Article

Honeydew Deposition by the Giant Willow Aphid

(Tuberolachnus salignus) Affects Soil Biota and Soil
Biochemical Properties
Kyaw Min Tun 1, Andrea Clavijo-McCormick 1, Trevor Jones 2, Stanislav Garbuz 1 and
Maria Minor 1,*
1 Wildlife and Ecology Group, School of Agriculture and Environment, Massey University, Private Bag
11222, Palmerston North 4410, New Zealand; [email protected] (K.M.T.);
[email protected] (A.C.-M.); [email protected] (S.G.)
2 Plant & Food Research, Fitzherbert Science Centre, Batchelar Road, Palmerston North 4410, New Zealand;

[email protected]
* Correspondence: [email protected]; Tel.: +64-6-356-9099 (ext. 84833)

Received: 30 June 2020; Accepted: 19 July 2020; Published: 22 July 2020

Abstract: Infestation of willow plants by the giant willow aphid Tuberolachnus salignus (Hemiptera:
Aphididae) is associated with copious deposition of sugar-rich honeydew under the plant canopy.
We explored the effect of aphid honeydew on the soil biota and biochemical indicators in a two-
year field trial. Soil samples from under aphid-infested and control willow trees, as well as samples
from black sooty mould spots under the aphid-infested willows were compared; soil samples before
aphid inoculation were used as a baseline. The honeydew deposition had a positive effect on the
total soil carbon (C), but not on the total soil nitrogen content or soil pH. Microbial biomass C, basal
respiration, number of yeast colony forming units, and the geometric mean of activities for six
enzymes were significantly higher in honeydew-affected soils than in the control treatment on both
years. The honeydew deposition also increased soil meso-fauna abundance, especially in the black
sooty mould spots. The soil biochemical properties, which differed before and after aphid
infestation, showed considerable overlap between the first and second year post-infestation. The
results highlight the cascading effects of T. salignus on soil biological activity and the importance of
using a multitrophic approach to explore similar scenarios.

Keywords: Tuberolachnus salignus; honeydew; soil biochemical indicators; soil enzymes; yeasts;
meso-fauna

1. Introduction
The giant willow aphid, Tuberolachnus salignus (Gmelin) (Hemiptera: Aphididae), is an invasive
stem-feeding pest of willow trees, which has recently arrived in New Zealand [1]. Willows (Salix spp.)
are important multi-purpose farm trees used for biomass production, bioremediation, erosion
control, and soil nutrient management [2,3]. As T. salignus is a new species in New Zealand, not much
is known about the ecological consequences associated with its presence in willow growing systems,
such as its effects on the soil biota.
One of the prominent features of infested willow plantings is the deposition of copious amounts
of honeydew by aphid colonies, and the growth of black sooty mould on the leaves, stems, and on
the soil surface ([1]; see also Figure 1). A single adult T. salignus can exude 1.71–2.08 mm3 of honeydew
per hour [4,5]. Chemically, the honeydew is a mixture of water, carbohydrates (90–95% dry weight),
amino acids (< 5%), lipids and other nutrients [6,7]. When this energy-rich liquid is deposited on the

Insects 2020, 11, 460; doi:10.3390/insects11080460 www.mdpi.com/journal/insects

Insects 2020, 11, 460 2 of 21

leaves and understory plants, it is splashed onto the soil surface by rainfall [8]. It can be hypothesized
that deposition of T. salignus honeydew on the soil surface will initiate a cascade of changes in soil
processes, causing modifications in soil chemical properties, microbial activities and in the
abundance of soil microbivores. Previous studies have linked the labile carbon (C) input from
aboveground aphid herbivory to changes in belowground biochemical properties [9–12]. These
effects are linked to aphid population density [13] and the identity of the aphid species [14].

Figure 1. Black sooty mould spots under the canopy of willow plants.

Aphid honeydew deposition on the soil surface is expected to increase nutrient availability, fuel
the growth of microbial communities in belowground systems [11,15], and influence the soil
decomposition processes [16,17]. Microorganisms (bacteria, fungi, and other taxa) contribute to the
functioning of soil ecosystems [18], regulating the processes of organic matter decomposition and
nutrient cycling [19]. Soil microorganisms are assumed to be energy-limited, and as a result, mostly
remain dormant when C resources are scarce [20]. The daily addition of sugar to the soil can cause a
2.5-fold increase in bacterial diversity, compared to control treatments, as sugar supplementation
encourages the soil microbes to become active [21]. Aphid honeydew is a suitable growing medium
for various saprophytic microbes [22] and has been shown to increase the activities of soil
microorganisms [11,16]. Among soil microbes, yeasts are important degraders and saprotrophs
[23,24], utilizing various C and nitrogen (N) sources [25]. Soil yeasts are ubiquitously present in many
agroecosystems [26] and in nutrient-rich forest environments [23], and exhibit a quick response to
changes in soil nutrient content [27].
Changes in soil microbial activity can also be reflected in the activities of soil enzymes [28]. Soil
enzymes activity is a commonly used soil bioindicator [29], because of their quick response to subtle
changes in available resources such as organic C input [30,31], and the ease of enzyme quantification
[32]. So far, soil enzyme activities have not been used to explore honeydew-mediated changes in soil
quality. Measuring the changes in the activities of soil enzymes following aphid infestation can
provide a good tool to quantify the soil microbial responses to honeydew deposition [19,33].
An increase in microbial biomass could have potential consequences for soil meso-fauna, as their
abundance is likely to be affected through food web interactions [14,34]. Soil meso-fauna (Collembola
and Acari) live in top soil layers and play different functional roles in soil processes and nutrient
cycling [35]. Collembola, Astigmata, and Oribatida are dominant soil microbivores [36–41], while
Gamasida are mobile predators of meso-fauna [42]. Although some studies have been conducted to
Insects 2020, 11, 460 3 of 21

explore the effect of honeydew deposition on soil meso-fauna abundance [14,34], the results are
inconclusive as both increases and decreases in meso-fauna abundance have been reported in
honeydew-affected soils.
The aim of this study was to investigate the cascading effects of honeydew deposition by T.
salignus on soil chemical properties (pH, C and N), soil microbial biomass and basal respiration, soil
yeasts, abundance of soil meso-fauna (Acari and Collembola), and soil enzyme activity. Six enzymes
were selected based on their sensitivities and importance in the electron transport system
(dehydrogenase), cycling of C (glucosidase, invertase and amylase), and N (amidase and urease) in
the soil. We analysed and compared soil biochemical properties and biota under control plants, under
aphid-infested plants and in black sooty mould spots under aphid-infested plants.

2. Materials and Methods

2.1. Willow Field Trial

A willow field trial with an area of 4000 m2 (50 m × 80 m) was established at the Orchard Block,
Plant Growth Unit, Massey University, Palmerston North, New Zealand (40°22′41.70″ S, 175°36′30.67″
E, 30 m a.s.l). Average annual rainfall at the study site is 980 mm, ranging from 64 mm in the driest
month (February) to 99 mm in the wettest month (July). Average annual temperature is 13.3 °C,
fluctuating from 8.6 (July) to 18.1 °C (February) [43]. The soil type in the experimental area is a
Manawatu fine sandy loam (Weathered Fluvial Recent Soil [44]). Prior to planting willows, weeds
were killed with glyphosate herbicide on 16 May 2017 and on 25 May 2017 the soil was rotary hoed
in six rows. Each row was 1 m wide and 75 m long, with 4.0 m spacing between rows. The field trial
was arranged in a split-plot layout, with three replicated blocks. Each block included two rows of
willows; each row contained row plots of 12 ramets of fifteen willow cultivars. Willow cuttings (20
cm in length and 13 ± 2.6 mm in diameter) were planted on 16 June 2017, with 0.4 m spacing between
cuttings within rows. After planting, the weeds were controlled by manual weeding and by spraying
with Buster® herbicide. The two treatments, presence of T. salignus and aphid-free control, were
randomly allocated to the two rows within each block.

2.2. Aphids
Willow plants in the aphid-infested rows were inoculated with five adult aphids per plant on
25–27 January 2018 and 6–7 December 2019. Additional inoculations with ten adult aphids per plant
were performed on 13–14 February 2018 and 30 January 2019. The willow plants in the control rows
were inspected for colonising aphids on a weekly basis, and any aphids found were removed
manually. The plants in control rows were sprayed with Mavrik® insecticide on 28 February 2018 and
on 17 January 2019, when manual control was impractical due to high population densities of T.
salignus.

2.3. Soil Sampling

Soil samples were collected on the willow field trial site on 16 May 2017 prior to willow planting
to assess spatial heterogeneity of the site. Following willow planting, samples were collected under
the canopy of willow plants, in the 1.0 m wide cultivation zone, before aphid inoculation on 24
January 2018, after aphid inoculation on 22 June 2018, and on 2 July 2019. Three sampling points that
were 20 m equidistant from each other, were marked along each of the six rows of the field trial
(Figure S1).
Eighteen samples (one per sampling point), consisting of nine replicates from the aphid-infested
and control rows, were collected during each sampling visit. Although all the plants in infested rows
were inoculated with aphids, the honeydew was unevenly deposited, reflecting the distribution
pattern of the aphid colonies. Therefore, additional soil samples were taken from black sooty mould
spots (Figure 1) on the soil surface in the aphid-infested rows. Three samples (one per row) were
collected on 22 June 2018, and six samples (two per row) were collected on 2 July 2019. Soil moisture
content was measured three times at each sampling point using a TDR 300 Soil Moisture Probe
Insects 2020, 11, 460 4 of 21

(Spectrum Technologies Inc., Aurora, IL, USA), and the average of the three readings was then
recorded. Soil temperature at 5 cm depth was measured with a QM7216 Digital Stem Thermometer.
At each sampling point, two samples were collected, one for soil fauna extraction and another for
analysing the soil chemical properties, microbial respiration and enzyme activities. The samples were
put into labelled plastic bags, placed in an ice chest and immediately brought to the laboratory.
The samples used to quantify the soil meso-fauna (Collembola and Acari) were taken using a 25
cm2 soil corer to 5 cm depth. The sample (300–500 g) for soil chemical properties, microorganisms
and enzymes was collected using a spade to 5 cm depth from five spots within a 1.0 m diameter circle
around each sampling point, and then mixed thoroughly in a plastic tray to get a homogenous
sample. Earthworms, plant roots, moss, stones, and other debris were removed before sieving the soil
through a 2 mm drum sieve. The sieved soil was then divided into two subsamples. The subsample
for analysing the soil chemical properties and enzyme activities was air-dried at room temperature,
ground and sieved through a 1 mm mesh drum sieve. The subsample for determining the yeast
colony forming unit (CFU), microbial respiration and biomass was frozen at −20 °C.

2.3.1. Soil Chemical Properties

The soil pH was measured in a slurry containing 5 g of soil and 12.5 mL of distilled water, using
an Orion Star™ A214 pH/ISE Benchtop Meter (Thermo Scientific, Waltham, MA, USA). The total C
and N content were determined by Vario Macro Cube (Elementar Analysensysteme GmbH,
Langenselbold, Germany) from the mixture of soil (75–100 mg) and tungsten oxide powder (25–50
mg).

2.3.2. Soil Fauna

The soil samples for meso-fauna extraction were processed within 24 h. The fauna were
extracted from the soil cores using a modified Berlese-Tullgren apparatus, as described by Oliver and
Beattie [45]. Extraction was performed under 15 W light bulbs (Sylvania, OH, USA, 240–250 W) in a
17 to 30 °C temperature gradient in a temperature-controlled room for 7 days. The animals were
collected into 70% ethanol and examined using an Olympus SZX12 stereomicroscope (Spach Optics
Inc., Rochester, NY, USA). The Collembola were identified to an order level. The soil Acari were
assigned to three suborders: Oribatida, Astigmata and Gamasida. The other meso- and macro-fauna,
including small insects, spiders, centipedes, Isopoda, Diplura, Symphyla, annelid worms and
Pauropoda, were grouped as “others”. The densities of the fauna were expressed as the number of
individuals per m2.

2.3.3. Soil Enzymes

The urease (EC 3.5.1.5), invertase (EC 3.2.1.26), β-amylase (EC 3.2.1.2), α-glucosidase (EC
3.2.1.20), dehydrogenase (EC 1.1.1.1) and amidase (EC 3.5.1.4) activities were assessed according to
the protocols developed by Shcherbakova [46], Frankenberger and Johanson [47], Ross [48], Mfombep
and Senwo [49], Serra-Wittling, et al. [50], Alef and Nannipieri [51] and Frankenberger [52], with
slight modifications. Moist soil (1 g dry weight equivalent) was treated with 1.6 mL of
triphenyltetrazolium chloride (TTC) before incubating at 30 °C for 24 h; 5 mL of acetone was added
followed by incubation in the dark for 2 h to measure the dehydrogenase activity [51]. After
incubating 0.25 g of soil with 2 mL of urea in phosphate buffer, and 20 µL of toluene at 37 °C for 4 h,
the urease activity was assayed using a Genova Nano Micro-Spectrophotometer (Jenway, Stone,
Staffordshire, UK) as the amount of nitrate released from urea [46]. The amidase activity was assessed
using formamide substrate, and the amount of ammonia released during hydrolysis of the enzyme
was measured at a wavelength of 400 nm in the above-mentioned spectrophotometer [52]. The
invertase activity was estimated by measuring the amount of glucose and fructose released from
sucrose, after incubating soil samples (0.3 g) with toluene and modified universal buffer at pH 5 [47].
The amylase activity was measured using starch solution as a substrate, according to Wainwright, et
al. [53]. Determination of the α-glucosidase consisted of incubating soil samples (1 g) with toluene
Insects 2020, 11, 460 5 of 21

(0.2 mL), 67 mM sodium acetate buffer (4.3 mL, pH 5.0) and 50 mM maltose (0.5 mL) in plastic tubes
at 37 °C for 1 h; the activity of this enzyme was assayed after placing the tubes in a boiling water bath
for 5 min [49]. The activity of each enzyme was expressed based on 1 g of dry soil. One gram of each
fresh soil sample was used to estimate the dry-weight equivalent conversion factor. The samples were
oven-dried at 80 °C for 72 h until constant weight was achieved, and the dry weight measured.
The geometric mean of the enzyme activities (GMea) is regarded as a sensitive indicator for soil
quality and soil health assessment [54–56]. The GMea calculation is based on the activities of all the
assayed enzymes [54], and it is a more reliable index than any of the specific enzyme activities alone
[57]. In this study, the GMea for six enzyme activities in the aphid-infested and control rows, and the
black sooty mould soil spots was calculated according to Paz-Ferreiro, et al. [57] as follows:
6
the GMea= ඥUre × Inv × Amy × Glu × Dehy × Ami (1)
where Ure, Inv, Amy, Glu, Dehy and Ami represent urease, invertase, β-amylase, α-glucosidase,
dehydrogenease and amidase, respectively.

2.3.4. Yeasts
The frozen soil samples were incubated at 25 °C for 24 h. Fresh soil was divided into three plastic
tubes (1 g dry soil equivalent each), and suspended in Milli Q water to obtain three dilutions (v/w,
1:5, 1:10 and 1:20) according to Yurkov, et al. [58]. After shaking the suspensions on an orbital shaker
for 1 h, 0.1 mL aliquots were plated in triplicate on casein-peptone glucose yeast extract agar,
supplemented with chloramphenicol (0.1 g L−1). Lactic acid was added to acidify the medium to pH
4.5. The plates were incubated at 25 °C for 2 days and then transferred to a chiller (5 °C) to prevent
mould development. Visible colonies were counted weekly for three consecutive weeks. The yeast
counts were expressed as the colony forming units (CFU) per 1 g of dry soil, multiplied by the dilution
factor (5, 10 and 20).

2.3.5. Microbial Properties

The microbial biomass C (Cmic) and basal respiration (BR) were determined by the substrate-
induced respiration (SIR) method [59]. The frozen soil samples were incubated for 24 h at 25 °C. The
samples (1 g dry weight equivalent) were weighed and placed into 22 mL glass vials. After dropwise
addition of 0.1 mL glucose solution (8 mg g−1 soil), the vials were closed with airtight lids containing
a septum in the centre. The vials were incubated at 22 °C for 3–5 h. Air samples were collected using
a syringe, and then injected into the CO2 analyser (HP 3396 Series II Integrator, Hewlett Packard, Palo
Alto, CA, USA). The Cmic (µg C g−1 soil) was calculated as: Cmic = SIR (µL CO2 g−1 soil h−1) × 40.04 +
0.37, according to Anderson and Domsch [60]. A similar procedure was used for determining the BR,
but only 0.1 mL of water was added to the vials prior to the 24 h incubation period at 22 °C. The BR
of the soil samples was measured as µg CO2-C g−1 dry soil h−1. The microbial metabolic quotient
(qCO2) was calculated by dividing the BR by the Cmic, and expressed in µg CO2-C mg−1 Cmic h−1 [61].

2.4. Data Analysis

All analyses were performed in R version 3.5.1 [62]. A Shapiro–Wilk test was used to check
whether the data distributions met the assumption of normality. Generalized linear models (GLMs)
were used for analysis of the treatment effects on the soil chemical and microbial properties, and
enzyme activities. The gamma distribution with log-link function was used for non-normally
distributed variables, while the normally distributed data were fitted using the gaussian distribution
with identity link. Count data (meso-fauna and yeast CFU counts) were analysed in the GLM using
the Poisson and quasi-poisson error distributions. The GLMs were fitted separately for the two
sampling times, after the willow plants were inoculated with aphids. The pre-treatment sampling on
24 January 2018, prior to the aphid inoculation, was used as the baseline measurement, but was not
included in the analysis of the aphid infestation treatment vs. control. The library “multcomp” was
used for multiple comparisons using Tukey’s HSD test, whenever the GLM results showed global
Insects 2020, 11, 460 6 of 21

significant differences of the means. Full results of all GLM tests are provided in the Supplemental
Table S1.
The principal component analysis-linear discriminant analysis (PCA-LDA) was used to visualise
the data and maximize the treatment segregation [63,64]. All variables were square-root transformed,
scaled, and centred (divided by their respective standard errors) to assure equal variance, before
conducting the PCA-LDA [65]. First, the PCA was performed to produce the principal components
(PCs) in the “FactoMineR” and “factoextra” packages. The first eight PCs together explained more than
80% of the variance in the data and were used in the linear discriminant analysis (LDA). The “caret”,
“MASS” and “tidyverse” packages [63] were used to perform LDA.
In the PCA-LDA evaluating the honeydew-related changes in the soil biochemical properties
among the control, aphid-infested treatments, and black sooty mould spots, the baseline
measurements (collected before aphid inoculation) were excluded, as the aphid absence on the plants
meant no honeydew was deposited on the soil surface. In the PCA-LDA, which compared the
selected soil indicators over time, the baseline measurements as well as data from first and second
year after aphid inoculation were included. Soil temperature and moisture measurements were
excluded from the time analysis to remove bias due to weather.
In the current study, the effect of the willow cultivars was excluded from consideration. Previous
research had showed the total sugar content in the honeydew of T. salignus was not statistically
different among the willow cultivars [66]. The soil samples were taken from under the canopies of
the same willow cultivars, in the aphid-infested and control rows.

2.5. Structural Equation Modelling

Structural equation modelling (SEM) is a more reliable approach than univariate correlations
and regressions, because it provides path coefficients to examine the multiple associations in a multi-
layered system [67]. SEM was constructed to explore how honeydew deposition could influence soil
biochemical processes and functions, and to quantify the relative contribution of the different
variables, which form a network of causal relationships [68].
The a priori hypothetical model was first constructed to describe the causal relationships for the
effects of aphid honeydew on the soil environment, linking T. salignus honeydew deposition to Cmic,
specific enzyme activities, meso-fauna abundance, and the geometric mean of the enzyme activities
(GMea). This was based on a modification of the path model constructed by Milcu, et al. [14].
Variables for the model were selected based on the PC scores (Figure S2) and previous literature. We
then used SEM to calculate the coefficients associated with each path in SPSS Amos 25 (IBM, Armonk,
NY, USA) [69].
The data on the soil analysis from the two sampling dates after aphid inoculation were pooled
for this analysis; the pre-treatment data, prior to aphid inoculation, were excluded. The honeydew
input was coded as a categorical (ordinal) variable, with 0 for control, 1 for aphid-infested, and 2 for
black sooty mould spots. The Cmic, total C and N contents were selected to evaluate the direct effect
of honeydew deposition. The Cmic was chosen to estimate the indirect effect of honeydew deposition
on the Gmea and meso-fauna abundance, as Cmic has been shown to associate with soil fauna [14],
soil chemical properties (total C, total N) and enzyme activities [70].
The critical ratio of the multivariate kurtosis and squared Mahalanobis distance were checked
for multivariate normality and the presence of outliers [71]. The pooled dataset, containing the
selected variables over the two sampling times, was square-root transformed to meet the assumption
of multivariate normality. The maximum likelihood estimation was used to test the path coefficients
in the SEM models. Standardized coefficients were calculated for all the variables in the paths
diagram [72,73]. The Chi-square test, probability value of the likelihood ratio test, comparative fit
index (CFI), root mean square error of approximation (RMSEA), and Akaike’s information criterion
(AIC) were used to evaluate the model fit.
Insects 2020, 11, 460 7 of 21

3. Results

3.1. Soil Chemical Properties

Before the willows were planted, the soil of the field trial site had a mean pH value of 6.1 ± 0.05,
2.4 ± 0.08% total C and 0.25 ± 0.01% total N. None of these parameters exhibited any significant spatial
differences prior to willow planting. The baseline measurements for soil chemical properties after
planting prior to aphid infestation are included in Table 1.

Table 1. Soil chemical properties before aphid inoculation (baseline) and after aphid inoculation
(control, aphid-infested and black sooty mould spots), during the first and second year of the
experiment. Values are the means ± SE. Different letters in each column indicate significant differences
between the treatments at each sampling time (Tukey’s HSD test, α = 0.05).

Sampling Time
Parameter Treatment Before Aphid Infestation First Year Second Year
Baseline 5.75 ± 0.03
Control 6.102 ± 0.032a 6.073 ± 0.029a
pH
Aphid-infested 6.177 ± 0.052a 6.058 ± 0.065a
Black sooty mould spots 6.163 ± 0.091a 6.067 ± 0.040a

Baseline 2.40 ± 0.06

Total C (%) Control 2.146 ± 0.076b 2.278 ± 0.110b
Aphid-infested 2.260 ± 0.061b 2.297 ± 0.084b
Black sooty mould spots 2.490 ± 0.023a 2.547 ± 0.070a

Baseline 0.27 ± 0.01

Total N (%) Control 0.238 ± 0.006a 0.247 ± 0.011a
Aphid-infested 0.249 ±0.005a 0.242 ±0.008a
Black sooty mould spots 0.237 ± 0.009a 0.247 ± 0.006a

Baseline 8.79 ± 0.13

C:N Control 9.008 ± 0.136b 9.207 ± 0.178b
Aphid-infested 9.076 ± 0.107b 9.483 ± 0.216b
Black sooty mould spots 10.547 ± 0.306a 10.302 ± 0.140a

Honeydew deposition resulted in a higher total C content in the black sooty mould spots
compared to the control and aphid infestation treatments in both first and second year after aphid
infestation (Table 1). There was no effect of the treatments on the soil pH values or total N content in
both years. The C:N ratio was significantly higher in the black sooty mould spots in both years (Table
1).

3.2. Soil Meso-Fauna

Of the 4.80 × 106 soil meso-fauna collected in samples, Collembola was the most dominant taxon,
comprising 75.5% of the total. Significantly higher Collembola densities were observed in the black
sooty mould spots in the first year, and in both the aphid-infested rows and black sooty mould spots
in the second year, compared to the control treatments (Figure 2a).
Insects 2020, 11, 460 8 of 21

Figure 2. The effect of T. salignus honeydew deposition on the abundance of soil meso-fauna:
Collembola (a), Gamasida (b), Astigmata (c), Oribatida (d) and others (e), prior to aphid inoculation
(before infestation), and after aphid inoculation (control, aphid-infested, and black sooty mould spots)
in the first and second year. The values are the means ± SE. Different letters indicate statistically
significant differences between treatments within each sampling time (Tukey’s HSD test, α = 0.05).

The soil Acari (0.92 × 106 individuals) accounted for 19.3% of the soil meso-fauna. Gamasida was
the most abundant taxon, comprising 67.9% of the Acari. The aphid infestation had a significant effect
on gamasid mites only in the first year, with higher densities in the black sooty mould spots (Figure
2b). Astigmata accounted for 19.3% of the total soil mites. Their densities were higher in the black
sooty mould spots than the control and aphid infestation treatments in first year (Figure 2c). Oribatida
was the least abundant group (12.8%) of soil mites. In the second year, significantly higher densities
of Oribatida were recorded in the black sooty mould spots than in aphid infestation treatment but
not in the control treatment (Figure 2d). The black sooty mould spots also had higher population
densities of the ‘other’ fauna in the second year (Figure 2e).

3.3. Soil Enzymes

In general, honeydew deposition affected the soil enzyme activities, which tended to be higher
in black sooty mould spots than in aphid-infested and control treatments (Figure 3). The
dehydrogenase and β-amylase had significantly higher activities in the black sooty mould spots in
both years; in the second year the activity of these enzymes was also higher in the aphid infestation
treatment than in the control (Figure 3a,d). The soil urease activity was consistent across the two years
and showed a significant response to honeydew deposition in the order: black sooty mould spots >
aphid-infested > control (Figure 3b). Both the amidase and invertase had significantly higher activities
in the black sooty mould spots in the first year; in the second year the trend remained, but the
differences were not significant (Figure 3c,e). The activity of soil α-glucosidase was significantly
higher in black sooty mould spots than in the control treatment in both years, but there was no
difference between the control and aphid-infested treatments (Figure 3f). Similar to the specific
Insects 2020, 11, 460 9 of 21

enzyme activities, the Gmea for the six enzymes was significantly influenced by the honeydew
deposition; the Gmea in the first and second year was in the order: black sooty mould spots > aphid-
infested > control (Figure 4).

Figure 3. The activity of the soil enzymes: dehydrogenase (a), urease (b), amidase (c), β-amylase (d),
invertase (e) and α-glucosidase (f) under the canopies of willow plants, prior to aphid inoculation
(before infestation), and after aphid inoculation (control, aphid-infested, and black sooty mould spots)
in the first and second year. The values are the means ± SE. Different letters indicate significant
differences between the treatments at each sampling time (Tukey’s HSD test, α = 0.05).
Insects 2020, 11, 460 10 of 21

Figure 4. The effect of T. salignus honeydew deposition on the geometric mean of soil enzyme
activities, prior to aphid inoculation (before infestation), and after aphid inoculation (control, aphid-
infested, and black sooty mould spots) in the first and second year. The values are the mean ± SE.
Different letters represent statistically significant differences between the treatments at each sampling
time (Tukey’s HSD test, α = 0.05).

3.4. Soil Microbial Properties and Yeast CFU

In both years, soil Cmic (Figure 5a), basal respiration (Figure 5b) and yeast CFU (Figure 6)
increased in the order: control > aphid-infested > sooty mould spots, with all differences being
significant. The microbial quotient (qCO2) was significantly higher in the black sooty mould spots
than in the aphid-infested and control treatments only in the second year (Figure 5c).
Insects 2020, 11, 460 11 of 21

Figure 5. The effect of T. salignus honeydew deposition on (a) soil microbial biomass (µg C g−1 soil),
(b) basal respiration (µg CO2-C g−1 soil h−1), and (c) metabolic quotient over time (µg CO2-C µg−1 Cmic
h−1), prior to aphid inoculation (before infestation), and after aphid inoculation (control, aphid-
infested, and black sooty mould spots) in the first and second year. The values are the means ± SE.
Different letters indicate statistically significant differences at each sampling time (Tukey’s HSD test,
α = 0.05).
Insects 2020, 11, 460 12 of 21

Figure 6. The yeast colony forming units (CFU) per gram of dry soil, collected under the canopies of
willow plants prior to aphid inoculation (before infestation), and after aphid inoculation (control,
aphid-infested, and black sooty mould spots) in the first and second year. The values are the means ±
SE. Different letters indicate statistically significant differences at each sampling time (Tukey’s HSD
test, α = 0.05).

3.5. Principal Component Analysis-Linear Discriminant Analysis (PCA-LDA)

The PCA-LDA showed the localized effect of the honeydew deposition in the black sooty mould
spots, and the change after aphid infestation (Figure 7).
The first PCA-LDA clearly separated the black sooty mould spots from the other two treatments,
but there was some overlap between the control and the aphid-infested treatment (Figure 7a). For the
three treatments, the first (LD1) and second (LD2) discriminant functions explained 95.8 and 4.2% of
the total variability, respectively. The GMea, BR, Cmic, C:N ratio, urease, β-amylase, qCO2, and yeast
CFU were the variables that contributed the most to the separations along the PC1 (Figure S2a), which
had the highest discriminant coefficient in the first linear discriminant (LD1).
The second PCA-LDA clearly separated the sampling times prior to aphid inoculation (before
aphid infestation), and after aphid inoculation in the first and second years, but there was
considerable overlap between the first and second year post-infestation (Figure 7b). The GMea, BR,
Cmic, C:N ratio, urease, β-amylase, qCO2, and yeast CFU were also the variables that contributed the
most to the separation along PC1 (Figure S2b) which had highest weight in separating sampling times
in LD1 (84.9% of the total variability) (Figure 7b).
Insects 2020, 11, 460 13 of 21

Figure 7. PCA-LDA bi-plots of the soil biochemical variables, classified (a) by treatment, and (b) by
sampling time. A PCA was run to reduce the dimensions, followed by LDA to separate the treatments
and sampling times. The variables were scaled and centred prior to the analysis. The shaded ellipses
represent the 95% confidence areas.
Insects 2020, 11, 460 14 of 21

3.6. Structural Equation Modelling (SEM)

The SEM for the soil enzymatic response revealed a fairly good fit to the data (χ2 = 125.7, df (66–28)
= 38, p < 0.001, RMSEA = 0.00, CFI = 1, AIC = 181.7). The honeydew deposition had a positive direct
effect on the total soil C and microbial biomass C (Cmic), but not on the total N (Figure 8a). Total soil
C also increased the Cmic, while the total N had significant negative effect on Cmic. The Cmic
increased the activities of all six assayed enzymes (Figure 8a), but the degree of influence was largest
for β-amylase, urease, and dehydrogenase. The increased activities of urease, α-glucosidase and
invertase contributed most to the geometric mean of the enzyme activities (GMea).
The SEM for the soil meso-fauna abundance had a better fit to the data (χ2 = 87.3, df(36–19) = 17, p
< 0.001, RMSEA = 0.00, CFI = 1, AIC = 72.0). The honeydew deposition increased the Cmic, which
increased the abundance of the Collembola and Astigmata, but no significant effect was found for
Oribatida mites (Figure 8b). The abundance of Gamasida was positively correlated with the
abundance of their prey—Collembola, Astigmata and Oribatida.
Insects 2020, 11, 460 15 of 21

(a)

(b)
Figure 8. Path diagrams for the effects of T. salignus honeydew deposition on (a) soil biochemical
properties, and (b) meso-fauna communities. The circles above the rectangles indicate the error terms.
The solid and dotted arrows represent significant and non-significant associations, respectively. The
path coefficients are the standard regression weights, with asterisks showing different levels of
significance (* p < 0.05, ** p < 0.01, *** p < 0.001). The squared multiple correlations (r2) are expressed
above the top right corner of each rectangle.
Insects 2020, 11, 460 16 of 21

4. Discussion
In the willow field trial, the development of black sooty mould spots on the soil surface (Figure
1) is an indication of a high population density of T. salignus. As expected, we found that the copious
deposition of honeydew on the soil surface affected the soil biological and biochemical properties,
especially in the spots marked by the black sooty mould. Overall, the soil biological indicators
(microbial properties, enzyme activities and meso-fauna abundance) were found to be more sensitive
to aphid honeydew deposition than the soil chemical properties.
In our study, T. salignus honeydew deposition increased the soil total C content but did not
change soil total N content. Stadler, et al. [16] found that honeydew input increased the dissolved
organic C in litter, but reduced inorganic N content, and suggested that net N immobilization had
occurred. Aphid honeydew is a C-rich but N-poor resource [5], inducing the population of soil
microorganisms to increase and then compete for the limited N, which can increase the N
immobilization rate and result in the depletion of inorganic N [16,74]. Thus, the honeydew deposition
could indirectly decrease the soil N content through enhanced microbial activity [15,75], where
microorganisms could emerge as potential competitors of willow plants for nitrogen resources [76].
However, there is some evidence that N limitations can be compensated through increased non-
symbiotic N2-fixation by soil microorganisms [77].
The results of our study showed that sugar supplementation in the honeydew increased the
yeast CFU count, microbial biomass C and microbial respiration. Soil microorganisms are mostly
energy limited and remain dormant in the absence of a suitable substrate [20], so honeydew addition
could increase their population numbers and respiration rate by promoting favourable growth
conditions [78]. Soil yeasts prefer nutrient-rich environments and are known to utilize low molecular
weight sugars [23] that are the major components of aphid honeydew. The increase in microbial
biomass C (Cmic) and basal respiration are in accordance with the study of Milcu, et al. [14], who
found that Cmic and basal respiration increased by 330% and 58.4%, respectively, in honeydew
treatments. However, care should be taken in interpreting the microbial response to honeydew
addition, as the sugar component of honeydew can shut down the metabolism of some microbes [79].
Further studies using molecular techniques are advised to determine the changes in the soil microbial
community structure following honeydew deposition.
The activity of soil enzymes is regarded as a direct measurement of the metabolic response of
the soil microbial communities to nutrient availability [28]. Our results show a significant effect of
nutrient supplementation from aphid honeydew on the soil enzyme activities. Although honeydew
contains an unbalanced ratio of C to N, we found that both C-hydrolysing (β-amylase, invertase, α-
glucosidase), and N-hydrolysing (urease and amidase) enzymes positively responded to the
honeydew deposition. Dehydrogenase was also found to be a sensitive indicator of increased
microbial activity [80,81] as a result of the supplementary C input from honeydew. The interpretation
of enzyme activity results should be treated cautiously as enzyme assays generate the highest
potential estimates, under optimum substrate, pH and temperature conditions, rather than the actual
values [51]. However, both the specific enzyme activities and the GMea were suitable indicators
discriminating the black sooty mould spots from the control, with GMea being the best predictor.
Soil meso-fauna (Collembola and Acari) normally live in the topsoil layers, and play different
functional roles in the soil processes and nutrient cycling [35]. We observed that Collembola counts
in the honeydew-affected soil were higher than in control. Sinka, et al. [34] found no significant
influence of honeydew deposition on Collembola abundance, while Milcu, et al. [14] reported the
decline in Collembola and mite numbers in soil treated with synthetic honeydew. Seeger and Filser
[82] noted that the effect varied for different collembolan taxa. The mite groups Astigmata and
Oribatida are fungivores and saprophages [83], while the Gamasida are predators of other soil mites
and Collembola [84]. In our study, the abundance of Oribatida and Gamasida varied over time, while
that of Astigmata was fairly consistent. All three mite groups are known to respond to external
resources [85,86], with the degrees of response to the aphid honeydew reflecting their different life
histories. The Astigmata are known for their rapid response to the changing environment, due to
their faster metabolism, shorter generation time, and higher fecundity than the Oribatida [83]. On the
Insects 2020, 11, 460 17 of 21

other hand, the higher population density of Gamasida reflects the presence of the prey on which
they feed [87].
The SEMs were a useful tool to assess the multiple linkages between the honeydew and the soil
biological and biochemical indicators, linking the aboveground herbivory to the below-ground soil
processes. The path diagrams show that honeydew deposition by T. salignus has a multitrophic
cascading effect on the soil biota, similarly as in Michalzik, et al. [13], Milcu, et al. [14] and Stadler, et
al. [88]. In the current study, the Cmic was positively correlated with the soil total C content, but not
with the total N. Our results are in line with those of Cheng, et al. [70] and Johnson, et al. [89], who
suggested that the Cmic was mainly dependent on the soil C source, and additional N input could
decrease the Cmic.
In our study system, the willow plants, aphids, soil chemical properties and soil biota are
interacting with each other, highlighting the need for a multitrophic approach to investigate similar
settings. It is important to mention that this study was conducted over two years only, so the long-
term effects of the honeydew deposition on the soil remain unknown and require further
investigation. Likewise, the impacts of the other sources of variation, including the different insect
and host plant species [14], weather conditions [90] and seasonality [91], require additional attention.

5. Conclusions
The deposition of T. salignus honeydew affect the various soil biotic and abiotic properties
through a multitrophic cascade. The aphid honeydew provides an energy-rich source for the soil
microbes, causing an increase in the Cmic, that leads to increased soil enzyme activities. These
processes affect the abundance of soil meso-fauna microbivores and their predators. This example
illustrates the importance of multitrophic interactions, and the cascading effects of an aphid herbivore
on soil chemical properties and soil biological communities.
Supplementary Materials: The following are available online at www.mdpi.com/2075-4450/11/8/460/s1, Figure
S1: Sampling layout for the soil samples, Figure S2: Correlation plots for contributing variables to each dimension
in the PCA for treatments (a), and sampling times (b), Table S1: Summary of treatment effects of T. salignus
honeydew deposition on soil biochemical properties, soil enzymes, and soil meso-fauna abundance in first and
second year of the experiment.

Author Contributions: Conceptualization, M.M., A.C.-M. and T.J.; methodology, M.M., A.C.-M., S.G. and
K.M.T.; validation, M.M., A.C.-M. and T.J.; formal analysis, K.M.T. and S.G.; investigation, M.M., K.M.T. and
S.G.; resources, M.M., and S.G.; writing—original draft preparation, K.M.T.; writing—review and editing, S.G.,
M.M., A.C.-M. and T.J.; visualisation, K.M.T.; supervision, M.M., A.C.-M. and T.J.; project administration, M.M.
All authors have read and agreed to the published version of the manuscript.

Funding: This was a part of PhD project funded by the New Zealand Government Scholarship to K.M.T.

Acknowledgments: The authors wish to acknowledge the contribution to the willow field trial from the
Sustainable Farming Fund Project, Ministry for Primary Industries (MPI), New Zealand. Thanks also go to Evans
Effah, Michelle Guerrero, Mari Nakano, Claire Zucchetta and Shaun Nelson for assisting in aphid inoculation
and soil sampling. We thank Paul Barrett, Ian Furkert, Kay Sinclair, Sunmeet Bhatia, Sue Nicholson and Peter
Jeffery for their assistance with laboratory work. We are also grateful to anonymous reviewers for improving
the earlier draft of manuscript.

Conflicts of Interest: The authors declare that they have no potential conflicts of interest.

References
1. Sopow, S.; Jones, T.; Mclvor, I.; McLean, J.; Pawson, S. Potential impacts of Tuberolachnus salignus (giant
willow aphid) in New Zealand and options for control. Agr. Forest Entomol. 2017, 19, 225–234,
doi:10.1111/afe.12211.
2. McIvor, I. Willows for the farm - Specially selected for New Zealand conditions; Plant and Food Research: New
Zealand, 2013.
Insects 2020, 11, 460 18 of 21

3. Smart, L.B.; Volk, T.A.; Lin, J.; Kopp, R.F.; Phillips, I.S.; Cameron, K.D.; White, E.A.; Abrahamson, L.P.
Genetic improvement of shrub willow (Salix spp.) crops for bioenergy and environmental applications in the United
States; Unasylva 221, 2005.
4. Mittler, T.E. Studies on the feeding and nutrition of Tuberolachnus salignus(Gmelin) (Homoptera:
Aphididae) I. The uptake of phloem sap. J. Exp. Bot. 1957, 34, 334–341.
5. Mittler, T.E. Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin) (Homoptera:
Aphididae) III. The nitrogen economy. J. Exp. Bio. 1958, 35, 626–638.
6. Dhami, M.K.; Gardner-Gee, R.; Van Houtte, J.; Villas-Bôas, S.G.; Beggs, J.R. Species-specific chemical
signatures in scale insect honeydew. J. Chem. Ecol. 2011, 37, 1231–1241, doi:10.1007/s10886-011-0030-5.
7. Byrne, D.N.; Miller, W.B. Carbohydrate and amino acid composition of phloem sap and honeydew
produced by Bemisia tabaci. J. Insect Physiol. 1990, 36, 433–439, doi:10.1016/0022-1910(90)90061-J.
8. Beggs, J.R.; Wardle, D.A. Keystone species: Competition for honeydew among exotic and indigenous
species. In Biological Invasions in New Zealand, Allen, R.B., Lee, W.G., Eds. Springer, Berlin, Heidelberg: 2006;
10.1007/3-540-30023-6_18pp. 281-294.
9. Hunter, M.D. Insect population dynamics meets ecosystem ecology: Effects of herbivory on soil nutrient
dynamics. Agr. Forest Entomol. 2001, 3, 77–84, doi:10.1046/j.1461-9563.2001.00100.x.
10. Reynolds, B.C.; Hunter, M.D. Responses of soil respiration, soil nutrients, and litter decomposition to
inputs from canopy herbivores. Soil Biol. Biochem. 2001, 33, 1641–1652, doi:10.1016/S0038-0717(01)00085-2.
11. Jílková, V.; Cajthaml, T.; Frouz, J. Relative importance of honeydew and resin for the microbial activity in
wood ant nest and forest floor substrate–a laboratory study. Soil Biol. Biochem. 2018, 117, 1–4.
12. Jílková, V.; Jandová, K.; Cajthaml, T.; Devetter, M.; Kukla, J.; Starý, J.; Vacířová, A. Organic matter
decomposition and carbon content in soil fractions as affected by a gradient of labile carbon input to a
temperate forest soil. Biol. Fertil. Soils 2020, 56, 411–421, doi:10.1007/s00374-020-01433-4.
13. Michalzik, B.; Müller, T.; Stadler, B. Aphids on Norway spruce and their effects on forest-floor solution
chemistry. Forest Ecol. Manag. 1999, 118, 1–10, doi:10.1016/S0378-1127(98)00481-2.
14. Milcu, A.; Bonkowski, M.; Collins, C.; Crawley, M. Aphid honeydew-induced changes in soil biota can
cascade up to tree crown architecture. Pedobiologia 2015, 58, 119–127, doi:10.1016/j.pedobi.2015.07.002.
15. Domisch, T.; Finer, L.; Neuvonen, S.; Niemelä, P.; Risch, A.C.; Kilpeläinen, J.; Ohashi, M.; Jurgensen, M.F.
Foraging activity and dietary spectrum of wood ants (Formica rufa group) and their role in nutrient fluxes
in boreal forests. Ecol. Entomol. 2009, 34, 369–377.
16. Stadler, B.; Schramm, A.; Kalbitz, K. Ant-mediated effects on spruce litter decomposition, solution
chemistry, and microbial activity. Soil Biol. Biochem. 2006, 38, 561–572, doi:10.1016/j.soilbio.2005.06.010.
17. Rousk, J.; Brookes, P.C.; Bååth, E. Contrasting soil pH effects on fungal and bacterial growth suggest
functional redundancy in carbon mineralization. Appl. Environ. Microbiol. 2009, 75, 1589–1596,
doi:10.1128/aem.02775-08.
18. Nannipieri, P.; Ascher, J.; Ceccherini, M.T.; Landi, L.; Pietramellara, G.; Renella, G. Microbial diversity and
soil functions. Eur. J. Soil Sci. 2017, 68, 12–26, doi:10.1111/ejss.4_12398.
19. Cregger, M.A.; Schadt, C.W.; McDowell, N.G.; Pockman, W.T.; Classen, A.T. Response of the soil microbial
community to changes in precipitation in a semiarid ecosystem. Appl. Environ. Microbiol. 2012, 78, 8587–
8594, doi:10.1128/aem.02050-12.
20. Blagodatskaya, E.; Kuzyakov, Y. Active microorganisms in soil: Critical review of estimation criteria and
approaches. Soil Biol. Biochem. 2013, 67, 192–211.
21. Shi, S.; Richardson, A.E.; O'Callaghan, M.; DeAngelis, K.M.; Jones, E.E.; Stewart, A.; Firestone, M.K.;
Condron, L.M. Effects of selected root exudate components on soil bacterial communities. FEMS Microbiol.
Ecol. 2011, 77, 600–610.
22. Stadler, B.; Müller, T. Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst.
Oecologia 1996, 108, 771–776.
23. Mašínová, T.; Yurkov, A.; Baldrian, P. Forest soil yeasts: Decomposition potential and the utilization of
carbon sources. Fungal Ecol. 2018, 34, 10–19, doi:10.1016/j.funeco.2018.03.005.
24. Connell, L.; Redman, R.; Craig, S.; Scorzetti, G.; Iszard, M.; Rodriguez, R. Diversity of soil yeasts isolated
from South Victoria Land, Antarctica. Microb. Ecol. 2008, 56, 448–459, doi:10.1007/s00248-008-9363-1.
25. Yurkov, A.M. Yeasts of the soil – obscure but precious. Yeast 2018, 35, 369–378, doi:10.1002/yea.3310.
26. Elena, S.; Renata, V. The diversity of yeasts in the agricultural soil. J. Basic Microbiol. 2003, 43, 430–436,
doi:10.1002/jobm.200310277.
Insects 2020, 11, 460 19 of 21

27. Birkhofer, K.; Schöning, I.; Alt, F.; Herold, N.; Klarner, B.; Maraun, M.; Marhan, S.; Oelmann, Y.; Wubet, T.;
Yurkov, A. General relationships between abiotic soil properties and soil biota across spatial scales and
different land-use types. PLoS ONE 2012, 7, e43292.
28. García-Ruiz, R.; Ochoa, V.; Hinojosa, M.B.; Carreira, J.A. Suitability of enzyme activities for the monitoring
of soil quality improvement in organic agricultural systems. Soil Biol. Biochem. 2008, 40, 2137–2145,
doi:10.1016/j.soilbio.2008.03.023.
29. Nosrati, K.; Govers, G.; Ahmadi, H.; Sharifi, F.; Amoozegar, M.A.; Merckx, R.; Vanmaercke, M. An
exploratory study on the use of enzyme activities as sediment tracers: Biochemical fingerprints? Int. J.
Sediment. Res. 2011, 26, 136–151, doi:10.1016/S1001-6279(11)60082-6.
30. Torres, I.F.; Bastida, F.; Hernández, T.; Albaladejo, J.; García, C. Enzyme activity, microbial biomass and
community structure in a long-term restored soil under semi-arid conditions. Soil Res. 2015, 53, 553–560,
doi:10.1071/SR14297.
31. Wei, S.; Emily, D.; Daniel, B.; Kannan, I. Soil enzyme activities and organic matter composition in a turfgrass
chronosequence. Plant. Soil 2006, 288, 285–296.
32. Rao, M.A.; Scelza, R.; Acevedo, F.; Diez, M.C.; Gianfreda, L. Enzymes as useful tools for environmental
purposes. Chemosphere 2014, 107, 145–162, doi:10.1016/j.chemosphere.2013.12.059.
33. Yu, P.; Liu, S.; Han, K.; Guan, S.; Zhou, D. Conversion of cropland to forage land and grassland increases
soil labile carbon and enzyme activities in northeastern China. Agri., Eco., & Envir. 2017, 245, 83–91,
doi:10.1016/j.agee.2017.05.013.
34. Sinka, M.; Jones, T.H.; Hartley, S.E. Collembola respond to aphid herbivory but not to honeydew addition.
Ecol. Entomol. 2009, 34, 588–594, doi:10.1111/j.1365-2311.2009.01106.x.
35. Coleman, D.C.; Callaham, M.A.; Crossley Jr, D. Fundamentals of soil ecology; Academic Press, Landon: 2017.
36. Schon, N.; Mackay, A.; Minor, M. Vulnerability of soil invertebrate communities to the influences of
livestock in three grasslands. Appl. Soil Ecol. 2012, 53, 98–107.
37. Hopkin, S.P. Biology of the springtails:(Insecta: Collembola); OUP Oxford: 1997.
38. Whalen, J.K.; Sampedro, L. Soil ecology and management; CABI: 2010.
39. Men’ko, E.; Chernov, I.Y.; Byzov, B. Interrelationships between yeast fungi and collembolans in soil.
Microbiology 2006, 75, 708–715.
40. Mylonakis, E.; Ausubel, F.M.; Perfect, J.R.; Heitman, J.; Calderwood, S.B. Killing of Caenorhabditis elegans
by Cryptococcus neoformans as a model of yeast pathogenesis. Proc. Natl. Acad. Sci. 2002, 99, 15675–15680.
41. Hoy, M.A. Soil mites (Acari: Oribatida and others). In Encyclopedia of Entomology, Capinera, J.L., Ed.
Springer, Dordrecht, Netherlands: 2008; 10.1007/978-1-4020-6359-6_4266pp. 3463-3466.
42. Walter, D.; Hunt, H.; Elliott, E. Guilds or functional groups? An analysis of predatory arthropods from a
shortgrass steppe soil. Pedobiologia 1988, 31, 247–260.
43. NIWA. CliFlo: National Institute of Water and Atmospheric Research's National Climate Database on the
Web. Availabe online: http://cliflo.niwa.co.nz/ (accessed on 12 December 2019).
44. Hewitt, A.E. New Zealand soil classification, 2nd ed.; Manaaki Whenua Press: Lincoln, Canterbury, New
Zealand., 1998; doi:10.7931/DL1-LRSS-1-2010.
45. Oliver, I.; Beattie, A.J. Designing a cost-effective invertebrate survey: A test of methods for rapid assessment
of biodiversity. Ecol. Appl. 1996, 6, 594–607, doi:10.2307/2269394.
46. Shcherbakova, T.A. Enzymatic activity of soil and transformation of organic matter. (In Russian). Nauka i
Tekhnika. Minsk.; Springer Science & Business Media: 1983.
47. Frankenberger, W.T.; Johanson, J.B. Factors affecting invertase activity in soils. Plant. Soil 1983, 74, 313–323,
doi:10.1007/bf02181349.
48. Ross, D.J. Invertase and amylase activities as influenced by clay minerals, soil-clay fractions and topsoils
under grassland. Soil Biol. Biochem. 1983, 15, 287–293.
49. Mfombep, P.M.; Senwo, Z.N. Soil maltase activity by a glucose oxidase–perioxidase system. 3 Biotech. 2012,
2, 225–231, doi:10.1007/s13205-012-0050-z.
50. Serra-Wittling, C.; Houot, S.; Barriuso, E. Soil enzymatic response to addition of municipal solid-waste
compost. Biol. Fertil. Soils 1995, 20, 226–236, doi:10.1007/bf00336082.
51. Alef, K.; Nannipieri, P. 7 - Enzyme activities. In Methods in Applied Soil Microbiology and Biochemistry, Alef,
K., Nannipieri, P., Eds. Academic Press, London: 1995; 10.1016/B978-012513840-6/50022-7pp. 311-373.
52. Frankenberger, W.T. Amidase activity in soils. Retrospective Theses and Dissertations. 6722. Ph.D, Iowa
State University, 1980.
Insects 2020, 11, 460 20 of 21

53. Wainwright, M.; Duddridge, J.E.; Killham, K. Assay of α-amylase in soil and river sediments: Its use to
determine the effects of heavy metals on starch degradation. Enzyme Microb. Technol. 1982, 4, 32–34,
doi:10.1016/0141-0229(82)90007-2.
54. García-Ruiz, R.; Ochoa, V.; Vinegla, B.; Hinojosa, M.; Pena-Santiago, R.; Liébanas, G.; Linares, J.; Carreira,
J. Soil enzymes, nematode community and selected physico-chemical properties as soil quality indicators
in organic and conventional olive oil farming: Influence of seasonality and site features. Appl. Soil Ecol.
2009, 41, 305–314.
55. Puglisi, E.; Del Re, A.; Rao, M.; Gianfreda, L. Development and validation of numerical indexes integrating
enzyme activities of soils. Soil Biol. Biochem. 2006, 38, 1673–1681.
56. Hinojosa, M.B.; García-Ruíz, R.; Viñegla, B.; Carreira, J.A. Microbiological rates and enzyme activities as
indicators of functionality in soils affected by the Aznalcóllar toxic spill. Soil Biol. Biochem. 2004, 36, 1637–
1644, doi:10.1016/j.soilbio.2004.07.006.
57. Paz-Ferreiro, J.; Fu, S.; Méndez, A.; Gascó, G. Interactive effects of biochar and the earthworm Pontoscolex
corethrurus on plant productivity and soil enzyme activities. J. Soils Sediments 2014, 14, 483–494,
doi:10.1007/s11368-013-0806-z.
58. Yurkov, A.M.; Kemler, M.; Begerow, D. Assessment of yeast diversity in soils under different management
regimes. Fungal Ecol. 2012, 5, 24–35, doi:10.1016/j.funeco.2011.07.004.
59. Ananyeva, N.D.; Susyan, E.A.; Gavrilenko, E.G. Determination of the soil microbial biomass carbon using
the method of substrate-induced respiration. Eurasian Soil Sci. 2011, 44, 1215–1221,
doi:10.1134/s1064229311030021.
60. Anderson, J.P.E.; Domsch, K.H. A physiological method for the quantitative measurement of microbial
biomass in soils. Soil Biol. Biochem. 1978, 10, 215–221, doi:10.1016/0038-0717(78)90099-8.
61. Ananyeva, N.D.; Rogovaya, S.V.; Ivashchenko, K.V.; Vasenev, V.I.; Sarzhanov, D.A.; Ryzhkov, O.V.;
Kudeyarov, V.N. Carbon dioxide emission and soil microbial respiration activity of Chernozems under
anthropogenic transformation of terrestrial ecosystems. Eurasian J. Soil Sci. 2016, 5, 146–154.
62. R Development Core Team R: A language and environment for statistical computing. R foundation for statistical
computing, Vienna, Austria, 2019.
63. Patel, I.I.; Shearer, D.A.; Fogarty, S.W.; Fullwood, N.J.; Quaroni, L.; Martin, F.L.; Weisz, J. Infrared
microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary
epithelium and stroma of breast tissues from healthy young women. Cancer Biol. Ther. 2014, 15, 225–235,
doi:10.4161/cbt.26748.
64. Walsh, M.J.; Singh, M.N.; Pollock, H.M.; Cooper, L.J.; German, M.J.; Stringfellow, H.F.; Fullwood, N.J.;
Paraskevaidis, E.; Martin-Hirsch, P.L.; Martin, F.L. ATR microspectroscopy with multivariate analysis
segregates grades of exfoliative cervical cytology. Biochem. Biophys. Res. Commun. 2007, 352, 213–219.
65. Stenberg, B.; Johansson, M.; Pell, M.; Sjödahl-Svensson, K.; Stenström, J.; Torstensson, L. Microbial biomass
and activities in soil as affected by frozen and cold storage. Soil Biol. Biochem. 1998, 30, 393–402.
66. Tun, K.M.; Jones, T.; Minor, M.; McCormick, A.C. Effect of willow cultivar and age on the melezitose
content of giant willow aphid (Tuberolachnus salignus) honeydew. Manuscript in preparation. Agr. Forest
Entomol. 2020.
67. Grace, J.B. Structural equation modeling and natural systems; Cambridge University Press: 2006.
68. Hatcher, L. Using SAS® PROC CALIS for path analysis: An introduction. Struct. Equ. Modeling 1996, 3, 176–
192, doi:10.1080/10705519609540037.
69. Arbuckle, J.L. Amos, 25; IBM SPSS: Chicago, 2014.
70. Cheng, F.; Peng, X.; Zhao, P.; Yuan, J.; Zhong, C.; Cheng, Y.; Cui, C.; Zhang, S. Soil microbial biomass, basal
respiration and enzyme activity of main forest types in the Qinling Mountains. PLoS ONE 2013, 8, e67353,
doi:10.1371/journal.pone.0067353.
71. Sutton-Grier, A.E.; Kenney, M.A.; Richardson, C.J. Examining the relationship between ecosystem
structure and function using structural equation modelling: A case study examining denitrification
potential in restored wetland soils. Ecol. Modell. 2010, 221, 761–768, doi:10.1016/j.ecolmodel.2009.11.015.
72. Kwan, J.L.Y.; Chan, W. Comparing standardized coefficients in structural equation modeling: A model
reparameterization approach. Behav. Res. Methods 2011, 43, 730–745, doi:10.3758/s13428-011-0088-6.
73. Grace, J.B.; Bollen, K.A. Interpreting the results from multiple regression and structural equation models.
Bull. Ecol. Soc. Am. 2005, 86, 283–295, doi:10.1890/0012-9623(2005)86[283:itrfmr]2.0.co;2.
Insects 2020, 11, 460 21 of 21

74. Milcu, A.; Heim, A.; Ellis, R.J.; Scheu, S.; Manning, P. Identification of general patterns of nutrient and labile
carbon control on soil carbon dynamics across a successional gradient. Ecosystems 2011, 14, 710–719,
doi:10.1007/s10021-011-9440-z.
75. Stadler, B.; Mühlenberg, E.; Michalzik, B. The ecology driving nutrient fluxes in forests. In Insects and
Ecosystem Function, Weisser, W.W., Siemann, E., Eds. Springer, Berlin, Heidelberg: 2008; 10.1007/978-3-540-
74004-9_11pp. 213-239.
76. Kaye, J.P.; Hart, S.C. Competition for nitrogen between plants and soil microorganisms. Trends Ecol. Evol.
1997, 12, 139–143, doi:10.1016/S0169-5347(97)01001-X.
77. Petelle, M. Aphid honeydew sugars and soil nitrogen fixation. Soil Biol. Biochem. 1984, 16, 203–206,
doi:https://doi.org/10.1016/0038-0717(84)90001-4.
78. Joergensen, G.R.; Stefan, S. Response of soil microorganisms to the addition of carbon, nitrogen and
phosphorus in a forest Rendzina. Soil Biol. Biochem. 1999, 31, 859–866, doi:10.1016/S0038-0717(98)00185-0.
79. Islam, R.; Wright, S.R. Microbial biomass measurement methods. In Encyclopedia of soil science, 2nd ed.; Lal,
R., Ed. CRC, Boca Raton: 2004; pp. 1067–1070.
80. García-Ruiz, R.; Ochoa, M.V.; Hinojosa, M.B.; Gómez-Muñoz, B. Improved soil quality after 16 years of
olive mill pomace application in olive oil groves. Agron. Sustain. Dev. 2012, 32, 803–810.
81. Ruzhen, W.; Timothy, R.F.; Zhuwen, X.; Xue, W.; Mai-He, L.; Yuge, Z.; Wentao, L.; Yong, J. Coupled
response of soil carbon and nitrogen pools and enzyme activities to nitrogen and water addition in a semi-
arid grassland of Inner Mongolia. Plant. Soil 2014, 381, 323–336, doi:10.1007/s11104-014-2129-2.
82. Seeger, J.; Filser, J. Bottom-up down from the top: Honeydew as a carbon source for soil organisms. Eur. J.
Soil Biol. 2008, 44, 483–490, doi:10.1016/j.ejsobi.2008.07.008.
83. Behan-Pelletier, V.M. Oribatid mite biodiversity in agroecosystems: Role for bioindication. Agri., Eco., &
Envir. 1999, 74(1-3), 411-423.
84. Jung, C.; Kim, J.W.; Marquardt, T.; Kaczmarek, S. Species richness of soil gamasid mites (Acari:
Mesostigmata) in fire-damaged mountain sites. J. Asia Pac. Entomol. 2010, 13, 233–237,
doi:10.1016/j.aspen.2010.04.001.
85. Bedano, J.C.; Cantú, M.P.; Doucet, M.E. Influence of three different land management practices on soil mite
(Arachnida: Acari) densities in relation to a natural soil. Appl. Soil Ecol. 2006, 32, 293–304,
doi:10.1016/j.apsoil.2005.07.009.
86. Cao, Z.; Han, X.; Hu, C.; Chen, J.; Zhang, D.; Steinberger, Y. Changes in the abundance and structure of a
soil mite (Acari) community under long-term organic and chemical fertilizer treatments. Appl. Soil Ecol.
2011, 49, 131–138.
87. Walter, D.; Kethley, J.; Moore, J. Heptane flotation method for recovering microarthropods from semiarid
soils, with comparison to the Merchant-Crossley high-gradient extraction method and estimates of
microarthropod biomass. Pedobiologia 2013, 30 221-232.
88. Stadler, B.; Michalzik, B.; Müller, T. Linking aphid ecology with nutrient fluxes in a coniferous forest.
Ecology 1998, 79, 1514–1525.
89. Johnson, D.; Leake, J.R.; Read, D.J. Liming and nitrogen fertilization affects phosphatase activities,
microbial biomass and mycorrhizal colonisation in upland grassland. Plant. Soil 2005, 271, 157–164,
doi:10.1007/s11104-004-2267-z.
90. Stadler, B.; Michalzik, B. Aphid infested Norway spruce are "hot spots" in throughfall carbon chemistry in
coniferous forests. Can. J. For. Res. 1998, 28, 1717–1722.
91. McDowell, W.H.; Likens, G.E. Origin, composition, and flux of dissolved organic carbon in the Hubbard
Brook Valley. Ecol. Monogr. 1988, 58, 177–195.

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