
Table 5.3. Dependence of linear movement velocity (V) of D. viridis Tead. on the effect of external factors:
illuminance (Xl), electrical field (X2) and temperature (X3)
X, X2 X3 V, V2 V3 <V> S
L e T 30.900 32.000 34.900 32.600 4.270
L e T 33.200 33.100 31.700 32.667 0.703
L E T 36.700 40.600 39.900 39.067 4.323
L E T 38.000 36.900 38.600 37.833 0.743
L e T 44.200 42.300 43.000 43.500 0.390
L e T 39.900 40.600 40.100 40.200 0.130
L E T 37.700 38.000 36.900 37.533 0.323
L E T 36.909 38.000 36.700 37.200 0.490
Table 5.4. Dependence ofphototopotaxis F of D. sa/ina Teod. on external factors effect: light (Xl), electrical
field (X2) and temperature (Xj)
X, X2 X3 FJ F2 F3 <F> SF
57
Table 505.Dependence ofphototopotaxis F of D. viridis Teod. on external factors effect: light (Xl)' electrical
field (X2 ) and temperature (XJ)
Xl X2 XJ Fl F2 FJ <F> SF
L e t 0.240 0.260 0.200 0.233 0.001
L e t 0.250 0.270 0.180 0.233 0.002
L E t --{).280 --{).330 0.140 --{).250 0.010
L E T --{).570 --{).360 --{).I90 -{).373 0.036
L e T 0.100 0.330 --{).I1O 0.107 0.048
L e T --{).050 0.500 0.260 0.087 0.025
L E T 0.210 0.200 0200 0.203 0.000
L E T --{).200 0.180 0.260 0.080 0.060
In equation (5.3), the detennining factor inDo salina phototopotaxis was the effect of the elec-
trical field which had an inhibiting effect (coefficient -0.07). Increasing the temperature canceled
the inhibiting effect of the electrical field and stimulated phototopotaxis (+0.09). At the same
time, the effect of temperature on phototopotaxis is not determinative.
Equation (5.4) for D. viridis indicates that as with D. salina the electrical field inhibited photo-
topotaxis (-0.125) while increased temperature increased phototopotaxis (+0.079). The greatest
effect was due to the interaction ofboth factors (+0.148).
The rather complicated effect of external physical factors on photomovement para-
meters in both Dunaliella species can be explained in the following way. Since the cell move-
ment velocity of both species depends on illumination by white light, maximum cell move-
ment activity falls within a light intensity range of 150-550 Ix and the maximum movement
velocity between a temperature range of 20 to 25°C [Posudin et aI., 1988]. Maximum positive
phototopotaxis values were reached at a white light illuminance of 500 Ix; at 1500 Ix phototo-
potaxis is absent. With further increases in illumination, phototopotaxis becomes negative
[Posudin et aI., 1991]. The electrical field, applied to the sample (see Section 5.2) inhibited
phototopotaxis.
The upper and lower limits for sample illuminance (100 Ix and 500 Ix) and temper-
ature (l8°C and 30°C) used in the experiment were close to the outer limits for these external
factors with regard to photomovernent parameters and the range in which they can alter each
other or the effect of an electrical field. The effect of external factors on photomovement pa-
rameters can be increased or decreased through the interaction of these factors.
Similar complementing and opposing effects caused by the external factors can be ex-
plained by differences in the ecology and behavior of these species under conditions found in
nature [Massjuk, 1973].
58
stimulates ionization processes in the upper atmosphere though little reaches the Earth's sur-
face due to absorption by the ozone layer. However, UV-B radiation reaches the Earth's sur-
face and its intensity depends on the latitude, solar elevation, cloud cover, reflectivity of the
surface, and the thickness of the ozone layer. Ozone depletion has lead to increases in the UV-
B radiation reaching the Earth's surface. UV-B causes damage to living organisms due to its
absorption by nucleic acids, proteins and other labile molecules [Hader, 1996]. Absorption of
UV-A radiation, in contrast, is due to its interaction with conjugated double bonds and with
cyclic and polycyclic structures such as isoprenoids, flavines, quiniones, alkaloids, and photo-
synthetic pigments in phototrophic organisms [Garcia-Pichel, 1996].
Halldal (1976) has shown that near-UV radiation (310-390 nm) had little or no inju-
rious effects, being similar to visible light, whereas far-UV (190-310 nm) produced imme-
diate growth inhibition, delayed growth inhibition, or simulative growth effects. Halldal ex-
amined the action spectra for inhibition of chloroplast development, changes in diurnal
rhythm, motility, phototopotaxis, and chloroplast movements. UV-A (320-400 nm) exposure
caused an inhibition of photosynthesis, bleaching of photopigments, and a loss of biomass
[Hllder, 1991, 1995, 1996b; Hllder et al., 1995; Ekelund, 1996].
Both natural and artificial ultraviolet radiation alters the behavioural strategy and pro-
ductivity of algae [Hllder, 1994; Huovinen et ai., 2006]. A number of articles have assessed
the interaction of natural and artificial ultraviolet radiation on algal photosynthetic activity
and orientation, in particular, the motility and photoorientation of Euglena gracilis G.A.
Klebs [Hllder, 1985, 1986a; Hllder and Hllder, 1988]. In addition, pre- and post-treatment with
thiourea, caffeine and cysteine on UV-induced damage in desmids [Sarma and Chowdhury,
1985], motility in P. uncinatum [Hllder et ai., 1986], photomovement and motility in Astasia
longa [Hllder and Hllder, 1989a], photosynthesis, protein and pigment composition in E.
gracilis [Gerber and Hllder, 1992], gravitaxis in Euglena gracilis [Hllder and Shi-Mei Liu,
1990], photoorientation, motility and pigmentation in Peridinium gatunense Nygaard [Hllder
et ai., 1990] and Cryptomonas sp. [Hllder and Hllder, 1989b, 1990,1991], photomovement and
pigmentation in Gyrodinium dorsum Kofoid & Swezy [Ekelund and Bjorn, 1990], positive
phototaxis in Volvox aureus Ehrenberg [Blakefield and Calkins, 1992], damage of photore-
ceptor proteins in the paraflagellar body of E. gracilis [Brodhun and Hllder, 1993], growth
and motility of the flagellate, E. gracilis [Ekelund, 1993], photoorientation, motility, and
chlorophyll photosynthesis in Euglena sanguinea Ehrenberg [Gerber and Hllder, 1994], pig-
ments and assimilation of 1~ ammonium and 1~ nitrate by macroalgae [Doehler et al.,
1995], photosynthesis in Laminaria digitata (Hudson) J. V. Lamouroux [Forster and Liining,
1996] and Dictyota dichotoma [Flores-Moya et ai., 1999], motility in Dunaliella bardawil
[Jimenez et ai., 1996], flagellar apparatus in C. reinhardtii [Donk and Hessen, 1996], amino
acids in macroalgae [Kusten et al., 1998], growth and pigment composition of Ulva expansa
(Setch.) S. [Grobe and Murphy, 1998], photoinhibition of marine macrophytes [Aguirre-von-
Wobeser et ai., 2000], reproduction of Enteromorpha intestinalis E. [Cordi et ai., 2001], fixa-
tion of inorganic nitrogen in the marine alga Dunaliella tertiolecta Butcher [Beardall et al.,
2002], composition of photosynthetic and xanthophyll cycle pigments in Ulva lactuca L. [Bi-
schof et al., 2002], photomovement of the swarmers of the brown algae Scytosiphon lomenta-
ria (Lyngbye) Link and Petalonia fascia (O.F. Muller) Kutzing [Flores-Moya et al., 2002],
biochemical composition of Ulva sp. [2002], canopy structure of Ulva communities [Bischof
et al., 2002], UV-induced biochemical processes in Ulva canopies [Bischof et al., 2003], pho-
tosynthetic UV responses in Ulva species [Figueroa et al., 2003; Posudin et al., 2004a,b], tax-
onomic composition of phytoplankton [Xenopoulos and Frost, 2003], variation in sunscreen
compounds (mycosporine-like amino acids) in marine species [Lamare et ai., 2004], increas-
ing competition between marine macro-algae and micro-algae populations [Zhang et al.,
2005], growth interactions between Ulva pertusa Kjellman and Alexandrium tamarense
(Lebour, 1925) Balech, 1992 [Cai et al., 2005], oxidative stress and responses of the ascor-
59
bate-glutathione cycle in Ulva fasciata Delile [Shiu and Lee, 2005], UV-B protection in U.
pertusa [Han and Han, 2005], carbon and nitrogen metabolism in Fucus spiralis L. and Ulva
olivascens Dangeard [Vinegla et al., 2006], growth of U. pertusa and Platymonas helgolandi-
ca Kylin var. tsingtaoensis [Xu et al., 2006], vertical migration and photosynthesis in E. gra-
cilis [Richter et al., 2007], effective quantum yield of Ulva lactuca L. [Xu and Gao, 2007],
and photosynthesis in U. lactuca [Fredersdorf and Bischof, 2007] have been studied.
It has been assumed [Ghetti et aI., 1992] that nucleic acids and proteins present in
pigmented microorganisms can be damaged, not only by ultraviolet radiation, but also by
near-UV and visible radiation through photosensitization reactions that result in the genera-
tion of singlet oxygen and/or other noxious oxygen species. To address this question, we
studied the dependence of phototopotaxis in two species of Dunaliella Teod. and in Tet-
raselmis viridis (Rouch) Norris et al. (syn. Platymonas viridis Rouch), on the wavelength of
lateral stimulating light within the ultraviolet region of the electromagnetic spectrum to com-
pare differences in the action spectra between Dunaliella [Posudin et al., 1990] and T. viridis
[Halldal, 1961].
The absence of phototopotaxis in Dunaliella within the ultraviolet region of the spec-
trum where flavins and rodopsin have maximum absorption supports the hypothesis that caro-
tenes act as photoreceptor pigments and are responsible for phototopotaxis in the two species
in Dunaliella. It is possible, therefore, that phototopotaxis in the ultraviolet portion of spec-
trum is also operative in the genus Tetraselmis [Posudin et al., 1990].
This section addresses the effect of ultraviolet irradiation on the two Dunaliella spe-
cies using different intensities, wavelengths, and durations. Photomovement parameters were
assessed in algae exposed to lateral white light irradiation (500 lx) and at a temperature of 18-
20°C [Posudin et aI., 2004]. The application of artificial ultraviolet radiation allowed discern-
ing its possible role on the viability and photo-behaviour of Dunaliella and identifying the
optimal conditions for survival.
A mercury lamp with an emission spectrum in the 250-350 nm range was utilized as
the ultraviolet radiation source. The dependence of photomovement parameters, in particular
the linear velocity v of movement, phototopotaxis F and relative motility N,,/No (where N m-
quantity of motile cells, and No- total quantity of the cells) on the intensity of ultraviolet ra-
diation were measured at 0.76 to 11 W/m2 with the intensity monitored using a DAU-81 do-
simeter. The duration of exposure was 5 and 10 minutes and the spectral sensitivity of the
photomovement parameters determined using interference filters placed between the ultravio-
let radiation source and the algal suspension. The filters had a maximum transmission at 248
nm, 280, 302, 313, 334 and 365 nm. Untreated algae illuminated laterally with white light
(500 lx, 18-20 °C) were used as a control. Three replications of all measurements were used
to calculate mean values and errors of measurements.
The dependence of the linear velocity v of movement and phototopotaxis F in both
species on the intensity I of unfiltered ultraviolet radiation is presented in Fig. 5.4. The values
for v did not change with increasing intensity of radiation, while phototopotaxis F was inhib-
ited by high intensity ultraviolet radiation (i.e., 2 to 11 W/m2).
60
v, Ilm/s
10
F J".U. 0 2 4 6 8 10 12
04
03
02 b
0]
o 2 4 6 8 10 12
Fig. 5.4. Dependence of the linear velocity v of movement (a) and phototopotaxis F (b) of the cells of two spe-
cies of Dunaliella on the intensity (1) of preliminary exposure to nonfiltered ultraviolet radiation (wavelength
range 250-350 om, duration of irradiation 5 nnn) (-e- - Dunaliella salina; -A- - Dunaliella viridis; c - con-
trol) [posudin et a1., 2004].
The dependence of the linear velocity u of movement, phototopotaxis F and relative motility
N,,/No of the cells on exposure duration t to unfiltered ultraviolet irradiation (10 W/m2) is pre-
sented by Fig. 5.5.
61
D,llm/s
40
10
a 2 4 6 8 10 12 t, min
F, LU.
0.3
t, min
N"IN, r.u.
10
0811Y=----------.::---------------:-:-:-:..-c
0.6 I iI
a4 I
0.2
a 2 4 6 t, min'
Fig. 5.5. Dependence of the linear velocity v of movement (a) and phototopotaxis F (b) and relative motility
N,,/No(c) of the cells of two species of Dunaliella on the duration t of preliminary exposure to nonfiltered ultra-
violet radiation (wavelength range 250-350 nm; intensity of radiation 10 W/m 2 (-e- - Dunaliella salina; -.A.-
- Dunaliella viridis; c - control) [posudin et aI., 2004].
The velocity v did not differ from the control during 10 minutes of irradiation, while pho-
totopotaxis F and relative motility N,,/No were inhibited by 7-10 minutes of irradiation. The
results support the position that different mechanisms govern the velocity of linear movement
(parameter v) versus photoorientation (parameter F) and motility (parameter N,,/No). It was
not possible, however, to discern differences in the structure and size of the photoreceptor
systems responsible for the velocity of linear movement and photoorientation of the cells. The
results are in agreement with previous investigations assessing the effect of medium pH and
ionizing radiation on photomovement parameters in Duna/iella [Posudin et aI., 1992; Massjuk
and Posudin, 2007] (see Sections 5.3 and 5.6).
62
The effect of natural solar and ultraviolet radiation on the velocity of linear movement
and phototopotaxis in E. gracilis has been previously described in the literature [Hilder,
1986a]. The mean value for linear movement was 120 J.UD!s; the velocity and phototopotaxis
declined to zero after 1.5-2 hours exposure to solar irradiation. Use of an ozone filter (cuvette
with 45 f.lg/mL ozone in air) decreased the intensity of the UV-B (290-320 nm) component of
solar radiation (estimated to be 1.2 W/m2) by 5.0 % which lead to an increase in cell viability
(i.e., 50 % of motile cells remained after 3 hours of irradiation) and a decrease in the velocity
of linear movement of up to 80 % of the initial value. The use of a glass lid that blocked ultra-
violet radiation made it possible to reach a level of phototopotaxis comparable to control
samples [Hilder, 1986a]. This dependence of photomovement parameters in E. gracilis could
not be explained by simply exceeding the light energy absorption capacity of chlorophyll
since cells of Astasia longa Pringsheim devoid of pigment and bleached cells of E. gracilis
demonstrate the same response to ultraviolet radiation [Hilder, 1986a].
Inhibition of motility and linear velocity of movement due to unfiltered solar irradia-
tion is also exhibited by the algal species Peridinium gatunense Nygaard, Cryptomonas spp.,
Gyrodimium dorsum Kofoid & Swezy, and Cyanophora paradoxa KorshiJrov [Hilder, 1991].
The radiation leads to immobilization of Cryptomonas maculata Ehr. cells after 140 minutes
exposure and by unfiltered artificial ultraviolet exposure after only 60 minutes [Hilder et al.,
1987; Hilder and Hilder, 1991].
The difference between our data and that ofHllder appears to be due to the fact that we
used a higher intensity of ultraviolet radiation (Le., up to 10 W/m2). Phototopotaxis F and
relative motility N"INo were inhibited by a 10 minute exposure indicating that irradiation is
the primary factor affecting the photomovement parameters. The dose was calculated as
D=J.t, where I is the intensity of ultraviolet radiation and t the duration. The dosage used by
Hilder was D = 1 W/m2 ·120 min and in the same order of magnitude as in our experiments
(i.e., D = 10 W/m2 ·10 min).
Differences in the velocity of linear movement in Dunaliella between our results and
those of Hilder and Hilder (1991) are most likely due to differences in the way the measure-
ments were made. We measured the velocity of individual cells that were mobile, not the
mean velocity estimated by videomicrography. The cells maintained a constant velocity in our
experiments even when there were only a small number of cells.
The dependence of velocity u and Plwtotopotaxis F in the two Dunaliella species on
the ultraviolet radiation wavelength (2 W/m2 intensity) is presented in Fig. 5.6 (5 min expo-
sure duration) and Fig. 5.7 (10 min exposure duration).
The effect of ultraviolet radiation wavelength on the linear velocity v ofD. salina (Fig.
5.6a) and D. viridis (Fig. 5.6c) was not established. The effect of monochromatic ultraviolet
radiation on phototopotaxis F in the two species was not straight forward. A 5 minute expo-
sure decreased the level of photoorientation (parameter F) below control values until reaching
negative values for phototopotaxis F near 248-334 nm in D. salina (Fig. 5.6b) and near 248
nm in D. viridis (Fig. 5.6d). Prolonged exposure (Le., 10 min) lead to a complete inhibition of
phototopotaxis near 248-280 nm for D. salina (Fig. 5.7b) and 248-334 nm for D. viridis (Fig.
5.7d).
Ultraviolet irradiation in the 302-365 nm (D. salina) and 365 nm regions (D. viridis)
for 10 minutes resulted in negative phototopotaxis in both species. The transition from posi-
tive to negative phototopotaxis in response to ultraviolet radiation with increased duration of
exposure has not been previously reported. Exposure of E. gracilis to ultraviolet radiation at
295 nm and below, however, resulted in inhibitory effects on motility and photoorientation of
the cells [Hilder, 1985].
63
v. ~,m's
DUIII/liellu ,alina
v. !,m! I
Dill/alieNa viridis
40 40
30 30
20 20 --I----I--n--I---I-- -,
10 10
248 280 302313 334 365 i., nm 248 280 302313334 365 J, om
F. cu. F. r.u.
Dl'no/idla Jolina Dumrliel!a viridis
04
------------------ -- -- C b
U4 --------I----r--r-- C
02 0.2 I'
o f - - - +T-l-T-tT_+-_
-0.2
I
Ir 1 -0.2
o
I
-0.4 -04
1
''--1 I I l
248 280 302 J13 334 365 i, Hill 248 280 302 3J 334 365 i, nm
Fig.5.6. Dependence of the linear velocity v of movement (a, b) and phototopotaxis F (c,d) of the cells
of two species of Dunaliella on the wavelength ). of ultraviolet radiation (intensity of radiation is 2 W1m2 ;
duration of irradiation 5 min; c - control) [posudin et aI., 2004].
V, !lm/s. v,'!1m/s
Duna/ie/fa \'iridis
40 OUl1a'Jella salina
40
i • , I. I i
248 280 102313334 365 ;,l1rn 2S0 302313334 365 J..l1rn
F. r.ll.
0.4
DunaJidlu s(,/Iina
.10~; ~ Duna/ie/la viridis
----------------------- c
0.2 02·
o Hrf--Tt-----4~--
i o .tt-I-- - r - d
-~I~:,-------
1 ~
-0.2 -0.2
-0.4 -0.4
Fig.5.7. Dependence of the linear velocity v of movement (a,b) and phototopotaxis F (c,d) of the cells of two
species of Dunaliella on thc wavelength). of ultraviolet radiation (intensity of radiation is 2 W/m2 ; duration of
irradiation 10 min; c - control) [posudin et aI., 2004].
64
It is noteworthy that the effect of ultraviolet exposure on the parameter F was reversible im-
mediately after the ultraviolet treatment (2 W/m2 intensity) and resulted in F values reaching
those of control at all wavelengths with the exception of 248 nm after a 2 hour exposure (Fig.
5.8). Dunaliella bardawil (=D. salina) cells recovered within 24 hours after the cessation of
10 hours of visible + UV-A radiation (26.01 W/m2 intensity) and visible + UV-A + UV-B
radiation (39.72 W/m2) [Jimenez et aI., 1996].
F, r.u.
Dunaliella salina
0.4
02 i
o a
-0.2
I
-0.4
1 i i i I
Duna/Iella viridis
0.4 ------,=--"!T--------- c
0.2 . ~ ! I
o b
-0.2 -
t
-0.4
Fig. 5.8. Phototopotaxis of Dunaliella salina (a) and D. viridis (b) 2 hours after cessation of a 10 min
pulse ofultravio1et radiation (intensity of radiation is 2 W/m2) [posudin et al., 2004].
There are several hypotheses concerning the mechanism of action of ultraviolet radiation on
algae. The first proposes that the primary impact of ultraviolet irradiation is on DNA mole-
cules and is based on the similarity in the absorption spectrum of DNA to the action spectrum
for the inhibition of microorganisms [Yammamoto et a!., 1983]. It was subsequently shown,
however, that the fine structure of the action spectrum for the inhibition of motility in E.
gracilis was characterised by a principal maximum near 270 nm (UV-C region), a smaller
peak: at 305 urn, and a shoulder at 290 nm (both in UV-B region) [Hader, 1991], a spectrum
that does not resemble the absorption spectrum for DNA [Jagger, 1983]. In addition, the very
fast effect of the radiation on motility and the absence of photo-repair, further argues against
an effect on DNA being the primary avenue for the inhibition by ultraviolet radiation [Hader
and Hader, 1988].
65
A second hypothesis is that ultraviolet damage of cells in through a photodynamic ef-
fect caused by the simultaneous action of ultraviolet radiation and chemical compounds. ill-
traviolet radiation is thought to be absorbed by a photoreceptor molecule and if the excited
molecule does not expend this additional energy via photochemical reactions or dissipative
yrocesses, the energy can be transferred to the triplet state and the formation of singlet oxygen
02 [Maurette et al., 1983] or free radicals [Spikes, 1977]. Free radicals have highly reactive
properties and can destroy membranes and other cellular components.
Arguments against this hypothesis are based on the application of specific diagnostic
reagents and quenchers of singlet oxygen and free radicals and the absence of viability in al-
gae exposed to radiation [Hader et al., 1986; Hader and Hlider, 1988b]. Likewise, the addition
of D20, which increases the half-life of singlet oxygen, does not inhibit the effect ofultravio-
let radiation on the algae [Hlider, 1991].
A more plausible explanation for the effect of ultraviolet radiation on photomovement
parameters in algae may be through the effect of radiation on the proteins governing the activ-
ity of the flagellar apparatus orland the photoreceptor system. Impairment of these proteins
when detached from the paraflagellar body of E. gracilis by ultraviolet radiation supports this
possibility [Hader, 1991]. The differential effect of varying levels of ultraviolet radiation on
photomovement parameters in Dunaliella also supports this hypothesis. It is possible that the
photoreceptor systems responsible for linear velocity and photoorientation differ in size and
structure. The dependence of phototopotaxis and motility of algal cells on the intensity, wave-
length, and duration of exposure to ultraviolet radiation can be used in biotesting of natural
ultraviolet radiation.
66
medimn. Measurement of v and F were carried out on the first and 16th day after irradiation
[Posudin et aI., 1992].
The velocity of cell movement v decreased linearly up to 600 Gr after the first day of
irradiation, reaching 20 % (Fig. 5.9a) and decreased sharply at 1000 Gr. Phototopotaxis F was
also affected linearly up to 600 Gr, but exhibited a different slope in comparison with parame-
ter v. Above 600 Gr (Le., 1000 Gr) inhibited both velocity v and phototopotaxis F.
The parameter v decreased linearly up to 1000 Gr in D. viridis at a slope similar to D.
salina. Parameter F decreased to the 10 % level at 30 Gr but exhibited considerable variation
at higher levels (Fig. 5.9b). Differences in the dependence of parameter F between species can
be explained by differences in cell size. The character of the dose-response curves were essen-
tially the same at the end of the first, seventh, and eleventh day indicating an irreversible ef-
fect of ionizing radiation. Dilution of the cultures with fresh medium did not appear to alter
the affect of irradiation on parameters v and F in comparison with undiluted cultures on the
16th day, confirming the absence of an inhibitory effect by the medium on the photomovement
parameters.
It is possible that there was a change in dimensions (or shape) of the cells due to the
action of ionising radiation. Shifts in the maxima and changes in amplitudes of the histo-
grams, characterized by either scattering of laser radiation by the cells or chlorophyll fluores-
cence due to the treatment, support this possibility (Fig. 5.10, 5.11). Thus, the histograms
(Fig. 5.10, 5.11) indicate possible irreversible damage to the cells of both species (D. salina
and D. viridis) and their photosynthetic apparatus due to exposure to the above levels of r-
irradiation.
v, .um/s F. LU.
80 \- 0.8 a
)'-- __ 1
J - - - - ........ - - - - - - -
7 -------1, I
2~ r02 --f-<~ ~_~
30 600 1000 D,Ge
U, ,tm/s F. LU.
80 ~ 08 b
t----+-------__
20 r ~'2""f ----:--T-L
I
Fig. 5.9. Dependence of the linear velocity v (1) and phototopotaxis F (2) on the dose of ionizing radiation after
one day of irradiation: a-D. salina; b - D. viridis. Axis of abscisses is the dose of irradiation in Gr [posudin et
aI.,1992].
67
.",~ 01
~"
't
,
l
---, 0
Ll-~
Fig. 5.10. Histograms which characterize relation Fig. 5.11. Histograms that characterize the relation
between scattering and fluorescence ( a ) of the cells, among scattering and fluorescence ( a ) of the cells,
fluorescence of the cells ( b ), scattering oflaser radia- fluorescence of the cells ( b ), scattering of laser
tion on the cells ( c), and scattering at an angle of 90° radiation on the cells ( c), and scattering at an angle
( d) for D. salina: A - before r-irradiation; B - after r- of 90° ( d) for D. viridis: A - before r-irradiation; B
irradiation [posudin et aI., 1992]. - after r-irradiation [Posudin et aI., 1992].
Ascertaining the location and the nature of photoreceptor that is responsible for light reception
and the transduction of the signal controlling flagellar beating are critical questions in the
study of photomovement. It is thought that in green algae such as Chlamydomonas the photo-
receptor system is located near surface of the cell in the plasmalemma [Nultsch, 1983]. It is
known that photoreceptor in E. gracilis is located inside the cell, at the basis of flagellum in
the paraflagellar body [Colombetti et ai, 1982].
In Dunaliella, there is at this time an absence of sufficiently precise data indicating
the location of photoreceptor. One might assume that if the photoreceptor was located at the
cell surface (as in Chlamydomonas) and the mechanisms responsible for controlling velocity
and direction of movement were in different locations, there would be different characteristic
dose-response curves for v and F. Our results demonstrate that v and F in Dunaliella display
different slopes for their dose-response curves (see Fig. 5.9) which supports the idea of differ-
ent mechanism(s) govern the linear velocity of movement and phototopotaxis. The possible
loss of protein conformation due to ionizing radiation can not be excluded. It is known that
membrane proteins control ion transport, in particular, Ca2+ ions are modulated by light condi-
tions [Posudin and Suprun, 1992]. Photoreception can be viewed as an excitation of certain
membrane proteins due to the absorption of light by a photoreceptor molecule and conforma-
tional alterations in the protein that lead to the activation of ion channels. Changes in the con-
centration of CcJ+ ions around the flagella modulates their beating and in tum, the photoori-
entation of the cell.
68
The damage caused by r-radiation exposure on the linear velocity of movement and
phototopotaxis in Dunaliella differ. The photoreceptor that is responsible for photoorientation
appears to be more sensitive to ionising radiation than the apparatus that controls the velocity
of linear movement. There are, for example, several differences in the dose-response curves
for F between D. salina and D. viridis that may be related to differences in the dimensions of
the respective photoreceptor systems or their sensitivity to ionising radiation. The linear char-
acter of the dose-response curves (i.e., for parameter v) indicates that algae might be useful as
a biological sensor for ionizing radiation, at least up to 1000 Gr.
5.7. Summary
The effects of illuminance, temperature, pH, electrical field, ultraviolet and ionizing radiation
on photomovement parameters such as motility [relative quantity of mobile cells N,,/No), ve-
locity oflinear movement (v, Ilffi/s)], and phototopotaxis (F) in two species of Dunaliella and
the interaction of several external factors (illuminance, temperature, electrical field) on pho-
tomovement were studied. Maximum values for motility, velocity, and positive and negative
phototopotaxis were observed for the temperature range between 20 and 30°C and a pH range
of 6.50 to 8.47. Optimum pH for the various photomovement parameters differed as did the
sensitivity of these parameters to pH extremes.
Exposure of cells to an external electrical field results in suppression of phototopotaxis
in Dunaliella, similar to other green algae (e.g., Chlamydomonas reinhardtii and Haemato-
coccus pluvialis) underscoring the role of bioelectrical potentials in photomovement.
The species D. salina and D. viridis differed in their motile behavior in response to
adverse external factors and combinations thereof. Increasing the temperature from 18 to 30°C
overcame the inhibition of the electrical field and stimulated phototopotaxis in both species.
With an i11urninat:ion of 100 and 500 Ix and temperatures of 18 and 30°C, there were levels in which
photomovement parameters are highly dependent upon the external factors tested. The response
may be positive or negative, altering the photomovement accordingly. In nature, differences in the
ecology and behavior of Dunaliella species may be in part due to complimentary or opposing ef-
fects ofthese external factors [Massjuk, 1973].
Ionizing and UV irradiation inhibit phototopotaxis in both D. salina and D. viridis.
The inhibiting effect depends on the dose of irradiation and in the case ofUV-irradiation, wave-
length. Ionizing radiation (up to 600 Gr) and UV-irradiation did not affect the velocity of
movement. We found a previously unreported transformation ofpositive to negative phototopo-
taxis due to UV-irradiation, with an inhibition ofphototopotaxis down to a zero value. Effects of
UV radiation on photomovement of Dunaliella were reversible, while the effects of ionizing
radiation were irreversible. The two species of Dunaliella displayed a differential in sensitivi-
ty to UV and ionizing radiation.
The relative number of motile cells (N,,/No) varied among populations of Dunaliella
from 0 to 100 % and displayed the same dependence on characteristics of the light stimulus
and environmental conditions as phototopotaxis. Phototopotaxis differs considerably, howev-
er, depending upon its presence or absence and by its degree of dependence on factors such as
pH, intensity, wavelength, dose of preliminary ionizing and UV radiation. There also appears
to be differences in the structure and dimensions of photoreceptor systems that are responsible
for variation in the various photomovement parameters within and among algal species.
69
Chapter 6
Structure of the Photoreceptor System
Fig. 6.1. Interaction oflight with a quarter-wave stack of altcmaling layers ofhigh and low refractive indices
wavelenglh; light reflects from such a multilayer Sylllcm without changing phase and producing an interference
maximum at the Bite ofpossible photoreceptor [adapted from Foste:r and Smyth, 1980].
Based on microphotographies in the literature, the stigma ofother species of green algae [e.g.,
Volvulilla pringsheimii Starr, Platydorina caudata Kofoid, Volvox aureus Ehrenb., V. tertius
Meyer, PteromollQS tenuis Belcher and Swale, Pyramimonas mOlltana Geitl., Eudorilla illi-
noisensis (Kofoid) Pascher] also function as a quarter-wave reflector [Foster and Smyth,
1980]. A nmtation of C. reinhardtii without a cell wall displayed a high level of specificity
72
(Melkonian and Robenek, 1980; 1984). The plasmalemma near the stigma was characterized
by a dense arrangement of intramembranous particles in comparison with other parts of its
surface. Smaller particles (8-12 om diameter) prevailed while larger particles (16-20 om) were
located outside the stigma area. The plasmalemma and chlorophyll envelope outer membrane
adjacent to the stigma were only 20-30 om from each other. It is thought that regions of both
membranes take part in photoreception and the primary sensory transduction [Melkonian and
Robenek, 1980]. Thus, the photoreceptor in green algae is thought to be located in the mem-
brane between the stigma and the adjacent part of the chloroplast envelope.
The structural organization of intramembraneous particles on the cytoplasmic surface
of the plasmalemma ["protoplasmic face" as per the terminology of Branton et aI., 1975] near
the eyespot was found in all species of algae under investigation. The plasmalemma of
C. reinhardtii near the eyespot has a specific chemical composition of proteins and lipids
[Melkonian, 1981; Melkonian and Robenek, 1980].
There are several variations in the outer membrane of the chloroplast envelope ranging
from specialized to the absence thereof. Specialization of the intramembraneous particles,
however, was not found in the inner membrane of the chloroplast envelope and thylakoid
membranes near the eyespot [see review by Melkonian and Robenek, 1980]. If light beams
enter the lateral side of the cell where the stigma is located, the photoreceptor receives a sig-
nal double in intensity due to an amplification that is equal to the sum of the incident and re-
fleeted intensities. If the opposite side of the cell is illuminated, the signal received by the
photoreceptor is attenuated due to the absorption of light by internal cellular constituents and
the stigma as well as due to reflection of light by the stigma. The stigma, therefore, modulates
the light and acts like a directional antenna, determining the spatial positioning of the light.
This effect is intensified due to the interchange of pigmented and non-pigmented layers in the
stigma by about a quarter of a wavelength. If the cell is illuminated from the side of the outer
surface of the stigma, a series of intensity maxima are produced. The location of these
maxima coincides with the plasmalemma and thylakoid membranes inside the stigma. How-
ever, if the light strikes the stigma from the opposite side, a series of minima at the same posi-
tion are produced within the stigma increasing the maximum contrast in the light received
from both opposite sides. This suggests that the photoreceptor pigments are located on the
outer membranes (plasmalemma and outer membrane of the chloroplast envelope) as well as
the thylakoid membranes [Foster and Smyth, 1980]. Such a location for the photoreceptor
(Le., in the plasmalemma in the region of the stigma) was proposed earlier based on the fact
that such a position would provide a direct linkage of the photoreceptor with the locomotor
apparatus since the plasmalemma is continuous with the flagellar membrane [Amott and
Brown, 1967; Walne and Amott, 1967]. The connection of internal membranes with the fla-
gella is less evident. Therefore, the location of the photoreceptor pigments not only in outer
membranes but inside the multilayer stigma on the surface of thylakoid membranes, appears
plausable. Use of either outer membrane (plasmalemma and chloroplast envelope membrane)
or the thylakoid membranes increases the surface area for photoreception and the intensifica-
tion efficiency [Foster and Smyth, 1980].
The structure of the eyespot apparatus for almost 90 species of green algae has been
studied (see review by Melkonian and Robenek, 1984) and categorized using 30 physical
traits e.g., the shape of the eyespot (ellipsoid, egg-shaped, spherical, etc.), the shape of outer
surface ~flat, concave, salient), the size of outer surface of the eyespot (which ranged from
0.28 J.llIl in zoospores of Chlorosarcinopsis gelatinosa Chantanachat and Bold, to 9 !J.m2 in
vegetative cells of Volvox (L.) Ehrenb. and female gametes of Bryopsis lyngbye Lyngb. = B.
plumosa (Hudson) C.Ag., and the size (80-130 om, sometimes 200 om), chemical composi-
tion, arrangement of packing (hexagonal or not), and electron density of the pigmented glob-
ules.
73
There are three main variations in the layers of pigmented globules and thylakoid
membranes within the stigma.
1) Chlamydomonas Ehrenb. possesses a single thylakoid membrane between the lay-
ers of pigmented globules. The thylakoid is in close proximity to the back side of the globular
layer. Between the thylakoid and the subsequent layer of globules there is an extensive space
that is filled with a granulated substance. This variation is found in the multilayer stigma of
Chlamydomonadaceae and the four-flagellar Hafniomonas Ettl and Moestrup [Foster and
Smyth, 1980; Ettl and Moestrup, 1980; Melkonian and Robenek, 1984].
2) Tetraselmis Stein. possesses exaggerated thylakoids located between globular layers
and contacting both adjacent globular layers. This variation is found in Tetraselmis Stein. and
Carteria Dies., and zoospores of Schizomeris leibleinii Klitz., Uronema belkae G.M
Lokhorst, Ulothrix zonata Klitz., and gametes of Acetabularia mediterranea Lamour. [Man-
ton and Parke, 1965; Parke and Manton, 1965, 1967; Crawley, 1966, 1970; McLachlan and
Parke, 1967; Birkbeck et aI., 1974; Melkonian and Robenek, 1979, 1984; Foster and Smyth,
1980; Hertz et aI., 1981].
3) Pyramimonas Schrnarda is characterised by the absence of thylakoids. The inter-
vals between the pigmented layers are electron transparent and contain a fibrous substance
that connects to the back surface of the globular layers. This type of structure is found only in
species of a genus of coloured flagellates, except for the colourless representatives of Poly-
toma Ehrenb. and Polytomella Aragao. Their eyespots are located in leucoplasts that are de-
void of thylakoids. The back of the second globular layer is connected to the thylakoids or
with the opposite side of the chloroplast exterior in the two-layer eyes of Pyramimonas orien-
talis Butcher [Moestrup and Thomsen, 1974] located in the very thin anterior parts of the
chloroplast. Even the most inner back globular layer does not contact the thylakoids within
the two-layered eyes [Moestrup and Thomsen, 1974]. The various globular layers are con-
nected to each other by a continuous transition at their edges. This results in a rigid design
between layers with constant intervals between separate layers and the absence of any exter-
nal supporting structures between these layers (e.g., thylakoids) [see review by Melkonian
and Robenek, 1984].
One additional type of eyespot structure, where the globular layers are located inside
the thylakoids, has been described in the literature, however, this variant is thought to be an
erroneous [Foster and Smyth, 1980] interpretation of the micrographs of the eyes of Carteria
turfosa Fott [Joyon and Fott, 1964], Carteria crucifera Korsch. [Lembi and Lang, 1965] and
an unusual sea algae Chlamydomonas reginae Ettl and Green [Ettl and Green, 1973]. Based
on their structure, the eyes of these species actually belong to Tetraselmis Stein. [Melkonian
and Robenek, 1984].
The stigma of many green algae contains only one layer of pigmented globules that
are located at the chloroplast surface and closely adjoin the plasmalemma. Such single-
layered stigma are found in Mantoniella squamata (Manton and Parke) Desikach., Mono-
mastix Scherff., Nephroselmis Stein., in some species of Chlamydomanas, and in mobile re-
productive cells of many representatives of Chlorococcales, Chlorosarcinales, Bryopsidales,
Ulvales, Ulotrichales, and Chaetophorales [reviews see: Foster and Smyth, 1980; Melkonian
and Robenek, 1984]. The pigmented globules of single-layered stigma are separated by either
the chloroplast lamella or a thylakoid-less granular substance. Despite differences in mor-
phology and stigma structure in green algae, all appear to function based on the principle of a
simple or quarter-wave interference reflector [reviews see: Foster and Smyth, 1980; Melk-
onian and Robenek, 1984].
The distance between the external chloroplast membrane and the plasmalemma of the
stigma associated in a cross-section of species of algae (-50) [Melkonian and Robenek,
1984]) ranges from 10 to 53 nm (24.5 nm average). The interval is wider (30 to 53 nm) in
single celled species of Pedinomonas Korsch., Mesostigma Lauterbom, and Nephroselmis,
74
while the mobile reproductive cells of multicellular species Microthamniales, Wotrichales
and Wvales are characterised by a narrow (10 to 17 nm) interval.
Species of algae can be separated into 5 principal categories base on the stigma loca-
tion near the cell surface. These are type I (Prasinophyceae (Mantoniella Desikach., Mamiella
Moestrup) [Melkonian and Robenek, 1984], type II (Pedinophyceae [Melkonian and Ro-
benek, 1984; Ettl and Manton, 1964]), type III (Chlorodendrophyceae and Prasinophyceae
with four-flaggelar cells of Tetraselmis and Pyramimonas) [Melkonian and Robenek, 1984],
type N (Nephroselmis and Prasinophyceae), and type V (many green algae of class Chloro-
phyceae sensu Mattox and Stewart, Wvophyceae Stewart and Mattox, and Microthamniales
sensu Melkonian [Watson, 1975; Melkonian, 1984; Melkonian and Robenek, 1984].
During reproduction, the photoreceptor apparatus in green algae can be transferred
three ways [Barlow and Cattolico, 1980; Melkonian, 1981; Melkonian and Robenek 1984].
While a discussion of possible combinations of pigmented globules and thylakoids in the
morphology, fine structure and location of the stigma of green algae, historical development
of photoreceptor systems, and photobehaviour of green algae during evolution is not within
the scope of this book, useful information can be found in the following references [Foster
and Smyth, 1980; Ettl and Moestrup, 1980; Melkonian and Robenek, 1984; Massjuk et aI.,
2007].
The complexity and variety in the structure and location of photoreceptor systems in
green algae allow their use in taxonomic systematic and phylogenetics. The following charac-
teristics are the most useful for these purposes: the shape and a location of the stigma, quan-
tity and the size of globules, character of their arrangement in layers, number of layers, spe-
cialization of photoreceptor membranes, and method of inheritance of the photoreceptor sys-
tem by daughter cells [Melkonian and Robenek, 1984].
The greatest diversity in photoreceptor systems is found among representatives of the
Prasinophyceae and correlates with other ultrastructural traits (e.g., structure of the locomotor
apparatus, mechanisms of mitosis, cytokinesis) reiterating the heterogeneity of this taxon.
Four different types of photoreceptor systems can actually be observed within the species of
Pyramimonas. Such correlations underscore the high phylogenetic weight of the traits.
An absolutely unique position in the complex of morphological and ultrastructural
traits (including peculiarities in structure, location, and photoreceptor inheritance) is held by
the green alga Pedinophyceae. Likewise, there is a unique type of photoreceptor system in
representatives of Microthamniales [Melkonian and Robenek, 1984]. Various types of photo-
receptor systems are also found in species of Carteria and Chlamydomonas. Distinctions in
the structure of stigma are found in species of the genus Chlamydomonas (e.g.. C. reinhardtii
PA Dang, C. moewusii Gerloff), that are characterized by differences in the structure of a
cellular envelope, flagellar apparatus, and reproductive behaviour [see review by Melkonian
and Robenek, 1984].
The photophobic responses of the unicellular alga Haematococcus pluvialis Flotow
and stimulus-response curves at four different wavelengths, were interpreted with regard to
the structure of the photoreceptor apparatus [Cecconi et al., 1996]. The authors confirmed that
the photoreceptors are located in the stigma region and suggested the presence of two photo-
receptors.
Each of these examples is relevant with regard to the evolution and classification of
green algae. Unfortunately, the functional value of the structural types of photoreceptor sys-
tems has not been elucidated until now, since information on the photobehaviour of green
algae has been based on only few species. Of particular interest are small, asymmetric green
flagellates that are considered an early evolutionary stage in green algae [Mattox and Stewart,
1984; Melkonian, 1984]. Despite a scarcity of data, an attempt has been made to construct the
probable evolutionary sequence in the development of photoreceptor systems and photo-
behaviour in green algae and while speculative, it is of considerable interest [Melkonian and
75
Robenek, 1984]. Photoreceptor pigments (such as rhodopsin or baeteriorodopsin [Foster and
Smyth, 1980; Foster et al., 1984]) that are spread chaotically in the plasmalemma were inher-
ent to ancestors of green algae (bacteriorodopsin found in cytoplasmatic membranes was al-
ready present in the prokaryotic Halobacteria belonging to the Archaebacteria group that is
connected to the predecessors of eukaryotes [Fox et al., 1980; George et al., 1983; Schnabel et
al., 1983]). Such colourless flagellates, with one or two flagella that produce wavy move-
ments and contain photoreceptor pigments within the plasmalemma, are capable of undergo-
ing elementary photophobic reactions [Melkonian, 1983, 1984]. In addition, the concentra-
tions of photoreceptor pigments led to strengthening of the light signal, and localization in the
cell opposite the flagella attachment. The presence of a stigma that contains carotenoid pig-
ments with absorption properties similar to rhodopsin and the phototopotaxis ability of the
cell were thought to have evolved after the inclusion of chloroplasts. The photoreceptor sys-
tem, with the arrangement of the photosynthetic organelle found under optimum light condi-
tions, probably evolved earlier in the evolution of green algae. Originally it included one layer
of pigmented globules that provided shading for the photoreceptor and increasing contrast in
light illuminance at different positions of the organism relative to the light source. This layer
was likely formed by chloroplast lipid globules (plastoglobules) and it did not have a fixed
position. Subsequently the size of the globules and the distance between them and a photore-
ceptor became fixed forming the photoreceptor system.
Transfer of flagella from an apical position and changes of their movement character
(from wavy to a rowing movement) resulted in a transfer of the photoreceptor system to a
lateral position in relation to the attachment site of the flagella and perpendicular to the direc-
tion of movement of a cell.
The occurrence of communication between the microtubular system that provides a
fixed position in relation to a plane of flagellar beatings and the subsequent occurrence of
multilayered stigmata represent further developments in the evolution of photoreceptor sys-
tems in green algae.
There was a subsequent loss of the photoreceptor systems in mobile reproductive cells
of many green algae (including representatives of the class Charophyceae sensu Stewart and
Mattox), especially in connection with the transition to a terrestrial way of life [Melkonian
and Robenek, 1984].
Many evolutionary schematics are based on hypothetical guesses as to the course of
evolution and development of the photoreceptor systems in green algae that require verifica-
tion and confrrmation. It is impossible to reject the possibility of numerous occurrences of
photoreceptor systems in green algae in various evolutionary branches of the monophyloge-
netic kingdoms of green plants Viridiplantae. The fact that photoreceptor systems similar to
those of green algae were found within other lineages in eucaryotic algae (e.g., Dinophyta)
argues in favour of such a position. The independent occurrence of such photoreceptor sys-
tems in these departments is quite evident based on differences in the structure of the chloro-
plasts and composition of the photosynthetic pigments.The scientific value of such specula-
tion lies in the possibility of increasing the precision of selecting models for further investiga-
tion that may lead to a greater understanding of the basic biology operative.
76
principle of a simple or quarter-wave reflector (see reviews by Foster and Smyth [1980] and
Melkonian and Robenek [1984]).
Stigma of Dunaliella species that have been studied consists of one- or two-layer of
pigmented globules between 100-200 nm in diameter. The number of globules can increase
during ontogenesis. The stigma is located in the subapical, subplasmalemma portion of the
chloroplast stroma, free from thylakoids, though sometimes part of the globules maybe lo-
cated between lamella [Eyden, 1975; Hoshaw and Maluf, 1981]. Thus, based on the location
of the stigma and its fine structure, Dunaliella does not differ from other green algae.
77
.... ~·f'OIj),li1t'(llit=ht
I
P" .sfU~J IIf'" .
-'"
...
r
A/
j'l,
f=
\ ttt /
t t ..
,-_t_~
\~~II\~
i,,s~">CI",1
'h
rI~
-------~
\"'.... I'1"inn
i.. .;;.ln.cnl
0,
" I.' 0'
~t~
L
r..
~
.Jr
I'J, t t t Pi,
t~t
L.-{Ulllf'OllC'1l1 /;-.....1>1 rar.cn:
I ~~Jo(lrtot·tI ]<..t~..mcd
(l-t,)
(.1 10,
"
Fig. 6.2. S<:hematic ofthe relative orientation of dipole moments of photoreceptor molecules inside the ph0tore-
ceptor Ph (vertical arrows) and direction of propagation if of stimul&ting ligbt: a -non-po1Brilled light (vectms
Ex and Eyare perpendicular to dipole moments); b - non-polarised light (vector E. is perpendicular to di-
pole moment and vector Ey is parallel to dipole moment); c - polarised light (vector Ey is perpendicular to
dipole moments); d - polarized light (vector E.
is parallel to dipole moments); a and c: absorption is absent (A
= 0); b: one component from both is abBarbed(A = 0.5); ti: the single component is absorbed (A = I).
Maximal absorption of light takes place during movement of the cell parallel with the direc-
tion of light propagation [Creutz and Diehn. 1976]. Such a dichroism was found in E. gracilis
[Hl!der, 1987b] and in C. reinhardtii [Yoshimura, 1994]. A simple mathematical model for
the signal received by the dichroic photoreceptor molecules in E. gracilis when irradiated by
polarized light is described by Hill and Plumpton [2000] and can be used to explain the expe-
rimental results ofHl!der [1987b].
Analysis of the angular distribution of Dunaliella cells moving under polarised light
with an electric vector that is parallel to the plane of cell movement indicates that the
distribution has a random character. The dominant directions of cell movement, which could
be changed after rotation of the plane ofpolarisation, were absent [posurlin, 1992].
AI!. mentioned earlier in Section 4.3.1, we did not find significant changes in cell linear
velocity of either species in response to polarized and non-polarized white light at the same
intensity (see Fig. 4.3). The same conclusion (i.e., the relative number N,,/No of motile cells
does not depend on the plane ofpolarization ofthe light) can be reached.
The absence of a dominating direction in histograms of angular distribution of the
cells in polarised light and the dependence of the linear velocity of movement and the relative
78
number of motile cells on the rotation plane of polarization also support the idea of a non-
dichroic nature for the photoreceptor system. It is therefore evident that Duna/iella has a quite
different photoreceptor system structure in comparison to E. gracilis.
AL''''~''1
0"
40
20
270" 90°¢:J
10 20
£,
£,
Fig. 6.3. Fourier-analysis of the angular distribution of Dunaliella salina cells due to two light flows ofmoderate
illuminance (£} = £1 = 500 Ix) (see text for further explantion) [Posudin et aI., 1991].
79
A
0°
40
20
~_ _ 900
10 20
d
0
0 E,= 10,000 Ix
£2~ 60,000 Ix
:\'=7
270
360
180 ~,uliilL
90
c::} 270 ---T'----1---:i'----
0
90°
"
10 20
[;,
180~
11 £.
Fig. 6.4. Fourier-analysis of the angular distribution of Dunaliella salina cells due to two light flows of high
illuminance (E, = 10,000 Ix andE2 = 60,000 Ix) (see text for further explantion) [Posudin et aI., 1991].
Comparison of our results with data that have been obtained for E. gracilis [Lebert and 1985;
Hllder, 1986b; Lebert and Hader, 2000] indicates a difference between D. salina and E.
gracilis in photomovement when exposed to two perpendicular low illuminance beams. At
low illuminance, the cells of E. gracilis display positive phototopotaxis and a bimodal distri-
bution. The population splits into two fractions moving toward both light sources. In two
strong light beams perpendicular to each other, the cells of both D. salina and E. gracilis
move away from the light beams. These distinctions indicate two different mechanisms of
photoreception at the moderate intensity. D. salina most likely exhibits periodical amplifica-
tion or attenuation of the light signal by way of the stigma - a mechanism typical for green
phytomonads. The photoreceptor system is located perpendicular to the surface ofthe cell and
the direction of movement, and it has non-dichroic nature.
80
6.4. Summary
The photoreceptor system in the species of Dunaliel/a, as well as that of other green algae, con-
sists of a photoreceptor, preswnably located in the plasmalemma and in the chloroplast mem-
branes near the stigma. The stigma, consisting of one-two layers of lipid globules in different
species is located in the peripheral zone of the plastid. It has been shown that, in contrast to
certain other algae species (e.g., E. gracilis), Dunaliel/a do not possess a photoreceptor with
a dichroic structure.
As indicated in Chapter 5, the effect of ionizing and ultraviolet radiation indicates dif-
ferent mechanisms govern the linear velocity of movement and phototopotaxis in Dunaliel/a
and the possible existence of two different photoreceptors that are responsible for different pho-
tomovement parameters.
81
Chapter 7
Identification of Photoreceptor Pigments
84
Microspectrophotometry was used to investigate the paraflagellar bodies of isolated
flagella from E. gracilis [Schmidt et al., 1990]. Flagella with attached paraflagellar bodies
were separated from the cell bodies using a short exposure to near-UV light. Fluorescence
emission spectra (excitation at 365 nm) of single paraflagellar bodies had maxima near 470
and 520 nm, indicating the presence ofpterins and flavins. The occurrence ofpterin-like fluo-
rescence in the paraflagellar body lends further support to the earlier proposal that pterins as
well as flaYins may function as photoreceptor pigments for near-UV and blue light [Schmidt
et al., 1990]. The fluorescence spectra of isolated paraflagellar bodies from E. gracilis excited
at different wavelengths indicated the presence of more than one (flavin and pterin) photore-
ceptor pigment [Sineshchekov et al., 1994].
Fluorescence spectroscopy has indicated the presence of an energy transfer between
pterins (pigments absorbing in the ultraviolet portion of the spectrum) and flavins [Sineshche-
kov et al., 1994]. With the exception of fluorescence at 440-520 nm, long-wave fluorescence
at 580 nm (excitation wavelength 520 nm) and 620 nm (excitation wavelength 550 nm) was
observed. The role of long-wave fluorescence is not clear. The presence of a third photorecep-
tor pigment was postulated by the authors. The participation of rhodopsin, however, was ex-
cluded due to the insignificant fluorescence quantum that is indicative of rhodopsins [Sinesh-
chekov and Litvin, 1987]. Likewise, contrasting the temporal kinetics of rhodopsin fluores-
cence at 397 nm (excitation at 365 nm) that had a photocycle duration of about 18 s [Barsanti
et al., 1997] with the real fluorescence kinetics at a frequency of 1-2 Hz induced by rotation of
the cells [Ascoli et al., 1978] argues against this position.
Flavins were found not only in the paraflagellar body but also in the stigma of E. gra-
cilis [Sperling et al., 1973]. The absence of stigma absorption dependence on the plane of
stimulated light polarization argues against the participation of the stigma in photoreception
[Benedetti et al., 1977]. In contrast, phototopotaxis is sensitive to changes in the polarization
plane [Bound and Tollin, 1967].
The preceding experimental methods used by advocates of the flavin-pterin hypothesis
supported the conclusions that: 1) the paraflagellar body of E. gracilis is the location of the
photoreceptor; 2) the stigma is not a photoreceptor; 3) flavins are the primary chromophores
for photoreception; and 4) the pterins function as a "light antenna" for the flaYins.
High-performance liquid chromatography was used to identify the pigments associated
with the flagellum of E. gracilis. A wild-type strain that contains FMN and FAD was com-
pared with two mutant strains that did not display phototactic reactions and photoaccumula-
tion in response to blue light. Flagella of the wild type contained flavin isoalloxazine deriva-
tives, FMN and FAD. The mutants, which lacked the stigma but retained a small paraxonem-
al body, contained less flavins. The white mutant (FB), which retained a residual stigma and a
paraxonemal body and the white phytoflagellate A. longa, a close relative of Euglena, had
normal amounts of flagellar flaYins. An unidentified pterin-like pigment (PtI6) was found in
the cells and flagella of Euglena. Small amounts of the pigment were found in Astasia and the
Euglena mutants. A third pigment (P528) was present mainly in the flagella. The three pig-
ments probably participate in photoreception [Geiss et al., 1997].
Another group [Gualtieri et al., 1988] believed, based on microspectrofluorometry of
the paraflagellar body (E. gracilis), that rhodopsin was responsible for photoreception. The
close similarity of the absorption spectrum of the paraflagellar body with that of rhodopsin
with a maxima at 500 nm supports this position [Gualtieri et al., 1989; James et al., 1992].
The intensity of the absorption band of the paraflagellar body depended on the relative orien-
tation of the polarization plane and longitudinal axis of the cell. This suggested that a rhodop-
sin protein located in the quasi-crystalline structure of the paraflagellar body functions as the
phototeceptor [Gualtieri et al., 1989; James et al., 1992].
The possible contribution of other cellular components that absorb light has been indi-
cated as a possible weakness of the methods. In addition, the absorption bands of practically
85
all of the pigments in the visible region of spectrum are superimposed to some degree (Fig.
7.1). However, extraction of retinal from intact cells and the photoreceptor after isolation
from membrane-less cells, further supports rhodopsin as the photoreceptor of E. gracilis
[Gualtieri et al., 1992]. A photochromic pigment was found in the paraflagellar body demon-
strated a reversion of fluorescence [Barsanti et aI., 1997].
Rh
,\
I I
c
I
\ ,./\.
\
J \
Fig. 7.1. Absorption spc:ctra ofthc photoreceptor pignlmIts: F - tlavins [Britton, 1986], Ph - rbDdopsin [Bensas-
son, 1980], C - carotenoids [BeIlSlISSon, 1975],1IIld Pt -pterins [Britton, 1986].
Thus, the paraflagellar body is characterized by optical bistability - the primary nonfluores-
cent and secondary fluorescent forms appear after light excitation, further substantiating the
paratlagellar body as photoreceptor in E. gracilis. However, since other fluorophores may be
located in the photoreceptor or near organelles, the precise identification of the photoreceptor
pigments is difficult.
86
photophobic reactions exhibited almost identical action spectra with peaks at 480 run and 375
run. Diehn [1969] supposed that these action spectra probably represent a composite of the
absorption spectra for the photoreceptor and the screening pigments.
A number of authors indicate that the shape of the spectrum reflects the absorption
properties of flavin-containing proteins. In general the experiments have focused on the mea-
surement of action spectra of E. gracilis [Wolken, 1960, 1977; Bensasson, 1975; Lenci, 1975;
Nultsch and Hader, 1979, 1988; Co10mbetti and Lenci, 1980; Galland et al., 1990] and have
indicated the presence of more than one photoreceptor pigment (e.g, flavins, carotenoids, pte-
rins).
A novel flavin-binding photoactivated adenylyl cyclase (EC 4.6.1.1, also known as
adenylate cyclase and adenyl cyclase) consisting of two subunits has been isolated from the
paraxonemal body [Ntefidou et al., 2003a]. This photoactivated adenylyl cyclase controls
phototaxis in E. gracilis [Ntefidou et al., 2003b] and is the blue-light receptor flavoprotein
identified as a sensor for photomovement in E. gracilis [Iseki and Watanabe, 2004; Iseki et
al.,2006].
A chlorophyll-free strain of E. gracilis var. bacillaris, possessing stigma and a photo-
receptor, displays negative phototopotaxis. The action spectrum for the reaction is characte-
rized by a maxima at 410 run and secondary peaks at 449 and 476 run; above 530 run no reac-
tion was detected [Gossel,1957]. Another strain of E. gracilis with a photoreceptor but with-
out a stigma displayed both positive and negative phototactic reactions. The maxima for posi-
tive phototopotaxis occurred at 410 and 425 run, while negative phototopotaxis was at 449
and 476 run [Gossel, I., 1957].
The colorless euglenoid flagellate, Astasia fritschii Pringsheim and Hovassee, exhibits
a pronounced step-up photophobic response (i.e., elicited by a sudden increase in light intensi-
ty) and rather weak photokinesis. The action spectrum has a major peak at 450 run suggesting
that flavins may be the pigments responsible for the step-up response [Mikolajczyk and
Walne, 1990].
In principle, the spectral sensitivity of photoreactions reflects the absorption properties
of the key pigments operative, however, the analysis of pigments is much more complicated.
For example, the possible participation of pigments that are present in shading structures (e.g.,
the stigma) or the presence more than one pigments in the photoreceptor structure confound
the direct interpretation of the action spectrum obtained. In addition, the action spectra ofpho-
toreactions are seldom of high resolution and other pigments may have overlapping absorp-
tion bands in the visible region of the spectrum. As a consequence, precise correspondence in
the patterns of the photoreceptor pigment and the action spectrum do not often occur. Finally,
ascertaining the photoreceptor pigments by comparing the action spectra for photomovement
parameters with the pigment absorption spectra is further complicated by alterations in the
pigments due to their surroundings.
87
7.2.6. Effect of Exogenous Chemicals on Photomovement
The application of compounds that prevent oxidative phosphorylation (e.g., cyanide, azide,
rotenone, 2,4-dinitrophenol, 2,6-dichloroindophenol) in the photomovement of E. gracilis did
not affect the parameters of motility and positive phototopotaxis equally. In contrast, com-
pounds that prevent photophosphorylation (e.g., salicylaldoxime, tetramethylthiuram disul-
fide) inhibit both motility and phototopotaxis. Other compounds [e.g., ethanol, (dichloro-
phenyl)dimethylurea, methyl octanoate, carbonyl cyanide p-(trifluoromethoxy) phenylhydra-
zone)] have been shown to specifically inhibit positive phototopotaxis [Diehn and Tollin,
1967]. Based on these results, it was concluded that the primary energy source for positive
photopotaxis is the photophosphorylation system of photosynthesis.
Additional confirmation of flavins functioning as the photoreceptor pigments was ob-
tained using chemicals that affected their excitation state (e.g., potassium iodide). There are,
however, some discrepancies in the results from different researchers. Several observed an
effect of potassium iodide only on positive phototopotaxis in E. gracilis [Mikolajczyk, Diehn,
1975], while others found a strong effect on either positive or negative phototopotaxis [Lenci
et aI., 1983]. These contradictory results may be due to the effect of circadian rhythm and/or
the age of the cultures on the response [Hilder and Lebert, 2001].
The effect of nicotine, a highly biologically active compound, was tested at various
concentrations on the photoreceptor in E. gracilis using phase-contrast microscopy, fluores-
cence microscopy, transmission electron microscopy and photobehaviour. Nicotine inhibited
the photoaccumulation of the cells. The authors [Barsanti et aI., 1993] concluded that the re-
sults strongly confIrmed the presence of a carotenoid or carotenoid-like chromophore as the
photoreceptor. However, the application of nicotine (that inhibits the biosynthesis of retinal)
and hydroxylamine (that responds to retinal, either free or opsin-bonded forms) inhibits the
formation of the paraflagellar body and photoorientation of the cells. Collectively the results
suggest that retinal in the paraflagellar body acts as the photoreceptor pigment [Barsanti et aI.,
1992, 1993]. However, Foster [2001] argues that the inhibition of the formation of the paraf-
lagellar body does not adequately support the participation of rhodopsin in photoreception, a
position supported by the inhibition of phototopotaxis in Chlamydomonas by nicotine and
norflurazon. Even though rhodopsin is present in Chlamydomonas cells, it does not substan-
tiate its role as the photoreceptor pigment in that the effect of the action of specific inhibitors
can be obtained by other means.
Formation of the paraflagellar body in E. gracilis can be dependent on rhodopsin. A
more precise analysis demonstrated the absence of the effect of the inhibitor on both positive
and negative phototopotaxis during extended exposure (>6 months) to nicotine, while retinal
was identified in the cells using chromatography [Gualtieri et aI., 1992].
Evidence for flaYins and/or pterins as the photoreceptor pigments in E. gracilis includes:
1). The action spectra for phototopotaxis (i.e., wavelength dependence for response sensi-
tivity) very closely resembles the absorption properties of flaYins and there is a close relation
88
between the absorption properties of the paraflagellar body and the known action spectra, thus
further substantiating this structure as the location for the photoreceptor;
2) Fluorim.etric measurements such as microspectrofluorometry of the pigments in vivo
indicate an energy transfer from pterins to flavins in intact paraflagellar bodies; and 3). Sever-
al proteins containing flavins and pterins have been identified in the structure [Lenci and
Ghetti, 1989; Cubedduet aI., 1991; Lebert, 2001].
The following arguments are presented by supporters of the rhodopsin hypothesis
[Gualtieri et al., 1989, 1992; Gualtieri, 1993; Barsanti and Gualtieri, 2007]. These include
theoretical considerations, absorption microspectroscopic measurements, experiments inhibit-
ing the biosynthesis of carotenoids, the photobehaviour in the presence of hydroxylamine hy-
drochloride, and the identification of all-trans-retinal by thin layer chromatography, high per-
formance liquid chromatography (HPLC), absorption spectroscopy, and gas chromatography-
mass spectrometry (GC-MS), each of which supports rhodopsin as the photoreceptive mole-
cule inE. gracilis.
Neither supporters of flavin (pterins) nor rhodopsin as the photoreceptor believe the
opposing group has adequately substantiated their case, regardless of the diverse range of ana-
lytical methods employed. It is evident that a lively scientific debate will continue until the
question is finally resolved.
89
axonemes to respond to changes in Ca2+ concentration, while the others exhibited a defective
flagellar Ca2+ channel. As a consequence, some mutants displayed one reaction (phototaxis)
but not the other (photoshock) in response to Ca2+ treatment.
Mutants pprl, ppr2, ppr3, and ppr4 do not display a photophobic response but display
phototactic activity at nearly the same level of sensitivity as wild type cells [Matsuda et aI.,
1998]. It was shown that with the all-or-nothing flagellar current, a Ca2+ current generated by
depolarization of the flagellar membrane was absent or seriously impaired in the mutants.
These findings clearly indicated that the all-or-nothing flagellar current is necessary for the
photophobic response but not for phototopotaxis. Similarly, a complementary DNA sequence
from C. reinhardtii that encodes a microbial opsin-related protein was studied by Nagel et aI.
[2002]. This protein, termed channel rhodopsin-I, is believed to be involved in the phototactic
activity of the algae.
In C. reinhardtii, the pigmented eye comprises the optical system and has at least five
different rhodopsin photoreceptors [Kateriya et aI., 2004]. Two of them, the channel rhodop-
sins, are rhodopsin-ion channel hybrids that switch between closed and open states by photoi-
somerization of the attached retinal chromophore. Channel rhodopsin-2, a light mediated
gated cation channel, switches a cation-selective ion channel. The system employs II-cis re-
tinal as its chromophore [Flannery and Greenberg, 2006].
Rhodopsins, identified from cDNA sequences, function as low- and high-light-
intensity phototactic receptors in C. reinhardtii [Ebrey, 2002; Sineshchekov et aI., 2002; Go-
vorunova et aI., 2004]. The function of the two rhodopsins (CSRA and CSRB) phototactic
receptors was demonstrated by in vivo analysis of photoreceptor electrical currents and motili-
ty responses in transformants using RNA interference (RNAi) directed against each of the
rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor
currents differ greatly among transformants depending upon the relative amount of photore-
ceptor pigments that are expressed. The action spectrum of CSRA has an absorption maxi-
mum at 510 nm and mediates a fast photoreceptor current. The action spectrum of CSRB, in
contrast, has a maximum at 470 nm and generates a slow photoreceptor current. The relative
wavelength dependence of CSRA and CSRB activity for inducing phototactic responses pre-
cisely matches the wavelength dependence of the CSRA and CSRB generated currents, de-
monstrating that either receptor can mediate phototopotaxis. The discovery of two distinct
sensory receptors (sensory rhodopsins A and B) responsible for phototactic activity of the
alga was also reported [Ridge, 2004].
The effect of hydroxylamine on the phototopotactic activity of C. reinhardtii and the
restoration of phototactic sensitivity after its removal appears to be due to the bleaching of
rhodopsin and subsequent replacement via de novo synthesis or by added retinal [Hegemann
et aI., 1987, 1988].
Several researchers believe that the abundant retinal protein in the stigma of Chlamy-
domonas is not the photoreceptor for phototopotaxis and photophobic responses and that the
responses are triggered by a yet to be identified rhodopsin species [Fuhrmann et aI., 2001].
Incubation of the organism with retinal analogs shifts the phototopotaxis action spectraI max-
imum in a manner similar to the shifts encountered in the bovine visual pigment [Nakanishi,
1985]. The author believes that the algal photoreceptor may be closely related to the bovine
rhodopsin but not the bacterial rhodopsin.
The ability of different retinal isomers and analogs to restore photosensitivity in blind
Chamydomonas cells (strain CC2359) was tested using flash-induced light scattering tran-
sients and by measuring phototaxis using a taxigraph [Hegemann et aI., 1991]. They con-
cluded that rhodopsin bears a closer resemblance to bacterial rhodopsins than to the visual
rhodopsins in higher animals. The blind mutant FN68 lacks retinal and therefore cannot dis-
play phototopotaxis, however, with incubation in a media containing retinal analogs, phototo-
potaxis is restored [Nakanishi et aI., 1989]
90
A real-time automated method was developed for simultaneously measuring phototac-
tic orientation and the step-up photophobic response in flagellated microorganisms [Takaha-
shi et al., 1991]. Addition of all-trans retinal restored both photoresponses in a carotenoid-
deficient mutant strain of C. reinhardtii in a dose-dependent manner. The results strongly
suggested that isomerization of the 13-14 double bond is important for photobehavioral signal
transduction and that a single retinal-dependent photoreceptor controls both phototactic and
photophobic responses. zacks et al. [1993] concluded that although phototaxis and the photo-
phobic response are mediated by retinal-containing receptors displaying a close similarity in
chromophore structural requirements for both behaviors, the results indicate that differences
exist between the two responses in their photoreceptor proteins and/or transduction processes.
Experiments with a C. reinhardtii mutant into which retinal and retinal analogs were
incorporated [Takahashi et al., 1992] were interpreted as indicating negative and positive pho-
totopotaxis are mediated by two rhodopsin species differing in their affmity for the exogenous
chromophores. However, a more reasonable explanation, that requires fewer assumptions, is
that the sign for phototaxis depends on a delay in intracellular photosignal transduction.
Around the same time, Kroeger and Hegemann [1994] proposed that photophobic res-
ponses and phototaxis in C. reinhardtii are triggered by a single rhodopsin photoreceptor.
According to their hypothesis, chlamyrhodopsin triggers a photoreceptor current in the eyes-
pot region, resulting in directional changes or phototopotaxis, however, when the light stimu-
lus exceeds a critical level, flagellar currents appear and are the basis for the photophobic re-
sponse.
The principal methodological approach that has been used in the investigation of
photoreceptor systems in green algae was similar to studies on membrane potential or electric
current that passed through cell membranes. Ions that pass through the membranes carry
electrical charges that induce an electric current in the membrane of about 10-12 A. It is
possible to measure these currents using microelectrodes within thin glass tubes. This
technology is called the patch clamp technique and uses a micropipette attached to the cell
membrane to allow recording a single ion charinel. The micropipette has tip diameter about
0.5-1.0 flm which is placed on the surface of the cell membrane. Gentle suction is applied to
the micropipette drawing a small area of the membrane (the "patch") into the microelectrode
tip. The presence of the micropipette tip against the membrane and the suction forms a
resistance "seal" between the glass and the cell membrane, electronically isolating the current
across the membrane patch. The current measurements are expressed either for the entire
protoplast or the portion that has been drawn into the microelectrode tip (Fig. 7.2). Thus, very
small diameter micropipettes allow measuring the electric currents through separate mem-
brane ion channels.
91
rip of mi(TOpipCllC SO!luil)l1
in l11icropipcll<.:
PrOlOp)aSI
Cell membrane
N1easufcmCIll MC,ISllfL'Ill<.:nl
with wholc cell. \\iLh p;l\ch
The specific membrane potential features in C. reinhardtii and H pluvialis are the
input resistance, 200-250 Mil (106 il) for C. reinhardtii and several hundred Mil for H plu-
vialis, and input resistances of hundreds of Gil (i.e., 109 il). The clamp patch technique to-
gether with micro-irradiation of the cell has made it possible to establish that light-induced
electric signals only appeared during irradiation of the stigma, further solidifying the presence
of the photoreceptor in this area [Ristori et al., 1981; Sineshchekov et al., 1991a]. The tech-
nique has also allowed determining the action spectrum by monitoring the generation of the
electric signal. Harz et al. [1992] concluded that the magnitude of the electric signal depends
on the orientation of the cell and its photoreceptor relative to the light stimulus. The ratio of
signals during irradiation of the photoreceptor and the posterior portion of the cell is approx-
imately 8 for C. reinhardtii and 3 for H. pluvialis [Harz et aI., 1992]. Differences in the ratio
may be explained by structural differences in the stigma - four-layers in C. reinhardtii versus
one in H pluvialis. In addition, the stigma of Chlamydomonas functions as a quarter-wave
plate that provides amplification of the light signal due to the reflection of the light waves
from the layers and interference. Finally, the shape of the action spectrum for the generation
of the electric signal is similar to the absorption spectrum for rhodopsin. The fine structure of
the spectrum indicates a fixed location for the chromophore in a protein and the possible par-
ticipation of more than one pigment in photoreception.
The microelectrode pipette technique was also applied to cell-wall-deficient Chlamy-
domonas cells [Holland et aI., 1996] stimulated by short but intensive flashes of light. Three
types of photocurrent were observed. 1) A transient current localized in the stigma that was
identified as the photoreceptor current. The photoreceptor current consists of at least two
components activated by different mechanisms. The first mechanism most likely involves
translocation of ions across the membrane either by rhodopsin itself or through an ion channel
directly coupled to it. 2) The mechanism of photoreceptor current generation appears to occur
via a biochemical amplification cascade. Membrane depolarization, induced by the photore-
92
ceptor current, leads to an unbalanced motor response by the flagella, which is the basis for
phototactic activity. This second transient current localized in the flagella is named the fast
flagellar current. 3) The third current is a slow flagellar current, which has a very small ampli-
tude. With an intense light stimulus, the photoreceptor current triggers a fast and a slow fla-
gellar current that results in backward movement, stopping the organism. The photoreceptor
current appears with a delay of less than 50 ~s, suggesting that rhodopsin and the photorecep-
tor channel are closely coupled or form one ion channel complex.
It is believed that receptor photoexcitation in green flagellates triggers a cascade of
rapid electric events in the cell membrane [Sineshchekov and Govorunova, 2001a,b,c]. The
photoreceptor current has been the earliest process detectable thus far in the cascade. Mea-
surement of the photoreceptor current at present has been the best approach for investigating
the photoreceptor pigment, since the low receptor concentration in the cell makes existing
optical and biochemical methods unsuitable. Existing physiological evidence indicates the
photoreceptor in green flagellate algae is a unique rhodopsin-type protein.
Proteomic analysis (the study of the structure and function of proteins) of the stigma of
C. reinhardtii identified 202 different proteins, in particular photoreceptors, retina(1)-related
proteins, and members of putative signaling pathways for phototaxis such as casein kinase 1
[Schmidt et al., 2006].
93
(syn. Platymonas viridis Rouch.) strain N 68 from the collection at Institute of Botany of
Ukrainian Academy of Science to compare T. hazenii and T. viridis.
An illuminator (LOS-2) with a 1 kW xenon lamp displaying a spectrum similar to so-
lar radiation was used. The radiation was directed at the angle 30° to the plane of the cells
positioned on a microscope stage. The radiation passed an infrared filter (a layer of distillated
water) and interference filters with transmission maxima at 249,279,304,310,335,364,407,
434,497,541, and 655 nm. A wide-band ultraviolet filter (lNW) with a spectral band at 240-
410 nm was also used. The spectral characteristics of these filters in comparison with absorp-
tion spectra offlavins and carotenoids are presented in Fig. 7.3. A unique feature of the expe-
riments was the use of a 1.75 mm quartz plate (that does not filter out the ultraviolet region of
the spectrum) as a cover slip [Posudin et a1., 1990].
D,T. I'll.
(\ (\
___ I
\
I \. ---2
\
I \
--3
\ \
\ I
l: 'v \\
4 f',
'
1I \
\
// './
'v
2
\
"
200 250 300 400 500 )" nm
Fig. 7.3. Absorption spectra of pigments (1- flavins; 2 - carotenoids) and transmission spectra (3) ofinterfe-
renee filters (figures correspond to maximum transmission) in the ultraviolet and visible portion of the spectrum;
D - absorption of pigments; T - transmission of filters [posudin et aI., 1990].
Measurement of phototopotaxis in two species of Dunaliella and T. viridis (Table 7.1) estab-
lished that the F parameter values for both Dunalie/la species in response to a light stimulus
at 249,279, and 310 nm differed significantly from those of T. viridis [posudin et al., 1990].
The values of this parameter are approximately the same for all three algae around 304
nm. Such a similarity can be explained by coincidence of absorption maxima of either f1aYins
or carotenoids.
There were no significant differences in parameter F in the near ultraviolet (335 and
364 nm) and visible (407-655 nm) regions of the spectrum among the three species. The ef-
fect of the ultraviolet region on photomovement of Dunaliella indicates that both species do
not display wavelength maxima for the dependence of parameter F. In the ultraviolet region
of the spectrum there are absorption maxima for flaYins and rhodopsin. At the same time, a
significant increase in parameter F was observed in the ultraviolet region of the spectrum (Le.,
94
249,279, and 310 nm). The results obtained with a wide-band (240-410 nm) ultraviolet filter
are similar (Table 7.1).
Table 7.1. Phototopotaxis of algae in the ultraviolet aod visible portions of the electromagnetic spectrum.
Comparison of the results with the action spectra for photomovement parameters of
other algae (e.g., C. reinhardtii [Foster and Smyth, 1980], H pluvialis [Sineshchekov, 1988],
T. hazenii [Halldal, 1961] and E. gracilis [Foster and Smyth, 1980]) are presented in Fig 7.4.
Our results for parameter F in the ultraviolet region of the spectrum for both
Dunaliella species and T. viridis in comparison with Hal1dal's [1961] indicates that the
presence of phototopotaxis is inherent only to the latter species. Halldal believed that carote-
noproteins function as the pigment. The complete absence of phototopotaxis in both species
of Dunaliella in the ultraviolet region of the spectrum (i.e., 240-400 nm) suggests that flavins
and rhodopsin cannot function in photoreception in these species [Posudin et ai., 1990].
95
F(A)
1,0
0,8
\,, I
0,6 I
I
1\
I
"
I
,I \
v \
0,4
\
I
0,2
,_/ I
\
,-------r--r_-'_r~-r_..........'_r~-T_.==.-_.__
A, HM
Fig.7.4. Action spectra of: 1 - positive phototopotaxis in Platymonas subcordiformis [HallOOl, 1961]; 2 - photo-
topotaxis in Chlamydomonas reinhardlii [Nultsch et aI., 1971]; 3 -phototopotaxis in Dunaliel/a spp. [posudin
et aI., 1991]; 4 - photoinduction of phototopotaxis potential in Haematococcus pluvialis [Sineshchekov, Litvin,
1988]; 5 - phototopotaxis in Euglena gracilis [Foster, Smyth, 1980].
Existing data on the photomovement of Dunaliella [Posudin et aI., 1991; Posudin et aI., 1992]
indicates that neither species displays a significant maxima for phototopotaxis in the ultravio-
let region of spectrum where absorption maxima by flavins, pterins, and rhodopsin takes
place. The absence of phototopotaxis in the ultraviolet region and the effect of the visible (Le.,
400-520 nm) region of the spectrum point toward carotenoids, that absorb between 350-500
nm, acting as the photoreceptor pigments. There is also evidence that carotenoproteins and
rhodopsin participate in the photoreception of D. salina [Wayne et aI., 1991].
7.5. Summary
The action spectrum for phototopotaxis in the two hyperhalobic species of Dunaliella is iden-
tical and within the 400-520 nm range, exhibiting maxima at 410-415 and 465-475 nm.
The spectrum differs somewhat from those of Chlamydomonas reinhardtii and Haematococ-
cus pluvialis, that display a wide band in the 400-600 nm region and a maximum at 500 nm.
In contrast, in representatives of the class Chlorophyceae, such as Tetraselmis viridis (Chloro-
dendrophyceae), phototopotaxis occurs in the ultraviolet region of the spectrum. Phototopo-
taxis in Euglena gracilis (Euglenophyta) is in the 300-550 nm region with principal maxima at
385 and 460 nm and two smaller maxima at 410 and 490 nm. Thus, representatives of various
genera, classes, and divisions of algae differ in the region of the spectrum that modulates pho-
totopotaxis, indicating some degree of diversity in photoreceptor pigments among species.
96
Chapter 8
Mechanisms of Photoreception and Photoorientation
in Dunaliella
!i'IilX:~ '1-
a
Fig. 8.1. Modulation mechanism for the photoorientation of Euglena gracilis. The rotational movement of the
cell modulates the light striking the photoreceptor (a); the amplitude of modulation depends on the direction of
movement of the cell relative to the direction ofpropagation of light and varies from maximal value (Imax) to zero
(10) with subsequent flagellar beating. If the light is directed along the 10ngitudinaI axis of the cell (b), light mod-
ulation and flagella beating are absent (adapted from [Colombetti et aI., 1982]).
97
Y. I. Posudin et al., Photomovement of Dunaliella Teod.,
DOI 10.1007/978-3-8348-9765-7_9,
© Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH 2010
Some algae possess a non-reflective stigma that acts as a shading device (Euglenophyceae)
[Colombetti et al., 1982; Lebert and Hader, 2000] or a stigma which focuses the reflected
light on the photoreceptor and acts as a shading device (e.g., Chrysophyceae, Phaeophyceae,
some species of Dinophyceae, zoospores of Eustigmatophyceae) [Kreimer, 1994, 2001].
Normal movement of motile eukaryotic algae is accompanied by a rotational movement
around the axis of the cell, a mechanism that is probably common to most algae. Therefore, it
would appear that the mechanism was inherited from prokaryotic organisms and common
ancestors. It is the primary event in photoreception in essentially all motile eukaryotic algae
and their zoospores and it is possible to distinguish among various taxons secondary mechan-
isms that amplify the effect of the modulation.
2) Waveguide mechanism. Lateral light that is perpendicular to the longitudinal
axis of the cell is believed to pass through coupled thylakoid disks. These disks contain pho-
topigments and are perpendicular to the pigmented layer ofthe stigma and longitudinal axis of
the cell. Thylakoids have a high refractive index in comparison to the light-coloured intervals
between them. Therefore, the coupled thylakoids and intervals function as a waveguide for the
light. The cell responds to the light stimulus due to its incidence on certain abdominal parts of
the cell (Fig. 8.2). Such a mechanism is found in the Cryptophyta Chroomonas Hansg. and
Cryptomonas Ehrenb. [Foster and Smyth, 1980].
Coupled thylakoids
Pyrenoid
Fig. 8.2. Structure and location of photoreceptor system in Chroomonas Hansg.: 1 - part of chloroplast where
coupled thylakoids and pigmented globular stigma are located; 2 - location of the photoreceptor system inside
the cell (adapted from [Foster and Smyth, 1980]).
98
The structure of the ocelloid in Nematodinium annatum (Dogiel, 1906) Kofoid & Swezy,
1921, is illustrated in Fig.8.3.
It has an egg-shaped structure with a lens that converge parallel beams of light deeply
into the retinoid that is covered underneath by a layer of lipid globules. The ocellar chamber
lies between the lens and the retinoid. Objects within about 50 IJ.m are focused on the retinoid;
therefore, as an aquatic object moves near the cell, the ocelloid produces a signal, the intensity
of which depends on the distance, size, and direction of the object [Omodeo, 1975]. Optical
processes in the ocelloid system include focusing the light by the lens, limiting light flux by
structural elements in the ocelloid, refraction and interference.
D Light
Pigmented
globules
Fig.8.3. MOIphology of the ocelloid in Nematodiurn annaturn (modified from [Omodeo, 1975]).
99
side the stigma. However, light falling on the opposite side of the cell results in several inter-
ference minima at the same location inside stigma. Thus, the contrast in the perception of light
that falls on opposite sides of the cell is increased.
Assessment of the stigma in Chlamydomonas reinhardtii P.A. Dang. using confocal
laser scanning microscopy and photoelectric measurements demonstrated the importance of
an intact stigma for interference reflection and the absorption of phototactically active light,
and therefore, the directional sensitivity of the stigma [Kreimer et al., 1992]. It is assumed that
interference mechanism is present in the Chlorophyceae, Prasinophyceae and some species of
Dinophyceae that have multilayered stigma [Foster and Smyth, 1980; Hegemann and Fischer,
2001]. If the thickness of the pigmented and non-pigmented layers equals one quarter of a
wavelength, amplification of the light takes place [Hegemann and Harz, 1998]. Therefore, an
interference quarter-wave mechanism is based on the assumption that there are several pig-
mented layers in the stigma. The presence of pigmented globules in continuous layers is a
critical component of the hypothesis of Foster and Smyth (1980).
100
IJ" : e.
D J c
.' - 0
Fig. 8.4. Schematic of the optical phenomena that occur during the interaction of light with the structure fonned
by spherical (a,b) or hexagonal (c) globules that are densely packed due to mutual compression, where: ()o and
()m are the angles of incidence and diffraction, respectively; d = D + s - period of diffraction grating (D is di-
ameter of the globule and s is the interval between globules); I - incident beam of light; 2,3 - diffracted beams
oflight[Posudin and Massjuk, 1996].
Light interacts with periodically arranged pigmented globules in the stigma that form the diffiac..
tion grating which can be described by the following equation [Born and Wolf, 1968]:
where p is the parameter of diffraction; d - the period of diffraction grating (d = D + s), where
D is the diameter of the globules and s - the distance between them; Om - the angle of diffrac-
tion; 0 0 - the angle of incidence of light on the plane of the diffraction grating; and m - an in-
teger (m= 0, ±1, ±2,....).
The intensity of diffraction maxima of m-order is described by the normalized function
F(P) of light distribution during diffraction as:
where k = 2m is a wave number and N - the number of elements in the diffraction grating (in
this case, the number of globules in the stigma). The function F(P) depends in a complex
manner on the diffraction parameter p. A quantitative estimation of the diffraction mechanism
was derived using electron microscopy to characterize the structure of the stigma monolayer of
D. salina Teod. [Vladimirova, 1978]. The diameter of the pigmented globules (D) was 91.5 nm,
the mean distance between globules (s) 3.5 nm, and the number of globules in the stigma (N)
18. These quantitative values are typical for all green algae [Melkonian and Robenek, 1984].
101
The diffraction parameters D, S, and N were used to calculate the dependence of the
intensity of diffraction maxima on the parameter of diffraction p [Born and Wolf, 1968]. The
dependence of the intensity F(p) of diffraction maxima on parameter p and, hence, on the an-
gle of diffraction Om for the normal incidence of light (0 0 = 0) and wavelength}, = 480 run is
presented in Fig. 8.5.
The positions of diffraction maxima are determined by the values of parameter p = Aid,
2A1d, 3A1d, etc. The dependence of the intensity F(P) of diffraction maximum of the first (m = 1)
order on the wavelength A. of the light is presented in Fig. 8.6 (here eo
= 0). It is clear that the
positions of these maxima depend strongly on the wavelength A. of the light.
F,
Dunaliella
.8
.6
..,
.2
<J- J .1".
"""
::~
l'"'In>,,;.
;J~~;:;
;::: ~
~~~ 0>-1-'"
..- ('1 P
. ..
"' ,< t<t~
('~
Fig. 8.5. The dependence of a function F(P) of the light intensity diffracting on pigmented globules of Duna/iella on
the parameterp of difliaction. The nonnal incidence (8 0 = 0) of the light on the plane of the stigma is considered;
the parameters of stigma as diffraction grating are: D = 91.5 nm; s = 3.5 nm; N = 18; the wavelength of light .l =
480.
The calculations support the hypothesis that there is a diffraction mechanism for photoreception
in unicellular green algae containing a stigma with periodical structure. The mechanism allows
the cells to respond to the angle of incidence and wavelength of the light. Changes in the direc-
tion of movement result in alterations in the angle of light incidence on the stigma along with
spatial and temporal positions of diffraction maxima. This leads to changes in illuminance of
the photoreceptor and a corresponding correction in the direction of movement (photoorienta-
tion).
102
F Dunaliella A, om
, .
--+- 400
---f:}- 450
-"'- 480
B·
2 -
Fig.8.6. The dependence of a function F(P) of the light intensity on the wavelength A of the light fulling on stigma
of Dunaliella, where: p - parameter of diffraction; 80 = 0; D = 91.5 nm; s = 3.5 run; N = 18; m = 1 - the order of
diffraction [posudin and Masssjuk, 1996, 1997].
The proposed diffraction mechanism is believed to be present across all monad biological
objects that possess a periodic stigma structure. This does not exclude the simultaneous action
of other photoreception and photoorientation mechanisms that are based on modulation, interfe-
rence, dichroism or other phenomena that provide light signal amplification and increased pho-
toorientation accuracy.
103
of transfer is close to that of sound and it can propagate a significant distance within the a-
spiral [Davydov and Suprun, 1974]. It is known that either exciton or soliton states are the
result of symmetrical or anti-symmetrical excitations. Symmetrical excitation is characterized
by the contraction of all three peptide chains in a a-spiral, while the distance between the
chains is increased symmetrically. Thus, the molecule contracts and thickens. These processes
occur locally, within a small region (Fig. 8.7a). Anti-symmetrical excitation provides the con-
traction of a two peptide chains, while the third does not deform. This process leads to bend-
ing of the entire molecule in response to exciton excitation or local bending under soliton ex-
citation (Fig. 8.7b).
Fig 8.7. Defonnation of peptide groups under a - symmetrical; b - antisymmetrical excitation. After Davydov
and Suprun, 1974
The deformations result in conformational changes in the protein molecules that are accom-
panied by the activation of ion channels and flagellar beating. Thus, functioning of the mem-
brane is tied to the activity of membrane proteins. Excitation of these proteins causes an alte-
ration in their configuration and of ion channels that control the diffusion of calcium ions into
the cell, stimulating motile activity of the algae [Posudin and Suprun, 1992].
8.4. Summary
The unique properties of the photoreceptor system of algae, in particular the structure of the
stigma, suggest that all monad flagellates, with the exception of those devoid of a stigma,
have possessed this mechanism for modulating photoreception since prehistoric times. It is
proposed that the mechanism was inherited from prokaryotic organisms and their common
ancestors. Certain green algae can simultaneously have three mechanisms (modulation, dif-
fraction, and interference) that increase the intensity of the light signal absorbed by the photo-
receptor. Proteins playa very important role in the photoregulation of movement in flagel-
lates. They are a component of the photoreceptor system and flagellar apparatus and confor-
mational changes in these proteins occur in response to a light stimulus.
104
Chapter 9
Sensory Transduction
106
At this time, the very limited amount of data on the control of the primary stages in
sensory transduction in E. gracilis has limited our understanding such that the process re-
mains a proverbial "black box" [Lebert and Hader, 2000].
107
Sineshchekov et al. [1989] established that phototactically inactive red light induces a
fast change in phototopotaxis from positive to negative in Chlamydomas cells exposed to
short-wave irradiation (450-500 nm). The stimulation of negative phototopotaxis was readily
reversible in the dark thereby excluding the participation of phytochrome. The long-wave
boundary was near 700 nm and was inhibited with diuron (N'-(3,4-dichlorophenyl)-N,N-
dimethyl-urea). These facts indicate the possible existence of a fast (i.e., seconds) control of
the positive or negative phototaxis sign by photosynthesis. The spectral sensitivity of photo-
topotaxis was determined by the collective absorption spectra of the photoreceptor, photosyn-
thetic pigments, and stigma.
Takahashi and Watanabe [1993] confirmed that photosynthesis modulates the sign of
phototopotaxis in wild-type C. reinhardtii. This conclusion was based on: 1). The transient
nature of phototopotaxis was preferentially observed in blue-green actinic light rather than
green actinic light; 2). Red background lighting induced negative phototopotaxis under actin-
ic-light conditions, however, without background light, the cells exclusively display positive
phototaxis; and 3). Both the effect of red background light and the transient change in the sign
of phototaxis were inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, a relatively specific
inhibitor of photosynthesis. Their conclusion altered the accepted view in the early 1970s that
photosynthesis was not linked to phototaxis (e.g., see Stavis and Hirshberg, 1973; Stavis,
1974).
Application of electrophysiological methods (microelectrode recording of electric sig-
nals on the protoplast's surface) made it possible to identify high-frequency rhythmic
processes that are related to changes in electric potential of H. pluvialis. The duration of an
oscillation was several tenths of a second. The processes pointed toward the existence of two
independent oscillators in the cell and that the frequency change and phase shift between the
two parallel rhythms probably determined the phototopotaxis sign [Sineshchekov et al., 2001;
Sineshchekovand Govorunova, 2001a].
It is possible that there is a link between periodic processes and the functioning of con-
tractile vacuoles. There are two contractile vacuoles in C. reinhardtii that are located near the
basilar bodies of the flagella. Their behaviour has been analyzed on micropipette tips using
videomicrography. The time interval between two contractions of the vacuole was 30 s. Con-
traction of both vacuoles occurred with frequencies that were close in magnitude but shifted
in phase. The magnitude of the shift changed periodically [Sineshchekov and Govorunova,
2001a].
These processes play a role in the regulation of cell movement. It is possible to ob-
serve periodic spontaneous changes of the direction of cell movement during prolonged moni-
toring of the movement trajectories of individual cells of H. pluvialis and C. reinhardtii.
These changes are similar to periodic changes in electric potential at the cell surface and in
the micromovement of chloroplasts, indicating the possibility of a common origin [Sinesh-
chekovand Govorunova, 2001a]. The interaction of both oscillators in the cell, the function of
which is controlled by the contractile vacuoles, is believed to be the basis for the mechanism
regulating the phototopotaxis sign [Sineshchekov and Govorunova, 2001a].
It was shown that the two flagella display different levels of response to light (i.e.,
changes in frequency and beating plane) [Sineshchekov, 1991a, b]. Free-motile cells were
characterized by the preferential reaction of the cis-flagellum, located on the side of the pho-
toreceptor that leads to rotation away from the source oflight (negative phototopotaxis), while
the preferential reaction of the trans-flagellum, located on the opposite side from the photo-
receptor, lead to rotation of the cell toward the source of light (positive phototopotaxis).
The trajectories of movement of individual cells under extended illumination change
depending upon the source of the light. The frequency of such changes can be compared with
the frequency of electric pulses on the cell surface. The sign for phototopotaxis is determined
by the level of the phase shift between the rhythms of two oscillators - the contractile va-
108
cuoles. The level depends on many factors, such as the intensity of illumination, aeration, ion
composition of medium, and age of the culture. Differences in the sensitivity of the oscillators
to these factors are thought to be the probable cause [Sineshchekov and, Govorunova, 2001a].
An electrochemical system using dual electrodes allowed the simultaneous monitoring of al-
gal motility and phototopotaxis in an investigation of photomovement in C. reinhardtii. The
effect of diltiazem (3-(acetyloxy)-5-[2-(dimethylarnino)ethyl]-2,3-dihydro-2-(4-methoxy-
phenyl)-I,5-benzothiazepin-4(5H)-one hydrochloride), sodium azide, or ethanol on the redox
currents were assessed as indices of photoinduced behavior [Shitanda and Tatsuma, 2006].
C. reinhardtii mutants with defects in thier dynein arm structure (ida4 and 0da22), ptx
mutants deficient in axonernal sensitivity to calcium ions, and ppr mutants lacking an ionic
channel specific for the photophobic response were analyzed [Ermilova et al., 2000]. The
mechanism of cell orientation in phototactic and chemotactic responses depended on func-
tional differences between cis- and trans-flagella that differed in beat frequency and in the
sensitivity of their axonernes to submicromolar calcium concentration. These responses are
distinct from the photophobic response.
Using DNA sequencing, overlapping segments of cDNA have been identified in C.
reinhardtii that encode two channelopsins proteins [Nagel et aI., 2005]. Nagel et al. proposed
that phototopotaxis and photophobic responses in green algae were mediated by rhodopsins
with microbial type chromophores, i.e. all-trans-retinal in the ground state.
The effect of copper ions on phototactic orientation in H pluvialis (syn. H lacustris
(Girod) Rostafinski) [Braune et aI., 1994] indicated that phototopotaxis was inhibited at con-
centrations that did not impair the velocity of cell movement. The effects of chromium (Cr6+,
the more toxic species) on the photoreceptive apparatus in C. reinhardtii [Rodriguez et al.
2007] was studied by in vivo absorption microspectroscopy of both the thylakoid compart-
ments and the stigma. Decomposition of the overall absorption spectra of the pigment consti-
tuents indicated that Cr6+ induced a modification of the carotenoids present in the stigma. It is
therefore possible that C. reinhardtii might be useful as a bioindicator of~.
109
F,r.lI. F,Lll.
0 0
0 0
0" 0 0 0,3 0
0
0
0
0,1
0.1
I 1 I 1 1 0 1 1 I 1 I
"
10' 10" 10· IO'~ 10" IO·~ 10·' 10· 10·J 10. 2
V, pm/s
V. pm/s.
• • • • • • • • • •
30 30
10 10
----,----.--,---,------" 0
10·" 10··' 10" 10·.1 10. 2 K 10,(, 10) 10-1 10·-' 10
Conctillralion C, M
Concentration C, M
Fig. 9.1. Dependence ofpbotomovement parameters F and U in a - Dunaliella salina and b - Duna/iella
viridis on the concentration of CaCI2'6H20 in the water [Posudin et aI., 1993].
Maximum values for parameter F were between 10-6_10-4 M for both species; the value of F
decreased to 80-90 % at a higher (_10'2 M) concentration in comparison with control values
(without calcium ions in the medium). There was not a significant difference among the Ca
concentrations on the velocity of movement U and control values.
In spite of the evident participation of calcium in algal photomovement (Le., activation
of calcium channels, triggering the ion pumps, changes in membrane permeability), a number
of aspects of its role remains to be determined. The dependence of photomovement parame-
ters in the two species was characterized by a maximum for parameter F in the concentration
range of 10'6_10-4 M Velocity u, however, did not appear to be affected by calcium ions in
the concentration range tested. These results are similar to those reported by Avron and Ben-
Arnotz (1992), who assessed motility, velocity of movement, and linearity of trajectory in D.
salina and Dunalie/la bioculata Butcher and found that Ca had little or no effect. Our results
in part coincided with the those obtained by a number of authors for C. reinhardtii.
The effect of the pesticide lindane (1,2,3,4,5,6-hexachloro-cyclohexane) on motility in
D. bioculata is related to a specific interaction between the chemical and Ca transport that
results in an increase in cytoplasmic Ca. The pronounced effect of lindane on ciliary beating
is probably related to a modification of the Ca balance within the cell [Marano et al., 1988].
Maximum phototopotaxis occurs at a Ca concentration of 5,10,5 M ~Dolle et aI., 1987;
Nultsch, 1979, 1983]; the level of phototopotaxis decreases to 50 % at 10' M and to 25 % at
2·10-4 M in comparison to control values. In contrast, phototopotaxis in Chlamydomonas is
completely inhibited at 10.3 M Phototopotaxis in Chlamydomonas is very sensitive to the in-
110
tensity of the light stimulus [Dolle et aI., 1987; Morel-Laurens, 1987]. In contrast, the velocity
of movement was dependent upon the concentration of calcium ions and the intensity of light
stimulus [Morel-Laurens, 1987].
The Dunaliella species differ from Chlamydomonas in that the velocity of movement
of the cells is not dependent upon the concentration of calcium ions.
'~" t
F fU
01 0 0.3 0
t
0 0
01 0,1 0
0
1
4 S
1 1
12
1
16
1
22
,
24
0
4
"
8 12 16 22 24
1
10 10
, 1 1 1 1 1
; 1 1 1 1 1
4 8 12 16 22 24 8 12 16 22 24
K tA2::;1S? K 11\23187 I, hours
I. hours
Fig. 9.2. Temporal dependence ofpbotomovement parameters F and v in two species of Dunaliella (a - D. sali-
na and b - D. viridis) on the addition of ionopbore A23187 to the medium (moment of addition indicated by
arrow) [Posudin et aI., 1993].
Insertion of 10-5 M ionophore into the medium induced practically complete inhibition of
photopotaxis in D. salina and D. viridis. Recovery of parameter F to the initial control value
was reached after 12 hours for D. salina and 24 hours D. viridis. Addition of calcium (10-3
M) 24 hours after introduction of the ionophore did not change parameter F and there were no
changes in the velocity of movement of the cells. The addition of the ionophore at 10-5 M
supports the role of calcium entering the cell in that phototopotaxis of Dunaliella was
inhibited practically instantly. The absence of its effect on the velocity of movement can be
explained by the increased permeability of the membrane to calcium ions.
Calcium ions can be present in the water used for the medium without the addition of
calcium salts. Likewise, microscope slides and cover glasses can be a source of calcium ions
[Dolle et al., 1987]). The fact that the velocity of movement in Dunaliella did not change in
111
response to the ionophore in comparison with other green Chlamydomonas species [Pfau et
aI., 1983] demonstrated inhibition of either phototopotaxis or velocity of movement by the
chemical (10- 5 M). The inhibitory effect of the ionophore on the velocity of movement in
Chlamydomonas was thought to be due to contraction (or detachment) of the flagella and their
subsequent restoration (regeneration).
The effect of the pesticide lindane on D. bioculata was tested with an ionophore
(A23l87) that functions as a mobile ion carrier. The results were compared with those of Du-
na/ie//a at 15 ppm lindane and a combination of lindane (15 ppm) and ionophore A23l87
(10-5 M). The ionophore enhanced the effect of lindane on the motility indicating that the
chemical may interfere with intracellular calcium flux [Krishnaswamy-Chang, 1997]. Expe-
riments with inhibitors, ionophores, and drugs support the conclusion that Chlamydomonas
cells have an energy-dependent, outward-oriented Ca 2+ pump [Hutchinson and Hirschberg,
1985].
Comparing the distinctions and similarities of the action of the ionophore on photo-
movement parameters of representatives of two genera (Duna/ie//a and Chlamydomonas)
underscores the greater adaptive ability of Duna/ie//a for survival under extreme conditions.
Across taxons the chemical induces similar photomovement responses (e.g., direction of
movement in Duna/ie/la, direction and velocity of movement in Chlamydomonas, frequency
and duration of spatial tumblings in E. gracilis [Doughty and Diehn, 1979]) indicating it af-
fects a fundamental control mechanism.
112
F,LII. F,l'.\l.
0
0
0,3 0.3
0
0 0
0.1
0.1
0 0
0
I I , ,0 i ,
10·.' 10·' 10·) 10·! K 10' 10. 1 10-' 10")
10· 10·'
((,\1)
• • • •
)0
• • •
JO
•
10
to
,0
10.0 I O-~ 10·' 10·) 10. 2 K IO~ 10' 10-' 10·) 1O-1
COllc",lllr<lliOll C. M (OJlcenll<llioll C. M
Fig. 9.3. Dependence ofphotomovement parameters F and U in two species of Dunaliella (a - D. salina and b
-D. viridis) on the concentration ofeoCh [Posudin et aI., 1993].
Ions of cobalt, lanthanum, manganese and nickel are known to block membrane calcium
channels inducing reversible flagellar beating, whereby changing the direction of movement
in Chlamydomonas [Schmidt and Eckert, 1976; Doughty and Diehn, 1982]. The effect of the
presence of blockers on photomovement indicates the participation of calcium ions in the sen-
sory transduction of alga.
Differences among species and temporal changes in behaviour (Le., photophobic
reactions) of the cells were observed in E. gracilis due to the presence of calcium, barium,
cobalt, and magnesium ions [Colombetti et aI., 1982]. The inhibitory action of cobalt ions on
phototopotaxis in Dunaliella suggests the transport of Ca across the membrane and therefore,
the ionic nature of sensory transduction in Dunaliella.
113
F.r.lI.
F,LlI.
'"
OJ 0 0,3 0
0
0 0
0.1 0,'
, , , $ ,0 , , q,
10< I o·~ 10" 10') 10"' 10' IO-~
10" IO·J 10.1
C(M)
C(M)
V,IUn/s
V,I-Imls
3D • • 3D
• •
• •
10
10
,0
10< 10'! 10' 10") 10.1 K 10< 10J 10" 10') 10.1
ConcclHrnlion C, M COllccnlnl.lionc'M
Fig. 9.4. Dependence of photomovement parameters F and v in two species of Dunaliella (a - D, salina and
b - D, viridis) on the concentration of cinnarizine [posudin et a!., 1993].
", I
I ," I
F,r,lI.
OJ
0 0
0
0,3
'" 0
0
0 0
0,'
0,1
,0
10< I O·~ 10" 10" I O'~ '0" 10'J 10""
• •
I', !J1l1/~
JO
• 30
• • •
10 10
1 0
10.1 IO~' 10'\ 10" 10'/ 10< 10" 10"
('ollcclltr;llioll C, [\'1 COIKL'lItr>llion C, LvI
a
Fig. 9.5. Dependence of photomovement parameters F and v of two species of Dunaliella (a - D. salina and
b - D, viridis) on the concentration ofisoptin [Posudin et aI., 1993].
114
Application of calcium channel blockers demonstrated an inhibition of both
photomovement parameters (v and F) in the two species of Dunaliella. Comparing these
results with those using calcium channel blockers such as flunarizine (1-[bis(4-
fluorophenyl)methyl]-4-[(2E)-3-phenyl-2-propenyl]-piperazine), verapamil (a-[3-[[2-(3,4-
dimethoxyphenyl)ethyl] methylamino] propyl]-3,4-dimethoxy-a-(I-methylethyl)-benzene-
acetonitrile), diltiazem, and nimodipine (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3-(2-
methoxyethyl) 5-(I-methylethyl) ester 3,5-pyridinedicarboxylic acid) [Nultsch et al., 1986]
provides additional insight into the mechanisms involved. For example, increasing the
concentration of flunarizine from an initial level of 10-6 M decreased phototopotaxis and
motility in Chlamydomonas [Nultsch et al., 1986]. At a concentration of 5.10-5 M both
parameters were completely inhibited. The authors considered the effect specific (i.e., induced
the inhibition of one parameter only).
Microscopic analysis indicated that the loss of motility was related to the contraction
or detachment of the flagella as is exhibited by sodium azide or ionophore A23187 [Pfau et
al., 1983]. Phototopotaxis and motility were recovered 6 hours after the application of cinna-
rizine indicating a regeneration of the flagella.
Verapamil inhibits phototopotaxis and motility at a concentration 2.10-5 M in varying
degrees (e.g., phototopotaxis up to 40 %, and motility up to 50-60 %) indicating a gradual
detachment of the flagella [Nultsch et ai., 1986]. Up to 20 % motility was recovered from its
initial level, while phototopotaxis was not recovered. The results suggest a specific action for
verapamil on phototopotaxis in Chlamydomonas.
Diltiazem and nimodipine, in contrast, affect phototopotaxis in Chlamydomonas with-
out an effect on motility. Both chemicals inhibited phototopotaxis up to 30-35 %. Recovery
was observed 6 hours after inoculation with diltiazem and 10 hours after inoculation with ni-
modipine. Collectively, the results indicate that the affect of these blockers (verapamil, diltia-
zem, and nimodipine) on photomovement parameters in Chlamydomonas are specific in that
the chemicals acted on phototopotaxis and motility to a different degree.
In our experiments with Dunaliella, cinnarizine and isoptin both acted on photo-
movement parameters of Dunaliella, inhibiting completely the velocity of movement and
phototopotaxis at a concentration 10-3 M.
115
V, IJm/s F o r.u.
V, ~Im/S F,r.ll.
! !I t f I
f
40 40
30 0.3 ?! ?! f 1 30 0.3 11 ~ 1
20 0.2 20 0.2
jJ !J
10 0.1 10 0.1
14
-0.1
-0,] 14
-0.2 -0.2
-0.] -0.3
-0.4 -o..J
Concentration C, M Concentration C, M
a b
Fig. 9.6. Effect of somum azide on the velocity (v) ofmovemevt and positive and negative phototopotaxis (F) in D.
salina (a) and D. viridis (b) illuminated with white light: 1 and 2 - V at 500 and 40,000 Ix, respectively; 3 and
4 - F at 500 and 40,000 Ix, respectively. Vertical bars inmcate the standard error [Posudin et aI., 1995].
116
9.5. Summary
Distinctions were found in response to certain biologically active chemicals (e.g., ionophore
A23187) between two species DunalieUa. In contrast, C. reinhardtii often reacts non-
specifically (autotomy of flagella) to chemicals that stimulate or block ionic channels while Du-
nalieUa cells did not lose their flagella. As a consequence, their locomotory reactions can be
regarded as specific in response to the blocking/stimulating of ionic processes. Likewise, distinc-
tions in photomovement responses to specific substances on hyperhalobic species of Duna-
lieUa and fresh-water E. graci/is and C. reinhardtii indicate a higher tolerance in the hyperha-
lobic species to external inhibitors.
Maximum values for phototopotaxis in the hyperhalobic species of DunalieUa are ob-
served at 10-5_10-3 M CaCh·6H20. Increasing the calcium chloride concentration to 10-2 M sup-
pressed phototopotaxis by 10-20 %. The addition of the ionophore A23187, that increases the
permeability of the cellular membrane to calcium ions, completely inhibited phototopotaxis in
both D. salina and D. viridis. The addition of CoCh, that blocks membrane calcium channels at
10-6_10-3 M, suppressed phototopotaxis in both species. Other calcium channel blockers (e.g.,
cinnarizine, isoptin, and sodium azide) similarly inhibited phototopotaxis. In contrast, ouabain,
which stimulates Na+-K+-ATPase, did not influence phototopotaxis.
The effect of substances that act specifically on photomovement parameters in Duna-
lieUa indicates that sensory transduction in the two DunalieUa species, as well as in Chlamy-
domonas, is of ionic nature and that photoregu1ation of the processes governing the velocity of
movement of the cells and phototopotaxis occurs through separate sensory transduction path-
ways in response to light that triggers flagella beatings. The absence of an effect by ouabain
on photomovement in DunalieUa indicates the absence of a Na+-K+-ATPase system participating
in the photoregulation of movement.
117
Chapter 10
Flagella Apparatus
10.1. Structure
Flagella are organelle that are typically located at the apical end of the cell. Their lengths are
equal and they are a slightly shorter or longer than the length of the cell [Massjuk, 1973].
Each flagellum presents a whip-like structure that is 0.2 ~m in diameter. The flagella provide
the cell with linear mobility through a pulling or pushing action mediated by active bending
and a simultaneous rotation around its longitudinal axis. The flagellum that is closest to the
stigma is called cis-flagellum, while the more distant one is the trans-flagellum.
The flagella apparatus consists of three main parts: the flagellum proper, the basal
body, and structures that are associated with basal body (i.e., connecting fibers and flagellar
roots). Each flagellum in tum consists of three parts: the flagellar top, shaft, and transition
region [Melkonian, 1984]. The flagellum is enclosed within a membrane that is continuous
with the plasmalemma. The axoneme is the central core of the flagellum and consists of two
central microtubules surrounded by nine peripheral microtubule pairs. The microtubules are
arranged in a 9+2 pattern and are immersed in an amorphous matrix. The basal bodies of both
flagella form a V-like figure that is connected by distal and proximal striated fibers. The basal
bodies are connected with the flagellar roots by a system of microtubules and microfibrils
[Ringo, 1967]. The fine structure of flagellur apparatus has been described by Ringo [1967],
Kvitko et al. [1978], and Melkonian [1982, 1984], while the flagellar apparatus, from the
point of view of the phylogeny of green algae, is described by O'Kelly and Floyd [1983-
1984].
Flagellar mediated algal motility in response to light (e.g., phototopotaxis and other
photoresponses, the role of calcium channels, and photoreceptors) has been reviewed by
Kreimer (1995).
Ca2+-dependent flagellar dominance in Chlamydomonas has been studied in a newly
isolated mutant (Ispl) that displays weak phototo~otaxis. The trans-flagellum in the mutant
beats more strongly than the cis-flagellum in a 10- -10-6 M Ca2+concentration range, a range
in which wild-type cells display a switching of flagellar dominance. When compared with
other mutants, Ca2+-dependent flagellar dominance control and inner-arm dynein subspecies
appear to be important for phototopotaxis, but not absolutely necessary [Okita et aI., 2005].
Fig. 10.1. Flagellar beatings in Chlamydomonas; a - forward motion (breast stroke, ciliary-like style); b - back-
ward motion (return stroke, ciliary-like style) according to [Ringo, 1967]; c - reverse beatings (flagellar-like
style) according to [Colombetti and Marangoni, 1991].
The velocity of rotation of the cell around the longitudinal axis during its linear movement is
2 Hz [RUffer and Nultsch, 1985]. Turning ofe. reinhardtii cells relative to the source oflight
occurs in the following manner [RUffer and Nultsch, 1990; Nultsch, 1991]. Light is detected
by a photoreceptor that is located in the plasmalemma opposite the stigma and asymmetrically
in relation to the longitudinal axis of the cell. Illumination of the photoreceptor is accompa-
120
nied by a flux of calcium ions across the flagellar membrane. The cis-flagellum (closest to the
stigma) increases the amplitude of its beating in response to the influx of calcium ions relative
to the beating of trans-flagellum. The differential in response between the two flagella to
changes in the calcium ion concentration within the intracellular space results in the change in
direction of movement toward the light source (i.e., phototopopotaxis).
Analysis of the movement of Chlamydomonas flagella using the radial-spoke system
between wild-type and mutant flagella was described by Brokaw et al. (1982). The flagellar
beat frequency of the biflagellated green alga C. reinhardtii was measured using fast Fourier
transform analysis of light intensity fluctuation in microscope images of swimming cells.
Live cells had a mean beat frequency of 48-53 Hz at 20 DC. However, detergent-extracted
"cell models", when reactivated in the presence of I mM ATP, appeared to have two different
beat frequencies of about 30 and 45 Hz. These observations suggest that the two flagella of
Chlamydomonas have different intrinsic beat frequencies but that they are somehow synchro-
nized and beat together in swimming cells [Kamiya and Hasegawa, 1987].
Although the two flagella of Chlamydomonas appear similar to each other, they differ
in the beat frequency. The trans-flagellum beats at a 3Q-40 % higher frequency than the cis-
flagellum in demembranated and reactivated cell models. Experiments with a set of mutants
(oda) suggested that the attachment site for the outer dynein arm is important in determining
the flagellar beat frequency and the basal portion of the outer arm dynein is important in regu-
lating the flagellar activity and therefore the behavior of the cell [Saeko and Ritsu, 1997].
C. reinhardtii cells are able to change the beating frequency, pattern, and synchrony of
the trans- and cis- flagella in response to light stimulation and the response of each flagellum
is quite different. The trans-flagellum responds with less delay than the cis for both beating
frequency and stroke velocity. With light stimulation at 2 Hz for the critical cell-rotation
frequency, the trans- and cis-flagella responses are about 180 degrees out of phase. The
trans-flagellar beating frequency peaks at a stimulus frequency of 5-6 Hz, higher than the cis
at 1-2 Hz. The stroke velocities of the trans-flagella and cis-flagella have the same stimulus-
frequency response (2 Hz), however, the trans-flagellum has a shorter delay than the cis. The
length of time to reach a maximum response is much shorter than the time required for a
single rotation of the cell. The use of two different mechanisms to enable the trans-flagellum
to respond ahead of the cis-flagellum in both the beating frequency and stroke velocity
responses suggests the importance of both responses in phototopotaxis [Josef et ai., 2006].
The same peculiarity in flagellar function that is characterized by a differential in the influx of
calcium ions between the cis- versus the trans-flagellum is found in Haematococcus pluvialis
Flotow [Sineshchekov, 1991a].
It is possible to observe a transfer in the undulation from cilia-type to a flagella-type.
Such changes of character of flagellar beating are observed during photophobic reactions in
response to an electrical reaction triggered by the opening of potential-dependent calcium
channels in the flagellar membrane [Sineshchekov et al., 1978; Beck and Uhl, 1994; Holland
et al., 1997].
Using electro-optic monitoring of flagella beating in C. reinhardtii, it was possible to
record the beating frequency, stroke velocity, and stroke duration of each flagellum and the
relative phase of the cis- and trans- flagella. Each beat cycle was resolved such that each
asynchronous beat was detected [ Josef et al., 2005].
121
There is a unique function of the flagellar apparatus in comparison with Ch/amydomo-
nas in that just before a change in movement direction of the cell, one of the two flagella re-
mains transiently immobile while the other rotates the cell via ciliary beating. As a result, the
cell moves in the new direction with both flagella active. In addition, there is a discrepancy in
flagella beating frequency in D. biocu/ata. One flagellum beats at 60 Hz and the other at 50
Hz. This changes the angle between the planes of beating causing rotation of the cell and its
helicoidal movement [Shoevaert et aI., 1988; Marano, 1992]. Analysis of the flagellar assem-
bly in D. biocu/ata is discussed in the work Marano et aI. [1988] and Schoppmeier and Lech-
treck [2002].
122
4f~
2v . e
-sm-cosqJ. ( 10.1 )
A. 2
Investigation of the interaction of laser radiation with cells of E. gracilis using Dopp-
ler frequency shifts allowed estimating the velocity of linear movement (100 llm/S), the fre-
quency of the cell rotation (-2 Hz), and the flagellar beating frequency (about 30-50 Hz) [As-
coli et aI., 1978; Ascoli and Frediani, 1980; Ascoli and Petracchi, 1991]. Similarly, Doppler
shifts in laser light scattered by the cells of Haematococcus pluvialis Flotow using heterodyne
detection techniques allowed measuring the swimming velocities of cells along the light sti-
mulus axis [Cantatoreet aI., 1989]. The time taken by a cell population to change orientation
was about 1 s.
Using laser Doppler spectroscopy, the linear velocity of D. bioculata cells was deter-
mined to be 109 ± 5 llm/S [Marano, 1992] and the frequency of flagellar beating in D. salina
approximately 25 Hz [Ascoli et aI., 1980]. The method identified two different frequencies for
E. gracilis; 30-50 Hz that corresponded to the flagella beating and 2 Hz that was related to the
cell body rotation [Angelini et aI., 1986; Ascoli and Petracchi, 1991]. The motility of D. bio-
culata under the influence of pesticides was also quantified by Doppler laser velocimetry
[Krishnaswamy-Chang, 1997].
Fig. 10.2. Principle of microphotometry for the study of flagella beating [Posudin, 2009, Patent of Ukraine 1'&
45376, Registration N U200905299]
123
The algal cell] is placed between the slide 2 and cover glass 3. Optical radiation of
the source 4 passes though the bases of the flagella thus modulating the intensity oflight. This
modulated light enters the objective of microscope 5 and photodetector 6. The electric output
signal of the photodetector then enters the amplifier-converter that amplifies the signal and
filters it in the 3-100 Hz range. The signal is elaborated due to the Schmidt trigger which pro-
duces a standard pulse of certain duration if the intensity of signal exceeds a certain level. The
pulse is indicated by the pulse counter 9. Individual pulses corresponding to flagellar beating
enter the analog-digital transformer 10 that estimates the frequency of flagellar beating. We
have used microphotometry to measure the frequency of flagella beating in D. salina which
was found to be in the 20-50 Hz range [Posudin, 2009].
10.4. Summary
Green algae such Chlamydomonas and Dunalie//a differ during the process of photomove-
ment in the character of their flagellar beating. Progressive movement of Chlamydomonas is
due to ciliary beating of the flagella and backward movement by undulate beating. Since cells
of Dunalie//a are not capable of photophobic reactions, the undulate mode of flagella beating is
not found in the genus. Turning of Chlamydomonas cells occurs due to an unequal frequency
in beating between the cis- and trans-flagella. In contrast, the cells of Duna/ie//a temporarily
cease beating in one flagellum, thereby initiating a tum.
The frequency of flagellar beating in seawater and hyperhalobic species of Dunalie//a
(20-50 Hz) is of the same order as in freshwater species ofHaematococcus and Chlamydomonas.
124
Chapter 11
Applied Aspects of Aquatic Biomonitoring Using the
Photomovement of Dunaliella
Observation of the state of the Earth's biotic component, its response to anthropogenic effects,
and deviations from a normal state assessed at various organizational levels (e.g., molecular,
cellular, organismal, populational, biocenotical) is called biological monitoring. A test-object
(biomonitor) is an organism, part of an organism, or a community of organisms that contains
quantitative information on the quality of the environment [Butterworth et aI., 1995]. A num-
ber of organisms (e.g., bacteria, fungi, actinomyces, algae, protozoa, invertebrates, fish,
amphibians, aquatic plants) can be used as test-objects [Shubert, 1984, 1984; Krajnyukova,
1988; Rosenberg and Resh, 1993; Markert et al., 2003].
A test-jUnction is a physiological or behavioral response of an organism to changes in
the quality of the environment. The intensity of reproduction, immobilization of cells, motili-
ty, photosynthetic activity, membrane permeability, bioluminescence, impedance of cell sus-
pension, bioelectrical reaction [Barenboim and Malenkov, 1986; Krajnyukova, 1988]; growth,
reproduction, mobility and energy potential of the cells [Parshikova, 2003; Parshikovaet aI.,
2004]; protoplasmic streaming [Mustacich and Ware, 1976; Evdokimov et al., 1982; Pileri,
1987], and phototopotaxis of organisms [Simone et al., 1978; Wang et aI., 2001; Wu et aI.,
2006] are examples of typical test-functions that can be used for biomonitoring.
The proposed use of photomovement of high density algae (Euglena gracilis G.A.
Klebs) for the removal of nutritive substances in photo-bioreaetors is of considerable interest
[Nakajima and Takahashi, 1991]. A number of articles and reviews have focused on the use
algae as test-objects. For example, a review on the development of a biosensor for monitoring
flagellar movement and phototopotaxis in algal cells for the detection of chemical toxicity in
wastewater was proposed by Shitanda et al. [2006]. Waste water toxicity monitoring is also
critiqued by Diao [2007].
The principal advantages ofbiomonitoring are high sensitivity, rapidity, reliability and
the possibility of creating automated systems for collecting and processing environmental
information. The very limited availability of useful biomonitors is related to the absence of
adequate quantitative measures of individual toxicants present in aquatic media and the possi-
ble interaction between separate toxic components that are present in a mixture. Test-objects
used under natural conditions are termed passive while in the laboratory they are considered
active.
H eavy M etals
Pollutant Test-Qbiect Test-Function Reference
copper and lead Dunaliella salina, D. biocu- growth and pigment content [Pace et aI., 1977]
lata and D. tertiolecta
copper, lead, cad- D. salina cell growth [Barghigiani et aI.,
IDium and mercury 1981, 1983; Serritti
et aI., 1981]
Hg, Cu, Cd, Pb D. viridis, D. tertiolecta uptake and intracellular accumu- [Tzvylev and Tka-
larion of heavy metals chenko, 1981]
orthovanadate D.parva motility [Gilmourct aI.,
19851
copper Dunaliella salina, D. terti- absorption of copper [Lustigrnan et aI.,
olecta, and D. viridis 19851
copper D. salina glycerol production [Lustigrnan et aI.,
19871
copper D. tertiolecta fluorescence induction [Samson et aI.,
19881
mercury, copper D. tertiolecta fluorescence induction [Samson and Po-
povic, 19881
boron D. tertiolecta growth [Ahmedetal.,
19881-
copper D. salina thermostahility, [Veselova et aI.,
photosynthesis rate, motility 1990]
AI, La, Cu, Cd, D. acidophila, photosynthesis and growth [Gimmler et aI.,
Hg,W D.parva 1991]
heavy metals, D. tertiolecta analitical and biotoxicity tests [BaIdi et aI., 1993)
aromatic hydro-
carbons and saIts
copper D. tertiolecta loss of flagella, changes in cell [Khristoforova et
shape, lack of motility, and col- aI.,1996]
lapse
Cd, Cr, Cu, Pb, Ni D. tertiolecta algaI inhibition (Pun et aI., 1995]
andZn
Cd, Cr, Cu, Pb in D. tertiolecta inhibition of growth [Wongetal.,1999)
coastal sediments
Zn D. tertiolecta synthesis of phytochelatins [Tsujiet aI., 2002]
copper D. salina and total chlorophyll and carotenoids [Nikookar et aI.,
D. tertiolecta 2005]
lead and aIumi- D. tertiolecta growth response and ultrastruc- [Sacan et aI.,2007] )
nium ture
copper D. tertiolecta inhibition of growth rate (Levy et aI., 2007]
126
Pesticides
PoUntant Test-Qbject Test-Function Reference
organophosphates D. euchlora photosynthesis [Derby and Ruber,
Baytex and Abate; 1970]
carbamate Baygon;
and the chlorinated
hydrocarbon; DDT
chlorinated hydro- Dunaliella photosynthesis and growth [Menzel, et al.,
carbons (DDT, 19701
dieldrin, and en-
drin)
organochlorine D. bioculata growth and structure [Levain and Mara-
insecticide lindane no-LeBaron, 19731
lindane D. bioculata division, cell cycles, and biosyn- [Jeanne, 1979]
thesis
hexachlorocyclo- D. tertiolecta population growth, death of cells [Walsh, 1983]
pentadiene, EPN,
chlorpyrifos, car-
bophenothion,
atrazine
atrazine, DCMU, D. tertiolecta fluorescence induction [Samson and Po-
Dutox, and Soil- povic, 1988]
=d
pesticide residues D. salina [Yarden et aI.,
19931
lindane D. bioculata motility [Krishnaswamy-
Chan.g, S., 19971
organophosphorus D.salina photosynthesis, growth, biochem- [Cai et aI., 1999]
pesticide ical compounds
organophosphorus D. salina growth, photosynthetic rate and [Xie et aI., 1999]
pesticide methami- biochemical compounds
dophos
profenofos D.salina tolerance to pesticide rXie et aI., 19991
pyrethroids (cy- D. salina growth and some cellular rna- [Noaman et al.,
permethrin and cromolecules (chlorophyll a, 2002]
fenvalerate) and proteins, carbohydrates, RNA
organophosphorus and DNA)
insecticides (diazi-
non and cyano-
Dhos)
herbicide atrazine, D. tertiolecta population growth rate [DeLorenzo and
the insecticide Serrano, 2003]
chlorpyrifos, and
the fungicide chlo-
rothaloni
atrazine, chlorpyri- D. tertiolecta population growth rate [DeLorenzo and
fos, and chlorotha- Serrano, 2003]
lonil
127
pesticides D. salina motility, biochemical parame- [Onne, Kegley,
ters, cell size, chlorophyll con- 2004]
tent, population growth rate,
biomass
irgarol, fungicide D. tertiolecta growth rate [DeLorenzo and
chlorothalonil, and Serrano, 2006]
herbicides atrazine
and2,4-D
cigarette smoke (solid and gas D. bioculata ciliostatic activity [Izard et aI., 1967,
phases) a; Izard and Testa,
19681
sodium chloride, sodium sulfate, D. acidophila growth, photosynthesis, [Fuggi et aI., 1988]
sulfuric acid and glucose and respiration
phenols: hydroquinone, pyrocatechol, D. salina motility [Stoma and Roth,
phenol, guaiacol, resorcinol 19811
pyrocatechol and p-benzoquinone D. salina loss of motility [Stom et al., 19841
porphyrin derivatives, furcoumarins, Dunaliella motor inactivation and [posudin and Re-
acridines loss of viability petskii, 19881
salt D. salina photosynthetic pig- [Heidari et aI.,
ments and proteins 20001
salt D. salina photosynthetic pig- [Heidari et aI.,
ments and proteins 20001
surface-active substances D. salina. chlorophyll fluores- [parshikova, 2004]
D. minuta cence,
photomovement
nitrobenzenes D. salina grow inhibition [Shen et al., 20061
pharmaceuticals and personal care D. tertiolecta toxicity threshold [DeLorenzo et aI.,
products (pPCPs) 2008]
There are also reports detailing the possible use of Dunalie//a species in the phytore-
mediation of aquatic media. The effectiveness of Dunalie//a in removing copper and nickel
from solutions was demonstrated by Abdel-Raouf and Ibraheem [2001]. The highest concen-
tration of the tested substances that did not inhibit the growth rate of the organism was 10
ppm of copper and 20 ppm of nickel. The bioaccumulation and toxicity of germanium in
Dunalie//a salina Teod. were investigated by Zhu and Wang (2001). The authors believed that
most of the germanium in the algae was associated with proteins and amino acids-dissolved
carbohydrate that may represent detoxicated storage forms of germanium. Several research
groups have described the accumulation of copper, lead, selenium [Sacan et aI., 2000], and
aluminum [Sacan et aI., 2001] by Dunalie//a tertiolecta Butcher and chlororganic substances
and oil products from water [Tzvylev and Tkachenko, 1981] by Dunalie//a viridis (Snow)
Printz and D. tertiolecta.
At present, there is considerable interest in developing new diagnostic methods for in-
dicating the need for surgery in humaos that require only a small amount of a test organism
and could provide a rapid assessment of the state of the patient. The cells of Dunalie//a
bioculata Butcher have been used as cellular models and test-objects for monitoring the effi-
128
ciency of gossypol, a chemical known for its contraceptive properties in humans and several
other marn:mals, through its induction of spermatogenesis disorders and the inhibition of
spermatozoal motility [Druez et al., 1989]. Inhibition of D. bioculata motility by gossypol
was observed at the same concentrations modulating spermatozoa.
Another possible medical application of Dunaliella is the introduction of aqueous ex-
tracts from D. tertiolecta into a subject and assessing the central nervous system, spontaneous
motor activity, rectal temperature, exploratory behaviour, muscle relaxation, catalepsy, and
conditioned avoidance responses. The extracts can be used as a central nervous system de-
pressant and a potential muscle relaxant [Villaret al., 1992]. Monocultures of D. viridis have
also been used for estimating the level of cytotoxical compounds in the blood using changes
in the relative motility of their cells as a rapid indicator [Dmitriev et al., 2005].
129
of the cells (from 0 to 55 funiS) were grouped in 11 categories and statistically analyzed using
biometric methods [Lakin, 1973].
Two three-factorial complexes were investigated. The application of the fIrst complex
made it possible to study the effect of such factors as type of surface-active substances
(CSAS, ASAS, NSAS), their combinations (CA - cation-active-anion-active surface-active
substances, cation-active-nonion-active surface-active substances, AN - anion-active-nonion-
active surface-active substances, CAN - cation-active-anion-active-nonion-active surface-
active substances), type of algae (D. salina and D. viridis) and duration of surface-active sub-
stances action (in 0.5; 1; 2; 3 and 4 hours after inoculation) on the velocity of cell movement.
The second complex was used to study the effect of different concentrations of sur-
face-active substances (1, 5, 10, 20, 30, and 40 mg/l), types of surface-active substances
(CSAS, ASAS, NSAS, PSAS), and type of algae (D. salina and D. viridis) on the velocity of
cell movement.
The effects of the following pesticides were studied: acetal (l,l-diethoxy-ethane) (55
%), acetazine (1-[1O-[3-(dimethylamino)propyl]-lOH-phenothiazin-2-yl]-ethanone) (50 %),
alachlor (2-ch1oro-N-(2,6-diethylphenyl)-N-(methoxymethyl)-acetamide) (45 %), arylon (75
%), basta (2-amino-4-(hydroxymethylphosphinyl)-butanoic acid) (20 %), dual (2-chloro-N-(2-
ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)-acetamide) (96 %), DPC (20%), harmo-
ni (75 %) and tecto (2-(4-thiazolll)-IH-benzimidazole) (45 %). The concentrations of the
pesticides ranged from 10-7 to 10- M. The effect of heavy metals was also determined using
the salts of copper (CuS04·5H20), cadmium (CdCh) and lead (Pb(N03h) in a concentration
range of 10-7 to 10-2M.
130
et al. [1989] reported the toxic effect of anion-active surface-active substances (sodium
dodecyl sulfate) on D. viridis and its concentration dependant effect on cell size distribution.
Some surface-active substances are also known to stimulate the growth of algae-
macrophytes in aquaculture [Kalugina-Gutnik and Belyayev, 1987] which is of significant
practical interest with regard to the industrial production of various macroalgae species. As a
consequence, it is essential to determine the interaction of various surface-active substances
on the growth and behavior of algae in both natural and cultivated conditions.
Table 11.2. A three-factorial dispersive analysis of the effect of type and duration of action ofSAS on the ve-
locity of movement by different species of Dunaliella.
131
Table 11.3. A three-factorial dispersive analysis of the effect of various factors on the velocity of movement by
different species of Dunaliel/a.
As shown in Fig. 11.1 a, CSAS and ASAS at 1 mgll result in a stimulating effect on
phototopotaxis. At higher concentrations (up to 20 mgll), the substances suppressed phototo-
potaxis in both species of Duna/ie//a through immobilization of the cells and changes in the
direction of their movement. NSAS and PSAS between 1-10 mgll stimulated phototopotaxis
though at 40 mgll phototopotaxis decreased more than twofold by 4 hours after inoculation
(Fig. 11.1 b).
The inhibitory action of CSAS and ASAS at up to 20 mgll on linear velocity is illu-
strated in Fig. 11.2 a. The inhibitory effect of NSAS and PSAS was less pronounced and was
accompanied by a decrease in the velocity of movement to 50 % ofthe initial level.
The dependence of photomovement parameters on the duration of action of various
types of surface-active substances and their combinations (at 10 mgll) demonstrated a toxic
effect on motility (N."INo) with both species decreasing in the following sequence: CSAS >
CSAS + ASAS > ASAS > CSAS + NSAS > ASAS + NSAS > CSAS + ASAS + NSAS >
NSAS > NSAS + PSAS > PSAS. CSAS resulted in complete immobilization of the cells 1
hour after exposure while NSAS and PSAS led to only a 30 % decreases in motility after 3
hours exposure.
132
:0
-
../
'x q. r CS,t<;. .-ISAS
I
II 0'" q,
I
CL
0 ~i
0 b)
-0
~ " "" ('onc~nlration "of SA':'. mgtl
:i
~
.0
x
b '"
Q I I
NS4S,t'S.·I.<;
0-
C
f 1
I
~ , j Ie ,-/1 .Iii _q
I
Fig. 11.1. Dependence of linear velocity of two species of DJmaJiella on the cancenttation of: a - eSAS IUld
ASAS; and b - NSAS and PSAS, during 4 hours of contact [parsbikova et a1., 1990].
133
-
~.
c
"
2) 10 C'IAS A.5AS
<- JI)
Q
c
~ .. 10 £ £
'.) ,0 I
~
>'"
T
If) .0
--'"
~
Concentration ofSA~. mg/l
i LVS.fS
::1.
iij
h PS.IS
'::. I [
~ '. "[ I
~. I I
OJ
Q
!!/f/f}
.-'
;• • O.J
Concentration of SAS. mg/l
Fig. 11.2. Dependence of phototopolJlXia in two species of DtmaJiella on the concentrlltWn: a - CSAS ll!ld
ASAS; b - NSAS ll!ld PSAS. duriIlg 4 haUl'S ofcontact [p1lIlIhikov& et aI., 1990].
The type of surface-active substance affects the motility, velocity, and direction ofmovem.ent
of the cells in both species.. The response depends on the chemical composition of compound,
its concentration, and its duration of action on the cells. CSAS that possess positive ions, de-
creased or completely stopped photomovem.ent at 1-20 mgII. ASAS, which has negative ions
in contrast to NSAS and PSAS that do not, induced smaller changes in the velocity and direc-
tion of movement of the cells. The higher sensitivity to positive ions indicates the possible
influence of surface-active substances on the cell's ,-potential and motile reactions that are
related to it. To date, however, there is not enough experimental data to adequately understand
the mechanisms involved in these interactions.
134
D. salina and D. viridis control and experimental (with toxicant) suspensions were ir-
radiated and fluctuations in light scattering by the motile cells monitored. The toxic effects of
substances were estimated using changes in energetic expenses W - a parameter determined
as follows [Begma et al., 1989; Vlasenko, 2004]:
m(v."r
W=-2- N,. {vOll/·N.. (11.1)
m(vS N.. (V.)2. Nfm '
2
m(v..,,)1 m(v.)2
where - - 2 - and --2- are the average kinetic energies of the cells in experimental
and control samples, respectively; (vG/» and (v.) are the average velocities of the cells in
experimental and control samples, respectively; N", and Ntm are the number of mobile and
immobile celts.
The dependence of the energetic expense of Dunaliella cells on the duration of action
of the toxicant (Cu2) at 10 mg/l is presented in Fig. 11.4. Parameter Wtbat characterizes the
energy expense changed sufficiently in comparison with its initial value showing that it can be
used as a quantitative criterion for evaluating the toxicant
Fig. 11.3. Doppler correlation spectrometer: 1 - laser; 2 - power source; 3 - diaphragm; 4 - lens; 5 - cuvette
with the II8IIlple; 6 - thermostllt; 7 - photodetec:tor; 8 - diaphragm; 9 - power soun:e; 10 - amplifier; 11 - COIrO-
Iator; 12-compull:r; 13 -timer; 14-readmrtsyatem [Begmaetsl., 1989].
135
The dependence of parameter Won the concentration of toxicants (Cu2+ and triton X-IOO) is
presented in Fig. 11.5.
::i
if>
~ 1.6
"0x .
~ 1.2
2-D
"c
w..l 0.8
0.4
o '-----'---~~---'I0~0-~~-~-2""'00
Time, min
Fig. 11.4. Dependence of energy expense W in the cells of Dunaliella on the duration exposure to the toxicant
(Cu2) at a concentration of 10 mgll [Begma et aI., 1989].
1.0 J:2~~~
=> \
..: 0.8 \
~ ~
if> \
~
c 0.6 2\
\
"
0.
x \.... f
'" 0.4 \
'"
2!J \
'"
c
w..l 0.2 \ \
a ~
-8 -7 -6 -5 -4 -3 -2 -I
IgC
Fig.11.S. The dependence ofparameter Won the concentration of two toxicants (Cu2+ and triton X-IOO) [Begma
et aI., 1989].
136
The distinctions in parameter W between experimental and control samples are readily evi-
dent. Laser Doppler spectroscopy, based on recording the frequency changes of scattered opt-
ical radiation by a moving object, provides a rapid and concise means for evaluating the toxic
effect of chemical substances on motile algae in an aquatic medium.
where Xi and Xic are parameters of movement of the test-objects in experimental and control
samples, respectively (i = 1,2).
R
Fig. 11.6. Value r and direction () of vector in a two-dimensional system of coordinates (v/vc,F/Fc). "e" indi-
cates the control sample [Posudin et al., 1996].
In a three-dimensional system of coordinates (N = 3), the value r and directions (f}l and f}2) are
determined as follows (Fig. 11.7):
137
(11.4 )
( 11.5 )
(11.6)
The value r and the direction ((1], O2 and ( 3) of the vector R in the four-dimensional
system of coordinates (N=4) are determined from the following equations:
(11.7 )
(11.8 )
( 11.9 )
(IUD)
(11.11)
A N-dimensional system of coordinates can also be used, but in such situations (e.g.,
N>4) a graphical description of the vector R is impossible; its value and direction can only be
tabulated.
I,D -----------------------------------,~~::--
0,8
0.6
0.4
0.2
,4 ! 0.6 O:~./";,O
/
""'" /,/
______________________ :::i,-'
Fig. 11.7. Value r and direction (OJ and 8:J) ofvector R in a three-dimensional system of coordinates (vlv e; FIFe;
(Nm/Na}I{Nm/NQJc) [Posudin et al., 1996].
138
11.5.1. Dependence of Vector R on the Type and Concentration of Surface-Active
Substances
Table 11.4. Effect of type and concentration of SAS on photomovement parameters of Duna/iella viridis
[posudin et aI., 1996]
139
ViV, V//< V/I< v/v e 11
R, ii, 1,0
N 1,0
1.0 1,0
~:~, #':
~
FIF,I,O F/F,> 1.0 FlI~. 1.0 F/F~ 1,0
Fig. 11.8. The dependence ofvalue r and the direction e ofvector R, in a two-dimensial system of coordinates
(lillie; FIFe), on the type and concentration ofSAS. [CSAS - cation-active SAS (calamine); ASAS - anion-active
SAS - sodium salt of dodecyl sulfoacid; NSAS - nonion-active SAS - hydropol; PSAS - a natural compound of
polysaccharide nature extracted from cyanobacteria; SAS concentrations were: I; 5; 10; 20; 30; 40 mg/1. Re
(Fig. I 1.9-1 1.10,1 1.14-1 1.16) indicates the control sample [posudin et aI., 1996a,b].
Graphical depiction of thek dependence is presented in Fig. 11.9. The data was used for the
construction of the vector R in two-, three-, and four-dimensional systems of coordinates.
a b c
e
Fig. 11.9 Dependence ofthe value r and direction ofvector R
for Dunaliella viridis in two-dimensional sys-
tem of coordinates (lillie; FIFe) on the type and concentration of heavy metal salts: a - CuSO.·5H20; b - CdC!,;
3 5 6
c - Pb(N~h at concentrations of 10- ; 10-4; 10- ; 10- M [Posudin et aI., 1996a, b].
140
11.5.2. Dependence of vector R on the type and concentration of heavy metals
The effect of various heavy metals and concentrations thereof on photomovement parameters
in D. viridis (e.g., velocity v, phototopotaxis F, relative number NinlNo of immobile cells,
velocity n of rotation of the cells) is presented in Table 11.5 with averages and error values
given.
Table 11.5. Effect of the type and concentration of heavy metal salts on photomovement parameters in Dunaliel-
la viridis [posudin et aI., 19900, b].
141
The dependence of value r and direction () of vector Ron the type and concentration of heavy
metal salts at a 10-4 M is presented in Table 11.6. All possible pairs of photomovement para-
meters responded to the action of copper.
Table 11.6. Dependence of tile value r and directioo (J of vector Rin a three-ilimeosiooal system of coordinates 00 the
type and concentration of heavy metal salts[posudin et aI., 19900, b].
Concentration ofsalt, M
Photomovement r
Salt and
parameters
0 0
(control) 10.7 10-6 10" 10-4 10" 10.2
CuSO.·5H2O v/v, and F/F, r 1.41 1.35 1.22 Ll7 0.97 0.52 0
0 45.0 46.5 52.3 52.6 70.8 82.3 -
CuSO.,5H2O
vlv, and r 1.41 1.48 1.52 2.03 2.85 5.42 -
(NulNo)/ (NulNol,
0 45.0 41.7 37.8 27.3 18.8 5.5 -
n/nk r 1.41 1.50 1.58 2.08 2.91 5.59 0
CuSO.,5H2O and
(NulNol/(NulNol, 0 45.0 47.4 49.3 60.0 68.0 75.0 -
Pb(NO,n vlv, and F/F, r 1.41 1.35 1.31 1.26 1.20 0.99 0.78
76.7
0 45.0 47.1 47.8 49.8 52.1 68.1
CdCl2 vAl, and F/F, r 1.41 1.36 1.30 Ll9 1.02 0.91 0
0 45.0 46.8 48.7 62.5 79.0 82.6 -
Noles.Here and further in Tables 11.6-11.7: v -linear velocity; F - pbototopotaxis; NulNo - relative number of immobile
cells; n - velocity of rotatioo of the cells in the experimental sample; v, - linear velocity; F, - pbototopotaxis; (NulNj,-
relative number of immobile cells; n, - velocity of rotation oflbe cells in the cootrol sample; defies means the uncertainty of
the angle 0 under zero vslue of r .
Fig. lLlO displays the value r and direction () of vector R in response to various heavy metal
salts at a 10-4 M concentration. The errors of measurements are also indicated. The behavior
of the vector Rdepends on the level of toxicity to the photomovement parameter in question.
_ The principal tendencies for alterations in the value r and direction () of the vec-
tor R are the following (Fig. ILl 1): both parameters are decreasing but the first parameter
(X/Xl c) decreases at a greater rate than the second (X2I'X2c) (see. Fig. 11.11 a); the first para-
meter is increasing and the second is decreasing (see Fig. ILl 1 b); the first parameter is de-
creasing and the second is increasing (see Fig. ILl 1 c); both parameters are decreasing, but
the first parameter is decreasing with slower rate than the second (see Fig. 11.11 d); and both
parameters are increasing but the first parameter is increasing with a greater rate than the
second (see Fig. ILl 1 e).
142
V/J~ CdCI; CuSOr5H;O Pb(NO,h
I
Fig.ll.10. Dependence of the value r and direction 80fvector that is constructed in two-dimensional system R,
of coordinates, on the type of the salts of heavy metals [CuS04 ·5H20, CdC!, and Pb(N03hJ at the same (1O""M)
concentration [Posudin et al., 1996, a,b].
_ "\ PiP"L~
,
~
..
:!
'.
------ -- .0_.- i ~ d
, , !,
• 'i·
~
e ,
: : ' .
I::' 4.5 6 7 s P/P., \::'.,,j.5 6 7 8 P;'P..,
Fig. 11.11. The primary tendencies for changes in the value r and direction 8 of the vector R in a two-
dimensional system of coordinates (P/Pe; P;/Pe) [Posudin et al., 1996].
143
Construction of the vector R in a three-dimensional system of coordinates using the simulta-
neous measurement of three photomovement parameters [vlv c, FIFe and (Nu./No)/(Nu./No)c] is
presented in Table 11.7.
Table 11.7. Dependence of the value r and direction ((JJ and (J2) of vector R in a three-dimensional system of coordi-
nates on the type and concentration of heavy metal salts[posudin et aI., 1996a,b].
144
0;
v/v,
Fig. 11.12. The dependence of the value r and direction «()/ and 8,) of the vector R in a three-dimensional sys-
tem of coordinates (vlv e;FIFe; (NmIN,JI(NmIN,Je) [Posudin et aI., 1996].
FiF, I
VIV,
Fig. 11.13. The dependence of the value r and direction «(}1 and (}2) of a vector;' on the concentration of heavy
metal salts in a three-dimensional system of coordinates (vlve; FIF,,; (NmIN,JI(NmIN,Je). Figures -7. --{). -5•...
correspond to the concentrations 10.7• 10'<;. 10·'•... 10·' M [Posudin et al.. 1996].
145
VIV,
CuSO,.5H,Q
ii,
10
10
()
n/n, CuSo.r5H!(j
146
Table 11.8. The dependence of value r and the direction «(JI, (J2, (JJ) of vector R in a four-dimensional system of
coordinates on the type and concentration of heavy metals using the simultaneous measurement of four photo-
movement parameters in Dunaliella viridis [Posudin et al.• 1996].
Pb(NO,n vlve• FIFo r 2.00 2.04 2.11 3.05 4.31 5.63 7.01
(NtnlNo)/(NtnlNo)e (JI 60.00 61.04 62.59 71.63 77.26 80.60 83.78
and nine (J, 54.73 58.97 61.98 73.75 79.86 96.18 88.52
(J, 45.24 41.82 39.27 22.35 22.35 11.52 10.04
It is possible to present the minimum tendencies in behavior of vector Rin respect to the con-
centration of a heavy metal. If both parameters XllXl c and X21X2c are decreasing with an in-
creasing heavy metal concentration but the first parameter decreases at a greater rate thll!} the
second, the value r is decreasing and the direction () changes in such a way that vector R de-
viates from the axis of the first parameter (see Fig. IUla). If one parameter is increasing
with increasing heavy metal concentration and the second is decreasing, and the change of
both parameters occurs at the same rate, the value r is increasing and the direction () changes
in such a way that vector Rmoves nearer the axis of the first parameter (see Fig. IUlb,c).
If both parameters are decreasing but the first parameter decreases more slowly, the
value r is decreasing and the direction () changes in such a way that vector R deviates from
the axis of the second parameter (see Fig. IUld). When there is a simultaneous increase of
both parameters but the first increases more rapidly than the second with increasing heavy
metal c.smcentration, the increase in the value r and the direction () change in such a way that
vector R deviates from the axis of the second parameter (see Fig. IUle). The simultaneous
increase of both parameters but the first increases more slowly than the second with increas-
ing heavy metal concentration, the increase in the value r and the direction () change in such a
way that the vector Rdeviates from the axis of the first parameter (see. Fig. lUI./).
The level of accuracy in detennining the effect of heavy metals can be increased by
increasing the number of parameters that are measured. The application of the vector method
in our experiments allowed estimating the toxicity of three heavy metals (Pb > Cu > Cd) and
determinin the most toxic concentration for their salts (i.e., 10'2 M for CuS04·5H20 and
f
CdC!z, 10' M for Pb(N03)z).
The concentrations (10-4_ 10,1 M) of the salts of the heavy metals [CuS04,5H20;
CdC!z; Pb(N03)z] tested are comparable to those found in municipal sewage. It is important to
note that the metals themselves do not have a direct toxic effect, however, a number of chem-
147
icals act as antagonists or synergists. Likewise, there is the possible formation of complexes
between the metals and other compounds that lead to a loss in toxicity.
VIV,
10
e
Fig. 11.16. The dependence of the value r and direction of vector R in a two-dimensional system of coordi-
nates using simultaneous monitoring of two parameters (vlv,) and (FIF,) on the type and concentration (10-9;
10-8 ; 10-7 ; 10-'\ 10-'; 10-4; 10-3 ; 10-2 M) of pesticides [Posudin et aI., 1996].
148
Table 11.9 .Effect of type and concentyration ofpesticieds on photomovement parameters of Dunaliella viridis
[posudin et aI., 1996, a,b]
149
Dual v 48+4 48±4 47±3 46±4 41±4 3.3±3
P 0.4±0.05 0.38±0.04 0.35±0.04 0.33±0.04 0.30±0.05 0.24±0.06
r 1.4145 1.07 1.04 1.02 0.90 0.73 0 0 0
fJ 47.1 49.1 50.5 49.3 49.9 -
DPC v 46±5 46±5 46±5 46±5 46±5 46±5 46±6 46±5 46±7
P 0.38±0.04 0.38±0.04 0.38±0.04 0.38±0.04 0.38±O.04 0.38±0.05 0.33±O. 0.30±0.05 O.l8±0.06
r 1.41 1.41 1.41 1.41 1.41 1.41 04 1.27 1.10
fJ 45 45 45 45 45 45 1.32 51.7 64.8
Har- v 45±5 45±5 45±5 45±5 45±5 45±5 45±4 45±5 45±5
moni P 0.40±0.05 0.40±0.05 0.40±0.05 0.40±0.05 0.4O±O.05 0.40+0.04 0.35±O. 0.10±0.04 0
r 1.41 1.41 1.41 1.41 1.41 1.41 04 1.03 1.00
fJ 45 45 45 45 45 45 1.32 75.9 90
48.9
11.6. Summary
The high sensitivity of the two species of Dunaliella to environmental factors enhances their
potential for use as test-objects in biomonitoring. The great advantage of these organisms is
their microscopic size, ability to reproduce at high temperatures, active movement, photoki-
netic and photovector reactions, salt-tolerance and euryhalinity.
The sensitivity of various photomovement parameters of Dunaliella salina and D. vi-
ridis to the presence of surface-active substances in an aquatic medium [e.g., cation-active
catamine (CSAS), anion-active sodium salt of dodecyl sulphoacid (ASAS), non-ionogenic
hydropol (NSAS) and natural surface-active substances of polysaccharide origin (PSAS) that
were extracted from a cyanobacteria], salts of heavy metals [e.g., copper
(CUS04 ·5H20), cadmium (CdCIz) and lead (Pb(N03)z) at concentration from 10 -7 to 10-2 M],
and pesticides [e.g., acetal (55 %), acetazine (50 %), alachlor (45 %), arylon (75 %), basta (20
%), dual (96 %), DPC (20 %), harmoni (75 %) and tecto (45 %) at 10 -7 to 10-2 M] are de-
scribed. The data indicates the possibility of using the linear and rotational velocities of the
cell, frequency of flagella beating, and phototopotaxis values in Dunaliella species as test-
functions during biomonitoring of aquatic environments.
The use of simultaneous measurement of several photomovem.ent parameters is pro-
posed in that it allows increasing the sensitivity of biomonitoring. The vector method for
biomonitoring is recommended for estimating the effect of toxicant concentration in aquatic
environments using simultaneous measurement of two or more parameters of movement.
This method facilitates processing of data from large-scale measurements, allows the quan-
titative estimation ofthe effect of toxicant concentration, and can also facilitate toxicant identifi-
cation.
150
Chapter 12
Dunaliella Biotechnology
Dunalie//a salina Teod. and Dunalie//a viridis Teod. are very interesting from scientific and
practical points of view in that they represent models for studying mechanisms of tolerance to
extreme conditions of salinity, temperature, and pH. Identifying the genes that encode pro-
teins responsible for the species remarkable tolerance to extreme conditions may facilitate
increasing resistance to these conditions in other plants via transgenic means. The species can
also be cultivated in large volumes and may therefore be a viable source for the industrial
production of ~-carotene (provitamin A), ascorbic and dehydroascorbic acids, glycerol, and fo-
rage for piscine industry and other uses [Massjuk, 1973].
D. salina, an exceptionally rich source of ~-carotene [Drokova, 1961; Ben-Amotz et
aI., 1982a], could be used for the prevention and treatment of cardiovascular and ophthalmic
diseases, avitaminosis, arthrosis, cancer (skin, liver, stomach and leukemia), macular degene-
ration, and asthma. ~-Carotene containing preparations from this species are known to pro-
mote an increase in appetite and decrease sleeplessness. It has also been proposed using
D. salina as hepatopathy inhibitors. Powders produced from this alga are claimed to act as
inhibitors that are useful in the treatment of hepatitis, liver cirrhosis, and fatty liver [Mizogu-
chi, 2006].
The cultivation of Dunalie//a for biomass in reservoirs for various uses has been de-
scribed in a number of publications [Massjuk, 1966, 1967, 1973; Massjuk and Abdulla, 1969;
Nosova et aI., 1979; Avron and Ben-Amotz, 1992; Ben-Amotz and Avron, 1982, 1989, 1990;
Borowitzka et aI., 1984, 1986; Moulton et aI., 1987; Rashkova and Vlakhov, 1988; Borowitz-
ka and Borowitzka, 1988, 1990; Moho and Contreras, 1990; Ben-Amotz et aI., 1991; Marko-
vits et aI., 1993; Zhou, Q., 1995; Ventosa and Nieto, 1995; Ben-Amotz, 1995, 1996; Krol et
aI., 1997; Hong et aI., 1998; Orset and Young, 1999; Jin and Melis, 2003; Mohammad R.R
and Mansour, S., 2003; Borowitzka, 2005; Pisal and Lele, 2005; Raja et aI., 2007; Del Cam-
po et aI., 2007; Prasanna et aI., 2007].
DOI 10.1007/978-3-8348-9765-7_13,
© Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH 2010
Prasanna et al., 2007]. The direct chemical synthesis of l3-earotene was first reported in 1956.
The molecular formula is C4()lIs6 with a mass of 536.9. It is violet-red in color in the crystalline
state. The cost of synthetic l3-carotene is approximately $500/kg which has in part been respon-
sible for an increasing demand for the chemical from. natural sources [Borowitzka and Boro-
witzka, 1990].
The presence of stereoisometric isomers is a well-known peculiarity of l3-carotene.
Each double bond in the aliphatic chain of l3-carotene can exist in two configurations. As result,
272 cis/trans-isomers of l3-earotene can theoretically be fonned, 12 of which have been identi-
fied in nature [Ben-Arnotz and Shaish, 1992, in: Avron and Ben-Arnotz, 1992]. Light absorp-
tion by the carotenoids is altered by isomerisation. For example, cis-stereoisomers are characte-
rized by a shift in the absorption maximum with the peak in ultraviolet portion of the spectrum.
In nature the trans-forms predominate. Naturall3-carotene, extracted from various sources, con-
tains significant mono- bi- and poly-cis-forms [Avron and Ben-Arnotz, 1992].
The content of !3-carotene in plants ranges from 0.01 to 10 mg/100 g with green leaves
(e.g., parsley, spinach, broccoli), yellow-orange fruits (e.g., mandarin, mango, peach), and
certain vegetables (e.g., carrots, sweetpotato, pumpkin) being rich sources. Some species of
microorganisms, such as the fungus Phycomyces blakesleanus (Bgtt). Arch. and the yeast Rho-
dotorula, accumulate large quantities of l3-carotene (5 and 0.5 mg/g dry weight, respectively).
These natural sources generally contain a mixture of carotenoids, carotenoid esters, carotenoid
isomers together with varying amounts of l3-carotene.
D. salina can accumulate a great quantity of l3-carotene and is considered the most dense
natural source of provitamin A known [Drokova, 1961; Milko, 1963; Aansen et al., 1969;
Ben-Arnotz et aI., 1982a; Loeblich, 1982; Orset and Young, 1999; Tm and Melis, 2003; Pisal
and Lele, 2005]. A number of methods for extracting carotenoids and l3-carotene, in particular,
have been reported [Yamaoka, 1994; Garcia Gonzalez et al., 2003; Chen et aI., 2008].
I3-Carotene that accumulates in D. salina, consists mainly of two stereoisomers, the ratio of
which depends on quantity of light absorbed during a cell cycle [Ben-Arnotz et aI., 1982a,
1987, 1988; Tsukida et aI., 1982].
The biosynthesis of l3-carotene in D. salina proceeds in four stages [Ben-Arnotz and
Shaish, 1992, in: Avron and Ben-Arnotz, 1992]: 1) formation of geranylgeranyl diphosphate
(GGDP) from mevalonic acid; 2) condensation with the formation of phytoene; 3) desatura-
tion of phytoene to lycopene and 4) cyclization of lycopene with formation of l3-carotene. The
intermediates in l3-carotene synthesis such as phytoene, phytofluene, ~-carotene, neurosporene,
l3-zeacarotene, lycopene, y-carotene were formed between prephytoene and l3-carotene [Ben-
Arnotz et al., 1987; Ben-Arnotz and Shaish, 1992, in: Avron and Ben-Arnotz, 1992].
The cells of D. salina have a green coloration under conditions suitable for growth and
reproduction and have 0.3 % l3-carotene on a dry weight basis, similar to the content in plant
leaves and the cells of carotene-containing algae. l3-carotene accumulates, under conditions that
delay growth and reproduction of the cells, within the orange oily globules located in the inter-
thylakoid space of the chloroplast.
The important parameters modulating the growth, reproduction and formation of caro-
tene are light intensity and duration, salt concentration, temperature, and nutrient availability
[Milko, 1963; Massjuk, 1966, 1973; Massjuk and Abdula, 1969; Semenenko and Abdulaev,
1980; Ben-Amotz et aI., 1982a; Loeblich, 1982]. High light intensity and slow growth in D.
salina result in a higher rate of carotenogenesis. Elevated salt concentrations (i.e., >4 M
NaCl) that osmotically alter the growth medium, extreme temperatures, and nutrient deficien-
cies (nitrogen, in particular) reduce the accumulation of l3-carotene in the cells.
The possibility of cultivating D. salina in commercial salt water ponds used for NaCl
production (Societe Scherifienne des sels, Larache, Morocco) was assessed [Riyahi et al.,
2006]. D. salina was the only algal species surviving at salinity levels up to 25 % (w/v). The
152
biosynthesis of l3-earotene in D. salina can easily be regulated. It is probable that the produc-
tion costs for l3-earotene derived from. algae grown in open, non-sterile conditions will decrease
with new technological improvements [Borowitzka and Borowitzka, 1990; Borowitzka, 1990,
2005]. At the same time, mass cultivation in closed industrial production systems with rigidly
controlled conditions for l3-carotene by the pharmaceutical industry is also a viable option.
153
, ZS
I
~I ::c"
r
<::
I v·
I <::
1500 '.2 /5 ~
"2 a
u
S ',DO /2
'f>
~
'g."" !!JO
-=
:6
6fJfJ
li
U JOO 3
o 5IU/52025jl1.
Time, days
Fig. 12.1. Accumulation of algae and carotene in biomass of Dunaliella salina wben produced in mass culture:
1 - density of the cells; 2 - content of carotene in the cells; 3 - carotene content/I L of suspension [Massjuk,
1973].
Since the main biological processes that help to maximize the yield of carotene (reproduction
of the cells and biosynthesis of ~-carotene) require different conditions, a two-stage method of
cultivation was proposed for carotene production. The first stage facilitates the accumulation
of biomass by using a 2M NaCI medium with appropriate biogenic elements at 25-30 °C and
a light intensity of 5,000-6,000 lx. The second stage of growth is in open reservoirs 4-5 M
NaCl, 35-40 °C and illuminance around 100,000 Ix without the addition of biogenic elements
[Massjuk, 1965a,b,c, 1966, 1967; Massjuk and Abdula, 1969, 1971]. We tested the method
near Kiev in 1963-1965 and obtained greater than 30 kl¥h!l of carotene per vegetation season
(5 months) or an average carotene production of24 mg m-2 day-t [Massjuk, 1966]. This semi-
industrial method for the mass cultivation of D. salina for ~-carotene was tested using an expe-
rimental area of 0.5 ha at the Saksky chemical plant in Crimea using an inexpensive chlorine-
magnesium brine and supplemental fertilizer [e.g., superphosphate, ammoniac saltpeter (nitric
acid potassium salt), potassium salt]. The station was equipped with 15 plastic 200 1 trays, 4
concrete pools of 4 m and 4 pools of 5 m3 where the algae was bred (photo 12, I) and the caro-
tene accumulated.
Experiments in 1965-1968 demonstrated the potential for production in the southern part
of the Ukraine, with yields of up to 120 kg of carotene per hectare during a growing season of 7
months [Massjuk and Abdula, 1969; Massjuk et aI., 1970; Massjuk, 1973]. The technology for
the repeated utilization of the brackish water produced during cyclic production of the algae
has also been studied [Massjuk, 1973].
154
Photograph 12.1. Experimental carotene production station at the 8aksky chemical plant, Crimea, 1965-1969:
A - trays and concrete pools where the first stage of Dunaliella salina cultivation occurred; B - industrial pools
with a ground floor [Massjuk, 1973].
The technology for the extraction of the carotene using plant oils was also developed. It in-
volves four stages: 1) mechanical fragmentation of the cells; 2) flotation of the cell residue
that includes carotene; 3) co-sedimentation of the residues using ferrum oxide hydrates and
the removal of the sediment from the rapa; and 4) extraction of the pigment using organic
solvents [Vendt et ai., 1965; Geleskul, 1966, cited by: Massjuk, 1973]. While the methods and
economics appeared to be attractive, industrial production of J3-carotene in Ukraine has not
yet occurred. While research on the production of carotene-containing algae in Ukraine has
been terminated, there continues to be considerable interest elsewhere. For example, this area
of research continued in the 1970s in Israel, USA, Australia, China and other countries where
the mechanisms of salt tolerance, osmoregulation, and the biosynthesis of glycerol, and /3-
carotene were studied [Ben-Amotz et aI., 1982a,b; see reviews: Borowitzka and Borowitzka,
1990; Avron and Ben-Amotz, 1992].
At the end of the 1970s, several commercial companies were interested in the possibil-
ity of production of D. sa/ina for J3-carotene which accelerated this area of research. At the
beginning of the 1980s, a carotene experiment station was established in a sea lagoon in
Western Australia near Perth. The first experimental ponds were 10, 100,250, and 600 m2 in
size. Another five industrial ponds of much greater size (i.e., 5 x) were built in 1986 and the
carotene manufacturing plant officially opened. The number of the ponds was doubled in
1988. At the same time, biotechnological and engineering research improved productivity
[Borowitzka and Borowitzka, 1990].
At present, the cultivation of Dunalie//a has expanded in volume and geographical 10-
cation. For example, production has been undertaken in hypersaline lakes, ponds and lagoons
155
under a range of climatic conditions in various countries (e.g., Larache, Maroc [Haouazine et
aI., 1999]; the north of Chile [Gomez et aI., 1999]; East Coast of Thailand [Bhumibhamon et
aI., 2003]; southern Spain [Garcia-Gonzalez, 2003]; Venezuela (Araya, Coche, Peonia, Cuma-
raguas. and Boca Chica) [Guevara et aI., 2005]; Urmia hypersaline lake, northwest of Iran
[Fazeli et aI., 2006] and central region of Iran [Tafreshi and Shariati, 2006]).
Separation of the algae from its aqueous environment is the most difficult, labor-
consuming and expensive stage in the technology of carotene production. Likewise, methods
for the separation of the ~-carotene such as filtration, centrifugation, flotation and floccula-
tion, sedimentation, and the concentrating of the cells in a salinity gradient have been tested
[Vendt et aI.,1965; Geleskul, 1966, cited by: Massjuk, 1973; Borowitzka and Borowitzka,
1990; Mohn and Contreras, 1990].
D. salina is typically processed into three general products: I) a capsulated solution of
1.6-4 % ~-carotene in plant oil for use as a dietary supplement; 2) a 30 % suspension of crys-
talline ~-carotene in plant oil that is used as a dye in the food industry; and 3) a dried product
that contains 2-3 % ~-carotene for use as a additive in animal feeds [Borowitzka and Boro-
witzka,1990].
Similar carotene factories were created in South Australia and the USA (California).
There were also smaller scale companies producing ~-carotene from D. salina in Israel (see
photographs 12.3, 12.4i, Chile, and Great Britain [Borowitzka, 1990; Borowitzka and Boro-
witzka, 1990; Mohn and Contreras, 1990].
Photograph 12.2. A general view of the reactor used for biomass production of Dunaliella near the city Eilat
(courtesy of Prof. A. Ben-Arnotz).
156
Photograph 12.3. Details of the reactor used for biomass production of Duna/iella near the city Eilat (courtesy
of Prof. A. Ben-Amotz).
Worldwide the country producing the greatest quantity of fl-carotene is Australia. Glycerol,
which can reach 30 % of the algal dried biomass, high-quality protein which remains after
extraction of fl-carotene, ascorbic and dehydroascorbic acids and other valuable biologically
active organic compounds are by-products of fl-carotene production [Massjuk, 1973; Ben-
Amotz et ai., 1982a, b; Borowitzka and Borowitzka, 1990; Avron and Ben-Amotz, 1992].
The current state of fl-carotene production from Dunaliella, based on either open-pond
systems or closed photobioreactors, is discussed in the following articles: [Yamaoka et ai.,
1994; Ben-Amotz, 1995; Yamaoka et ai., 1996, 1997; Chen and Wang, 2003; Leon et al.,
2003; Hejazi et al., 2003; Chai et ai., 2004; Hejazi and Wijffels, 2004]. Likewise, the effect of
various abiotic and chemical factors on Dunaliella cell growth and carotene content is re-
viewed in a number of papers. This includes the effect of high light intensity, pH, NaCl con-
centration, temperature, and trace nutrients [Yamaoka et ai., 1992]; high irradiance and areal
densities [Grobbelaar, 1995]; differences in the chemical composition of the media [Cifuentes
et ai., 1996]; various stress conditions (nutrient deficiency or high salt concentration) [Haoua-
zine et ai., 1999]; temperature, pH and illumination [Markovits et ai., 1993]; high light inten-
sity, high salt concentration, and nitrate deficiency [Bhumibhamon et ai., 2003]; mixing, flow
rate, culture depth, cell density, and dilution cycles [Garcia-Gonzalez, 2003]; salinity [Mo-
hammad and Mansour, 2003]; temperature and irradiance [Gomez and Gonzalez, 2005]; dif-
ferent salt concentrations [Fazeli et ai., 2006]; and high light intensity, high salinity, tempera-
ture and availability of nutrients [Raja et ai., 2007]. Laser mutagenesis of the organism and its
effect on fl-carotene production was investigated by Zhao et ai. (1992).
157
Utilization of Dunaliella by the food industry includes the introduction of ~-carotene
into foods as a source of vitamin A and as an antioxidant, the production of ~-carotene tablets,
and as a component of mixed carotenoid products are described by Honda [1997]. Dunaliella
spp. have significant amounts of lipid, protein, chlorophyll, carotenoids, vitamins, minerals,
and unique pigments and are used as nutrient-dense foods and sources of fine chemicals [Kay,
1991; Honda, 1997; Hatanaka, 2002]. Certain strains of Dunaliella are harvested to produce
dried algal meals for the extraction of polyunsaturated fatty acids and oils for the health food
industry and for coloring agents to be used in the food and cosmetic industries [Jin and Melis,
2003]. Due to their health benefits, algal pigments have great commercial value as natural
colorants in food industry [Prasanna et al., 2007].
Interest in ~-carotene has increased significantly due to the discovery of its anti-tumor
activity and its role as an antioxidant in human nutrition. At the present time D. salina is cul-
tivated as a source of ~-carotene on an industrial scale in Australia, USA, Japan, Taiwan,
China, and Indonesia [Borowitzka, 2005]. ~-Carotene, astaxanthin, fucoxanthin, halocynthax-
anthin and peridinin are believed to inhibit lung, breast, buccal pouch, and nerve cell cancers
[Hong et ai., 1998].
The problems of production, functions, and quality standards of D. salina powder and
soft capsules are discussed by Guo et ai. (2003). The preliminary experiments with laboratory
animals have shown that the capsules containing the powder inhibit radiation damage and
tumor development and enhance immunity. Certain carotenoids in Dunaliella (e.g., lutein) are
thought to be effective agents in the prevention and treatment of a variety of degenerative
diseases [Campo et ai., 2007].
Preparations of ~-carotene are exported from Australia to Japan, USA, Korea, Taiwan
and other countries with total exports to Japan and USA in 1990 of 2 million Australia dollars
[Borowitzka, 1990]. The cost of ~-carotene from D. salina in the 1990s was estimated by
Australian manufacturers as 50-100 Australia dollars per kilogram while the price of natural
~-carotene on the world market at the same time varied between 500-1000 dollars per kg. It is
estimated that the worldwide market value of carotenoids will exceed US $1,000 million by
the end of the decade [Campo et ai., 2007].
It is worth mentioning the potential application of D. salina as an educational model
for biotechnology students [Bosma and Rouke, 2003]. Students can develop strategies for
optimization of the growth rate, and how and when to stress the alga to initiate ~-carotene
production. The students should be responsible for their own results and creating a competi-
tion among the students can stimulate their interest. The approach also provides considerable
practical experience for the students.
12.3. Summary
Several species of Dunaliella can be cultivated as a natural source of !3-carotene, ascorbic and
dehydroascorbic acids, glycerol, and other valuable organic compounds. In addition, the genes
that encode for proteins that confer exceptional tolerance to extreme environmental conditions
are viable candidates for facilitating the resilience of other organisms to environmental stress
using recombinant DNA technology.
Mass cultures of Dunaliella can be used to provide a nutritionally excellent forage for
the piscine industry, especially for the cultivation of valuable breeds of sturgeon. D. salina is
the richest source of ~-carotene in the plant kingdom. Mass cultivation of selected strains is
utilized in a number of countries for the industrial production of !3-carotene preparations that
are used in food and pharmaceutical industries and in medicine for the prevention and treatment
of tumors, cardiovascular, ophthalmic diseases, avitaminosis, arthrosis, and other diseases. Fur-
ther study of photomovement regularities and peculiarities in Dunaliella species can facilitate
solving certain technological problems associated with carotene production.
158
Chapter 13
General Results and Perspectives of Further
Investigations
The senior author of the present work started her studies of the genus Dunaliella at the N.G.
Kholodny Institute of Botany, the National Academy of Science of Ukraine in the late 1950s in
the context of projects on surveying algae as prospective sources of ~-earotene (provitamin A).
In the 1960s-l970s, the composition and ecology of the genus in watersheds of the Ukraine,
the Trans-Caucasian region, Central Asia, and the Far East were investigated in detail along
with issues of phylogeny and taxonomy. A collection of Dunaliella strains was created and
peculiarities in their morphology, physiology, and biochemistry were investigated. In addition,
methods for their laboratory and semi-industrial commercial cultivation [Massjuk 1973] were
developed In the 1980's, several species of Dunaliella that had attracted the attention of a bio-
physicist were selected as possible models for studying photomovement using a series of com-
plex optical, spectroscopic, and laser methods [Posudin et al., 1992]. An experimentallaborato-
ry was established and the instrumentation needed for studying the photomovement of cells of
Dunaliella developed in the Department of Biophysics at the National Agricultural Universi-
ty in Kiev (now National University of Life and Environmetal Sciences of Ukraine).
The main objective of the present work is to summarize our research on the photo-
movement of Dunaliella and to compare our results with data of other scientists that have studied
similar phenomena in other organisms, identifying both common and specific peculiarities
in photomovement in representatives of various taxa and assess differences relative to phyloge-
netic relationships. Experimental studies were preceded by a detailed analysis of previous
results published by the photobiology community on the photomovement of flagellates. Par-
ticular attention has been focused on discrepancies in terminology covering the various aspects
of photomovement in microorganisms.
13.3. Photoreactions
Species of Dunalie//a are capable of photokinetic and photovector reactions. In contrast, the
presence of photokinetic reactions in Chlamydomonas has been challenged in the literature. We
did not observe photophobic reactions in Dunalielfa, though they are present in Chlamydomo-
nas and Euglena. There are, however, several publications indicating the possibility of such
reactions in Dunalie//a.
13.4. Photokinesis
The locomotion velocity of Dunalie//a cells (photokinesis) depends on the intensity of the light
stimulus and the environmental conditions, such as temperature, strength of electric and elec-
tromagnetic fields, intensity and duration of ionizing radiation, and the presence and concentra-
160
tion of calcium channel blockers (such as isoptin and cinnarizine), sodium aside, surfactants,
salts of heavy metals, and pesticides. Likewise, a combination of factors can modulate velocity.
We did not find the locomotion velocity of individual Dunaliella cells depended on the wave-
length of the light, dosage of preliminary UV-irradiation, concentration of cobalt and calcium
ions, the presence of ionophores that increase the permeability of the cellular membrane to
calcium ions, and the presence of ionotropic preparations that stimulate N a+-K+-ATPase.
The average cell velocity of translational movement of hyperhalobic species of
Dunaliella was 36 ± 2 IlITh's for Dunaliella viridis and 48 ± 2 IlITh's for D. salina. The average
velocity of movement for these two closely related species among experiments varied over a
wide range, indicating that distinctions between the species based on velocity probably are not
valid. The modal value for the average velocity of movement of the marine species Dunaliel-
la bioculata Butcher was 105 ± 5 Ilm/s. The values for the Dunaliella species exceed by 1-3
orders of magnitude those of microorganisms not possessing a flagellar apparatus and are within
the limits known for others flagellates, both prokaryotic and eukaryotic. However, the average
locomotion velocity of hyperhalobic species of Dunaliella is lower (sometimes by an order of
magnitude) than in the marine species D. bioculata and the freshwater species C. reinhardtii and
E. gracilis. These differences may be caused by variation in the viscosity of the media.
The average cell velocities for rotary movement in hyperhalobic species of Duna-
liella were nearly identical [Le., 0.52±0.04 rotations (revolutions) per second in D. salina and
0.54±0.04 rotations per second in D. viridis]. These values, however, were lower than in the
freshwater algae C. reinhardtii. The maximum average velocity values for forward and rotary
cell photomovement in Dunaliella were observed under the following conditions: white-light
intensity of 0.22-0.81 W/m2, illuminance of 150-550 lx, temperatures of 20-30 °C, and
pH 8.
The rhythmic regulation of cell movement in Haematococcus pluvialis Flotow is
caused by rhythmic pulses, presumably related to the functioning of contractile vacuoles. How-
ever, in hyperhalobic species ofDunaliella, the contractile (pulsating) vacuoles are absent and
the beating rhythm of flagella is apparently related to other oscillators. The flagellar beating
frequency in hyperhalobic species of Dunaliella is 25-50 Hz while in freshwater
C. reinhardtii it is as much as 64 Hz.
13.5. Phototopotaxis
The direction of cell movement of Dunalie/la in relation to the light source (phototopotaxis) de-
pends on the parameters of the light signal (e.g., light intensity, spectral composition, gradients
of intensity in space and time, polarization) and environmental conditions (e.~., ~ ,
intensity of electric fields, level of ionizing and UV radiation, concentration of Ca +, Co ,cinna-
rizine, isoptin, sodium aside, surfactants, salts of heavy metals and pesticides), and combinations
of these factors. Phototopotaxis of Dunalie/la species, however, is not modulated by ionotropic
preparations that stimulate a membrane Na+-K+-ATPase.
Phototopotaxis of both hyperhalobic species of Dunalie/la in laboratory cultures was
observed at an illuminance of 500 Ix (positive) and 40,000 Ix (negative). Transition from
positive to negative phototopotaxis was observed at 1,500 Ix. These parameters are within the
limits known for other algae. However, sensitivity thresholds to weak and strong illuminance,
and transitions from positive to negative phototopotaxis differ substantially among algal spe-
cies. They allow assessing shade-tolerance, sun-tolerance, and resistance to high-level light
exposure of these species.
D. viridis is more sensitive to weak light (30 Ix) than D. salina, which corresponds to
behavioral peculiarities between the two species in nature. In laboratory culture, both species
161
are more sensitive to high illuminance than C. reinhardtii; in the latter, the transition to nega-
tive phototopotaxis occurred at 100,000 Ix.
The transition of Dunaliella species from positive to negative phototopotaxis differs
from that in chIamydomonads. In contrast to chlamydomonads, the change in flagellar beating
from a ciliary to an undulate mode was not observed in the Dunaliella species. The beating
of only one flagellum was observed, which caused turning of the cell followed by its subse-
quent movement in the opposite direction to the light source.
Under the conditions found in Crimean hyperhaline watersheds with high illuminance
(i.e., above 100,000 Ix), natural populations of D. salina are found in the ''red form" and dis-
playa complete absence of negative phototopotaxis. Thus, hyperhalobic species D. salina and
D. viridis differ in their degree of sensitivity to both high- and low-light intensity which ex-
plains differences in the ecological niches occupied by the species in nature.
The maximum values for positive and negative phototopotaxis were found at
20-30°C and the maximum value for positive phototopotaxis was at pH 7.35.
Electric fields suppress phototopotaxis in Dunaliella as well as in other algae, which in-
dicates the participation of bioelectric potentials in this process. Increasing the temperature from
18 °C up to 30 °C removes the inhibitory influence of the electric field density and stimulates
phototopotaxis in both hyperhalobic species of Dunaliella.
Ionizing and UV irradiation inhibit phototopotaxis in D. salina and D. viridis. The inhi-
biting effect depends on the irradiation dosage; in the case ofUV-irradiation, the response also
depends on the wavelength. We described for the first time in Dunaliella the transformation
from positive phototopotaxis to negative due to the influence ofUV-irradiation; at high levels of
irradiation, phototopotaxis can be completely blocked.
The maximum values for phototopotaxis in hyperhalobic species of Dunaliella were
observed when CaClz·6H20 was in the medium at concentrations between 10-5 and 10-3 M. In-
creasing the calcium chloride concentration up to 10-2 M suppressed phototopotaxis by
10-20 %. The addition of the ionophore A23187 to the medium increased the permeability of
the cellular membrane to calcium ions causing a complete inhibition of phototopotaxis both in
D. salina and D. viridis. The addition of CoClz, that blocks membrane calcium channels at con-
centrations from 10-6 to 10-3 M, suppressed phototopotaxis in both species of Dunaliella. Other
calcium channel blockers, such as cinnarizine, isoptin, and sodium aside, elicit a similar effect
on phototopotaxis in Dunaliella. Ouabain, which stimulates Na+-K+-ATPase, does not influence
phototopotaxis in Dunaliella.
The phototopotaxis action spectrum for the two hyperhalobic species of Dunaliella
was identical. It occurred between 400 and 520 nanometers (nm) and has maxima at
410-415 nm and 465-475 nm. The action spectrum for phototopotaxis in Dunaliella differs
somewhat from those in Chlamydomonas reinhardtii and Haematococcus pluvialis which dis-
playa wide band in the 400-600 nm range, with the maximum at 500 nm. In contrast to the
situation observed in representatives of the Chlorophyceae, Tetraselmis viridis Rouch. (Chloro-
dendrophyceae) exhibited phototopotaxis in the UV region of the electromagnetic spectrum. In
E. gracilis (Euglenophyta), phototopotaxis occurs in the 300-550 nm range with two basic
maxima at 385 nm and 460 nm and two smaller maxima at 410 nm and 490 nm.. Thus, repre-
sentatives of different genera, classes, and divisions differ distinctly in their action spectra for
phototopotaxis indicating differences in their photoreceptor systems and the composition of pho-
toreceptor pigments.
13.6. Motility
One parameter of photomovement is the motility of cells or the relative number of motile cells
(N,,/No), where N m is the number of motile cells, and No - the total number of motile and non-
162
motile cells. In populations of Dunaliella this varies from 0 to 100 % and displays the same de-
pendence on the characteristics of the light stimulus and environmental conditions as photo-
topotaxis. The number of motile cells differs with the presence or absence and degree of
dependence due to certain factors (e.g., wavelength of light, dose of preliminary ionizing and
UV irradiation, concentration of compounds opening or blocking calcium channels) on the
velocity of individual cells (photokinesis).
163
13.9. Sensory Transduction of the Light Signal
AI; it has been demonstrated with ions of calcium and cobalt, and substances blocking or stimu-
lating ion channels, sensory transduction of an absorbed quantum of light into a locomotory
reaction in species of Dunaliel/a, as well as in other green algae, is most likely of ionic nature,
thus confirming the critical role of ea2+ in these processes. At the same time, Na+-K+-ATPase
does not participate in the photoregulation of locomotion in Dunaliella cells nor does ouabain
have an effect on photomovement parameters. It is assumed that the intake of calcium ions into
the cells of these algae is controlled not by a Na+-K+ pump, as is the case in E. gracilis, but prob-
ably occurs directly, through light-induced membrane channels, as in Chlamydomonas.
In contrast to C. reinhardtii, in many cases the non-specific reaction (autotomy offlagel-
la) of Dunaliel/a cells to substances stimulating or blocking ionic channels does not result in
the loss of flagella. Therefore, their locomotory reactions can be regarded as a specific response
to the blocking or stimulating of ionic processes.
We demonstrated for the first time that different photomovement parameters in
Dunaliel/a (phototopotaxis and the relative number of motile cells, on the one hand, and pho-
tokinesis, on the other hand) are controlled by different mechanisms. This is confirmed by the
presence/absence or different degrees of dependence of these parameters on such environmental
factors as the wavelength of incident light, dose of preliminary ionizing and UV-irradiation, con-
centration of ions of calcium or cobalt, and compounds stimulating or blocking calcium chan-
nels, etc.
164
Critical differences in photobehaviour are found among representatives of various genera
belonging to different orders of green algae in the class Chlorophyceae: species of Dunaliella
(Dunaliellales) on the one hand, and C. reinhardtii and H. pluvialis (Chlamydomonadales), on the
other hand. Differences in the action spectra and maxima for phototopotaxis leads to the as-
sumption that there are different sets of photoreceptor pigments: carotenoids and caroteno-
proteins in Dunaliella and rhodopsin as the basic photoreceptor pigment in C. reinhardtii and H.
pluvialis.
Taking into account differences in the stigma structure between the species of Dunaliella
and C. reinhardtii, it is probable that photoreception in the former, in addition to the modula-
tion mechanism, light diffraction plays an essential role, while in the latter, the critical mechan-
ism involves interference in light flux.
Introduction of chemical compounds into the medium that stimulate or block membrane
ionic channels causes in the Dunaliella species specific locomotory reactions, while in C. rein-
hardtii the reactions are nonspecific (autotomy of flagella). Likewise, in the Dunaliella species
photophobic reactions like those in Chlamydomonas have not been observed, while the presence
of photokinetic reactions, so well expressed in Dunaliella, are questionable in Chlamydomonas.
Photomovement in Chlamydomonas and Dunaliella differ in the beating mode of their
flagella. Forward movement of a Chlamydomonas cell is caused by ciliary beating of the flagel-
la, while backward movement utilizes undulate beating. Since the cells of Dunaliella are not
capable of photophobic reactions, the undulate mode of flagella beating has not been observed
in the genus. Chlamydomonas cells change direction by using unequal beating frequencies
between the cis- and trans-flagella. In contrast, Dunaliella cells temporarily stop the beating of
one flagellum.
Thus, species of Dunaliella differ from C. reinhardtii in a complex array of fundamental
features that include the composition of their photoreceptor pigments, mechanisms of pho-
toreception, certain details in the sensory transduction of the light signal into a locomotory reac-
tion, the presence or absence of photophobic and photokinetic reactions, functioning of the fla-
gellar apparatus, etc. The results obtained correlate with data of molecular cladistics. The data of
molecular phylogeny demonstrate that C. reinhardtii, as a representative of the heteroge-
neous genus Chlamydomonas. belongs to the group of chlamydomonads (which is closely re-
lated to colonial Volvocales) that is very distant from another group that is closer to Dunaliella
(e.g., Chlamydomonas applanata Pringsh.). The evolutionary distance between these two groups
is comparable to the distance between soybeans and cycads.
Interesting data are available on photomovement of Tetraselmis viridis (Roukhiyajnen)
Norris et al., a representative of a separate class of green algae, the Chlorodendrophyceae [Mass-
juk, 2006]. Photomovernent in the ultraviolet region of the spectrum and the presence of a photo-
topotaxis maximum in that area indicates the possible presence of flavins and/or pterins as part of
the composition of photoreceptor pigments of this species. Thus, evolution of photoreceptor
systems, in particular sets of photoreceptor pigments, may occur in different ways and in various
branches (clades) of the phylogenetic tree of green plants.
Even greater differences in the structure of the photoreceptor were found between Du-
naliella spp. and E. gracilis, a representative of the division Euglenophyta. According to some
authors, Euglena and Dunaliella belong to different kingdoms: Euglenozoa Cavalier-Smith,
1981, or Euglenobionta Kussakin et Drozdov, and Plantae Leedale, 1974, or Viridiplantae Ca-
valier-Smith, 1981, respectively. Other authors place them in different super-kingdoms of the
organic world: Discicristata (Mirabdullaev) Leontiev and Akulov (2002) ex Zmitrovich, 2003
and Lamellicristata (Taylor) Starobogatov (1986) emend Zmitrovich (2003).
Dunaliella differs from Euglena in its low speed of cell movement, higher sensitivity to
low and high light intensities, and the lower threshold for the transition from positive to nega-
165
tive phototopotaxis, which is probably partly explained by the differing ecological requirements
at the species level. Until now the transition from positive to negative phototopotaxis with the
subsequent complete suppression of phototopotaxis in response to UV irradiation was observed
only in species of Dunaliella. Differences in the phototopotaxis action spectra also indicate dif-
ferences in photoreceptor pigments. There are also distinct differences in the structure of the
photoreceptor: crystal and dichroic in Euglena and non-crystal and non-dichroic in Dunaliella.
Taking into account the peculiarities of the photoreceptor system of euglenoid algae, in
particular, their stigma structure, it is possible to believe that they possess only the most an-
cient, basic modulation mechanism for photoreception inherited from prokaryotes or common
ancestors of these groups. In contrast, in green algae three mechanisms (modulation, diffrac-
tion, and interference) can function simultaneously, increasing the level of the light signal
absorbed by a photoreceptor. Though processes of sensory transduction of the light signal
into a locomotory reaction are most likely of an ion nature in all flagellate algae, in E. gracilis,
in contrast to green algae, a Na+-K+-pump participates in the control of these processes. Eug-
lena also differs from species of Dunaliella and C. reinhardtii in the unique functioning of its
flagellar apparatus. During beating, the flagellum of E. gracilis looks like a "broken (inter-
rupted) spiral" where the spiral parts of the flagellum are interrupted by its straight parts. Neither
typical ciliary nor undulate type flagella beating are observed in euglenids.
Thus, the greater the phylogenetic distance between taxa, the greater number and degree
of distinctions such as differences in their photobehaviour, structure and functioning mechan-
isms in the photoreceptor systems, sensory transduction of the light signal into the different
types of locomotory reaction and the functioning of the flagellar apparatus. These differences
make it possible to use variation in the photomovement traits as additional diagnostic criteria in
the evolutionary biology,phylogenetics, systematics, and taxonamy of algae.
Along with the differences in photomovement processes and its photoregulation,
representatives of different taxa of flagellates have common features. These include the struc-
ture of the photoreceptor system (which, with a few exceptions, consists of the photorecep-
tor and stigma), the primarily modulation mechanism in photoreception (which in representa-
tives of different taxa, depending on the stigma structure, can be associated with additional
mechanisms, such as diffraction and interference), the ionic nature of photoreception and sen-
sory transduction of the light signal into a locomotory reaction coupled with the dominant role
of calcium ions in these processes, the possible participation in these processes in light-induced
changes in membrane electric potentials, conformational changes in protein molecules as the
basis of photoreception and sensory transduction, and the participation of centrin in photoorien-
tation of basal bodies.
At the same time, it is evident that the current experimental data on algal photomove-
ment are far from complete and are based on only a small number of model organisms. Too
often the studies are not carried out according to a coherent plan at an appropriate methodical
level. Therefore, the results obtained in studies using different model organisms and by repre-
sentatives of different photobiology schools are not always comparable. As a consequence, the
conclusions made here must continue to be considered preliminary and represent a critique of
the initial results of ongoing studies. It is anticipated that future studies will add to, modify, or
even negate one or more of these conclusions.
Studies of peculiarities in photomovement are of interest with respect to the ecology
and geography of algae, in particular, for autecology. Such studies allow specifying characte-
ristics of selected species focusing on their reaction to parameters of light, determining opti-
mum, maximum and minimum values of these parameters among species, promoting their sub-
dividing the species into groups of shade-preferring, shade-resistant, photophilous, and light-
resistant organisms, and enhancing our understanding of laws governing their distribution on
the planet Earth.
166
13.11. Applied Importance of Data on the Photomovement of Algae
The applied importance of studying photomovement parameters in species of Dunalie//a is de-
termined mainly by the position of the genus in the kingdom of green plants (Viridiplantae).
Plants dominate our planet and provide the everyday needs of mankind. Hyperhalobic species of
Dunalie//a are classical models for studying the mechanisms of salt tolerance, osmotic regula-
tion, permeability of membranes, and processes governing the biosynthesis of carotene in plants
and photomovement. Due to their copious synthesis of J3-carotene, osmotically active com-
pounds of strategic importance, and high content of additional physiologically active sub-
stances, species of Dunalie//a are valuable organisms for biotechnology. Their high sensitivity
to environmental factors provides an opportunity for their use as biomonitoring test organ-
isms. The advantages of these organisms include their microscopic sizes, high rate of reproduc-
tion, active motility, and photokinetic and photovector reactions.
In our experiments, the sensitivity of various parameters of photomovement in D. salina
and D. viridis were studied in the presence of surface-active substances in the environment
(surfactants: cation-active catamin, anion-active sodium dodecylsulfonate, non-ionactive hydro-
pol, a natural polysaccharide compound isolated from blue-green algae, agents "water bloom"
in the Dnieper reservoirs, and also their combinations in the concentration range from
1 mg/L to 40 mg/L), salts of heavy metals [CuS04·5H20, CdCh , and Pb(N03h in the
10-7_10-2 M concentration range] and pesticides (Acetal 55 %, Acetazine 50 %, Alachlor 45 %'1
Arylon 75 %, Basta 20 %, Dual 96 %, Harmoni 75 %, Tecto 45 % in concentrations from 10-
to 10-2 M). The data obtained indicate the possibility of using the velocity of forward and rota-
ry movement of the cells, the frequency of their flagella beating, and phototopotaxis values
in the species of Dunalie//a as biological monitors to assess the health of aquatic environ-
ments. The simultaneous assessment of several photomovement parameters has the potential to
significantly increase the sensitivity of the method. The vector method of biotesting is pro-
posed for assessing the concentration of various toxicants in aquatic environments and allows
simultaneously monitoring of two or more movement parameters. The method facilitates
processing the data from large-scale measurements, allows obtaining a quantitative estima-
tion oftoxicant concentration, and provides the potential of identifying the toxicant.
Species of Dunaliella are excellent sources of f3-carotene, ascorbic and dehydroascorbic
acids, glycerol and other valuable organic compounds and are therefore candidates for biotech-
nological manipulation. Strains of Dunaliella are grown in many countries on an industrial
scale for the production of J3-carotene for use in the food and pharmaceutical industries and in
medicine for the prevention and treatment of tumors, cardiovascular and ophthalmic diseases,
avitaminosis, arthrosis, and other pathologies. Studying photomovement regularities and pecu-
liarities in species of Dunalie//a can help solve certain technological problems currently con-
fronting the production of carotene from these algae.
The main tendencies and perspectives for further photomovement investigations in fla-
gellates are discussed.
167
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213
Index of Latin Names
216
Subject Index
218
Nickel 113 Photoresponse 13,22
Nicotine 88 Photoresponses 14
Nimodipine 115 Photoshock cell response 15
Non-ionogenic hydropol (NSAS) 129 Photoshock response 15
Ocelloid mechanism 98 Photosynthesis 2
Okazaki Large Spectrograph 9, 10 Phototactic movement 14
Omega conotoxin 107 Phototactic orientation 14
Orientation reactions 14 Phototactic reactivity 14
Orthokinesis 15, 17 Phototactic response 14
Ouabain 105,112 Phototaxigraphy 2
Paraflagellar body 6,7 Phototaxis 6, 13, 15, 18,20
Parametrical principle 19 Phototopotaxis 14,21,22,32, 161
Patch clamp technique 91 Phototropism 13
Periodic shading and illumination ofthe Phototropisms 6
photoreceptor by the stigma 97 Pimozide 107
Pesticide lindane 109 Pollutants 128
Pesticides 127 Positive kinesis 17
Phenomenology of photomovement 160 Positive phototopotaxis 14
Phobic responses 17 Potassium iodide 88
Phobism 15 Primary photoorientation 14
Photobehaviour 13 Projected microspectrum 6
Photobehaviour photomotion 14 Provitamin A 3,151
Photobehaviour response 13 Pseudopodia 5
Photocontrol of movement 21 Pterins 83
Photoinduced behaviour 13 Quarter-wave-stack 72
Photokinesis 13, 14, 15, 18,21,35,160 Rapa 149
Photokinetic response 32 Rayleigh test 33
Photoklinokinesis 22 Red eye 5
Photomotile response 13 Red flowering 23, 149
Photomotion 13,14 Regeneration offlagella 115
Photomovement 1,13,14,20 Related fields of sciences 164
Photomovement parameters as test- Response strategies 14
functions 129 Response type 14
Photomovement response 13 Responses 16
Photoorientation 6,14 Restoration of flagella 115
Photoorientation 97 Rhodopsin 83
Photoortokinesis 21 Role of proteins in mechanisms of
Photophobic reaction 15,22 photoregulation 103
Photophobic response 14,15,18 Roseoflavin 88
Photophobic, stop or ecclitic response 15 Rotenone 88
Photophobotaxis 15 Salicylaldoxime 88
Photoreaction 13,22 Schmidt trigger 124
Photoreception 1 Schreckbewegung 7
Photoreceptor pigments 83 Secondary photoorientation 14
Photoreceptor system 163 Semi-industrial commercial cultivation 149
Photorections 156 Sensory transduction 1,105,164
Photoregulation 1 Shock reactions 7
Photoregulation of movement 13 Simultaneous effect of several external
219
factors 55 Ultraviolet radiation 49
Sodium azide (NaN3) 105,115 Vector method ofbiomonitoring 137
Soliton excitation 103 Vector reaction 22
Statistical analysis 31 Velocity oflinear movement 35
Statistical methods 33 Velocity of movement 32
Steady-state (kineses) 17 Verapamil 115
Stigma 5,28 Videomicrography 2, 31
Stigma of Duna/iella 76 Visible radiation 49
Stimulus 16 V-test method 33
Stop response 15 Waveguide mechanism 98
Strophic phototaxis 14 X-rays 7
Strophokinesis 17 Zoospores 5
Structure ofDunaliella photoreceptor 77
Structure ofpbotoreceptor system 71
Substitutes of photoreceptor pigments 78
Sulphoacid (ASAS) 129
Surface-Active Substances 129
Taxis 14
Tecto 130
Telo-taxis 14
Temperature 7,45
Terminology 2,159
Test-function 125
Test-object 125
Tetramethylthiuram disulfide 88
Thermal fields 49
Thermotaxis 14
Toxicants 128
Trans-flagellum 121
Transient (phobic responses) 17
Transverse phototopotaxis 14
Tropo-taxis 14
Tunable dye-laser 9
Two-beam irradiation of the cells 79
Ultraviolet microirradiation 8
220
Author's Index
alachlor = 2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)-acetarnide
cinnarizine = 1-(diphenylmethyl)-4-(3-phenyl-2-propen-l-yl)-piperazine
diltiazem = 3-(acetyloxy)-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-
methoxyphenyl)- 1,5-benzothiazepin-4(5H)-one hydrochloride
diuron = N-(3,4-dich1orophenyl)-N,N-dimethyl-urea
DPC = diphenylcarbazide
dual = 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)-piperazine
eradicane = 2,2-dichloro-N,N-di-2-propen-l-yl-acetamide
mMX = isobutylmethylxanthine
223
Y. I. Posudin et al., Photomovement of Dunaliella Teod.,
DOI 10.1007/978-3-8348-9765-7,
© Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH 2010
isoptin= benzeneacetonitrile = a [3-[[2-(3,4-dimethoxyphenyl) ethyl] methylamino]
propyl]-3,4dimethoxy-a-( l-methylethyl) hydrochloride
lindane = 1,2,3,4,5,6-hexachloro-cyclohexane
tecto = 2-(4-thiazolyl)-lH-benzimidazole
224
About the Authors
Professor Yuriy Posudin, Doctor of Biological Sciences, National University of Life and
Environmental Sciences of Ukraine, Kiev, Ukraine. He graduated from the Radiophysical
Faculty of Kiev State University in 1969. Dr. Posudin's principal scientific interests are the
investigation of photobiological reactions of algae and plants, and the non-destructive quality
evaluation of agricultural products. He is an author of 18 textbooks, and over 120 scientific
papers and has 20 patents for inventions.
Professor Nadiya Massjuk, Doctor of Biological Sciences, Leading Research Fellow, MG.
Kholodny Institute of Botany of National Academy of Sciences of Ukraine, Kiev, Ukraine.
Dr. Massjuk has studied the biology of algae for many years (biodiversity, evolution, phylog-
eny, its place in the world of living organisms, and applied algology). She has been interested
in the biology of photomovement in relation to the study of the diversity, phylogeny ofphyto-
flagellates, classification, biotechnology of growth, and synthesis of carotene. Dr. Massjuk
has authored over 260 scientific papers.
Dr. Galyna Lilitskaya, Leading Engineer, M.G. Kholodny Institute of Botany of the
National Academy of Sciences of Ukraine, Kiev, Ukraine. Dr. Lilitskaya graduated from the
Biological Faculty of Kiev State University in 1986. She has authored over 40 scientific pa-
pers on algal photomovement, biodiversity, and flora.