A Comparison of Aboveground and Belowground Populations of Culex
pipiens (Diptera: Culicidae) Mosquitoes in Chicago, Illinois,
and New York City, New York, Using Microsatellites
LINDA KOTHERA,1MARVIN GODSEY, JOHN-PAUL MUTEBI, AND HARRY M. SAVAGE
Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, 3150 Rampart Road,
Fort Collins, CO 80521
J. Med. Entomol. 47(5): 805Ð813 (2010); DOI: 10.1603/ME10031
Aboveground and belowground populations of the mosquito Culex pipiens L. are
traditionally classiÞed as form pipiens and form molestus, respectively, and gene ßow between forms
is thought to be limited. Relatively few f. molestus populations have been found in the United States,
a newly discovered population of f. molestus in Chicago, IL, and compared levels of genetic diversity
and differentiation in aboveground and belowground populations from Chicago and New York City,
NY. Levels of genetic diversity, as measured by expected heterozygosity and allelic richness, were
markedly lower in both f. molestus populations. Allele frequencies were distinctly different between
the two f. molestus populations, and some alleles were present in one belowground population and
not the other. Pairwise FSTvalues between populations indicated that f. molestus populations were
highly divergent from each other, as well as from their associated aboveground populations. Cluster
analysis suggested the most likely number of groups was three, with the four f. pipiens populations
in one cluster, and each of the f. molestus populations in its own cluster. Admixture analysis detected
a low number of hybrids, 8%, between forms. We also tested the efÞcacy of two assays purported to
distinguish between the forms, the CQ11 assay and a restriction fragment-length polymorphism assay
of the COI gene, and found neither assay reliable in this regard. Our Þndings support the hypothesis
population genetics, Culex pipiens, Culex pipiens form molestus, microsatellites,
A hallmark of a subdivided population is the presence
of genetic structure, which arises from limited gene
ßow between subpopulations. Populations of Culex
pipiens may occupy open, aboveground habitats or
conÞned, belowground habitats, the latter of which is
with humans (Barr 1967, Spielman 1967, Chevillon et
al. 1995). Physiological characteristics associated with
produce the Þrst egg raft without a blood meal, and a
lack of winter diapause, despite living in a temperate
climate (Chevillon et al. 1995, 1998; Vinogradova
These populations are currently recognized as two
forms of Cx. pipiens: form pipiens and form molestus
Forska ¨l (Harbach et al. 1984). The f. molestus indi-
viduals are associated with belowground habitats and
are stenogamous (able to mate in conÞned spaces),
whereas f. pipiens occupy aboveground habitats and
are eurygamous (mate in swarms in large areas). Al-
though f. molestus has been studied often in Europe
and the Middle East, where it is thought to have
originated (Knight and Abdel Marek 1951, Urbanelli
et al. 1981, Nudelman et al. 1988, Byrne and Nichols
few populations have been found in the United States
et al. 2007, Huang et al. 2008; San Mateo County, CA,
McAbee et al. 2004; Philadelphia, PA, Kilpatrick et al.
2007; and Chicago, IL, Wray 1946, Mutebi and Savage
2009), which has impeded their study in North Amer-
Turell et al. 2006, Kramer et al. 2008).
The persistence of f. molestus in enclosed, isolated
habitats raises the question of how this form evolved.
Early cytological work by Laven (1959) was support-
ive of the idea that f. molestus populations did not
share a common origin. Although Byrne and Nichols
(1999) found little genetic similarity between popu-
1Corresponding author: Centers for Disease Control and Preven-
tion, Division of Vector-Borne Infectious Diseases, 3150 Rampart
Road, Fort Collins, CO 80521 (e-mail: LKothera@cdc.gov).
argued their results were still consistent with a single
colonization event by local f. pipiens. According to
their most parsimonious scenario, colonization was
followed by a subsequent increase in genetic differ-
entiation and a decrease in genetic diversity among
ined populations of both forms from locations in Eu-
rope and asserted that belowground f. molestus pop-
ulations could not have arisen from their associated
aboveground f. pipiens populations because the de-
gree of genetic divergence was lower among popula-
tions of f. molestus than between f. molestus and f.
pipiens. With regard to United States populations,
Huang et al. (2008) could not exclude the possibility
that the belowground f. molestus population on 91st
Street in New York City (designated NYMolG0in the
current study) originated from colonization by
aboveground f. pipiens. Fonseca et al. (2004) asserted
that in the United States: 1) f. pipiens populations
show a high degree of hybrid (with f. molestus) an-
f. molestus. However, that study failed to include
United States populations of f. molestus in the analy-
ses. If f. molestus in the United States is derived from
European populations, we would expect f. molestus
populations in the United States to be genetically
f. molestus arose independently from pipiens ances-
tors, we would expect f. molestus populations in the
United States to be genetically diverged from one
nearby aboveground populations.
to describe a heretofore uncharacterized f. molestus
population collected from Chicago, IL, and to com-
pare aboveground and belowground populations of
Cx. pipiens from Chicago and New York City, NY.
Materials and Methods
Specimens were collected from three locations
(two aboveground and one belowground), each in
Chicago and New York City between 2005 and 2009
(Table 1; Fig. 1). The low number of individuals (n ?
20) collected from the belowground location in New
York City precluded colonization attempts, so those
specimens were frozen and used for genetic analysis
as representative of the wild population and desig-
Table 1. Site and population information
Site nameN LocationLatitudeLongitude Site locationTrap methodAutogenous?
New York, NY
New York, NY
New York, NY
CDC light trap
CDC light trap
CDC light trap
CDC, Centers for Disease Control and Prevention; NT, not tested (see text for details).
Map showing locations of populations used in this study. Maps of individual sites are at the same scale.
806JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 47, no. 5
nated NYMolG0. For comparative purposes, we es-
tablished a colony of f. molestus from colony speci-
mens originally obtained from the NYMolG0site.
Aboveground specimens from New York City, col-
lected from Fort Totten in Queens (NYFT) and
Greenwood Cemetery in Brooklyn (NYGC), were
colonized, and a subsample of Þeld-collected individ-
uals from each site was used for this study. Similarly,
umet Water Reclamation Plant in Chicago (Mutebi
specimens from Chicago, designated Chi16 and
Chi17A, were collected as part of an earlier study
(Kothera et al. 2009). A total of 242 specimens was
examined in our study.
Initial Morphology and Genetic Screening. Field-
collected specimens were examined morphologically,
as described in Savage et al. (2007), to determine
species or lowest taxonomic unit. Specimens from the
two belowground populations were previously deter-
mined to be autogenous (Huang et al. 2008, Mutebi
and Savage 2009; Table 1). Diapausing females from
the aboveground populations in New York City were
collected in winter from hibernacula, and autogeny
was conÞrmed when in colony by delaying blood
meals for 2 wk after placing pupae into clean cages as
part of routine colony maintenance. Egg raft produc-
tion ceased until blood meals were resumed, so the
NYFT and NYGC populations were deemed anauto-
genous. The aboveground populations in Chicago,
Chi16 and Chi17A, were not tested for autogeny, as
DNA extraction and visualization of polymerase
chain reaction (PCR) fragments were described pre-
viously (Kothera et al. 2009). Brießy, individual mos-
quitoes were homogenized with BA-1 diluent, and
DNA was extracted from a portion of the sample.
Specimens were screened initially with the internal
transcribed spacer(ITS) assay (Crabtree et al. 1995,
Aspen et al. 2003) to conÞrm membership in the Cx.
pipiens species complex. A panel of eight microsatel-
lite loci was used to assess genetic diversity and dif-
ferentiation. All loci were from previously published
work (Fonseca et al. 1998, Keyghobadi et al. 2004,
Smith et al. 2005, Edillo et al. 2007; Table 2). PCR
conditions were as described in the original sources,
were visualized on a Beckman Coulter (Fullerton,
CA) CEQ8000 sequencer, and a multilocus genotype
was generated for each individual with the sequenc-
erÕs fragment analysis module. Approximately 10% of
the individuals in the study were analyzed twice, and
results from both runs were identical.
In addition, specimens were evaluated with two
assays that purportedly differentiate the two forms:
the CQ11 locus (Bahnck and Fonseca 2006), and
ShaikevichÕs (2007) restriction fragment-length poly-
morphism assay of the COI gene. CQ11 is a microsat-
ellite marker that reportedly produces form-speciÞc
bands, with both bands present in hybrids. The re-
striction fragment-length polymorphism assay takes
advantage of a reported one-nucleotide difference
between the forms in a region of the COI gene. PCR
was conducted as per their respective published pro-
tocols. Results of the CQ11 assay were visualized on
2% agarose gels, and individuals were scored as f.
restriction fragment-length polymorphism assay were
cloned and sequenced from one individual from each
of the aboveground populations in Chicago and New
the belowground populations (n ? 4).
format genotypic data for subsequent analyses in Ar-
et al. 2000), and FSTAT 2.9.3 (Goudet 1995), as well
as to generate tables of allele frequencies. Genetic
expected heterozygosity (HE) was calculated using
also used to evaluate departures from Hardy-Wein-
berg (HWE) and linkage equilibrium. FSTAT was
used to derive values for allelic richness, which were
calculated using a sample size corrected method. The
program Bottleneck (Piry et al. 1999) was used to
determine whether any of the populations displayed
signs of having undergone a recent reduction in ef-
fective population size. A statistically signiÞcant (P ?
0.05) Wilcoxon signed rank test is interpreted as ev-
idence of a genetic bottleneck. The program also uses
changes in the distribution of allele frequencies char-
acteristically associated with populations that have
An analysis of molecular variance (AMOVA) was
populations, and among populations. Arlequin was
also used to generate Þxation indices, or F statistics,
inbreeding and genetic differentiation in and among
populations. Statistical signiÞcance was obtained via
permutation tests. Microsatellite Analyzer (Dieringer
percent shared alleles between pairs of populations,
and a neighbor-joining tree was generated from the
resulting matrix using Phylip (Felsenstein 1993).
Table 2. Microsatellite loci used in this study
N, no. of alleles; HE, expected heterozygosity; HO, observed het-
a1, Fonseca et al. 1998; 2, Keyghobadi et al. 2004; 3, Smith et al.
2005; 4, Edillo et al. 2007.
September 2010KOTHERA ET AL.: Culex pipiens IN CHICAGO AND NEW YORK CITY
Structure was Þrst used to infer the mostly likely
number of clusters (K). Fifty runs using the default
parameters, without prior population information,
were performed for each value of K ranging from 2 to
8, and consisted of 500,000 burn-in steps, followed by
posterior probability associated with each number of
clusters, and used Evanno et al.Õs (2005) ?K method.
In addition, the Structure results were used to calcu-
late a similarity coefÞcient (Rosenberg et al. 2002),
which evaluated the stability of individual assign-
values closer to 1 indicate that individuals are consis-
tently placed in the same cluster.
Structure was also run to assign individuals to clus-
ters and to detect hybrids between the two forms. A
hybrid was deÞned as an individual with a qi(prob-
ability of membership) value of ?0.90 in any one
cluster (Vaha and Primer 2006), whereas pure f. pipi-
ens or f. molestus were deÞned as having qivalues of
to visualize Structure results.
Within Population Patterns of Genetic Diversity.
Two instances of signiÞcant (Bonferroni corrected
? ? 0.001; Rice 1989) departures from HWE were
found in the Chi16 population. As loci were generally
in HWE in the other populations in our study, as well
as in a previous study (Kothera et al. 2009), all loci
were retained for further analyses. Five instances (of
instance involved a different pair of loci, it was as-
sumed the loci were statistically independent of each
A total of 98 alleles was scored, and the number of
instances of private alleles were observed, but they
were all at a frequency of 4% or less. Allelic richness
the aboveground populations (range, 2.742Ð7.291),
and a similar trend was observed for the overall mea-
sure of genetic diversity, the average HEper site
(range, 0.383Ð0.670; Table 3).
Some marked differences in frequencies were ob-
served for several alleles in the belowground popula-
tions (Table 4). In four instances, a locus was mono-
morphic in one population and not the other. For
example, the 149-bp allele of locus CxqGT4 is mono-
morphic in the NYMolG0population, and present at a
frequency of 0.490 in the ChiMolG0population. In
other cases, an allele was present in one belowground
population and absent in the other, as in the 187-bp
allele of locus CxqCAG101, which is absent from the
ChiMolG0population, but present in the NYMolG0
population at a frequency of 0.250. There were 31
private alleles within the two belowground popula-
tions: 11 occurred at a frequency of ?5%, and 20
alleles found in each belowground population were
also found in the local aboveground populations.
Bottleneck results indicated the ChiMolG0popula-
tion showed a statistically signiÞcant departure from
coxonÕs test for an excess heterozygosity ? 0.020). In
addition, both the ChiMolG0and the NYMolG0pop-
ulations exhibited a mode-shift in their allele fre-
quency distributions, suggesting both populations
have experienced a genetic bottleneck (Table 5).
Among Population Differentiation. Results from
the AMOVA indicated the majority of genetic varia-
tion was found at the individual level (81.23%), and
and among (12.71%) populations (Table 6). Mean
Þxation indices were signiÞcantly different from 0
(P ? 0.0001) via permutation tests in Arlequin (FIS?
higher levels of inbreeding than the other sites, be-
cause of comparatively higher FISvalues at two loci.
ulations in Chicago and New York City were not
signiÞcantly different (Table 7). In contrast, the two
belowground populations were highly differentiated
Table 3.Among-population genetic diversity measures
0.670 0.654 0.4270.3830.6650.670
two f. molestus populations
Table 5.Results of genetic bottleneck analysis
Population Exp. H ex.H def. H ex.Wilcoxon Mode-shift
Exp. H ex., expected no. of loci having excess heterozygosity; H
def., observed no. of loci showing heterozygote deÞciency; H ex.,
observed no. of loci showing heterozygote excess; Wilcoxon, P value
for one-tailed WilcoxonÕs test; Mode-shift, whether a shift in allele
frequency distribution occurred. *, P ? 0.05.
808JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 47, no. 5
from each other (FST? 0.394) as well as from their
respective aboveground populations (FSTrange from
0.152 to 0.253).
The neighbor-joining tree based on the proportion
ulations clustering together, and the group of
2). The belowground populations are positioned on
the tree with a maximum of distance between each
other and the aboveground populations.
abilities, the most likely number of clusters was three,
with each of the two belowground populations in its
own cluster, and all four of the aboveground popula-
K, which are not suggested by either posterior prob-
abilities or similarity coefÞcients, the number of clus-
ters an individual belonged to increased for individ-
uals belonging to the four aboveground populations,
but remained the same for individuals in the below-
ground populations (Fig. 3, B and C). The similarity
coefÞcients had a value of 1 when K ? 2 or K ? 3,
indicating that individuals were placed into the same
cluster consistently with either value of K, and larger
values of K had successively smaller similarity coefÞ-
cients (Fig. 4). EvannoÕs ?K method suggested the
most likely number of clusters was two. When Struc-
ture was constrained to two clusters, the ChiMolG0
population occupied its own cluster, with the remain-
ing cluster comprised of all of the aboveground pop-
ulations as well as the NYMolG0population.
Twenty individuals (8%) were classiÞed as hybrids
between forms using qi? 0.90. Hybrids were most
common in the aboveground sites in New York City,
which each had Þve hybrid individuals (Table 8).
Comparison of Methods to Detect Forms and Hy-
brids. We were unable to use ShaikevichÕs (2007)
restriction fragment-length polymorphism assay be-
cause there were no sequence differences between
aboveground and belowground individuals in the rel-
evant region of the COI gene. Two clones were se-
quenced twice in each direction, and the results were
consistent over all runs. Whereas f. pipiens in the
Shaikevich (2007) study had a sequence of GGCC in
the relevant region, and f. molestus had AGCC, all of
the individuals sampled for our study had the se-
quence GGCC. We then direct sequenced an addi-
tional two f. molestus individuals (twice in both di-
rections) each from NYMolG0and ChiMolG0, and
those individuals also had the GGCC sequence. Se-
quences have been deposited on GenBank, received
15 January 2010 (accession numbers GU473997-
analysis detected about equal numbers of f. molestus
individuals in belowground populations (Table 8).
However, the CQ11 assay incorrectly classiÞed 14.3Ð
21.3% of individuals in each aboveground population
as f. molestus, whereas Structure detected no f. mo-
lestus in these populations. Overall, the CQ11 assay
the Structure analysis, with the exception of the
NYGC population, in which each analysis detected
Þve hybrid individuals.
Whereas aboveground populations in Chicago and
populations in both locations displayed reduced ge-
netic diversity and were highly divergent from their
corresponding aboveground populations and from
Inspection of pairwise FSTvalues (Table 7) dem-
onstrates several trends among populations. First,
there is a high degree of similarity among the
aboveground populations. This is notable, given that
Chicago and New York City are over 1200 km apart.
from all other populations, and the two belowground
populations are the most divergent of all. Noticeably
different allele frequencies, such as those shown in
Table 4, may account for the observed degree of di-
Pairwise FSTvalues for pairs of populations in this
Chi16 Chi17A ChiMolG0
aSigniÞcant pairwise FSTvalue.
Table 6.Results of AMOVA
Source of variation df
proportion of shared alleles. Belowground populations in-
clude “Mol” in the name, and aboveground populations in-
clude “pip” in the name.
September 2010KOTHERA ET AL.: Culex pipiens IN CHICAGO AND NEW YORK CITY
presence or absence of certain alleles. In addition,
these trends are supported by the population level
HEwere noticeably reduced in the two belowground
populations, whereas values associated with the four
aboveground populations were similar and compara-
tively larger. Given that the aboveground populations
are so similar, the high degree of genetic divergence
in belowground populations suggests they have per-
sisted over time with little, if any, gene ßow. Our
were linked to traits associated with adapting to an
underground environment, we would expect to ob-
serve more similarities than were seen in our study.
Finally, the neighbor-joining tree based on the pro-
assignments for the most likely number of clusters, K ? 3. B and C, Illustrate that f. molestus individuals remain in their
respective clusters, and the f. pipiens cluster arbitrarily divides into further clusters when K ? 4 and K ? 5, respectively.
Results from Structure analysis, with default settings and no prior population information. A, Shows individual
of clusters), ranging from 2 to 8. Also shown, on the second y-axis, is the similarity coefÞcient (Rosenberg et al. 2002) for
each value of K, which was calculated from the Structure results and plotted as a function of K.
810JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 47, no. 5
proportion of alleles, and its branch is the longest on
the tree (Fig. 2).
The lack of diagnostic sequence differences corre-
sponding to a restriction enzyme site in the target
region of the COI gene in the United States popula-
tions of f. pipiens and f. molestus that we analyzed is
in contrast to the Þndings of Shaikevich (2007) based
on analysis of Russian populations. All of the United
States populations that we tested contained sequence
indicative of f. pipiens. If the Shaikevich (2007) assay
is truly diagnostic of f. pipiens and f. molestus in the
Old World, our results suggest that the two United
States f. molestus populations we sampled are related
to and likely evolved from f. pipiens and not from Old
World f. molestus. Further analysis of COI sequence
in other populations, particularly those from North
The higher posterior probability values for K ? 3,
the high degree of genetic differentiation observed
(Table 7), indicate that the most likely number of
clusters in this system is three. Even though the Ev-
anno et al. (2005) method suggested that K ? 2, the
presence of two clusters is not supported by the pos-
is clear from Fig. 3 that the assignments of individuals
from the belowground populations to their clusters
are very stable, even when K is increased beyond 3.
The aboveground populations form one relatively ho-
mogeneous cluster, and increasing K beyond 3 does
not suggest any further genetic structuring in the sys-
tem. The small similarity coefÞcients associated with
K ? 4 and above (Fig. 4) fail to support additional
structuring among the aboveground f. pipiens popu-
lations. Indeed, when Structure assigns specimens to
a greater number of clusters than is supported by the
data (Fig. 3, B and C), the proportions of each indi-
vidual assigned to the additional clusters are roughly
symmetric, as pointed out by Pritchard et al. (2000).
The admixture analysis revealed a number of hy-
brids in all aboveground populations, which ranged
from 4 to 15%. It would appear from these results that
gene ßow between aboveground and belowground
populations is generally low, and usually unidirec-
The aboveground populations in New York City ex-
hibit slightly more hybridization than those in Chi-
cago, which underscores the degree to which the
ChiMolG0site was physically and genetically isolated
from its associated aboveground sites (Mutebi and
Several studies have documented a low degree of
hybridization between forms, as measured by the
presence of both stenogamy and autogeny in
United States (France, Pasteur et al. 1977; Italy, Ur-
banelli et al. 1981; Japan, Sasa et al. 1966; Russia, Vi-
nogradova 2000). One well-characterized autogenous
population in the United States was studied by Spiel-
man (1957, 1964, 1971, 1973), who also found limited
hybridization between forms and concluded that
whereas the two forms are interfertile in the labora-
tory, behavioral and ecological factors reduce oppor-
tunities for hybridization to occur, and thus serve as
effective reproductive isolating mechanisms.
Fonseca et al. (2004) examined the incidence of
hybridization between f. pipiens and f. molestus, and
the present work used some of the same markers as
proportion of hybrids between forms in the United
States differed greatly, with 40% of aboveground Cx.
identiÞed as hybrids in our study, a Þnding consistent
with studies by Huang et al. (2008) and Gomes et al.
(2009), which identiÞed 8Ð12% of aboveground Cx.
pipiens individuals as hybrids. Fonseca et al. (2004)
used a qivalue of 0.94 when interpreting the results
from their Structure analysis, which would result in a
but this difference could not account for the discrep-
the Fonseca study did not include any f. molestus
populations from the United States. It is possible that
f. molestus in the United States is unlike European f.
molestus, perhaps being more isolated and therefore
more genetically differentiated, although still closely
related to f. pipiens. The COI sequence data from the
current study support this assertion, as does our Þnd-
ing that the belowground populations had a subset of
the alleles found in the local aboveground popula-
from within and outside the United States could shed
light on this question.
Compared with the proportion of hybrids detected
by the full microsatellite analysis, the CQ11 assay
overestimated the number of hybrids and incorrectly
classiÞed a signiÞcant proportion of f. pipiens speci-
mens as f. molestus (Table 8). There are several pos-
locus assay and, as such, its inheritance pattern would
of hybrids are likely to be encountered, which could
microsatellite analysis that generates multilocus ge-
notypes uses more information than a single gene
single gene CQ11 assay
Comparison of hybrid detection by Structure and the
N, no. of individuals sampled; NH, no. of hybrids detected (see
text for criteria); NM, no. of form molestus detected.
aP ? form pipiens; M ? form molestus.
September 2010KOTHERA ET AL.: Culex pipiens IN CHICAGO AND NEW YORK CITY
of the population. Finally, because the CQ11 marker
itself is a microsatellite locus, it is possible that some
f. pipiens individuals have the same allele size that
Bahnck and Fonseca (2006) ascribed to f. molestus,
even if that allele is monomorphic in f. molestus. The
Þrst two points above were raised by Bahnck and
Fonseca (2006), but the current study raises the issue
of the CQ11 assay overestimating the number of in-
were collected from an aboveground population. In
to detect hybrids or f. molestus in aboveground pop-
This study describes the Þrst population genetic
study performed on a newly described population of
Cx. pipiens f. molestus from Chicago. Comparisons to
another f. molestus population in New York City, as
well as to aboveground populations in both cities,
indicate that these f. molestus populations are highly
genetically divergent from each other and their asso-
ciated aboveground populations.
The Andreadis laboratory at the Connecticut Agricultural
were used to establish the New York City colony of f. mo-
lestus. Chris Richards (United States Department of Agri-
culture, Center for Genetic Resources Preservation, Fort
commented on an earlier draft of this manuscript. Staff from
the OfÞce of Vector Surveillance and Control in the New
and provided safe access to specimens. Staff from the Met-
ropolitan Water Reclamation District of Greater Chicago
specimens. We appreciate the helpful comments of two
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Received 9 February 2010; accepted 11 April 2010.
September 2010KOTHERA ET AL.: Culex pipiens IN CHICAGO AND NEW YORK CITY