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Fluctuating Asymmetry as a Method of Assessing Environmental Stress in Two Predatory Carabid Species within Mediterranean Agroecosystems

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Fluctuating asymmetry (FA) is used in assessing the effect of environmental stress on the development stability of individuals by measuring small random deviations from perfect bilateral symmetry. Here, we checked for FA on two predatory carabid beetles, Pterostichus melas and Poecilus koyi, in order to evaluate species response to agricultural practices within Mediterranean agroecosystems, as well as FA as a method. The samples were collected in vineyards and olive groves, both under integrated pest management (IPM) and ecological pest management (EPM), and in pristine habitats in the Mediterranean region of Croatia. Geometric morphometrics (GMMs) were used to analyze the pronotum and abdomen shape variations and left-right asymmetries of each population. In respect to the FA measurements, analyzed species responded differently, with P. koyi displaying a lower intensity of FA than P. melas. On the other hand, P. melas beetles from vineyards showed a higher intensity of FA compared with populations from pristine habitats and olive groves. Accordingly, FA pointed out olive groves as potentially less adverse habitats to predatory carabids, keeping in mind the different levels of asymmetry between the two species. Our study singled out P. melas as a more suitable species for further research, in the effect that different agricultural practices can have their impact on non-target invertebrates analyzed by measuring the FA.
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symmetry
S
S
Article
Fluctuating Asymmetry as a Method of Assessing
Environmental Stress in Two Predatory Carabid
Species within Mediterranean Agroecosystems
Lara Ivankovi´c Tatalovi´c 1, Barbara An ¯
deli´c 1, Mišel Jeli´c 2, Tomislav Kos 3, Hugo A. Benítez 4
and Lucija Šeri´c Jelaska 1,*
1Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia;
lara.ivankovic@biol.pmf.hr (L.I.T.); barbara.andelic@biol.pmf.hr (B.A.)
2Varaždin City Museum, Šetalište Josipa Jurja Strossmayera 3, 42000 Varaždin, Croatia; misel.jelic@gmv.hr
3Department of Ecology, Agronomy and Aquaculture, University of Zadar, 23000 Zadar, Croatia;
tkos@unizd.hr
4Laboratorio de Ecología y Morfometría Evolutiva, Centro de Investigación de Estudios Avanzados del
Maule, Universidad Católica del Maule, Talca 3466706, Chile; hbenitez@ucm.cl
*Correspondence: slucija@biol.pmf.hr
Received: 5 October 2020; Accepted: 13 November 2020; Published: 17 November 2020


Abstract:
Fluctuating asymmetry (FA) is used in assessing the eect of environmental stress on the
development stability of individuals by measuring small random deviations from perfect bilateral
symmetry. Here, we checked for FA on two predatory carabid beetles, Pterostichus melas and
Poecilus koyi, in order to evaluate species response to agricultural practices within Mediterranean
agroecosystems, as well as FA as a method. The samples were collected in vineyards and olive
groves, both under integrated pest management (IPM) and ecological pest management (EPM), and in
pristine habitats in the Mediterranean region of Croatia. Geometric morphometrics (GMMs) were
used to analyze the pronotum and abdomen shape variations and left–right asymmetries of each
population. In respect to the FA measurements, analyzed species responded dierently, with P. koyi
displaying a lower intensity of FA than P. melas. On the other hand, P. melas beetles from vineyards
showed a higher intensity of FA compared with populations from pristine habitats and olive groves.
Accordingly, FA pointed out olive groves as potentially less adverse habitats to predatory carabids,
keeping in mind the dierent levels of asymmetry between the two species. Our study singled out
P. melas as a more suitable species for further research, in the eect that dierent agricultural practices
can have their impact on non-target invertebrates analyzed by measuring the FA.
Keywords:
agricultural practices; body shape; developmental instability; geometric
morphometrics; pesticides
1. Introduction
An increase in the application of plant protection products, due to the need for constant production
growth and following the eects of climate change, can be viewed as one of the major factors that
poses a threat to overall insect biodiversity [
1
,
2
]. Intensive use of pesticides has been proven to be
harmful to non-target insects [
3
]. In several cases, neonicotinoids were known to cause sublethal
eects to bees and other insects [
4
6
]. This eect of pesticides is not only harmful to pollinators,
but also to predator species such as ground beetles, who are important for controlling changes in prey
populations and thus in biocontrol in agroecosystems [
7
,
8
]. Furthermore, ground beetles are important
bio indicators of dierent human-induced environmental stresses, including agronomic production
as well as urbanization, forestry, landscape fragmentation, and other anthropogenic impacts [
9
13
].
Symmetry 2020,12, 1890; doi:10.3390/sym12111890 www.mdpi.com/journal/symmetry
Symmetry 2020,12, 1890 2 of 17
Intensive management in agricultural habitats provides constant stressors to organisms from the
developmental to adult stages, especially by adding a broad range of pesticides to the systems.
Fluctuating asymmetry (FA) is a common technique that measures random, small deviations from
bilateral symmetry in organisms and is used in the assessment of dierent levels of stress, environmental
or genomic, that aect organisms during their development, known as developmental instability
(DI) or developmental noise (DN) [
3
]. Sukhodolskaya et al. [
14
] showed that, in the case of ground
beetles, there is a species-specific sensitivity to FA, even among closely related species. Furthermore,
it was shown that some species cannot be used as indicators of environmentally induced stress for
measuring FA. Asymmetry of ground beetle populations was used in various studies to measure the
eects of dierent agricultural practices [
3
,
15
,
16
] and to estimate the eects of urbanization [
17
,
18
]
and intensive landscape changes on forestry [
19
22
]. There are dierences between various authors’
approaches; Elek et al. [
18
] demonstrated that ground beetles, common along a Danish urbanization
gradient, do not seem to indicate dierences in habitat quality by their level of FA, and Labrie et al. [
15
]
found no dierences in FA between sites with dierent agricultural practices. On the other hand,
Weller and Ganzhorn [
17
] showed that proximity to urban areas increases the FA in species which are
susceptible to urbanization. Furthermore, Weller and Ganzhorn [
17
] noticed that eurytopic species are
less prone to environmental changes and thus have smaller levels of FA. This led us to the conclusion
that disturbances in the environment, spanning from urbanization changes to pesticide application
in agriculture, sometimes do not cause changes to FA in a way that dierent levels of disturbances,
such as higher levels of pesticides, can be distinguished. It is also important to highlight the dierence
in FA between sexes, as it is a factor that has been omitted in many studies, but could have an impact
FA scores [18,2325].
Olive groves and vineyards are a vital part of agricultural production in the Mediterranean region.
Copper, as a plant protection product, has been used in this area for centuries and is still widely
used nowadays when numerous farmers are turning to organic production and ecologically based
pest management (EPM), in which the use of copper is permitted. On the other hand, integrated
pest management (IPM) and conventional management (CM) use a spectrum of chemically synthetic
pesticides that include active compounds, targeting dierent specific groups of organisms [
15
,
18
,
23
25
].
The level of impact on insects is dierent depending on the group of pesticides, but their joined
eect must not be neglected. Some research shows that organic farms have more species at three
trophic levels compared to conventional farming, but this did not improve the mortality of pest species
or, thus, the ecosystem service [
26
]. In addition, the meta-analysis research of Bengtsson et al. [
27
]
showed that there was a higher species richness in organic farming than IPM farming. Additionally,
not only the species richness, but the total fitness of predators, such as ground beetles, were revealed as
important when compared between pest management types [
28
31
]. On the other hand, some authors
reported no significant increase in carabid beetle diversity among dierently managed fields [
32
,
33
].
It is important to keep in mind that in the agroecosystem, there is a broad list of factors influencing the
predator dynamics and abundance, and that these results are sometimes due to an overall eect [29].
Even though olive groves and vineyards have been an integral part of the Mediterranean
landscape and its diversity for thousands of years, there is still a lack of studies regarding the impact
of management of stress on predatory invertebrate fauna. Therefore, we focused our research on
two predatory ground beetles, Poecilus koyi (Germar, 1824) and Pterostichus melas (Creutzer, 1799),
that are common in the studied sites, located in the Mediterranean area of Croatia. The selected
predatory carabids play an important role within agroecosystems in controlling their prey abundance,
including potential pest species, and thus contribute to biocontrol of pest populations [
34
37
]. Using FA,
we wanted to measure stress induced by dierent agricultural practices on these two species, assuming
that a higher intensity of FA could point out the type of practice that was more adverse toward
predatory carabid beetles. Besides checking for the presence of FA within populations across the sites
under dierent agricultural practices, we questioned if the selected species, both co-occurring in arable
land across southwest Europe [
38
40
], showed the same patterns of FA, or if one of them was more
Symmetry 2020,12, 1890 3 of 17
sensitive to environmental changes. In addition, the study aimed to analyze the variation in the body
shape and size between sexes and populations from dierent agroecosystems.
2. Materials and Methods
2.1. Sampling Sites
Sampling took place within four agricultural sites in Zadar County in the Mediterranean part
of Croatia: vineyards, one site with ecological pest management (EV) and one with integrated
pest management (IV), and olive groves with ecological pest management (EO) and integrated pest
management (IO) (Figure 1). They were in pristine habitats (C) with no histories of pesticide treatment,
represented by typical Mediterranean maquis and garrigue, with one site in Zadar County in 2019 and
one in nearby Šibenik-Knin County. The study sites have a long history in agricultural use, with EO
and EV being under EPM for a decade or more. Detailed information about agricultural practices
during 2018 in the selected sites is listed in Table 1. To estimate the intensity of the FA within the sites,
two predatory carabid species, P. koyi and P. melas, were sampled, and we proceeded with further
analyses (Table 2). At the control sites, only P. melas individuals were sampled in a substantial number
after pooling them from two control sites for the analyses (Table 2). Information on the amount of
pesticides applied on the studied sites in the year of sample collection is given in Figure 2.
Figure 1.
Map of Zadar County and Šibenik-Knin County with six study sites (Transverse Mercator
Projection, HTRS96/TM): (1) IV, a vineyard with integrated pest management (IPM) (located in Baštica,
Zadar County); (2) IO, an olive grove with IPM (located in Škabrnja, Zadar County); (3) EV, a vineyard
with ecological pest management (EPM) (located in Nadin, Zadar County); (4) EO, an olive grove
with EPM (located in Poliˇcnik, Zadar County); (5,6) C, control sites within unmanaged pristine
habitats (two locations, one in Suhovare, Zadar County and one in the Brnjica area within Krka NP,
Šibenik-Knin County).
Symmetry 2020,12, 1890 4 of 17
Figure 2.
The amount of pesticides added at each study site during 2018, shown as grams of active
substances applied per hectare. Copper was used either as copper (I) oxide or copper oxychloride
(H6Cl2Cu4O6). Site abbreviations are as follows: one vineyard with EPM (EV) and one with IPM (IV),
and one olive grove with EPM (EO) and one with IPM (IO).
Table 1.
Number of treatments, including the number of pesticides added and soil processing
during 2018 for four managed sites. Added pesticides have been grouped according to the main
active compounds, those being synthetic, biological, and copper ones. Details are given below.
Site abbreviations are as follows: one vineyard with EPM (EV) and one with IPM (IV), and one olive
grove with EPM (EO) and one with IPM (IO).
Number of Treatments in 2018
Treatments Sites IV EV IO EO
Pesticides added
Synthetic pesticides * 12 0 6 0
Biological pesticides ** 0 0 0 4
Copper compounds *** 3 6 3 5
Soil processing Mulching 2 0 5 4
Ploughing and shallow
disking 1 3 0 0
* Organochlorides and chlorinated hydrocarbons, organophosphates, pyrethroids, neonicotinoids, and ryanoids.
** Bt kurstaki and Spinosad. *** Copper (I) oxide or copper oxychloride.
Table 2.
Number of males (
) and females (
) used in the analyses of each species per site.
Site abbreviations are as follows: one vineyard with EPM (EV) and one with IPM (IV), and one
olive grove with EPM (EO) and one with IPM (IO), and a control (C).
Type of
Study Site
Site
(Mark)
Pest Management
Type
Number of Specimens for
Poecilus koyi
Number of Specimens for
Pterostichus melas
Olive groves EO Ecological 27 53 0 0
IO Integrated 26 53 50 50
Vineyards EV Ecological 25 74 50 50
IV Integrated 22 64 50 50
Control C unmanaged 0 0 20 9
Symmetry 2020,12, 1890 5 of 17
2.2. Collection of Specimens
Specimens were collected from mid-April to mid-July and from mid-September to mid-November
in 2018, using pitfall traps and actively searching and collecting by hand. Plastic containers with
a volume of 300 mL were placed in the ground and used as pitfall traps. Traps were covered with
elevated, flat stone slabs. The distance between traps was approximately 2 m in all sites, as they were
dug in under olive trees or under the stumps of vines, depending on the site. For the preservation of
trapped arthropods, an aqueous salt solution was used. Traps were emptied every two to three weeks,
and the material was transferred in 80% ethanol and analyzed in the laboratory.
From the total of the ground beetles sampled, two common and numerous species, P. koyi and
P. melas, were present at almost all sites, and for further analyses, a substantial number of individuals
were selected for FA measurements (Table 2). In the case of the P. melas from EO, there were not
enough specimens, even after additional sampling eorts, so this population was not included in
further analyses. This applied to P. koyi from the control site (C) as well. The control sample of P. melas
was a bulk sample, assembled from samples collected at two control sites. Samples were pooled and
analyzed as one. Specimens of P. koyi were photographed using an Epson Perfection V600 photo
scanner, while specimens of P. melas were photographed using a digital Nikon D60 camera with a
Sigma macro objective. In both species, males and females were separated, based on sex combs on the
first pair of legs or, in a case that feature was insucient or absent, external genital parts were used,
and they were analyzed separately. For all specimens of P. koyi and P. melas, the ventral part of the
body was photographed, while the dorsal part of the body was imprinted in clay to keep the specimen
horizontally levelled during the process.
2.3. Shape Analysis
A total of 16 landmarks on the ventral part of the body were used for P. koyi and P. melas (Figure 3).
Suggested landmarks were digitized using tpsDig 2.31 software [
41
]. To be able to evaluate the
significance of fluctuating asymmetry, landmarks were digitized twice in the case of P. koyi and three
times in the case of P. melas.
Figure 3.
Graphical representation of the position of 16 selected landmarks on the ventral side of
the body: (1) pygidium; (2) middle point of the metastern; (3,5,7) left lateral vertex of the abdominal
segment; (4,6,8) right lateral vertex of the abdominal segment; (9,10) metathorax; (11,12,13,14) coxa of
the first legs; (15) left vertex of the pronotal epimere; (16) right vertex of the pronotal epimere.
Symmetry 2020,12, 1890 6 of 17
Statistical analyses were performed in MorphoJ [
42
] and STATISTICA 13 (Statistica Inc. TIBCO
Software). After Procrustes superimposition was done in MorphoJ, the coordinates of each landmark
were used for further analyses [
43
]. To analyze the measurement error, Procrustes analysis of variance
(Procrustes ANOVA) was performed on combined datasets of repeated landmark digitization for
both P. koyi and P. melas. To estimate the dierences in size between specimens from dierent sites
and sexes, ANOVA and post hoc unequal honest significant dierence (HSD) was performed in
STATISTICA 13. Procrustes ANOVA was performed for the centroid size and shape, with sites and
sexes as extra eects. Canonical variate analysis (CVA) was used to determine the relationships
between groups of variables in our data set and for better visualization of separated (discriminated)
groups. CVA was performed using symmetric components of the shape variation. In order to assess
statistically significant dierences across the sites, a pairwise comparison after 10,000 permutations
was performed using Mahalanobis and Procrustes distances. The presence of FA in the species
was tested using Procrustes ANOVA by checking individual*side interactions and mean squares
of individual*side (MS
ind*side
) interactions, corrected for error variance, were used as a measure
representing FA. The T-test for independent samples was used to statistically confirm the dierences
in Procrustes FA scores between the species. In addition, to check if the dierences in Procrustes FA
scores (p<0.05) observed between the populations were significant, ANOVA and Tukey HSD post hoc
tests were performed after transformation, as using the natural logarithm had normalized the data
(Kolmogorov–Smirnov
p>0.05
). Levene’s test confirmed the null hypothesis of equal variances for
tested groups and populations (p>0.05).
3. Results
The Procrustes ANOVA, applied on repeated measures of individuals in order to assess the
measurement error (ME), showed that the ME was negligible (MSindiv >ME, MSind*side >ME,
with MS standing for mean squares) between sets of measurements for both P. melas and P. koyi (Table 3).
Subsequently, the same analysis indicated significantly high variations for both shape and size between
populations and sexes (p<0.0001) of P. melas, but just for shape in the case of P. koyi. Females were
larger than males at every site for P. melas (Figure 4). The post hoc test showed a significant dierence
in size between females from IV and the control (p=0.00216) and females from EV and the control
(p=0.00427),
in the case of P. melas. The P. koyi species showed no dierences between males and
females from dierent sites, or between the sexes within the same site.
Table 3.
Procrustes analysis of variance (ANOVA) performed for the centroid size and shape of P. melas
(325 individuals) and P. koyi (344 individuals). Sums of squares (SS) and mean squares (MS) are in units
of Procrustes distances (dimensionless). * denotes interaction eects.
Eect SS MS df F P (param.)
Pterostichus melas Centroid size
Individual 10.438037 0.037682 277 35.14 <0.0001
Error 1 0.585504 0.001072 546
Shape
Individual 0.62945837 0.000162315 3878 5.39 <0.0001
Side 0.0045157 0.00032255 14 10.71 <0.0001
Ind* Side 0.11677858 3.01131 ×1053878 28.63 <0.0001
Error 1 0.01608002 1.0518 ×10615288
Poecilus koyi Centroid size
Individual 5.802676 0.016674 348
409.65
<0.0001
Error 1 0.013676 0.000041 336
Shape
Individual 0.2256271 0.000046311 4872 3.25 <0.0001
Side 0.00885456 0.000632468 14 44.43 <0.0001
Ind* Side 0.06936083 1.42366E-05 4872 11.69 <0.0001
Error 1 0.01145416 1.2175E-06 9408
Symmetry 2020,12, 1890 7 of 17
Figure 4.
Average centroid size of male (M) and female (F) P. melas in four study sites. Vertical bars
denote +/standard errors. For site abbreviations, see Table 2.
A canonical variate analysis (CVA), applied to the symmetrical components of shape variation in
P. melas, showed that the Mahalanobis distance significantly diered (p<0.0001) between populations
from studied sites, except for males from the control sites, which did not dier from other groups.
Furthermore, there was a significant Mahalanobis distance between sexes within the same population
(p<0.0001) (Table 4, Figure 5). The results of the CVA with Mahalanobis and Procrustes distances
(Tables 4and 5, Figure 6) for P. koyi showed high morphological variability, especially in males,
across the sites (p<0.0001).
Figure 5.
A canonical variate analysis (CVA) applied to symmetrical components of the shape for
P. melas. Female (
A
) and male (
B
) individuals are shown separately (
males,
females). Individuals
from the IV site are presented by blue dots, those from EV by red dots, IO by green, and C by grey dots.
Symmetry 2020,12, 1890 8 of 17
Table 4.
Results after T-testing pairwise distances, reported as the Mahalanobis distance, and pvalues after 10,000 permutations of runs. For site abbreviations,
see Table 2. Symbols denote sexes: males () and females ().
Species Site and Sex Distance
pValue
C
Mahalanobis
pValue
IO
Mahalanobis
pValue
EV
Mahalanobis
pValue
IV
Mahalanobis
pValue
C
Mahalanobis
pValue
IO
Mahalanobis
pValue
EV
Mahalanobis
pValue
Pterostichus melas
IO 1.7771 0.0677
EV 2.3878 0.0057 2.0741 <0.0001
IV 2.2988 0.0007 2.3702 <0.0001 1.6511 <0.0001
C3.8665 <0.0001 4.7129 <0.0001 4.1761 <0.0001 4.1955 <0.0001
IO 2.781 <0.0001 2.9191 <0.0001 2.922 <0.0001 2.5668 <0.0001 3.0542 <0.0001
EV 3.2551 <0.0001 3.6275 <0.0001 2.7623 <0.0001 2.8527 <0.0001 2.6604 <0.0001 2.1433 <0.0001
IV 3.4221 <0.0001 3.9055 <0.0001 3.4544 <0.0001 2.7985 <0.0001 2.5228 <0.0001 1.5866 <0.0001 1.9401 <0.0001
Species Site and Sex Distance
pValue
EO
Mahalanobis
pValue
IO
Mahalanobis
pValue
EV
Mahalanobis
pValue
IV
Mahalanobis
pValue
EO
Mahalanobis
pValue
IO
Mahalanobis
pValue
EV
Mahalanobis
pValue
Poecilus koyi
IO 1.1751 0.206
EV 1.4212 0.0041 1.8508 <0.0001
IV 0.9435 0.6635 1.1502 0.3692 1.7277 0.0002
EO 2.9347 <0.0001 3.2679 <0.0001 3.3421 <0.0001 2.9529 <0.0001
IO 3.3318 <0.0001 3.3071 <0.0001 3.8005 <0.0001 3.2384 <0.0001 1.6127 <0.0001
EV 3.0853 <0.0001 3.3635 <0.0001 3.1367 <0.0001 3.0515 <0.0001 0.9469 0.0123 1.8619 <0.0001
IV 2.9559 <0.0001 3.0142 <0.0001 3.4758 <0.0001 2.7716 <0.0001 1.2945 0.0001 1.0131 0.0128 1.4896 <0.0001
Symmetry 2020,12, 1890 9 of 17
Table 5.
Results after T-testing pairwise distances, reported as the Procrustes distance, and pvalues after 10,000 permutations of runs. For site abbreviations, see Table 2.
Symbols denote sexes: males () and females ().
Species Site and Sex Distance
pValue
C
Procrustes
pValue
IO
Procrustes
pValue
EV
Procrustes
pValue
IV
Procrustes
pValue
C
Procrustes
pValue
IO
Procrustes
pValue
EV
Procrustes
pValue
Pterostichus melas
IO 0.0088 0.4891
EV 0.0152 0.0572 0.0189 <0.0001
IV 0.0187 0.001 0.0247 <0.0001 0.0152 <0.0001
C0.0275 0.0002 0.0344 <0.0001 0.0234 <0.0001 0.0243 <0.0001
IO 0.0161 0.0143 0.0219 <0.0001 0.0164 <0.0001 0.0147 <0.0001 0.0176 0.0001
EV 0.0237 0.0003 0.0294 <0.0001 0.0181 <0.0001 0.0227 <0.0001 0.0113 0.0228 0.0177 <0.0001
IV 0.025 0.0003 0.032 <0.0001 0.0217 <0.0001 0.016 <0.0001 0.0123 0.0063 0.0116 0.0001 0.0161 <0.0001
Species Site and Sex Distance
pValue
EO
Procrustes
pValue
IO
Procrustes
pValue
EV
Procrustes
pValue
IV
Procrustes
pValue
EO
Procrustes
pValue
IO
Procrustes
pValue
EV
Procrustes
pValue
Poecilus koyi
IO 0.0071 0.0672
EV 0.006 0.1352 0.01 0.0036
IV 0.0055 0.2602 0.0039 0.6577 0.0091 0.0169
EO 0.0109 0.0001 0.0155 <0.0001 0.0107 0.0002 0.014 <0.0001
IO 0.0133 <0.0001 0.0163 <0.0001 0.0136 <0.0001 0.0162 <0.0001 0.0079 0.0005
EV 0.0125 <0.0001 0.0171 <0.0001 0.0108 <0.0001 0.0156 <0.0001 0.0034 0.3014 0.0096 <0.0001
IV 0.0094 0.0014 0.0134 0.0001 0.0108 0.0002 0.0123 0.0001 0.0045 0.1098 0.0061 0.0057 0.007 0.0021
Symmetry 2020,12, 1890 10 of 17
Figure 6.
A CVA applied to symmetrical components of the shape for P. koyi. Female (
A
) and male (
B
)
individuals are shown separately (
males,
females). Individuals from the IV site are presented by
blue dots, those from EV by red dots, IO by green, and EO by grey dots.
FA values (mean squares value of ind*side interaction, corrected for error variance) diered
between the studied species, with P. melas displaying higher FA values (Figure 7, with the T-test on
normalized Procrustes FA scores yielding t-value =5.97, p<0.05). Regarding P. melas, populations
from all three agroecosystems had higher Procrustes FA scores than the control population, with the
FA of the population from EV being the highest and diering significantly from other populations
(ANOVA and Tukey HSD Post hoc, F =11.01, p<0.05). Individuals from the olive grove had lower FA
values than those from the vineyards, but a bit higher than individuals from the control sites (Figure 7).
There was no significant dierence in FA values between the males and females of P. melas within
or between the sites (Factorial ANOVA, p<0.05). In P. koyi, on the other hand, males were more
asymmetrical at every site except EV (Table 6, Figure 7), but there were no significant dierences in
FA values between its populations, neither for the site nor for sex and management (IPM vs. EPM)
eects (Factorial ANOVA, p>0.05). Regression of the Mahalanobis and Procrustes FAs with individual
shapes was performed in order to see the magnitude in FA values, showing olive groves as indeed less
influential on FA than vineyards (Figure 8).
Table 6.
Results of the Procrustres ANOVA on populations of P. melas and P. koyi. All values in the
table depict individual*side interaction (fluctuating asymmetry).
Species Population MS Error MS MS-Error (MS) df F P
Pterostichus melas
C1.67888 ×1051.7489 ×1061.50399 ×105308
11.58
<0.0001
C2.02496 ×1051.7489 ×1061.85007 ×105434
11.81
<0.0001
IO 2.03913 ×1057.246 ×1071.96667 ×105672
28.14
<0.0001
IO 2.07874 ×1056.739 ×1072.01135 ×105672
30.84
<0.0001
EV 3.85526 ×1051.1008 ×1063.74518 ×105672
35.02
<0.0001
EV 3.75624 ×1051.2696 ×1063.62928 ×105700
29.59
<0.0001
IV 2.90281 ×1051.147 ×1062.78811 ×105686
25.31
<0.0001
IV 2.83715 ×1051.1566 ×1062.72149 ×105686
24.53
<0.0001
Poecilus koyi
EO 1.01514 ×1059.295 ×1079.2219 ×106378
10.92
<0.0001
EO 1.48921 ×1051.0947 ×1061.37974 ×105742
13.6
<0.0001
IO 1.00047 ×1059.719 ×1079.0328 ×106350
10.29
<0.0001
IO 1.33458 ×1059.843 ×1071.23615 ×105728
13.56
<0.0001
EV 1.3171 ×1051.1953 ×1061.19757 ×105378
11.02
<0.0001
EV 1.26104 ×1051.3333 ×1061.16779 ×105
1022 9.01
<0.0001
IV 1.20838 ×1051.3234 ×1061.07604 ×105308
9.42
<0.0001
IV 1.42704 ×1051.5912 ×1061.26792 ×105896
8.97
<0.0001
Symmetry 2020,12, 1890 11 of 17
Figure 7.
Histogram of the intensity of fluctuating asymmetry (FA) in dierent populations and sexes
of P. melas and P. koyi. Blue bars show values obtained from populations of P. melas, and yellow bars
depict populations of P. koyi. Mean squares of ind*side interaction corrected for error variance were
used as a corrected measure of FA. (
Symmetry 2020, 12, x FOR PEER REVIEW 11 of 17
Table 6. Results of the Procrustres ANOVA on populations of P. melas and P. koyi. All values in the
table depict individual*side interaction (fluctuating asymmetry).
Species Population MS Error MS MS-Error (MS) df F P
Pterostichus
melas
C 1.67888 × 105 1.7489 × 106 1.50399 × 105 308 11.58 <0.0001
C 2.02496 × 105 1.7489 × 106 1.85007 × 105 434 11.81 <0.0001
IO 2.03913 × 105 7.246 × 107 1.96667 × 105 672 28.14 <0.0001
IO 2.07874 × 105 6.739 × 107 2.01135 × 105 672 30.84 <0.0001
EV 3.85526 × 105 1.1008 × 106 3.74518 × 105 672 35.02 <0.0001
EV 3.75624 × 105 1.2696 × 106 3.62928 × 105 700 29.59 <0.0001
IV 2.90281 × 105 1.147 × 106 2.78811 × 105 686 25.31 <0.0001
IV 2.83715 × 105 1.1566 × 106 2.72149 × 105 686 24.53 <0.0001
Poecilus koyi
EO 1.01514 × 105 9.295 × 107 9.2219 × 106 378 10.92 <0.0001
EO 1.48921 × 105 1.0947 × 106 1.37974 × 105 742 13.6 <0.0001
IO 1.00047 × 105 9.719 × 107 9.0328 × 106 350 10.29 <0.0001
IO 1.33458 × 105 9.843 × 107 1.23615 × 105 728 13.56 <0.0001
EV 1.3171 × 105 1.1953 × 106 1.19757 × 105 378 11.02 <0.0001
EV 1.26104 × 105 1.3333 × 106 1.16779 × 105 1022 9.01 <0.0001
IV 1.20838 × 105 1.3234 × 106 1.07604 × 105 308 9.42 <0.0001
IV 1.42704 × 105 1.5912 × 106 1.26792 × 105 896 8.97 <0.0001
Figure 7. Histogram of the intensity of fluctuating asymmetry (FA) in different populations and sexes
of P. melas and P. koyi. Blue bars show values obtained from populations of P. melas, and yellow bars
depict populations of P. koyi. Mean squares of ind*side interaction corrected for error variance were
used as a corrected measure of FA. ( denotes P. melas populations significantly different in
Procrustes FA scores from others, ANOVA, p < 0.05).
0
0.000005
0.00001
0.000015
0.00002
0.000025
0.00003
0.000035
0.00004
C
C
IO
IO
EV
EV
IV
IV
EO
EO
IO
IO
EV
EV
IV
IV
Pterostichus melas Poecilus koyi
FA corrected
denotes P. melas populations significantly dierent in Procrustes
FA scores from others, ANOVA, p<0.05).
Figure 8.
Regression of the Mahalanobis (
A
) and Procrustes (
B
) FAs for the shapes of P. melas individual
species from four sites (IO =green, C =black, IV =blue, and EV =red).
Symmetry 2020,12, 1890 12 of 17
4. Discussion
The FA, as a subtle deviation from bilateral symmetry, has long been of interest to researchers
studying the eects that changes in environmental quality have on organisms [
44
]. In this study,
we employed FA measurements on two predatory carabids, P. melas and P. koyi, which were common
and relatively abundant in the study area [
40
]. The FA was detected in every population, regardless
of species, site, or sex, but FA values diered between the species and between populations (Table 6,
Figure 7). Besides dierences in FA, Procrustes ANOVA and CVA revealed a significant variation in
shape and size of the tested populations of both species between study sites, as well between sexes for
P. melas.
Canonical variate analysis showed significant dierences in shape between populations for both
P. melas and P. koyi, with the exception of P. koyi females from the olive groves, which did not statistically
dier from each other (Tables 4and 5). Significant shape variation between sexes and among sites
was noted for other species of beetles [
18
,
45
49
]. In this study, P. melas females from both vineyards
were larger than females from the control. The same observation was made for males, but it was not
statistically significant. It is generally considered that environmental conditions cause variations in size
between populations, whereas variations in shape reflect variations in the genetic composition [
50
,
51
].
In addition, it is possible that P. melas, a species with weak dispersal power, developed intraspecific
variability in shape and size as a result of dierent habitat conditions at the study sites. The fact that the
control group for P. melas was pooled from two dierent locations likely increased the shape variation.
Alibert et al. [
46
] also found females to be larger than males, but there was no size dierence between
populations from dierent sites. Fitness in females is often size-related, and is profoundly influenced
by conditions during larval development [
52
]. Such female-biased sexual dimorphism is commonly
observed in invertebrate taxa, and it is hypothesized to be a result of a positive correlation between
fecundity and female abdomen size [45].
Higher FA scores within P. melas populations from vineyards than those in olive orchards may
indicate practices run within olive groves as potentially less adverse to predatory carabids. Contrary to
our assumptions that IPM would produce higher levels of asymmetry within populations due to a
broader spectrum of pesticides being applied [
23
,
53
,
54
], higher FA values were detected in both species
from sites with ecological pest control practices, although for P. koyi, those dierences were not as
prominent as in P. melas, which appeared to be the less robust species. However, these results corroborate
with findings from some previous research where FA levels did not dier between populations of
ground beetle species among continuous forest and fragmentation habitats in a plantation area [
22
],
or with dierent types of management in orchards [15].
A higher FA score for the P. melas from EV can be due to the frequency of mechanical tillage of the
soil being the highest at this study site (Table 1). Carabids, as a part of the soil fauna, can be aected by
mechanical disturbances of the soil, especially during their development. Ben
í
tez et al. [
3
] reported
higher FA scores in populations within annual arable lands as more unstable than in a perennial
agroecosystem. Additionally, these results are supported by the fact that this population has the highest
density at the stated study site, as higher densities in populations enhance intraspecific competition,
which reduces individual food availability and increases the FA [
22
,
55
]. Furthermore, Nattero et al. [
6
]
showed that pesticide treatment levels of asymmetry decreased due to the higher mortality rates of
less adapted individuals, often those with higher asymmetry levels. This potentially can explain
higher FA values at sites with EPM, compared with those using IPM, meaning that anthropogenic
factors (e.g., pesticide use and tillage) act as selective pressures that favor more symmetric individuals.
On the other hand, individuals within control populations from pristine habitats develop without
anthropogenic factors disrupting their developmental stability and, because of that, are the least
asymmetric. Floate and Fox [
56
] oered a dierential mortality hypothesis as an explanation for
this phenomenon. After laboratory experiments conducted on dipterans, they observed that only
individuals that survived stress could be captured later, which means our sample consisted of less
aected individuals. Several previous studies showed a correlation between survival probability
Symmetry 2020,12, 1890 13 of 17
and the level of FA in insects [
57
59
]. Keeping in mind that field conditions are even more variable
than laboratory conditions, more research is needed to establish FA as an indicator of the harmful
eects of pesticides on predatory organisms. Research on two dierent populations of P. melas in
Croatian agroecosystems did not reveal any significant dierence between FA values when compared
with the control [
3
], and [
60
] did not detect any relationship between FA and heavy metal pollution
in populations of the wolf spider Pirata piraticus. Furthermore, more common species can be less
susceptible to stress in their environment [
17
], which could also explain why there was little dierence
in the FA values between P. koyi populations, as it was one of the dominant species at our research sites.
The same observation was made for rodents from two dierent farms, with the conclusion that the
eects of the farming practice would aect generalist species less than it would aect specialists [
5
].
P. melas, as a predator from natural forest ecosystems, was able to adapt to conditions in Mediterranean
areas managed for agricultural purposes, such as olive groves [
13
]. It is considered that this adaptation
was boosted by the turnover of specimen from surrounding natural environments to agricultural
lands. This could also be the reason why P. melas was more susceptible to asymmetry changes, due to
agricultural practices in areas where this turnover was made more dicult. Furthermore, the reason for
this dierence in asymmetry levels between the two species could be due to the fact that P. koyi is more
adapted to dierent types of agricultural lands, where often this species is among the most abundant
ones. Dierent responses between species in terms of FA, and species sensitivity and adaptation to
agricultural practices, may be strongly connected to its historical presence in the natural environment
surrounding arable land [
17
,
61
]. Mazeed [
62
] demonstrated that a honeybee species not native to the
area had a higher FA than the native species, meaning that the geographical location and adaptation
to dierent environmental conditions, to which each organism is subjected, may aect the degree
of asymmetry.
Dierences in FA between sexes were noted in previous studies [
18
,
23
,
24
], where males of studied
species had been regularly less asymmetric compared with females. In our study, P. koyi males were
more asymmetrical at every site except EV, while no dierences between sexes were noted in P. melas
with the exception of the control group, where the males were a bit more asymmetrical. Since the FA in
P. koyi was much lower than in P. melas, and the lowest FA score was recorded for the P. melas control
group, we believe that the dierences in FA between sexes could be ignored in this case. This small
deviation in FA between sexes within the control group could be due to pulling P. melas from two
dierent pristine locations.
5. Conclusions
As expected, the lowest FA intensity was found in the control population of P. melas, which goes in
favor of FA as a method to be used in assessing certain environmental stresses caused by agricultural
practices. However, the results indicate that dierent species do not have the same response in respect
to FA. P. koyi showed no patterns in FA between populations from dierent agricultural sites, while the
populations of P. melas from olive groves displayed a lower FA than populations from vineyards,
and the FA values were closer to the control group from the pristine habitat. Thus, we recommend
P. melas as a test species for future studies on environmental stress using FA. Furthermore, FA singled
out olive groves as a potentially less adverse habitat to predatory carabids when compared with
vineyards, regardless of the pest management (IPM or EPM). In future studies, the FA of P. melas can
be used as a method to indicate agricultural practices that are closer to natural.
Author Contributions:
Conceptualization, L.Š.J. and H.A.B.; methodology, L.Š.J., H.A.B., and M.J.; formal analysis,
H.A.B., L.I.T., L.Š.J., and B.A.; sampling design and field sampling, L.Š.J., T.K., B.A., and L.I.T.; validation and
interpretation of data, H.A.B., L.Š.J., and L.I.T.; resources, L.Š.J.; writing—original draft preparation, L.I.T., B.A.,
and L.Š.J.; writing—review and editing, L.Š.J., H.A.B., T.K., and M.J.; visualization, H.A.B., L.I.T., and L.Š.J.
All authors have read and agreed to the published version of the manuscript.
Symmetry 2020,12, 1890 14 of 17
Funding:
This research was funded by The Croatian Science Foundation under the MEDITERATRI Project
(UIP-2017-05-1046) granted to Lucija Šeri´c Jelaska, and by the Department of Biology, Faculty of Science at the
University of Zagreb.
Acknowledgments:
We are thankful to Vedran Bahun for his help in the field and in sorting the samples in the
lab, to Karlo Vinkovi´c for creating the map of study sites, and to three reviewers for their valuable comments
on the manuscript. H.B. thanks the Fondecyt de Iniciacion 11180366 from the ANID of the Chilean government;
T.K. thanks the Interreg-IPA PESCAR project and M.J. thanks to the Varaždin City Museum.
Conflicts of Interest: The authors declare no conflict of interest.
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... Several studies have indicated that the main sources of stress that induce fluctuating asymmetry in reptile populations are due to human disturbances (Castillo & González-Rivas, 2023) which, together with genetic changes (Clarke, 1995;Vervust et al., 2008), are sources of the main phenotypic variations (Castillo & González-Rivas, 2023). However, extreme temperatures, lack of food, presence of predators and parasitism are also important sources of phenotypic variation and influence fluctuating asymmetry (Bonn et al., 1996;Sasal & Pampoulie, 2000;Brown & Brown, 2002;Pečínková et al., 2007;Tatalović et al., 2020). There is evidence for a relationship between parasites and fluctuating asymmetry, which shows that different levels of asymmetry in a population may be due to a strong ex-posure to parasites. ...
... The purpose of this study is to investigate whether there is a statistically significant association between fluctuating asymmetry levels and the presence of parasites. Overall, we endorse on a limited basis, as did previous researchers (Tatalović et al., 2020), for the idea that FA represents an index of individual quality. These types of analyses are relevant for a more integrated estimation of the ecological status of populations and help to understand the consequences at the community level (Almeida et al., 2023). ...
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A comparative analysis and investigation of the relationship between the parasite Physaloptera retusa (Physalopteridae) and the fluctuating asymmetry (FA) of the lizard Liolaemus darwinii from Argentina was carried out. In this study, the effect produced by the presence of P. retusa on the symmetry of the heads of lizards has been investigated. Thirty specimens (12 parasitised and 18 non-parasitised) from a population of L. darwinii were examined. The results obtained showed statistical differences in fluctuating asymmetry between parasitised and non-parasitised specimens; the parasitised L. darwinii specimens presented greater head asymmetry compared to the non-parasitised ones, confirming the initial hypothesis. The present study allowed us to consider the analysis of fluctuating asymmetry as a tool that, together with parasites, can be considered and be useful in studies related to reptile populations and in conservation biology.
... exposure to agrochemicals, shelter scarcity and microclimatic changes), FA has been used as a biomarker to assess the impact of intensive agriculture on their development. Although literature is not extensive, different studies have associated intensive agriculture with increased FA in wall lizards , carabids (Tatalovi c et al., 2020) and small mammals (Coda et al., 2016(Coda et al., , 2018. A recent review by Ben ıtez et al., 2020 supported the use of FA as a biomarker to assess the effect of stressors related to agriculture, also suggesting the use of landmark-based geometric morphometrics (GM, Bookstein, 1997) to considerably enhance such endeavours. ...
... These results suggest that sub-optimal conditions experienced by vipers inhabiting intensive agricultural habitats are affecting the efficiency of developmental processes, and thus, the increase in DI is reflected in the FA levels of dorsal head shape. This suggestion is in agreement with studies in other taxa (Coda et al., 2016(Coda et al., , 2018Tatalovi c et al., 2020), and supports the usefulness of GM to address FA of shape as an indicator of agriculture-linked environmental stress . Different stressors associated with intensive agriculture could be responsible for the increment of uAI in V. aspis zinnikeri dorsal head shape. ...
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Habitats resulting from agricultural intensification harbour a wide array of stressors for resident organisms. Environmental stress during development can leave imprints on the phenotype that are useful as indicators of its impairment. Among them, fluctuating asymmetry (FA), considered the morphological manifestation of developmental instability (DI), may reflect the increment of developmental noise and the inefficiency of buffering mechanisms. Here, we evaluated for the first time the impact of intensive agriculture on the development of a viviparous reptile, the asp viper, Vipera aspis (Linnaeus 1758), by means of FA. To this end, we first quantified unsigned asymmetry indexes (uAI) of dorsal head shape captured by geometric morphometrics (GM) and of seven pholidotic traits in 127 and 482 individuals, respectively. Using linear models, we then tested for variation in uAI according to habitat type. Furthermore, we tested the effect of climatic variables with a potential influence on development, and thus, on FA. We performed analyses at the V. aspis zinnikeri subspecies range and at the population level, including three well‐sampled natural localities (NAT) and three sites of intensive agriculture (AGR). We found significantly higher FA levels in dorsal head shape in individuals from agricultural habitats than in those from natural habitats, which suggests that asp viper development is impaired by stressors resulting from agricultural intensification. Our results also point to a lower sensitivity of traditional morphological tools to capture the signal that intensive agriculture leaves on development and a lack of capacity of climatic factors in isolation to predict variation in the degree of asymmetry. In addition to encouraging the use of high‐precision methods, we hypothesize on the overall effect of different stressors linked to intensive agriculture as the real constraint.
... Finally, the association between longevity and FA of wings in male and female flies is addressed with a strong positive association between the two. FA often represents developmental instability (Debat & Peronnet, 2013) where higher FA indicates greater asymmetry and poorer environmental or nutritional conditions (Ivanković Tatalović et al., 2020). However, the positive correlation between FA and longevity in the present study suggests a different inference. ...
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Longevity plays a significant role in determining an animal's overall health. Nutrition is a key factor that can influence an animal's longevity. Studying the relationship between wing symmetry and longevity provides a useful approach to understanding how nutrition can affect both longevity and symmetry. The effect of Pomegranate Juice (PJ) and Pomegranate Peel (PP) on longevity and Fluctuating Asymmetry (FA) of wings in male and female Drosophila melanogaster was analysed using Geometric Morphometric Analysis (GMA). The study revealed that PJ and PP-treated flies showed increased longevity and the highest FA compared to control in both males and females. Between PJ and PP-treated flies, PJ had the highest longevity in both males and females whereas PP flies had higher FA in both sexes. Between the sexes, females had lower longevity and FA values compared to males. Overall GMA study reveals shape variation for wings and the presence of a strong FA in PJ and PP flies. Pearson correlation revealed a strong positive association between longevity and wing FA.
... This small, random deviation from ideal symmetry measures developmental instability, a sensitive biomarker of environmental stress [24][25][26]35]. Higher FA has been proved to be a measure of susceptibility to pesticides in, for example, lizards [36], frogs [37], birds [38], fish [39,40], and invertebrates: Odonata [41], Apis [42], Triatoma [43,44], carabid beetles [45], and Drosophila [46,47]. ...
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Long-term exposure to low concentrations of toxic substances can cause several adverse consequences ranging from molecular to morphological. Sublethal doses may also lead to increased tolerance in the offspring of surviving individuals. One of the consequences of such stress is deviations from the ideal body symmetry during development, reflected by increased levels of fluctuating asymmetry (FA). This research aimed to verify FA in the wing veins of insects belonging to the Drosophilidae family—Drosophila suzukii, a fruit pest controlled by the insecticide acetamiprid, a neonicotinoid. To determine whether FA varied depending on insecticides present in the diet, multigenerational cultures of D. suzukii were carried out on media supplemented with different concentrations (below the LC50) of two insecticides. Nicotine was used as a positive control. Fecundity decreased, the number of insects decreased, and breeding did not continue beyond the tenth generation. However, the FA level at different concentrations was similar, and high FA values were observed even at lower acetamiprid concentrations. We did not see significant changes in FA levels in subsequent generations. D. suzukii proved extremely sensitive to acetamiprid, and FA is a good index of this sensitivity.
... Theoretically, it is assumed that FA levels increase under stress conditions and that the application of sub-lethal doses of insecticides destabilizes pest development. Indeed, in some cases, fluctuating asymmetry increases proportionally with the level of application rates [21,22,25,27,28,62,63]. Although increased FA is an expected acute response to stress, the adaptation to continuous stress, such as in the transgenerational exposure of beetles to insecticides, could change this relationship through the stabilization of development in order to reach the adaptive maximum in a certain environment. ...
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One method of crop protection is the application of a widely used group of pesticides—pyrethroids. As xenobiotics, sub-lethal doses of insecticides cause stress in pests, resulting in a change in the shape and size of their organs or bodies. The stress caused by pesticides may lead to acute destabilization of development, but also to transgenerational canalization through the process of genetic assimilation. Fluctuating asymmetry (FA), small random deviations between the right and left sides of bilaterally symmetrical traits, is an outcome of developmental instability and is a measurable indicator of phenotypic response to stress. We exposed four populations of the seed beetle Acanthoscelides obtectus to sub-lethal doses of cypermethrin for ten generations in a laboratory evolution experiment. Using geometric morphometrics, we analyzed size and shape changes and the level of fluctuating asymmetry in untreated beetles and in samples from the fifth and tenth generation. Exposure over ten generations led to an increase in the body size of the beetles, shortening of their pronotum and elongation of their thorax and abdomen. After ten generations of exposure to cypermethrin, FA levels decreased, indicating a canalization of development. This study provides new insights into the phenotypic markers of environmental pollution from agricultural activities.
... The exposure of parasitoids to stressful environments, whether due to natural causes such as drastic temperature fluctuations, prolonged periods of drought, long rainy periods, or constant exposure to insecticides, can result in an adaptive advantage for parasitoids, ensuring their ability to parasitize (Benítez et al., 2018;Tatalović et al., 2020). These effects can be results in evolutionary implications for individuals (Žikić et al., 2017). ...
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