Hylesia + HPLC
Hylesia + HPLC
RESUMEN El género “Hylesia” está conformado por un grupo de polillas neotropicales que abundan
desde el estado de Arizona de los Estados Unidos hasta el extremo sur del subcontinente. Una de las
especies de las polillas “Hylesia” encontados en Venezuela, la Guayana Francesa y Trinidad ha sido
identiÞcado como Hylesia metabus (Cramer, 1775) (Lepidoptera, Saturniidae). En Venezuela estas
polillas se encuentran en gran abundancia dentro de los manglares (Avicennia spp.) del Golfo Triste
(Golfo de Paria) y los caños adyacentes del Delta de Orinoco en el oriente del paṍs. Durante las épocas
de empareamiento las hembras adultas de este especie libera cantidades enormes de una minúscula
pelusa urticante (setas) en el medio ambiente, produciendo una dermatitis papulo-vesicular severo
entre los pobladores de las zonas adyacentes a los manglares. En el presente estudio hemos logrado
aislar y caracterizar sustancias protéicas con propiedades proinßamatorias de las setas urticantes de
las posturas de huevos de este especie usando electroforesis en poliacrilamida y cromatografṍa de
intercambio aniónico lṍquida de alta resolución (HPLC). Hemos estudiado la respuesta inßamatoria
del extracto de las posturas de huevos y sus componentes aislados en un modelo animal utilizando el
pabellón auricular del Acure (Cavia porcellus) mediante métodos de histopatologṍa. Los resultados del
presente estudio muestran que el veneno extraṍdo, y sus subcomponentes, dan origen a una reacción
inßamatoria intensa caracterizado por inÞltración masiva de células inßamatorias, echimosis, y de-
generación vascular. La separación cromatográÞca mostró que el veneno contiene proteṍnas con
propiedades selectivamente Þbrinolṍtico-vasodegenerativos o quṍmotactico-proinßamatorios.
KEY WORDS Hylesia metabus, erucism, proteolytic enzymes, inßammation
Caterpillars from !12 families of Lepidoptera species tion coagulopathy, renal failure, and intracerebral
worldwide can inßict serious injuries such as urticarial hemorrhage (Diaz 2005). The term erucism is used to
dermatitis, atopic asthma, osteochondritis, consump- describe the pruritic maculopapular to bulbous con-
tact dermatitis caused by exposure to urticating setae
1 Departamento de Biologṍa Estructural, Instituto Venezolano de
from the larvae or the adults (Benaim-Pinto et al. 1991,
Investigaciones CientṍÞcas, Carretera Panamericana. Km 11. Estado
Diaz 2005). In most cases, the urticating setae are
Miranda, Venezuela, Apdo. 21827, Caracas 1020A, Venezuela. present in the larval stage, and it is only in a few species
2 Corresponding author, e-mail: [email protected]. that they are found in adults. For example, in the
3 Centro de Biofṍsica y Bioquṍmica, Instituto Venezolano de Inves-
genera Acyphas and Euproctis in the Lymantridae and
tigaciones CientṍÞcas, Carretera Panamericana. Km 11. Estado
Miranda, Venezuela, Apdo. 21827, Caracas 1020A, Venezuela.
in the genus Hylesia of the Saturniidae (Rodriguez et
4 Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas, al. 2004). Approximately 115 species of the Hylesia
Universidad de Oriente, Cumana, Venezuela. genus have been described in the Americas, 25 of
which have been identiÞed in Venezuela (The Insect Materials and Methods
Company 2006). Most species identiÞed in Venezuela
Reagents. All reagents used were of the highest
do not seem to cause any discomfort with the notable
degree of purity available and obtained from Sigma-
exception of Hylesia metabus (Cramer 1775). This
Aldrich (St. Louis, MO) unless otherwise speciÞed.
moth is considered a major public health problem for
Insect Material. H. metabus samples were obtained
the populations living in the vicinity of the mangrove
by recollection of adults and egg-nests in various sites
(Avicennia spp.) swamps of the coast off the Gulf of
of the mangrove swamps in the Gulf of Paria and
Paria (Golfo Triste) and the Delta Orinoco in north- rapidly transferred to the Venezuelan Institute for
eastern Venezuela (Fornéz and Hernandez 2001). ScientiÞc Investigation (Instituto Venezolano de In-
The biological cycle of H. metabus lasts !101 d, vestigaciones CientṍÞcas, Caracas, Venezuela) where
producing 3.65 successive generations each year the material was stored at "80#C until use.
(Boyé 1932, Benaim-Pinto et al. 1991). Adult females Venom Extraction. Five grams of setae (equivalent
shed copious amounts of urticating arrow-like setae to !170 adults or 120 egg-nests) were extracted in a 50
during mating and oviposition at the end of each cycle. mM Tris buffer adjusted to pH 8.5 supplemented with
The females also use their urticating abdominal setae 0.5 M NaCl at a proportion of 10 ml of buffer per gram
to cover the egg masses to protect them from preda- of raw material for 24 h. The resulting material was
tors and parasites (the males do not possess urticating Þltered through Þve layers of gauze cloth and clariÞed
setae) (Rodriguez et al. 2004). Adults exhibit a posi- by centrifugation for 30 min at 10,000 rpm in a RC-5B
tive phototropism, and they are strongly attracted by centrifuge (Sorvall, Newton, CT) by using an HB4
the streetlights and other light sources at nighttime. rotor (DuPont, Wilmington, DE). ClariÞed extracts
Thus, during the ßight periods, all lights must be were concentrated by the addition of solid ammonium
turned off after sunset. Most activities of daily life are sulfate to reach a Þnal saturation of 60%. Precipitated
restricted, e.g., agricultural work, Þshing, commerce, proteins were harvested by centrifugation at 20,000
and educational activities. rpm for 120 min in a Sorvall RC-5B centrifuge using an
Contact with the urticating abdominal setae of the HB4 rotor. The pellets were redissolved in 50 mM
females, or accidental contact with the egg-nests of H. Tris-buffer, pH 8.5, supplemented with 0.05 M NaCl at
metabus, gives rise to an intense pruritic papuloery- a ratio of 0.5 ml/g raw material and dialyzed exten-
thematous dermatitis with discrete vesicular and vas- sively against the same buffer. For the chromato-
cular degeneration occurring without previous expo- graphic separation and subsequent histopathological
sure (Benaim-Pinto et al. 1992). The condition often studies, we used the venom extracted from the egg-
requires systemic administration of potent steroids nests, because of the relative lack of contaminating
and antihistamines to alleviate symptoms. The dermal proteins and lipids. The Bradford protein assay was
reaction lasts typically from 5 to 10 d. However, in used for the determination of total protein content
some cases the dermatitis may persist for considerably (Bradford 1976). Qualitative analysis of proteins was
longer periods (months). done by sodium dodecyl sulfate-polyacrylamide gel
Several studies have demonstrated the presence of electrophoresis (SDS-PAGE) in 10 and 12% gels (Lae-
proteins with proinßammatory properties in the urti- mmli 1970). Protein bands were visualized by the
cating setae (Benaim-Pinto et al. 1992, Lundberg et al. Coomassie Brilliant Blue R-250 method (Andrews
2002). These studies also reported the presence of a 1986) or by the periodic acid-Schiff reagent for glu-
proteolytic enzyme with a molecular mass of 32 kDa coprotein detection (Jay et al. 1990). The amidolytical
as determined by gel Þltration chromatography with a activity was determined using the chromogenic sub-
pH optimum of 8.5Ð9.0. The determination of K3/Km strates S-2288 and S-2302 (Instrumentation Labora-
values in different chromogenic substrates showed a tory, Milan, Italy). Initial p-nitroaniline (p-NA) for-
preference for the plasma kallikrein substrate S-2302 mation rate was monitored by absorbance at 410 nm.
(H-D-Pro-Phe-Arg-pNA) followed by the broad-spec- in a PerkinElmer Lambda Bio 20 spectrophotometer
trum serine protease substrate S-2288 (H-D-Ile-Pro- (PerkinElmer Life and Analytical Sciences, Boston,
Arg-pNA). In addition, the enzyme showed a marked MA). The concentration of chromogenic substrates
Þbrinolytic activity in vitro (Lundberg et al. 2002). was 80 !M. Fibrinolytic activity also was evaluated by
Substances with serine-protease activities also have the Þbrin lysis plate method (Marsh and Arocha-
been demonstrated from Euproctis spp. caterpillars Pinango 1972) and expressed in square millimeters of
(Bleumink et al. 1982). A protein with a molecular Þbrin lysis area.
mass of 28 kDa extracted from the setae of Thaumeto- Purification of Egg-Nest Extract. Analytical and pre-
poea pityocampa (Schiff) (Lepidoptera: Thaumeto- parative separation of venom components was per-
poeidae) caused similar dermal reactions as the eru- formed by anion exchange AE-HPLC on a Waters 626
cism produced by exposure to setae from H. metabus LC system using a Protein-Pak Q 8HR anion exchange
(Lamy et al. 1986). In the current study, we have column (Waters, Milford, MA). Separated fractions
explored the biochemical properties of the compo- from several high-performance liquid chromatogra-
nents obtained from the extracts of adult males, fe- phy (HPLC) runs were combined, concentrated by
males, and egg-nests from H. metabus, and we also lyophilization, and dissolved in a Þnal volume of 1 ml
implemented a sensitive in vivo assay to investigate the by the addition of 50 mM Tris, pH 8.5, supplemented
biological activities of the puriÞed proteins. with 0.05 M NaCl.
442 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3
Table 1. Protein concentration, specific amidolytic acticity, and fibrinolytic activity of crude 60% ammonium sulfate extract and
HPLC-purified fractions of H. metabus egg-nest extract
Sample Protein content (mg/ml) SpeciÞc activity in S-2288a Fibrin lysis area (mm2)b
"2 "3
Crude extract 1.309 ' 0.21 7.34 & 10 ' 2.49 & 10 45 ' 2.5
60% (NH4)2SO4 precipitate 1.569 ' 0.17 1.51 & 10"1 ' 3.3 & 10"3 58 ' 4.5
HPLC fraction 1 0.66 ' 0.033 0 0
HPLC fraction 2 0.07 ' 0.0073 1.51 & 10"3 ' " 4.5 & 10"5 24 ' 3.0
HPLC fraction 3 0.28 ' 0.0023 1.52 & 10 ' " 2.7 & 10"4
"2
24 ' 20.8
HPLC fraction 4 0.36 ' 0.001 8.4 & 10"3 ' 1.0 & 10"4 0
HPLC fraction 5 0.386 ' 0.002 3.14 & 10"2 ' " 3.5 & 10"4 0
Data are presented as mean ' SD from Þve cycles of puriÞcation of egg-nest extract.
a
SpeciÞc activity is expressed as moles of p-NA released from chromogenic substrate S-2288 (broad-spectrum serine protease substrate)
per mole of protein per minute.
b
For in vitro Þbrin lysis assay, 5 ! l of sample was loaded onto a preformed Þbrin gel.
444 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3
Fig. 4. Histopathological studies showing the inßammatory response of crude 60% ammonium sulfate precipitate of H. metabus
egg-nest extract. (A) Response of control animals in receiving sterile isotonic saline solution in 10 mM phosphate buffer, pH 7.4.
(BÐD) Massive inÞltration of PMNLs and mononuclear cells in the extracellular matrix and within the venules after receiving
egg-nest extract as described under Materials and Methods. Arrows shown in B and C indicate intravascular aggregates of mainly
PMNLs. D shows massive tissue cellular inÞltration. MagniÞcation, 100& (A) and 200& (BÐD).
from control animals with respect to cellularity (P ( Table 2. Cellularity determined from tissue samples taken
from guinea pigs auricles infiltrated with sterile normal saline (con-
0.05, P % 0.576008) and morphology. There was no trol), egg-nest extract precipitated with 60% ammonium sulphate
lysis of preformed Þbrin (Table 1). and HPLC-purified fractions of precipitate
Fraction 2. Inoculation of 5 !g of HPLC fraction 2
proteins (Fig. 2) on the dorsum of the auricle of the Fraction Cellularity (mean ' SD)a
guinea pig ear caused prolonged bleeding ((20 min.) Control 3.353 ' 0.418
and formation of macroscopic hematomas. The mi- Extract, inÞltrated zone 27.207 ' 9.780
croscopic study of the tissue showed no evidence of Extract, noninÞltrated zone 9.780 ' 3.040
mechanical damage to the vascular structures. There Extract, distal zone 6.233 ' 2.322
HPLC fraction 1 3.580 ' 0.322
was a marked degeneration of the structure of the HPLC fraction 2 13.080 ' 3.825
vessel walls as shown by the lobulation and engorge- HPLC fraction 3 11.887 ' 4.695
ment of venules and extensive extravasation of large HPLC fraction 4 10.380 ' 5.697
quantities of erythrocytes (Fig. 6A, arrow). Impor- HPLC fraction 5 15.580 ' 3.896
tantly, there was no evidence of Þbrin clot formation a
The area percentage occupied by nuclei as an indicator of the
in association with the massive erythrocyte accumu- number of nucleated cells, calculated as described under Materials
lation. There was a slight inßammatory reaction show- and Method. Values are average from 10 determinations.
May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus 445
Fig. 5. Histopathological studies showing the inßammatory response of crude 60% ammonium sulfate precipitate of
H. metabus egg-nest extract. (A and B) Massive intravascular aggregates and tissue inÞltration of leukocytes in the
connective tissue of the dorsal part of the auricle. Petechiae were a common Þnding in these samples. (C) Petechial
bleeding of the dorsal part of the auricle in vicinity to the lesion produced by the venom (arrow). Frank vasculitis was
also a common Þnding as shown in D (arrow). MagniÞcation, 100& (A) and 200& (BÐD).
ing PMNLs scattered in the tissue surrounding the 2 inoculation. Intravascular degradation of clots was
inÞltrated area. The cellularity value for inÞltrating observed. Fraction 3 proteins produced an in vitro
leukocytes was statistically different from the control Þbrin lysis of the same magnitude as fraction 2
tissue (P $ 0.05, P % 2.3269 " & 10"9). The fraction 2 proteins (Table 1).
proteins did produce a signiÞcant lysis of preformed Fraction 4. Inoculation with fraction 4 proteins pro-
Þbrin in vitro (Tables 1 and 2). duced a moderate but statistically signiÞcant increase
Fraction 3. The lesions caused by inÞltration with of intravascular leukocyte aggregation and tissue in-
fraction three proteins showed a moderate leuko- Þltration compared with the control group (P $ 0.05,
cyte inÞltration, statistically different from control P % 0.0003) (Fig. 6C; Table 2). A marked inÞltration
animals with regard to cellularity in connective tis- of leukocytes into the surrounding extravascular con-
sue (P $ 0.05, P % 0.0003) but no statistically dif- nective tissue with predominance of PMNLs could be
ferent from fractions 2 (P ( 0.05, P % 0.186189) and seen adhering to the endothelium and in the process
4 (P ( 0 0.05, P % 0.152094) (Fig. 6B; Table 2). The of diapedesis. No hemorrhage or damage to blood
animals inoculated with fraction 3 protein showed vessels was observed. Fraction four proteins did not
prolonged bleeding at the injection site and also a cause any in vitro lysis of preformed Þbrin (Table 1).
moderate number of areas with erythrocyte extrav- Fraction 5. Fraction 5 protein caused a marked
asation. However, the amount and severity of the tissue leukocyte inÞltration (Fig. 6D) compared
bleeding were far less than that seen from fraction with the control group (P $ 0.05, P % 0.0004). This
446 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3
Fig. 6. Histopathological studies showing the inßammatory response of HPLC-puriÞed fractions of H. metabus egg-nest
extract. (A) Structural derangement with lobulation and engorgement of the vessel with erythrocytes is clearly visible
(arrow), caused by inoculation of 5 !g of HPLC fraction 2 proteins (see Fig. 2). Note the absence of Þbrin clot formation
in association with the erythrocyte masses. (B) Effect of HPLC fraction 3 on the dorsal tissues of the auricle showing moderate
leukocyte inÞltration and erythrocyte extravasation. Note the absence of Þbrin clot formation associated with erythrocytes
(arrow). (C) Tissue response to HPLC fraction 4 proteins. A marked inÞltration of leukocytes with a predominance of PMNLs
in the perivascular connective tissue was evident. In addition, leukocytes adhering to the vascular endothelium and actively
migrating toward the perivascular tissue are clearly seen (arrow). (D) Effect of HPLC fraction 5 protein on the auricular
tissue. There was a high degree of tissue cellular inÞltration (signiÞcantly higher than HPLC fraction 4 proteins). SigniÞcant
aggregation of intravascular leukocytes was clearly observed (arrow). MagniÞcation, 200&.
cellular inÞltration was signiÞcantly higher than dermatitis. There is a consensus that it is the femalesÕ
fraction four proteins (P $ 0.05, P % 0.009). The high abdominal setae (ßéchettes) that are responsible
degree of tissue leukocyte inÞltration was the chief for the dermal lesions by carrying and introducing
characteristic of this fraction. Fraction 5 did not highly irritant substances into the dermisÐ epider-
cause any lysis of preformed Þbrin in in vitro assays mis (Hill et al. 1948, Vicente 1952, Gusmao et al.
(Tables 1 and 2). 1961, Dinehart et al. 1985). These dart-like ßéch-
Fraction 6. Fraction 6 did not produce any signs of ettes, 50 Ð100 !m in length and !1.5 !m in diameter,
inßammatory reaction or edema. There was no lysis of have been seen incrusted deeply in the dermal le-
preformed Þbrin in vitro assays (Table 1). sions (Dinehart et al. 1985). During oviposition, the
female sheds large amounts of setae, including the
ßéchettes originating from the last abdominal seg-
Discussion
ments (!50,000 per mm2) to cover and protect the
Dermatitis caused by exposure to the urticating eggs. These egg-nests have very pronounced urti-
setae of H. metabus gives rise to an intense pruritic cating properties (Tisseuil 1935, Benaim-Pinto et al.
May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus 447
1991). Adult females and egg-nests both have a 1985). However, the effect of histamine is of very
prominent band at !38.5 kDa in the SDS-PAGE short duration. Therefore, it seems unlikely that
analysis of extracts, which is absent in adult males histamine alone would be of importance in the long-
(Fig. 1). Because the adult males do not have any lasting inßammatory reactions that are typically
urticating properties, the 38.5-kDa band may be seen after exposure to the urticating setae of H.
associated with the proinßammatory activity ob- metabus. In addition, the extracts and chromato-
served in this study. graphic fractions used in this study were all negative
The papuloerythematous dermatitis produced by when assayed for the presence of histamine by using
exposure to the urticating setae of H. metabus varies a sensitive histamine ELISA test. Other studies also
in size from a few millimeters to several centimeters, have failed to detect histamine in extracts from H.
being solitary, or occurring in large numbers in metabus urticating setae (Benaim-Pinto et al. 1992).
exposed areas. In the latter case, papules are usually Therefore, we do not think that histamine present
conßuent. The onset of the dermal reaction is typ- in the urticating setae has any signiÞcant role in the
ically of a retarded nature starting from 1 to 6 h after extended inßammatory response.
exposure to urticating material in humans. The der- It has been suggested that there must be other
mal lesions give rise to an intense pruritus that inßammatory mediators present in the setae (Dine-
worsens with scratching (Benaim-Pinto et al. 1991, hart et al. 1985). Several studies have noted the
1992). Ocular complications are fortunately rare, presence of substances with highly antigenic and
but they require careful treatment when present enzymatic properties (Benaim-Pinto et al. 1992,
(Vicente 1952, Ducombs et al. 1983, Dinehart et al. Lundberg et al. 2002). In the current study, we have
1985). General malaise and arthralgia also have been shown in an animal model that the aqueous extracts
reported (Dinehart et al. 1985). Respiratory symp- from the egg-nests of H. metabus are capable of
toms are rare and usually seen only in predisposed reproducing the histopathological alterations de-
persons or in cases of direct exposure to the upper scribed previously. In addition, we have shown that
airways (Ducombs et al. 1983, Dinehart et al. 1985). this venom may be separated by anionic exchange
Histopathological examination of skin biopsies of chromatography into separate components having
skin lesions resulting from exposure to Hylesia different biological activities. Fractions 2 and 3 from
paulex (a Hylesia species reported exclusively from the chromatographic separation caused vascular
Brazil) revealed rounded areas of patchy necrosis of damage and petechial bleedings. There was clear
the dermis and epidermis with an inßammatory in- evidence of marked degradation of the vessel walls
Þltrate consisting of disintegrating PMNLs. Small evidenced by lobulation, engorgement of venules,
dermal vessels exhibited Þbrinoid necrosis. Ne- and extensive extravasation of large quantities of
crotic vessels were surrounded by an inßammatory erythrocytes. Both of these fractions also showed an
inÞltrate composed mainly of PMNLs and lympho- in vitro Þbrinolytic activity. There was no signiÞcant
cytes presenting nuclear pyknosis and karyorrhexis. increase of inÞltration of inßammatory cells in these
On the basis of these Þndings, the lesions were fractions. Fractions 4 and 5, however, induced a
characterized as a cutaneous leukocytoclastic vas- marked inÞltration of inßammatory cells with no
culitis (Benvenuti et al. 1998). The lesions produced signs of vascular damage or Þbrinolytic activity. It
by adult females of Hylesia metabus (Cramer) (east- should be noted that all fractions contained proteins
ern Venezuela) have been described clinically as an with a molecular mass between 31 and 41 kDa with
urticarial vesicular or papuloerythematous dermatitis an amidolytic activity in synthetic peptide substrate
with discrete vesicular degeneration and necrosis. Ec- S-2288 speciÞc for a broad range of serine proteases.
chymotic spots also have been reported. Histopatho- The molecular mechanisms by which the puriÞed
logical studies have shown hydropic degeneration of fractions of the venom bring about their respective
epidermal cells, spongiosis and cellular inÞltration by biological effects are at present not well understood.
lymphocytes, histocytes, and neutrophils. (Zaias et al. The results of the studies of cutaneous leukocytoclas-
1969, Benaim-Pinto et al. 1991). The results of the tic vasculitis resulting from exposure to H. paulex sug-
current study using an animal model are in clear ac- gests an antibody-mediated vascular inßammation
cordance with these Þndings. Thus, inoculation with with either deposition or formation in situ of immune
egg-nest extract caused a massive migration, inÞl- complexes in the vessel wall and complement activa-
tration, and aggregation of PMNLs and mononu- tion (Benvenuti et al. 1998). However, a wide range of
clear cells with predominance of PMNLs in the stimuli, e.g., cytokines, bioactive lipids, and even
extracellular matrix, veins, and venules. PMNLs agents such as monosodium urate crystals and serum-
could be observed in the process of diapedesis activated zymosan particles, are efÞcient activators of
through the vessel walls. Petechiaes and frank vas- PMNLs (Claesson et al. 1981; Serhan et al. 1984a,
culitis also was observed frequently. 1984b; Štvrtinová et al. 1995). In addition, a trypsin-
The nature of the urticating substances and their like activity has been described previously with ex-
mechanism of action in the etiology of these lesions tracts of setae from adult females that may be related
have been a matter of controversy. The presence of to the vasodegenerative and necrotic traits of the
histamine in the urticating hairs has been reported whole extract as well as fractions 2 and 3 (Benaim-
previously and has been implicated as a mediator Pinto et al., 1992). Apart from the direct toxic effect of
responsible for the dermal lesions (Dinehart et al. the components of the moth venom, the possibility of
448 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3
allergic reactions toward the moth must also be eval- the principle of protein-dye binding. Anal. Biochem. 72:
uated. Studies are presently being undertaken to de- 248 Ð254.
termine what immunological mechanisms and medi- Claesson, H. E., U. Lundberg, and C. Malmsten. 1981. Se-
ators may be involved in the pathophysiology of the rum-coated zymosan stimulates the synthesis of leuko-
cutaneous lesions caused by exposure to the urticating triene B4 in human polymorphonuclear leukocytes. In-
hibition by cAMP. Biochem. Biophys. Res. Commun. 99:
setae of H. metabus.
1230 Ð1237.
Cook, H. C. 1990. Carbohydrates en Theory and Practice of
Histological Techniques, pp. 188 Ð193. In J. D. Bancroft
Acknowledgments
and A. Stevens [eds.], Theory and practice of histological
We thank Frances Osborn of the “Instituto de Investiga- techniques, 3rd ed. Livingston Churchill. London, United
ciones en Biomedicina y Ciencias Aplicadas, Universidad de Kingdom.
Oriente, Cumana, Venezuela” and also the Þeld technicians Cramer, P. 1775. De Uitlandishe Kapellen Voorkomenede
of the H. metabus project for invaluable support in making in de Drie Waereld-Deelen Asia, Afrika en America/
this study possible. We deeply acknowledge the patience and Papillons Exotiques de Trois Parties du Monde lÕAsie,
spirit of collaboration of the people living in the affected lÕAfrique et lÕAmerique, Volume 1(7). Hylesia (Phalaena)
areas (municipalities of Benitez, Libertador, Cajigal, Mariño, metabus, pp. 117 (plate 74 Fig. D). S. J. Baalde. Amster-
and Valdéz of the state of Sucre). We also are indebted to the dam, Holland. Barthelemy Wild & J. Van Schoonhoven &
staff of FUNDACITE of the state of Sucre, Venezuela. We Comp., Utretcht, Holland.
acknowledge the support provided by the Department of Diaz, J. A. 2005. The evolving global epidemiology, syn-
Structural Biology of the Venezuelan Institute for ScientiÞc dromic classiÞcation, management, and prevention of
Investigation (Departamento de Biologṍa Estructural, Insti- caterpillar envenoming. Am. J. Trop. Med. Hyg. 72: 347Ð
tuto Venezolano de Investigaciones CientṍÞcas, IVIC). We 357.
also thank Antonio Aguado for assistance in the animal ex- Dinehart, S. M., M. E. Archer, J. E. Wolf, Jr., M. H. McGarran,
periments. Finally, we acknowledge the skillful help of C. Reitz, and E. B. Smith. 1985. caripito itch: dermatitis
Rebeka Powell de Vaz in the preparation of this manuscript. from contact with Hylesia moths. J. Am. Acad. Dermatol.
This project, identiÞed as “Proyecto Reto Hylesia metabus, 13: 743Ð747.
subproject Caracterización de la sustancia urticante de la Ducombs, G., M. Lamy, and M. Michel. 1983. La papillonite
mariposa Hylesia metabus (”Palometa Peluda“) y identiÞca- de Guyane Française. Étude clinique épidémiologique.
ción de inhibidores de la misma”, (Project Hylesia metabus, Ann. Dermatol. Vénérol. 100: 809 Ð 815.
sub-project: Characterization of the Urticating Substance of Fornéz, L., and J. V. Hernandez. 2001. Reseña histórica e
the butterßy Hylesia metabus and identiÞcation of its inhib- incidencia en la salud pública de Hylesia metabus (Cra-
itors) has been Þnanced by the Ministerio de Ciencia y mer) (Lepidoptera: Saturniidae) en Venezuela. Entomo-
Tecnologṍa de la República Bolivariana de Venezuela (“Min- trópica 16: 137Ð141.
istry of Science and Technology of the Boliviarian Republic Gao, J., F. Qian, A. Szymanski-Exner, N. Stowe, and J. Haaga.
of Venezuela”). 2002. In vivo drug distribution dynamics in thermoab-
lated and normal rabbit livers from biodegradable poly-
mers. J. Biomed. Mater. Res. 62: 308 Ð314.
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