19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
160 Journal of Vector Ecology June 2016

Genetic and phenotypic variation in central and northern European populations of

Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera, Culicidae)
Ljubinka Francuski1, Vesna Milankov*1, Jasmina Ludoški1, Bosiljka Krtinić2, Jan O. Lundström3,
Gábor Kemenesi4, and Jakab Ferenc4
1
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia,
vesna.milankov@dbe.uns.ac.rs
2
Ciklonizacija, Primorska 76, 21000 Novi Sad, Serbia
3
Department of Medical Biochemistry and Microbiology/Zoonotic Science Centre, Uppsala University, SE-75123 Uppsala, Sweden
4
Virological Research Group, Szentágothai Research Center, University of Pécs, Pécs, Hungary

Received 25 December 2015; Accepted 30 March 2016

ABSTRACT: The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the
vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies
worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic
variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape
genetics approach (FST, STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern
populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations,
while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial
analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on
spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation-by-Distance patterns.
Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low-to-moderate FST values on
a broader geographical scale indicate a continuous between-population exchange of individuals, including considerable gene flow on
the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed
data considering population structure in the light of vector control strategies of the mosquito from public health importance. Journal of
Vector Ecology 41 (1): 160-171. 2016.

Keyword Index: Landscape genetics, wing geometric morphometrics, vector control program, genetics, genotype-phenotype association.

INTRODUCTION useful (Cywinska et al. 2006). However, Krtinić et al. (2013)

The floodwater mosquito Aedes (Aedimorphus) vexans
(Meigen) is an aggressive species with long flight range and a
worldwide distribution (Knight and Stone 1977, Reinert et al.
2004). Before the implementation of efficient mosquito control,
this mosquito was a nuisance in large areas surrounding the flood
plains and deltas of the River Rhein, the River Danube, and the
River Rhone in mainland Europe (Becker et al. 2010). In addition,
Ae. vexans is the main vector of Tahyna virus in central Europe
(Lundström 1994, 1999) and a potential vector of dog heartworm
Dirofilaria immitis in Europe (Yildrim et al. 2011, Rudolf et al.
2014). In total, 30 arboviruses have been isolated from the species
worldwide (Horsfall and Novak 1991), including West Nile virus
and Rift Valley fever virus (Goddard et al. 2002, Miller et al. 2002).
Based on current taxonomy, this important and interesting
mosquito forms three subspecies worldwide: Aedes vexans vexans
(Meigen) found in East Asia, Aedes vexans arabiensis (Patton)
found in Africa and Europe, and Aedes vexans nipponii (Theobald)
found in Southeast Asia (Knight and Stone 1977, Reinert 1973,
Reinert et al. 2004). Distinction between subspecies was defined
by adult morphological traits, including tergites, mid-tibia, and
male palpi (Gutsevich 1971, White 1975) and ND4 mitochondrial
DNA (mtDNA), while the 5’ end of cytochrome oxidase I
mtDNA (‘barcoding’ fragment) was found to be only partially
used population diagnostic isozyme loci and observed a lack of
gene flow between Ae. vexans populations in North America and
Europe, indicating the presence of independent gene pools. In
addition to taxonomic issues, understanding the species potential
for adaptation to changeable environments, as well as knowing
population connectivity, is of interest for the long-term ability of
performing efficient control programs that could reduce nuisance
and the risk for spread of human pathogens by Ae. vexans in
Europe and elsewhere. Consequently, adaptability of pest and
invasive species and their vector capacities for transmission of
disease pathogens have been a focus in vector control programs
(Forsman 2014).
Therefore, we applied an integrative approach to study
phenotypic and genetic diversity of Ae. vexans across Europe.
Knowledge of the variation in wing traits can contribute to better
insights into the population structure of vector-borne pathogens
and provide necessary data on dispersion patterns, and thus
transmission pathogens (Ruiz-Garcia et al. 2003, Poolprasert et
al. 2008). Although it has been considered as a sensible method
for studying intraspecific (Krtinić et al. 2015, 2016, Petersen et
al. 2015) and interspecific (Jaramillo-O et al. 2015, Laurito et
al. 2015) phenotypic variation in mosquitoes, wing geometric
morphometrics has not yet been applied to Ae. vexans in Europe.
We are interested in whether populations from northern and
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Vol. 41, no. 1 Journal of Vector Ecology 161

central Europe are different based on wing size and shape. This
is of particular importance since these two traits are considered
to be genetically determined by two different gene clusters
(Carreira et al. 2011). In addition, wing size is thought to be a
more environmentally sensitive trait (Dujardin 2008, Gómez et al.
2014), and thus less heritable than wing shape (Jirakanjanakit et al.
2008). Similarly, we were interested in any genetic differentiation
between populations that originated from geographically distant
regions. Thus, we applied individual- and population-based
methods for assessing the spatial structure of the European Ae.
vexans.

MATERIALS AND METHODS

Mosquito collection
Five populations of Ae. vexans were sampled across Europe:
in Serbia (Novi Sad and Zrenjanin, Vojvodina Province), Hungary
(city Pécs), and Sweden (Gysinge, Deje). The Serbian mosquito
collections were performed in Novi Sad, Serbia (29 June 2013.)
and in Zrenjanin, Serbia (5 May 2013.). The Hungarian mosquito
collection was performed in 2012 in Pécs, Hungary. The Swedish
mosquito collections were performed in Gysinge, Gävleborg
Figure 1. Map of Europe showing the location of the five Aedes
County (20 October 2007), and in Deje, Värmland County (4 July
vexans populations studied. 1. Gysinge, Sweden (60°17′ N; 16°53′
2012). Populations were defined a priori based on sample origin
E); 2. Deje, Sweden (59°36′ N; 13°26′ E); 3. Pécs, Hungary (46°4′
and applying the biological definition of a population (Waples and
N; 18°13′ E); 4. Zrenjanin, Serbia (45°23′ N; 20°22′ E); 5. Novi Sad,
Gaggiotti 2006).
Serbia (45°15´ N; 19°50´ E).
Mosquito control, using organophosphates, pyrethroids,
insect-growth regulators, and biological agents have been
applied intensively in Novi Sad since the 1970s and early 1980s, 31, RSNS 14), and an additional 33 (SEGYS 9, SEDEJ 10, HUPEC
and occasionally against the population in Zrenjanin (not every 11, RSZR 3) and 54 (SEDEJ 7, RSNS 47) specimens were included
year and less intense during the season) since the 1980s. Prior in allozyme and geometric morphometric analyses, respectively.
to mosquito sampling, no mosquito control activities had been Species identification was performed based on the morphological
directed at the investigated Ae. vexans populations in Sweden and taxonomic characters defined in Becker et al. (2010) prior to
Hungary. However, since July, 2009, a mosquito control program preparation of wings and body tissue extracts, and nomenclature
using VectoBac G (containing Bacillus thuringiensis israelensis) follows the suggestion by Wilkerson et al. (2015). Microscopic
has been active against Aedes sticticus and other floodwater slides of wings and wing images used for morphological studies
mosquitoes in the Gysinge area of the River Dalälven flood plains. were deposited at the Department of Biology and Ecology, Faculty
Dry ice-baited miniature traps were used for sampling adult of Sciences, University of Novi Sad.
female mosquitoes during one night in each of the five study areas.
Specimens were stored at −20° C until use in allozyme analysis Allozyme analysis
and wing geometric morphometrics (Figure 1; sample codes A total of 133 specimens from Sweden (47), Hungary (38),
and numbers of collected specimens are provided in Table 1). A and Serbia (48) was included in the allozyme analysis to quantify
total of 100 specimens were used in both allozyme and geometric genetic diversity at the intra- and interpopulation levels (Figure 1,
morphometric analyses (SEGYS 11, SEDEJ 17, HUPEC 27, RSZR Table 1). Allozyme polymorphism was studied for ten allozymes

Table 1. The number of individual Aedes vexans females sampled from five populations in three European countries
and assayed by wing geometric morphometrics and allozyme analysis.
Wing geometric Allozyme Included in
Country Locality Abbrevation
morphometrics analysis both analyses
Sweden Gysinge SEGYS 11 20 11
Deje SEDEJ 24 27 17
Hungary Pécs HUPEC 27 38 27
Serbia Zrenjanin RSZR* 31 34 31
Novi Sad RSNS* 61 14 14
Total 154 133 100
*RSZR and RSNS in Serbia are insecticide-treated populations.

162 Journal of Vector Ecology June 2016
at 14 loci by vertical polyacrylamide gel electrophoresis. Enzymes
were extracted from body tissues in 0.05 M Tris-HCl (pH 7.1) and
homogenates were centrifuged at 13,000 r.p.m. for 3 min at 6° C. The
loci studied were: aspartate amino transferase (2.6.1.1. AAT; Aat),
aldehyde oxidase (E.C. 1.2.3.1. AO; Ao), esterase (E.C. 3.1.1.1. EST;
Est-6), glucosephosphate isomerase (5.3.1.9. GPI; Gpi), glycerol
3-phosphate dehydrogenase (1.1.1.8. GPD; Gpd-2), hexokinase
(2.7.1.1. HK; three loci: Hk-2, Hk-3, Hk-4), β-hydroxy acid
dehydrogenase (1.1.1.30. HAD; Had), isocitrate dehydrogenase
(1.1.1.42. IDH; two loci: Idh-1, Idh-2), malic enzyme (1.1.1.40.
ME; Me), and phosphoglucomutase (2.7.5.1. PGM; two loci: Pgm-
1, Pgm-2,). A tris-borate-ethylenediaminetetraacetic acid buffer
system (pH 8.9) was used to assay AO, EST, GPI, HK, ME, and
PGM, whereas a tris-citrate buffer system (pH 7.1) was used for
analysis of AAT, GPD, HAD, and IDH.
Electrophoresis was conducted at 90 mA (135–220 V) for
3–4 h. Specimens from all samples were separated concurrently
on all gels to facilitate comparisons of electrophoretic mobility.
Genotype and allele frequencies were calculated directly from
observed banding patterns based on the genetic interpretation of
zymograms.
Wing geometric morphometrics
Female mosquitoes from Sweden (35), Hungary (27), and
Serbia (92) were analyzed to study variation in wing size and
shape by applying geometric morphometrics (Table 1). Both
left and right wings of the 154 individuals were removed and
mounted in Hoyer’s medium between microscope slides, and
digital images were taken with a Leica DFC320 camera connected
to a Leica MZ12.5 stereomicroscope. Images of right wings were
used for the morphometric analysis, and a set of 16 landmarks
(type I) (Figure 2) were digitized using TpsDig 2.17 (Rohlf 2013).
To evaluate a measurement error due to the digitizing process,
each wing was digitized three times by the same person (JL) and
subjected to a Procrustes ANOVA (Klingenberg et al. 2002). The
results showed that the Procrustes mean squares for individual
variation substantially exceeded measurement error. Therefore,
for the remaining analyses, the shape data from the replicate
measurements of each specimen were averaged.
To explore wing size and shape variation, raw landmark
coordinates were superimposed using a full Procrustes fit
procedure (Dryden and Mardia 1998) that removes variation due
to the scale, position, and orientation of landmark configurations.
Wing size variation was examined using centroid size (the square
root of the sum of the squared distance between each landmark and
the wing centroid). Differences in wing size between populations
were assessed using one-way ANOVA followed by a multiple
Figure 2. Right wing of Aedes vexans showing the location of the
16 landmarks selected for geometric morphometric analysis.
19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
comparison test (Tukey’s pairwise post-hoc test). To test for the
presence of allometry, the relationship between size and shape, a
multivariate regression of Procrustes coordinates against centroid
size on pooled within-group (pooled by population) variation,
was conducted. The significance of the allometry was calculated
with a permutation test with 10,000 iterations.
To determine differences in wing shape, the set of shape
variables (a matrix of Procrustes coordinates) were subjected
to the Procrustes ANOVA (analogous to MANOVA) and
Canonical Variates Analysis (CVA) with 10,000 permutations.
Pairwise differences were quantified using Procrustes distances
and compared with a permutation test with 10,000 iterations.
To investigate phenetic relationships between populations, an
unweighted pair-group method using an arithmetic average
(UPGMA) cluster analysis was performed on the matrix of
Procrustes distances.
All statistical analyses were done using MorphoJ version
1.06d (Klingenberg 2011) and PAST (Paleontological Statistics)
version 3.07 (Hammer et al. 2001) software.
Population genetic structure and genetic differentiation
To assess if the sampling and the allozyme alleles used were
enough to detect a structure if there was one (i.e., power of the
tests), we conducted several simulations using the computer
program POWSIM version 4.1 (Ryman and Palm 2006). The
program detects significant differentiation (using Chi-squared
and Fisher’s exact tests) under a specified level of population
divergence given by 1 - (1 - 1/2Ne)t, where t is the time since
divergence and Ne is the effective population size. We simulated
scenarios assuming different sample sizes and levels of divergence,
with populations being separated for 100 generations, each with
Ne of 5,000. The values were used to simulate a case where the
split of populations was very recent and the populations were very
large. Hence, this was a very conservative test of power, as if time
was longer and Ne was smaller, it would be much easier to find
significant results. Significance estimates were based on 1,000
independent simulations.
Calculated parameters of the population genetic structure
were based on allozyme data and were corrected using Levene’s
(1949) formula for small samples using BIOSYS-2 (Swofford and
Selander 1989). The analysis included determining genotypic and
allelic frequencies, percentage of polymorphic loci (P), the mean
observed (Ho) and expected (He) heterozygosity, and the presence
of rare and major alleles. Rare alleles were defined as variants with
frequencies of ≤ 0.05 (Ayala et al. 1974, Munstermann 1994).
Although the distinction between commonness and rareness
is arbitrary, in this study major alleles are defined as present at
frequencies of ≥0.5 (Ghosh et al. 1999).
Genetic diversity was assessed by calculating the mean gene
diversity over loci using ARLEQUIN, version 3.11 (Excoffier et
al. 2005). Theta (θH) for the allozyme data was estimated from the
expected homozygosity (Ewens 1972). Estimates of θ (where θ =
2Nu, N is the effective population size and u is the mean mutation
rate per locus per generation) were based on the infinite alleles
model.
Genetic population structure at both population and
individual levels was carried out using a model-based clustering
method implemented in STRUCTURE software (Pritchard and

Vol. 41, no. 1 Journal of Vector Ecology 163
Wen 2003). The correlated allele frequencies model and the
admixture model were selected with a burn-in period of 100,000
and 100,000 Markov chain Monte Carlo. To check the consistency
of results between runs with the same K, five runs were performed
for each value of K. We estimated the number of clusters and the
assignment of individuals into clusters using two methods: (1)
the most likely number of clusters was estimated by determining
the change in the marginal likelihood of the data Pr(X|K) when
the numbers of clusters (K) was fixed to different values; (2)
the ΔK method sensu Evanno et al. (2005) was implemented to
detect the amount of structuring. ΔK is the second order rate of
change of the marginal likelihood function and takes into account
both the gain in posterior probabilities over a range of K-values
and the variance between independent runs at given values of
K. Results of all runs were summarized using STRUCTURE
HARVESTER, version 0.6.92 (Earl and vonHoldt 2012). We also
used the Bayesian clustering method implemented in the program
BAPS 5.3 (Corander et al. 2004, 2008). This method allows a
more hierarchical analysis treating the partition among groups of
individuals as the parameter of main interest. It treats both the
allele frequencies of the molecular markers and the number of
genetically diverged groups in the population as random variables
and uses stochastic optimization to infer the posterior mode of
the genetic structure. BAPS was run with the maximal number
of groups (K) set to 2 - 5 (i.e., a number equal to the number of
sampled populations). Each run was replicated ten times, and the
results were averaged according to the resultant likelihood scores.
To estimate potential locations of discrete population
structure in the data we also applied the Bayesian clustering
algorithm employed in GENELAND 4.0.3 (Guillot et al. 2005),
implemented in R software 2.15.1 (R Development Core Team
2008), which takes into account the spatial location of sampling
sites and estimates the optimal number of population clusters. We
ran ten replicates for two million iterations (thinning = 200) with
the number of possible clusters K ranging from two to five and
checked for consistency. We processed a final run on a landscape
of 100x100 cells and with a burn-in of 2,000 iterations, fixing
the maximum K to three, the value with the highest posterior
density from all preliminary runs (Leaché 2011). GENELAND
incorporates spatial data directly under the assumption that
populations are spatially organized. However, this model does
not assume admixture and any genetic boundaries found are
assumed to separate K random mating populations (Manel et al.
2007, Guillot et al. 2005). Improvements to the model suggest
the ability to increase the detection of clusters having low genetic
differentiation (Guillot et al. 2008). Finally. geo-referenced
phenotypic and genetic data were integrated in GENELAND
software. The dataset, consisting of 100 specimens genotyped
at allozyme loci and their morphometric data (individual CVA
scores), was analyzed using data combinations of phenotypic and
genetic data under the spatial model. Ten independent Markov
chain Monte Carlo runs of 100,000 iterations were performed,
discarding the first 10,000 iterations as burn-in (Guillot et al.
2005, 2008).
In addition, association among genetic and geographic
distances was assessed by Mantel test (Mantel 1967). Approximate
linear geographical distances (measured in km) between
population pairs were obtained with GoogleTM Earth software and
19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
prior to analysis, natural logarithm transformation of geographic
distances was obtained. The estimate of genetic distance between
pairwise populations was characterized as FST/(1-FST) (Rousset
1997). This analysis was performed using Isolation by distance
web service (Jensen et al. 2005) and significance levels of observed
associations were evaluated using a total of 10,000 permutations.
Interpopulation genetic differentiation, measured as Wright’s
FST (Weir and Cockerham 1984), was estimated by ARLEQUIN
software, version 3.11 (Excoffier et al. 2005) and the significance
between each comparison pair was evaluated through 1,000
permutation procedures. A hierarchical analysis of molecular
variance (AMOVA) was estimated using ARLEQUIN, version
3.11 (Excoffier et al. 2005). Ten thousand permutations were used
to determine the significance of variance components at several
levels: 1) between “treated” (RSZR and RSNS) and ”untreated”
(SEGYS, SEDEJ, and HUPEC) populations; 2) between two
genetically clustered groups according to STRUCTURE analysis; 3)
among three unique genetic clusters defined by both GENELAND
and BAPS (Cluster I: HUPEC, RSZR, and RSNS, Cluster II:
SEGYS and Cluster III: SEDEJ); and 4) among all populations
(ungrouped). Genetic differentiation among populations was also
assessed using the genetic distance coefficient (D) of Nei (1972).
RESULTS
Population genetic structure
Ten enzyme systems representing allelomorphs of 14 allozyme
loci were assayed in five populations of Ae. vexans. Analysis of
allozyme variation revealed the presence of 31 alleles, from which
28 were detected in populations SEDEJ and HUPEC, while 26, 25,
and 24 were registered in SEGYS, RSZR, and RSNS, respectively.
The Aat, Hk-2, Hk-3, and Hk-4 loci were monomorphic with
the same allele in all populations. In all populations, Ao, Est-6,
Gpi, and Idh-2 loci were polymorphic. By contrast to SEDEJ and
HUPEC, loci Gpd-2 and Pgm-1 were polymorphic in three other
populations. Additionally, loci Idh-1 and Pgm-2 were polymorphic
in all populations but SEGYS. The Me locus was monomorphic in
both Serbian populations as well as Had in the HUPEC population.
The largest number of alleles (6) and genotypes (16) was identified
at the Est-6 locus (Table 2).
Thirteen heterozygote genotypes were registered at Est-6 (10),
Had (2), and Idh-2 (1) loci (Table 2). The observed heterozygosity
(Ho) was lower than the expected heterozygosity (He). The
genotypic fixation index (Fis) indicated excess homozygosity (Fis >
0) at Had in SEDEJ, Idh-2 in HUPEC, Had and Idh-2 in RSZR, and
Idh-2 in RSNS populations (results not shown).
Analysis of population genetic structure parameters revealed
differences among the studied populations of Ae. vexans. The mean
number of alleles per locus (A) and the frequency of polymorphic
loci (P) were highest in the SEDEJ and HUPEC populations from
Sweden and Hungary, respectively. Differences in the amount of
genetic variation among the studied populations were connected
with the effective population size obtained by θH (Table 2).
Phenotypic variation
Multivariate regression of shape variables on centroid size
was significant (p<0.0001), although it accounted for only a small
amount (5%) of the overall shape variation. To remove allometric
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
164 Journal of Vector Ecology June 2016

effects, size-corrected shape variables (the residuals from the
regressions) were used in subsequent shape analyses.
Wing size varied significantly among populations
(F(4,149)=19.09, p<0.001). The two Serbian populations (RSZR and
RSNS) had significantly larger centroid size (Tukey test, p<0.001)
than one of the Swedish populations (SEDEJ) and the Hungarian
(HUPEC) population (Figure 3A). The Serbian population RSZR
had significantly larger centroid size (Tukey test, p<0.01) than the
Swedish population SEGYS.
Canonical variate analysis also revealed differences in wing
shape among populations (Figure 3B). Along the first canonical
variable (CV1), the two Serbian populations were separated from
the two Swedish populations. Shape changes along the CV1 were
associated with the displacement of the landmarks 1, 8, 9, 13, 14,
15, and 16, which influenced the length and width of the wing
(Figure 4). Permutation tests on Procrustes distances showed that
the Swedish SEGYS mean shape configurations were significantly
different both from the Hungarian HUPEC (Procrustes distance=
0.0126, p<0.05) and from the Serbian RSZR (Procrustes distance=
0.0137, p<0.05) and RSNS (Procrustes distance= 0.0134, p<0.01)
populations. The Swedish SEDEJ population was also significantly
different from the Hungarian HUPEC (Procrustes distance=
0.00152, p<0.001), and the Serbian RSZR (Procrustes distance=
0.0156, p<0.001) and RSNS populations (Procrustes distance=
0.0174, p<0.001).
Genetic differentiation and phenotypic relationships
The POWSIM analysis of statistical power for detecting
differentiation among samples showed 100% for both Chi-squared
and Fisher’s exact test where Ne/t combinations corresponded to
5,000/100. Therefore, the simulations indicate that the number
of individuals and the polymorphism of loci used in this study
provide strong support to identify genetic structure at low levels
of divergence with 100% probability of detecting FST values as low
as 0.02. The alpha error (the probability of obtaining significant
genetic structure when the true FST =0) was consistently about 5%
in all simulations.
The presence of major (≥ 0.5) and rare (≤ 0.05) alleles indicate
spatial patterns of genetic diversity. The same major alleles at all
loci were observed in all populations. However, different major
alleles were found at Gpd-2 in SEDEJ. Different rare alleles at Ao
(HUPEC), Had and Idh-2 in RSZR, and Pgm-1 (HUPEC) were
recorded as well (Table 2).
Bayesian genotypic clustering analysis implemented in
the STRUCTURE software assayed two clusters of genetically
related individuals. Average log-likelihoods across five replicate
STRUCTURE runs showed steadily increasing values with a
declining rate of increase in Pr(X|K) from K = 2 (empirical and
simulation evidence suggests that a biologically meaningful
number of K may be indicated by a declining rate of increase in
Pr(X|K) as K increases rather than by the absolute maximum
likelihood (Pritchard et al. 2000, Evanno et al. 2005)). Based on
two of Evanno’s proposed genetic clusters, one cluster was formed
by a majority of individuals from Swedish populations, while most
of the individuals from the three remaining populations clustered
together into another cluster (Figure 5). However, the Bayesian
analysis performed on the software GENELAND (Figure 6A) and
BAPS (Figure 6B) supported the existence of three unique genetic
clusters: Cluster I: HUPEC, RSZR, and RSNS, Cluster II: SEGYS
and Cluster III: SEDEJ. The final Bayesian implementation was
performed using GENELAND on geo-referenced phenotypic and
genetic data. The estimated number of clusters was three across
the ten independent runs. From this, integrated genetic and
morphometric data suggested groupings of populations defined
as Cluster I: SEGYS, Cluster II: SEDEJ and HUPEC, and Cluster
III: RSZR and RSNS (Figure 7).
Isolation-by-distance was significant based on the Mantel
test (r = 0.591, p < 0.05). Pairwise standardized variance of allelic

Figure 3. Visualization of the wing size and shape variation observed in populations of Aedes vexans from Sweden, Hungary, and Serbia;
A) Bar chart showing mean centroid size with standard deviation; B) Scaterplots of individual scores for the first two canonical variates
(CV1 and CV2) with the percentage of explained variance in parentheses.
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Vol. 41, no. 1 Journal of Vector Ecology 165

Table 2. Genotypic frequencies and estimates of genetic variability parameters for ten variable allozyme loci in five populations of Aedes
vexans from Sweden (SEGYS, SEDEJ), Hungary (HUPEC), and Serbia (RSZR, RSNS). Four additional allozyme loci (Aat, Hk-2, Hk-3,
and Hk-4) were monomorphic, with common alleles, and thus excluded from the table. Rare alleles with frequencies ≤ 0.05 are shown
in bold.
Locus Genotype SEGYS SEDEJ HUPEC RSZR RSNS
a/a 0.100 0.056 0.345 0.407 0.375
Ao b/b 0.900 0.833 0.621 0.593 0.625
c/c - 0.111 0.034 - -
a/a 0.077 - 0.171 0.111 0.223
b/b 0.154 0.143 0.113 0.075 0.111
c/c 0.154 0.071 0.086 0.184 0.222
d/d 0.154 0.143 0.143 0.111 0.222
e/e 0.077 0.428 0.143 0.147 -
f/f 0.230 0.215 0.057 0.075 0.111
a/b - - 0.029 - -
a/c - - - 0.075 -
Est-6
a/e - - 0.057 - -
a/f - - - 0.037 -
b/c - - 0.029 - -
b/e - - 0.057 - -
b/f - - 0.057 - -
c/e - - 0.029 0.111 0.111
c/f - - - 0.037 -
d/f 0.154 - 0.029 0.037 -
a/a 1.000 0.300 0.600 1.000 1.000
Gpd-2
b/b - 0.700 0.400 - -
a/a 0.077 0.143 0.138 0.182 0.182
Gpi
b/b 0.923 0.857 0.862 0.818 0.818
a/a 0.769 0.933 1.000 0.958 1.000
b/b 0.077 - - - -
Had
a/b - - - 0.042 -
a/c 0.154 0.067 - - -
a/a 1.000 0.760 0.789 0.882 0.643
Idh-1
b/b - 0.240 0.211 0.118 0.357
a/a 0.750 0.941 0.897 0.926 0.875
Idh-2 b/b 0.100 0.059 - - -
a/c 0.150 - 0.103 0.074 0.125
a/a 0.200 0.300 0.250 - -
Me
b/b 0.800 0.700 0.750 1.000 1.000
a/a 1.000 0.900 0.950 1.000 1.000
Pgm-1
b/b - 0.100 0.050 - -
a/a - 0.400 0.300 0.125 0.286
Pgm-2
b/b 1.000 0.600 0.700 0.875 0.714
n (SE) 9.1 (2.1) 11.2 (2.0) 17.6 (3.5) 17.0 (2.7) 7.0 (0.9)
A (SE) 1.9 (0.4) 2.0 (0.3) 2.0 (0.3) 1.8 (0.4) 1.7 (0.4)
P(0.95) 42.9 64.3 64.3 35.7 42.9
P(0.99) 42.9 71.4 64.3 50.0 42.9
Ho (SE) 0.033 (0.017) 0.005 (0.005) 0.028 (0.021) 0.029 (0.021) 0.017 (0.011)
He (SE) 0.144 (0.060) 0.236 (0.060) 0.240 (0.068) 0.152 (0.064) 0.182 (0.069)
θH 0.177 0.328 0.331 0.184 0.238
n = mean sample size per locus; A = Mean number of alleles per locus; He = Expected heterozygosity averaged over all loci; Ho = Average
frequency of observed heterozygosity; P(0.95) = Frequency of polymorphic loci based on the 0.95 criterion; P(0.99) = Frequency of
polymorphic loci based on the 0.99 criterion (standard errors in parentheses), θH = Mean locus θH.
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
166 Journal of Vector Ecology June 2016

Table 3. Pairwise genetic distance (above diagonal) and pairwise FST estimates (below diagonal) among five populations of Aedes vexans
from Sweden (SEGYS, SEDEJ), Hungary (HUPEC), and Serbia (RSZR, RSNS). The genetic distances were calculated after Nei (1972).
Shaded boxes indicate a significant pairwise difference (p < 0.05).
SEGYS SEDEJ HUPEC RSZR RSNS
SEGYS - 0.064 0.029 0.015 0.024
SEDEJ 0.019 - 0.009 0.067 0.063
HUPEC 0.049 -0.036 - 0.017 0.013
RSZR 0.084 0.087 0.016 - 0.000
RSNS 0.080 0.109 -0.012 -0.006 -

frequency, FST, indicated significant genetic differences among
RSZR and populations from Sweden as well as between RSNS
and SEDEJ (Table 3). The greatest degree of genetic divergence
according to Nei (1972) was found between SEDEJ and both
Serbian populations (Table 3). Results from analysis of molecular
variance (AMOVA) suggested that a majority of the genetic
variance was attributed to variation within populations (95%), and
the remaining variation (5%) was among populations (Table 4).
UPGMA cluster analyses of wing shape, based on Procrustes
distances, grouped in the same branch two Swedish population,
while the remaining three populations clustered together (Figure
8).
DISCUSSION
As far as we know, this is the first integrative study of genetic
and phenotypic variation in Ae. vexans on a large geographical scale,
and we observed geographically related variance and allozyme
allele occurrence as well as variance in wing size and shape.
Previous analysis of wing size and shape in Ae. vexans populations
from northeastern Turkey showed that interpopulation differences
in wing morphology varied with altitude, but the genetic basis
of the phenotypic variation was not studied (Kuclu et al. 2011).
In the present study, the Serbian populations of Ae. vexans had
significantly larger wings than Hungarian and northern European
populations. That pattern might be a case of a converse Bergmann
cline, which presumably occurred in half of all the insect species
(Shelomi 2012). However, whether wing size (as a measure of
body size) correlates with annual mean temperature, meaning that
body size decreases with increasing latitude, and consequently
decreasing mean temperature, season length, and development
time (Masaki 1978), is still open to study in ectoterms, including
mosquitoes. Furthermore, the inter-population variation in Ae.
vexans wing shape we estimated showed a spatial structure with
two clusters at the European scale; one included the two Swedish
populations and the other included the Hungarian and two
Serbian populations. However, canonical variate analysis of wing
shape separated two Serbian populations from the two Swedish
populations, implying intermediate status of the Hungarian
population. Spatial analysis based on integration of genetic and
phenotypic data clustered Hungarian and one Swedish populations
(SEDEJ) together. However, based on allozyme polymorphism,
we verified the strength of genetic structuring using both spatially
non-explicit (STRUCTURE) and spatially explicit models
(BAPS and GENELAND) by grouping Hungarian and Serbian
populations into one cluster. Thus, disagreement of the data, which
is mainly linked with the placement of the Hungarian population,
can be explained by the observed Isolation-by-Distance pattern.

Table 4. Results from AMOVA analysis on allozyme allele variation in five populations of Aedes vexans from Sweden, Hungary, and
Serbia.
Variance Variation
Groups of samples Source of variation Fixation index
components (%)
Between groups 0.032 4.75 FCT= 0.047
“treated” vs “untreated”* Between populations within groups 0.002 0.27 FSC= 0.003
Within populations 0.643 94.98 FST= 0.050
Between groups 0.025 3.81 FCT= 0.039
Structure (K2) Between populations within groups 0.006 0.86 FSC= 0.009
Within populations 0.643 95.33 FST= 0.047
Between groups 0.024 3.52 FCT= 0.035
Baps and Geneland (K3)** Between populations within groups 0.005 0.77 FSC= 0.008
Within populations 0.643 95.71 FST= 0.043
Among populations 0.02 3.15 FST= 0.030
All (nongrouped)
Within populations 0.64 96.85
FST fixation index within populations; FSC fixation index among populations within groups; FCT fixation index between groups.
*“treated” populations: RSZR, RSNS; “untreated” populations: SEGYS, SEDEJ, HUPEC.
**Cluster I: HUPEC, RSZR and RSNS; Cluster II: SEGYS; Cluster III: SEDEJ.
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Vol. 41, no. 1 Journal of Vector Ecology 167

Figure 4. The wireframe visualization of the average wing shape for populations of Aedes vexans from Sweden,
Hungary, and Serbia showing the shape changes (black lines) from the consensus configuration of landmarks (grey
lines) to each extreme negative (CV1-) and positive (CV1+) along the first canonical variate.

Figure 5. Membership of Aedes vexans individuals to a number of presumed clusters (K = 2)

determined by the a priori Bayesian cluster method in STRUCTURE. Each vertical line represents
an individual’s probability of belonging to one of the K clusters (represented by different colors).

Figure 6. Spatially derived genetic clusters of Aedes vexans from Sweden (SEGYS, SEDEJ), Hungary
(HUPEC), and Serbia (RSZR, RSNS) inferred with the program GENELAND (A) and BAPS (B).
Different colors of sample plots indicate heterogeneous genetic composition: medium gray-1. SEGYS,
light gray-2. SEDEJ and dark gray-3. HUPEC, 4. RSZR and 5. RSNS.

168 Journal of Vector Ecology June 2016
Figure 7. Patterns of population structure of Aedes vexans inferred
by the integration of geo-referenced genetic and morphometric
data using GENELAND software. Different colors of sample plots
indicate heterogeneous genetic composition: light gray -1. SEGYS,
medium gray -2. SEDEJ and -3. HUPEC, dark gray -4. RSZR and
5. RSNS.
Indeed, correlation between geographic distance and genetic
differentiation (based on Isolation-by-Distance model) further
confirmed the population structure of Ae. vexans across Europe.
Therefore, according to our findings, the Hungarian population
exhibits a transition between northern and central European
populations.
In addition, unlike the STRUCTURE results, both BAPS
and GENELAND identified additional sub-structuring between
the two Swedish populations. This substructuring observed for
Swedish populations might be the result of divergent selection
pressures and evolutionary processes that act on the mosquitoes
from the area studied. Since our study aimed to compare the
variation of genetic markers and phenotypic traits in populations
across Europe, associations between these variation and
environmental variables at finescale will be the subject of future
studies.
Furthermore, we observed lower genetic variation in both
Serbian populations that likely reflects the influence of genetic
19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Figure 8. UPGMA phenogram of five populations of Aedes vexans
from Sweden (SEGYS, SEDEJ), Hungary (HUPEC), and Serbia
(RSZR, RSNS) based on the Procrustes distances.
drift and divergent selection that stemmed from artificial selection
pressures (insecticide treatments have been applied intensively
since the 1970s). Data presented are similar to those observed in
samples of Ae. vexans collected 14 and 16 years ago in Novi Sad
(Milankov et al. 1999, Krtinić et al. 2013) and populations from
Germany and California, U.S.A. (Krtinić et al. 2013).
In summary, our study of genetic variation of European
populations of Ae. vexans showed that a large amount of genetic
variation occurred within populations, despite the registered
population structuring. These findings were further supported by
the low proportion of interpopulation vs intrapopulation variance
revealed by AMOVA and low-to-moderate FST values on a broader
geographical scale. We consider that continuous between-
population exchange of individuals, including considerable gene
flow on the regional scale, are likely to be responsible for the
maintenance of the observed population similarity in Ae. vexans.
Since the pattern of wing shape variation and genetic diversity
indicated a spatial structure (differentiation between northern
and central European samples separated by more than 2,000 km),
it is reasonable to conclude that gene flow is present across a broad
geographical area and that the presence of multiple populations
between the sampled sites (that were not sampled in this study) is
obviously contributing to gene flow across the European landmass.
Indeed, the ability of Ae. vexans to migrate long distances was
shown by Clarke (1943) and Briegel et al. (2001), including
urban areas (Szalanski et al. 2006). Likewise, extensive dispersal
capacity was registered based on small FST values (Solorzano et al.
2010). The dispersal capacity of mosquito vectors is an important
characteristic for understanding vector-borne disease spread
(Sayson et al. 2015). For instance, a correlation was documented
between genetic distances and variation in pathogen susceptibility
and insecticide resistance patterns (Poolprasert et al. 2008). Since
our findings indicated that Ae. vexans gene flow occurs over large
distances, vector control programs should require both local and
large-scale actions. However, to obtain a more comprehensive
understanding of the influence of divergent selection pressures on
the adaptive potential of Aе. vexans, an integrative approach of

Vol. 41, no. 1 Journal of Vector Ecology 169
the relation between environmental, genetic and morphometric
variation should be conducted.
Acknowledgments
The authors thank the two anonymous reviewers for
constructive comments on an earlier version of this manuscript.
We thank Dragana Vujković for English language editing. B.K. is
supported by Ciklonizacija d.o.o. Novi Sad. Lj. F., J.L., and V.M. are
supported in part by the Ministry of Science of Serbia (Dynamics
of gene pool, genetic and phenotypic variability of populations,
determined by the environmental changes, No. 173012), and the
Provincial Secretariat for Science and Technological Development
(Molecular and phenotypic diversity of taxa of economical and
epidemiological importance, and endangered and endemic
species in Europe).
REFERENCES CITED
Ayala, F., M. Tracey, L. Barr, J. McDonald, and S. Perez-Salas. 1974.
Genetic variation in natural populations of five Drosophila
species and the hypothesis of the selective neutrality of
protein polymorphisms. Genetics 77: 343–384.
Becker, N., D. Petrić, M. Zgomba, C. Boase, C. Dahl, J. Lane, and
A. Kaiser. 2010. Mosquitoes and Their Control. 2nd ed. Springer
Verlag, Berlin Heidelberg. 577 pp.
Briegel, H., A. Waltert, and R. Kuhn. 2001. Reproductive
physiology of Aedes (Aedimorphus) vexans (Diptera:
Culicidae) in relation to flight potential. J. Med. Entomol. 38:
557–565.
Carreira, V.P., I.M. Soto, J. Mensch, and J.J. Fanara. 2011.
Genetic basis of wing morphogenesis in Drosophila: sexual
dimorphism and non-allometric effects of shape variation.
BMC Dev. Biol. 11: 32.
Clarke, J.L. 1943. Studies of the flight range of mosquitoes. J. Econ.
Entomol. 36: 121-122.
Corander, J., P. Marttinen, J. Sirén, and J. Tang. 2008. Enhanced
Bayesian modelling in BAPS software for learning genetic
structures of populations. BMC Bioinformat. 9: 539.
Corander, J., P. Waldmann, P. Marttinen, and M. Sillanpaa. 2004.
BAPS 2: enhanced possibilities for the analysis of genetic
population structure. BMC Bioinformat. 20: 2363–2369.
Cywinska, A., F.F. Hunter, and P.D.N. Hebert. 2006. Identifying
Canadian mosquito species through DNA barcodes. Med.
Vet. Entomol. 20: 413–424.
Dryden, I.L. and K.V. Mardia. 1998. Statistical Shape Analysis.
Wiley, Chichester.
Dujardin, J.-P. 2008. Morphometrics applied to medical
entomology. Infect. Genet. Evol. 8: 875–890.
Earl, D.A. and B.M. Vonholdt. 2012. STRUCTURE HARVESTER:
a website and program for visualizing STRUCTURE output
and implementing the Evanno method. Conserv. Genet.
Resour. 4: 359-361.
Evanno, G., S. Regnaut, and J. Goudet. 2005. Detecting the number
of clusters of individuals using the software structure: a
simulation study. Mol. Ecol. 14: 2611–2620.
Ewens, W.J. 1972. The sampling theory of selectively neutral
alleles. Theor. Popul. Biol. 3: 87–112.
19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin (version
3.1): An integrated software package for population genetics
data analysis. Evol. Bioinform. 1: 47-50.
Forsman, A. 2014. Effects of genotypic and phenotypic variation
on establishment are important for conservation, invasion,
and infection biology. PNAS 111(1): 302-307.
Ghosh, K., J. Mukhopadhyay, H. Guzman, R. Tesh, and L.
Munstermann. 1999. Interspecific hybridization and genetic
variability of Phlebotomus sandflies. Med. Vet. Entomol. 13:
78–88.
Goddard, L., A. Roth, W. Reisen and T. Scott. 2002.Vector
competence of California mosquitoes for West Nile virus.
Emerg. Infect. Dis. 8: 1385–1391.
Gómez, G.F., E.J. Márquez, L.A. Gutiérrez, J. E. Conn, and
M.M. Correa. 2014. Geometric morphometric analysis of
Colombian Anopheles albimanus (Diptera: Culicidae) reveals
significant effect of environmental factors on wing traits and
presence of a metapopulation. Acta Trop. 135: 75–85.
Guillot, G., F. Mortier, and A. Estoup. 2005. GENELAND: a
computer package for landscape genetics. Mol. Ecol. Notes 5:
712–715.
Guillot, G., F. Santos, and A. Estoup. 2008. Analysing georeferenced
population genetics data with Geneland: a new algorithm to
deal with null alleles and a friendly graphical user interface.
Bioinformatics 24: 1406–1407.
Gutsevich, A.V., A.S. Monchadskii, and A.A. Shtakel’berg. 1971.
Fauna SSSR Vol. 3. (4). Family Culicidae. 384 pp. Leningrad.
Akad. Nauk. SSSR. Zool. Inst. N. S. No. 100. (English
translation: Israel Program for Scientific Translations,
Jerusalem 1974.).
Hammer, Ø., D.A.T. Harper, and P.D. Ryan. 2001. PAST:
Paleontological statistics software package for education and
data analysis. Palaeontol. Electron. 4: 9.
Horsfall, W.R. and R.J. Novak. 1991. The inland floodwater
mosquito, Aedes vexans: an annotated bibliography to world
literature, part I/part II. J. Am. Mosq. Contr. Assoc. 1–43.
Jaramillo-O, N., J.-P. Dujardin, D. Calle-Londoño, and I. Fonseca-
González. 2015. Geometric morphometrics for the taxonomy
of 11 species of Anopheles (Nyssorhynchus) mosquitoes. Med.
Vet. Entomol. 29: 26–36.
Jensen, J.L., A.J. Bohonak, and S.T. Kelley. 2005. Isolation by
distance, web service. BMC Genetics 6: 13.
Jirakanjanakit, N., S. Leemingsawat, and J.P. Dujardin. 2008.
The geometry of the wing of Aedes (Stegomyia) aegypti in
isofemale lines through successive generations. Infect. Genet.
Evol. 8: 414-21.
Klingenberg, C.P. 2011. MorphoJ: an integrated software package
for geometric morphometrics. Mol. Ecol. Resour. 11: 353-357.
Klingenberg, C.P., M. Barluenga, and A. Meyer. 2002. Shape
analysis of symmetric structures: quantifying variation
among individuals and asymmetry. Evolution 56: 1909-1920.
Knight, K.L. and A. Stone. 1977. A Catalog of the Mosquitoes of the
World (Diptera, Culicidae). 2nd ed. Thomas Say Foundation.
6: 1–610.
Krtinić, B., Lj. Francuski, D. Petrić, and V. Milankov. 2013. Genetic
diversity and differentiation between Palearctic and Nearctic
populations of Aedimorphus vexans [Aedes vexans] (Meigen,
1830) (Diptera, Culicidae). J. Vector Ecol. 38(1): 154-162.

170 Journal of Vector Ecology June 2016
Krtinić, B., Lj. Francuski, J. Ludoški, and V. Milankov. 2016.
Integrative approach revealed contrasting pattern of spatial
structuring within urban and rural biotypes of Culex pipiens.
J. Appl. Entomol. doi: 10.1111/jen.12307
Krtinić, B., J. Ludoški, and V. Milankov. 2015. Multi-character
approach reveals a discordant pattern of phenotypic
variation during ontogeny in Culex pipiens biotypes (Diptera:
Culicidae). B. Entomol. Res. 105: 129-138.
Kuclu, O., A. Aldemir, and B. Demirci. 2011. Altitudinal variation
in the morphometric characteristics of Aedes vexans Meigen
from northeastern Turkey. J. Vector Ecol. 36: 30-41.
Laurito, M., W.R. Almirón, and F.F. Ludueña-Almeida. 2015.
Discrimination of four Culex (Culex) species from
the Neotropics based on geometric morphometrics.
Zoomorphology 134: 447-455.
Leaché, A.D. 2011. Multi-locus estimates of population structure
and migration in a fence lizard hybrid zone. PLoS ONE 6:
e25827.
Levene, H. 1949. On a matching problem arising in genetics. Ann.
Math. Stat. 20: 91–94.
Lundström, J.O. 1994. Vector competence of western European
mosquitoes for arboviruses: A review of field and experimental
studies. Bull. Soc. Vec. Ecol. 19: 23-36.
Lundström, J.O. 1999. Mosquito-borne viruses in Western Europe:
A review. J. Vector Ecol. 24: 1-39.
Manel, S., F. Berthoud, E. Bellemain, M. Gaudeul, G. Luikart, J.E.
Swenson, L.P. Waits, and P. Taberlet. 2007. A new individual-
based spatial approach for identifying genetic discontinuities
in natural populations. Mol. Ecol. 16: 2031–2043.
Mantel, N. 1967. The detection of disease clustering and a
generalized regression approach. Cancer Res. 27: 209-220.
Masaki, S. 1978. Seasonal and latitudinal adaptations in life cycles
of crickets. In: H. Dingle (ed.). Evolution of Insect Migration
and Diapause. pp. 72-100. Springer-Verlag, NY.
Milankov, V., A. Vujić, S. Šimić, J. Radovanov and D. Milenković.
1999. Gene-enzyme variability in natural population of Aedes
vexans from Novi Sad. Arch. Biol. Sci. 51: 23–24.
Miller, B.R., M.S. Godsey, M.B. Crabtree, H.M. Savage, Y. Al-
Mazrao, M.H. Al-Jeffri, A.-M. M. Abdoon, S.M. Al-Seghayer,
A.M. Shahrani, and T.G. Ksiazek. 2002. Isolation and genetic
characterization of Rift Valley fever virus from Aedes vexans
arabiensis, Kingdom of Saudi Arabia. Emerg. Infect. Dis. 8:
1492–1494.
Munstermann, L.E. 1994. Unexpected genetic consequences of
colonization and inbreeding: allozyme tracking in Culicidae
(Diptera). Ann. Entomol. Soc. Am. 87: 157–164.
Nei, M. 1972. Genetic distance between populations. Am. Nat.
106: 283–292.
Petersen, V., M. Devicari, and L. Suesdek. 2015. High morphological
and genetic variabilities of Ochlerotatus scapularis, a potential
vector of filarias and arboviruses. Parasites & Vectors 8: 128.
Poolprasert, P., S. Manguin, M.J. Bangs, S. Sukhontabhirom, S.
Poolsomboon, P. Akaratanakul, and T. Chareonviriyaphap.
2008. Genetic structure and gene flow of Anopheles minimus
and Anopheles harrisoniin Kanchanaburi Province, Thailand.
J. Vector Ecol. 33: 159–165.
19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Pritchard, J.K. and W. Wen. 2003. Documentation for STRUCTURE
software: Version 2. Available from http://pritch.bsd.uchicago.
edu.
Pritchard, J.K., M. Stephens, and P. Donnelly. 2000. Inference
of population structure using multilocus genotype data.
Genetics 155: 945–959.
Reinert, J.F. 1973. Contribution to the Mosquito fauna of Southeast
Asia. XVI. Genus Aedes Meigen, Subgenus Aedimorphus
Theobald in Southeast Asia. Contrib. Am. Entomol. Inst. 9:
1-219.
Reinert, J.F., R.E. Harbach, and I.J. Kitching. 2004. Phylogeny
and classification of Aedini (Diptera: Culicidae) based on
morphological characters of all life stages. Zool. J. Linn. Soc.
142: 289–368.
Rohlf, F.J. 2013. tpsDig, version 2.17. Department of Ecology and
Evolution, State University of New York at Stony Brook. New
York, NY.
Rousset, F. 1997. Genetic differentiation and estimation of gene
flow from F-statistics under isolation by distance. Genetics
145: 1219-1228.
Rudolf, I., O. Šebesta, J. Mendel, L. Betášová, E. Bocková, P.
Jedličková, K. Venclíková, H. Blažejová, S. Šikutová, and
Z. Hubálek. 2014. Zoonotic Dirofilaria repens (Nematoda:
Filarioidea) in Aedes vexans mosquitoes, Czech Republic.
Parasitol. Res. 113: 4663-4667.
Ruiz-Garcia, M, D. Ramirez, F. Bello, and D. Alvarez. 2003.
Psorophora columbiae and Psorophora toltecum (Diptera:
Culicidae) Colombian populations cannot be differentiated
by isoenzymes. Genet. Mol. Res. 2: 229–259.
Ryman, N. and S. Palm. 2006. POWSIM: a computer program
for assessing statistical power when testing for genetic
differentiation. Mol. Ecol. Notes 6: 600-602.
Sayson, S.L., A. Gloria-Soria, J.R. Powell, and F.E. Edillo. 2015.
Seasonal genetic changes of Aedes aegypti (Diptera: Culicidae)
populations in selected sites of Cebu City, Philippines. J. Med.
Entomol. 52: 638-646.
Shelomi, M. 2012. Where are we now? Bergmann’s rule sensu lato
in insects. Am. Nat. 180: 511-519.
Solorzano, C.D., A.L. Szlanski, C.B. Owens, and C.D. Steelman.
2010. Genetic diversity of Aedes vexans (Diptera, Culicidae)
from New Orleans: Pre- and Post-Katrina. Biochem. Genet.
48: 711–726.
Swofford D.L. and R.B. Selander. 1989. BIOSYS-2: A computer
program for the analysis of allelic variation in genetics.
University of Illinois at Urbana-Champaign, Urbana, IL.
Szalanski, A.L., C.B. Owens, J.A. Lewter, and A.B. Broce. 2006.
Genetic structure of Aedes vexans (Diptera: Culicidae)
populations from central United States based on mitochondrial
ND5 Sequences. Ann. Entomol. Soc. Am. 99: 157-163.
Waples, R.S. and O. Gaggiotti 2006. What is a population? An
empirical evaluation of some genetic methods for identifying
the number of gene pools and their degree of connectivity.
Mol. Ecol. 15: 1419-1439.
Weir, B.S. and C.C. Cockerham. 1984. Estimating F-statistics for
the analysis of population structure. Evolution 38: 1358–1370.
White, G.B. 1975. Notes on a catalogue of Culicidae of the
Ethiopian Region. Mosq. Syst. 7: 303–338.
 19487134, 2016, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jvec.12208 by INASP - HONDURAS, Wiley Online Library on [24/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Vol. 41, no. 1 Journal of Vector Ecology 171

Wilkerson, R.C., Y-M. Linton, D.M. Fonseca, T.R. Schultz,
D.C. Price, and D.A. Strickland. 2015. Making mosquito
taxonomy useful: A stable classification of tribe Aedini that
balances utility with current knowledge of evolutionary
relationships. PLOS ONE, July 30, 26 pp. DOI:10.1371/
journal.pone.0133602.
Yildirim, A., A. Inci, O. Duzlu, Z. Biskin A. Ica, and I. Sahin.
2011. Aedes vexans and Culex pipiens as the potential vectors
of Dirofilaria immitis in Central Turkey. Vet. Parasitol. 178:
143–147.