Culex Pipiens Complex Research Articles

Oviposition signals and their neuroethological correlates in the Culex pipiens complex

Mosquitoes in the Culex pipiens complex (Diptera: Culicidae), especially Cx. pipiens and Culex quinquefasciatus, have successfully exploited the rapid growth of the human population and globalization to their advantage by successfully utilizing man-made habitats, particularly for oviposition. Culex spp. lay over 100 eggs together in a raft. Each egg in the raft produces an apical droplet containing an oviposition attractant, erythro-6-acetoxy-5-hexadecanolide, commonly referred to as Mosquito Oviposition Pheromone (MOP). Here we present a detailed gas chromatography–mass spectrometry (GC–MS) analysis of the apical droplets from six populations that revealed MOP as the most abundant constituent. Subjecting MOP and the remaining 17 most abundant chemical constituents of the droplets from these populations to a Principal Component Analysis (PCA) resolved the populations into two distinct clusters that contained two populations each of Cx. quinquefasciatus and Culex pipiens molestus. The two Culex pipiens pipiens, however, did not resolve into a single cluster, with the Shasta population sorting closer to Cx. quinquefasciatus. Comparing the PCA scores with the genetic evidence from adult females using available molecular markers that have earlier shown to sort various Culex forms, we found that the molecular data support the PCA clustering pattern. Behavioral investigation of the droplet-induced attraction tested in gravid Cx. quinquefasciatus elicited various degrees of oviposition to the droplets from each population. Overall, droplets from all six populations induced higher attraction compared to controls. A detailed time-course analysis of droplet composition in Cx. quinquefasciatus from 6 to 54h post egg-laying identified MOP again as the main constituent. Finally, our electrophysiological investigation identified MOP as the only biologically active constituent from of the droplets eliciting responses from female antennae. These studies will aid in global efforts to understand the vector biology and evolution that can be exploited to develop novel vector management strategies.

Morphological Studies onCulex molestusof theCulex pipiensComplex (Diptera: Culicidae) in Underground Parking Lots in Wuhan, Central China

Abstract Mosquito surveillance was conducted in 42 underground parking lots in the densely populated metropolitan area of Wuhan, Hubei Province, central China from Aug to Oct 2012. Autogenous Culex p. molestus mosquitoes, which inhabit underground sites elsewhere in China, were identified and recorded for the first time in Hubei Province. Also Culex quinquefasciatus and intermediates (presumably with Cx. p. pallens) were identified from the samples, but Cx. p. pallens, which is known to hybridize with Cx. quinquefasciatus, was not found during the study. We attempted to determine how to differentiate between the subspecies of the Culex pipiens complex in Wuhan. In the female wings of Cx. p molestus, the subcosta intersected the costa beyond or at the furcation of R2+3; however in Cx. quinquefasciatus and intermediates, the subcosta intersected the costa before the furcation of R2+3. In female abdomens of Cx. p. molestus, lateral patches on tergum II reached the posterior margins; however in Cx. quinquefas...

Detection and isolation of the α-proteobacterium Asaia in Culex mosquitoes.

Investigations of microbiota within mosquitoes continue to widen the spectrum of possible symbiont-based applications against vector-borne diseases. In this context, α-proteobacteria of the genus Asaia (Rhodospirillales: Acetobacteraceae) are emerging as possible endosymbiotic candidates, particularly in paratransgenic approaches aimed at interrupting pathogen transmission. Previous studies have shown that Asaia spp. distribution among Anopheles gambiae and Anopheles stephensi (Diptera: Culicidae) mosquitoes displayed positive rates of infection in isolated midguts, salivary glands and reproductive tissues. Similarly, Asaia has been detected in Aedes albopictus (Stegomyia albopicta) and Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) populations. Within the Culex pipiens complex (Diptera: Culicidae), Asaia infection is still largely unexplored. Here, we summarize a preliminary survey of laboratory-reared Cx. pipiens complex and field-collected Culex quinquefasciatus for the presence of Asaia spp., and present the first identification of Asaia in some of the members of the Cx. pipiens complex and the first description in West African populations of Cx. quinquefasciatus.

Phenotypic Variation among Culex pipiens Complex (Diptera: Culicidae) Populations from the Sacramento Valley, California: Horizontal and Vertical Transmission of West Nile Virus, Diapause Potential, Autogeny, and Host Selection

The vector competence and bionomics of Culex pipiens form pipiens L. and Cx. pipiens f. molestus Forskäl were evaluated for populations from the Sacramento Valley. Both f. pipiens and f. molestus females became infected, produced disseminated infections, and were able to transmit West Nile virus. Form molestus females also transmitted West Nile virus vertically to egg rafts and F1 progeny, whereas f. pipiens females only transmitted to egg rafts. Culex pipiens complex from urban Sacramento blood-fed on seven different avian species and two mammalian species. Structure analysis of blood-fed mosquitoes identified K = 4 genetic clusters: f. molestus, f. pipiens, a group of genetically similar hybrids (Cluster X), and admixed individuals. When females were exposed as larvae to midwinter conditions in bioenvironmental chambers, 85% (N = 79) of aboveground Cx. pipiens complex females and 100% (N = 34) of underground f. molestus females did not enter reproductive diapause.

Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species

BackgroundThe maternally inherited bacterium Wolbachia often acts as a subtle parasite that manipulates insect reproduction, resulting potentially in reproductive isolation between host populations. Whilst distinct Wolbachia strains are documented in a group of evolutionarily closely related mosquitoes known as the Culex pipiens complex, their impact on mosquito population genetics remains unclear. To this aim, we developed a PCR-RFLP test that discriminates the five known Wolbachia groups found in this host complex. We further examined the Wolbachia genetic diversity, the variability in the coinherited host mitochondria and their partitioning among members of the Cx. pipiens complex, in order to assess the impact of Wolbachia on host population structure.ResultsThere was a strong association between Wolbachia and mitochondrial haplotypes indicating a stable co-transmission in mosquito populations. Despite evidence that members of the Cx. pipiens complex are genetically distinct on the basis of nuclear DNA, the association of Wolbachia and mtDNA with members of the Cx. pipiens complex were limited. The Wolbachia wPip-I group, by far the most common, was associated with divergent Cx. pipiens members, including Cx. quinquefasciatus, Cx. pipiens pipiens form pipiens and Cx. pipiens pipiens form molestus. Four other wPip groups were also found in mosquito populations and all were shared between diverse Cx. pipiens members.ConclusionThis data overall supports the hypothesis that wPip infections, and their allied mitochondria, are associated with regular transfers between Cx. pipiens members rather than specific host associations. Overall, this is suggestive of a recent and likely ongoing cytoplasmic introgression through hybridization events across the Cx. pipiens complex.

Mosquitoes established in Lhasa city, Tibet, China

BackgroundIn 2009, residents of Lhasa city, Tibet Autonomous Region (TAR), China reported large numbers of mosquitoes and bites from these insects. It is unclear whether this was a new phenomenon, which species were involved, and whether these mosquitoes had established themselves in the local circumstances.MethodsThe present study was undertaken in six urban sites of Chengguan district Lhasa city, Tibet. Adult mosquitoes were collected by bed net trap, labor hour method and light trap in August 2009 and August 2012. The trapped adult mosquitoes were initially counted and identified according to morphological criteria, and a proportion of mosquitoes were examined more closely using a multiplex PCR assay.Results907 mosquitoes of the Culex pipiens complex were collected in this study. Among them, 595 were females and 312 were males. There was no significant difference in mosquito density monitored by bed net trap and labor hour method in 2009 and 2012. Of 105 mosquitoes identified by multiplex PCR, 36 were pure mosquitoes (34.29%) while 69 were hybrids (65.71%). The same subspecies of Culex pipiens complex were observed by bed net trap, labor hour method and light trap in 2009 and 2012.ConclusionThe local Culex pipiens complex comprises the subspecies Cx. pipiens pipiens, Cx. pipiens pallens, Cx. pipiens quinquefasciatus and its hybrids. Mosquitoes in the Cx. pipiens complex, known to be, potentially, vectors of periodic filariasis and encephalitis, are now present from one season to the next, and appear to be established in Lhasa City, TAR.

Overwintering Biology of <I>Culex</I> (Diptera: Culicidae) Mosquitoes in the Sacramento Valley of California

At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010-2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to >or=stage I-II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 +/- 30.7 and 162.85 +/- 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.

Identification of Culex complex species using SNP markers based on high‐resolution melting analysis

Mosquitoes belonging to the Culex pipiens complex are primary vectors for diseases such as West Nile encephalitis, Eastern equine encephalitis, many arboviruses, as well as lymphatic filariases. Despite sharing physiological characteristics, each mosquito species within the Culex complex has unique behavioural and reproductive traits that necessitate a proper method of identification. Unfortunately, morphometric methods of distinguishing members of this complex have failed to yield consistent results, giving rise to the need for molecular methods of identification. In this study, we propose a novel identification method using high-resolution melting (HRM) analysis by examining single-nucleotide polymorphisms in the acetylcholinesterase-2 (ace-2) locus. Our method provides a high confidence for species determination among the three Culex complex mosquitoes.

Predicting distribution of Aedes aegypti and Culex pipiens complex, potential vectors of Rift Valley fever virus in relation to disease epidemics in East Africa

BackgroundThe East African region has experienced several Rift Valley fever (RVF) outbreaks since the 1930s. The objective of this study was to identify distributions of potential disease vectors in relation to disease epidemics. Understanding disease vector potential distributions is a major concern for disease transmission dynamics.MethodsDiverse ecological niche modelling techniques have been developed for this purpose: we present a maximum entropy (Maxent) approach for estimating distributions of potential RVF vectors in un-sampled areas in East Africa. We modelled the distribution of two species of mosquitoes (Aedes aegypti and Culex pipiens complex) responsible for potential maintenance and amplification of the virus, respectively. Predicted distributions of environmentally suitable areas in East Africa were based on the presence-only occurrence data derived from our entomological study in Ngorongoro District in northern Tanzania.ResultsOur model predicted potential suitable areas with high success rates of 90.9% for A. aegypti and 91.6% for C. pipiens complex. Model performance was statistically significantly better than random for both species. Most suitable sites for the two vectors were predicted in central and northwestern Tanzania with previous disease epidemics. Other important risk areas include western Lake Victoria, northern parts of Lake Malawi, and the Rift Valley region of Kenya.ConclusionFindings from this study show distributions of vectors had biological and epidemiological significance in relation to disease outbreak hotspots, and hence provide guidance for the selection of sampling areas for RVF vectors during inter-epidemic periods.

Local Adaptation and Hybridization Across the Culex pipiens Complex in the USA

We used eight highly polymorphic molecular markers (microsatellites) to characterize the extent of the hybridization between Culex pipiens (both forms) and Cx. quinquefasciatus in the US. We examined populations from three longitudinal transects, two on the East coast and one on the West Coast. The markers used were chosen based on three criteria: (1) a sufficient number of markers were needed to insure statistical accuracy during the analyses; (2) the markers had to be amplifiable in all taxa (3) the markers had to include alleles unique to each taxa, allowing a clear separation between the genomes of pure populations and those of potential hybrids. We obtained specimens of Cx. pipiens complex by contacting entomologists in local Departments of Health, Vector Control Units, and Universities. We found much deeper introgression of alleles from each species than expected from previous classical analysis of male genitalia. A Bayesian multilocus cluster analysis without a priori identification of the specimen's geographical origin identified three distinct microsatellite signatures. We found virtually no individuals with a “pure” signature in the mid-latitudinal states, and found that the frequency of hybrid types shifts across elevation, at each latitude, likely associated with temperature. We examined how temperature may affect hybrid distribution by performing laboratory studies and developed a predictive model of the impact of hybridization on the population dynamics of Culex pipiens complex across the US latitudinal gradient.

Introduction to the Symposium Global Perspective on the Culex pipiens Complex in the 21stCentury: The Interrelationship of Culex pipiens, quinquefasciatus, molestus and Others

Mosquitoes in the Culex pipiens Complex, including Culex pipiens, Cx. quinquefasciatus, and Cx. molestus, are important pest species and vectors of human and animal diseases throughout the world's tropical, temperate, and Holarctic regions. Diseases transmitted by member of the Pipiens Complex include: St. Louis encephalitis and West Nile virus in North America, West Nile virus on several other continents, Rift Valley fever in Africa, lymphatic filariasis caused by Wuchereria bancrofti in the tropics, and Dirofilaria immitis globally. Here and in the following 14 papers, 3 abstracts and a summary paper are the proceedings of a symposium that gathered many of the world's experts on the Pipiens Complex to explore the current state of knowledge of the taxa. Information presented at the symposium will improve our knowledge of important members of the complex and enhance our ability to conduct efficient surveillance and efficacious control strategies. A background on previous discussions on the Pipiens Complex, and a brief description of current symposium contributors and their topics are discussed.

The Contribution of Culex pipiens Complex Mosquitoes to Transmission and Persistence of West Nile Virus in North America

Mosquitoes within the Culex pipiens complex have been implicated as major vectors of West Nile virus (WNV) in North America due to their seasonal abundance, vector competence and high field infection rates. However, the role of Cx. p. pipiens complex mosquitoes in enzootic amplification of WNV among avian hosts and epidemic transmission to humans varies throughout its geographical distribution. In the northeastern United States, Cx. p. pipiens is recognized as the primary enzootic vector responsible for amplification of virus among wild bird populations. However, because this mosquito is strongly ornithophilic, its role in transmission to humans appears to be more limited in this region. In the north central and Mid-Atlantic States by contrast, Cx. p. pipiens shows an increased affinity for human hosts and has been incriminated as a key bridge vector. In southern regions of the United States, Culex p. quinquefasciatus are more opportunistic feeders, and are thought to be principal enzootic and epidemic vectors. In western regions of the United States where Culex tarsalis predominates, especially in rural areas, Cx. p. pipiens and Cx. p. quinquefasciatus play roles that are more limited and are recognized as secondary vectors. In the southwestern United States Cx. p. quinquefasciatus also appears to be the predominant vector in urban habitats, but only a secondary vector in more rural environs. The direct involvement of Cx. p. pipiens form molestus in WNV transmission is largely unknown, but human-biting Cx. p. pipiens are more likely to have a probability of genetic ancestry with Cx. p. pipiens form molestus. The detection of WNV from overwintering populations of diapausing Cx. p. pipiens and non-diapausing Cx. p. quinquefaciatus and their role in local overwintering of WNV are addressed.

Summary of the Symposium Global Perspective on the Culex pipiens Complex in the 21stCentury: The Interrelationship of Culex pipiens, quinquefasciatus, molestus and Others

A symposium on the mosquitoes in the Culex pipiens Complex (Pipiens Complex), including Cx. pipiens, Cx. quinquefasciatus, and Cx. molestus, invited 17 of the world's experts on this species Complex to explore the current state of knowledge on the systematic status, speciation, ecology, biology, vector competence, population genetics, endosymbionts, and role in disease transmission of these species in various parts of the world. A Foreword, Introduction, 14 full papers, 3 abstracts and this Summary are published in this special issue of the Journal of the American Mosquito Control Association. A summary of findings, overall conclusions, and research needs will be discussed in relation to mosquito control and interrupting disease transmission caused by these species.

The Culex pipiens Complex in Europe

The present study examines the mating and breeding behavior as well as the genetic differentiation of Culex pipiens biotype pipiens and Cx. pipiens biotype molestus. Firstly, the mating behavior of Cx. pipiens s.l. originating from larval populations of various epigeous and hypogeous breeding sites in Germany was examined. Autogeny was prevailing in underground populations, occasionally found in semi-open water reservoirs like drains, rarely in containers, but never in ponds and ditches. Secondly, in a multilocus enzyme electrophoretic study the gene flow among seven geographic populations of Cx. pipiens biotype pipiens and the biotype molestus from several European countries was quantified. For comparison, five populations of Cx. quinquefasciatus from Asia, Africa and North America, three populations of Cx. torrentium (Germany) and other outgroup species were also examined. Thirdly, the mitochondrial cytochrome c oxidase submit I gene of both biotypes from Germany was analysed by a polymerase chain reaction - restriction fragment length polymorphism assay and the ascertained DNA-sequences were aligned with genebank data of Russian populations. The population genetic analyses revealed much higher genetic distances between local populations of Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus compared to the low differentiation between geographically remote populations within each taxon. The UPGMA (unweighted pair group method with arithmetic mean) analysis and F-statistics positioned the biotypes pipiens on one side and molestus on the other side in discrete monophyletic clusters. Gene flow between local populations of the biotypes pipiens and molestus could be shown to be lower than gene flow between geographically distant populations within each of the two groups, leading to the conclusion that Cx. pipiens biotype molestus could be a distinct taxon. Culex quinquefasciatus was genetically well-separated, in particular by the diagnostic enzyme marker malate dehydrogenase (nicotinamide adenine dinucleotide phosphate). The genetic markers adenylatekinase and hydrobutyrate dehydrogenase allowed to screen thousands of morphologically similar samples of either Cx. pipiens s.l. and Cx torrentium and it could be shown that Cx. torrentium is a very frequent species in central Europe.

The Culex pipiens Complex in the Mississippi River Basin: Identification, Distribution, and Bloodmeal Hosts

Members of the Culex pipiens complex are the primary vectors of St. Louis encephalitis virus and West Nile virus in the Mississippi River basin (MRB). The Cx. pipiens complex in the MRB is composed of 4 taxa: Cx. p. pipiens form pipiens, Cx. p. quinquefasciatus, hybrids between Cx. p. pipiens f. pipiens and Cx. p. quinquefasciatus, and Cx. p. pipiens form molestus. Three studies on bloodmeal hosts with large sample sizes have been conducted on members of the Cx. pipiens complex in the MRB including 1 each on Cx. p. quinquefasciatus from Louisiana, Cx. p. pipiens-quinquefasciatus hybrids from Tennessee, and Cx. p. pipiens from Illinois. The top 8 bloodmeal hosts from each of the 3 sites accounted for 68-92% of bloodmeals. Only 14 species accounted for the top 8 bloodmeal hosts at each of the 3 sites. The most often utilized bloodmeal hosts for members of the Culex pipiens complex within the MRB are the American robin, Northern cardinal, human, raccoon, common grackle, house sparrow, mourning dove, dog, Northern mockingbird, blue jay, opossum, domestic horse, house finch and European starling. Human feeding varied widely among sites from 1% to 15.7% of bloodmeals. The proportion of bloodmeals taken on humans is an important epidemiological variable and future studies are needed to define the primary genetic and environmental factors that influence host utilization by members of the Cx. pipiens complex.

Members of the Culex pipiens Complex as Vectors of Viruses1

Members of the Culex pipiens complex have been implicated as vectors of a number of arboviruses including St. Louis encephalitis, West Nile, Sindbis, and Rift Valley fever viruses. For some viruses, such as West Nile virus, laboratory studies have indicated that various members of this complex have a similar ability to become infected with and transmit virus, thus providing evidence for the similarity among the various members of this complex. On the other hand, although strains of Cx. pipiens from various parts of the world have all been relatively efficient vectors of Rift Valley fever virus, Cx. quinquefasciatus from Africa, Australia, and North America have been nearly refractory to this virus, thus indicating that the various members of this complex do not necessarily respond similarly to a particular arbovirus. Based on the similar response to some viruses and differing response to others, Cx. pipiens and Cx. quinquefasciatus appear to be closely related, but distinct species.

The Spielman Legacy: His Influence on the CX. pipiens Complex in the USA

Any modern discussion of the biology of the Culex pipiens complex bears an unshakable influence of the late Andrew Spielman. His accomplishments, with those of his former students, post docs and collaborators, were as diverse as they were significant. His contributions began with his doctoral work focused on the genetics of autogeny of Cx. pipiens. He embraced cutting edge technology of that era to uncover previously unrealized complexities in the physiology and population structure of this taxon. From the late 1950s through the late 1970s, Spielman published most of his dozen seminal papers that focused on aspects of Cx. pipiens biology. These covered aspects of physiology, autogeny, population structure, anatomy and ecology. His attention returned to this group with the emergence of West Nile virus (WNV). He then led a team of colleagues distributed throughout this mosquito's geographic range to probe latitudinal and diel influences on the distribution of the vector as well as on the force of transmission of WNV. He sought sound physiological bases to reveal all aspects of vector competence and vectorial capacity, and insisted that these findings be central to the design of any intervention. A review of Spielman's contributions and perspectives are pertinent when considering new findings as well as the challenges and opportunities that lie ahead.

The Forms of the Culex pipiens Complex in East Asia, with Ecological Thoughts on Their Origin and Interrelation

In East Asia, 4 forms of the Culex pipiens complex have been confirmed. A form pipiens s. s. (anautogeous pipiens) has been confirmed only in westernmost China. A temperate form pallens and a subtropical and tropical form quinquefasciatus are connected with intermediates in morphology and many aspects of ecology, but their difference is rather clear in climatic adaptation traits. The distribution of a form molestus overlaps with pallens and, in Taiwan, quinquefasciatus, and, in East Asia, this form requires artificial underground habitats for its persistence. The origin and interrelation of 3 forms other than pipiens s. s. are considered from ecological aspects, especially climatic adaptation. A hypothesis is presented that molestus was originally a form having adapted to the Mediterranean climate in the western Palaearctic, secondarily colonized artificial underground habitats, and reached East Asia in the early 20th century by ships from North America. At present, it is difficult to assign pallens with certainty to either the old or new groups of the Manchurian mosquito fauna. Three hypotheses about the interrelation between pallens and quinquefasciatus are compared, and the strengths and problems of each hypothesis are indicated. Finally, a map to show the distribution of the forms of the Culex pipiens complex before it was extensively changed by humans is presented as an initial trial. The Culex pipiens problems now troubling humans are largely human-made problems.

Characterization of Culex pipiens Complex (Diptera: Culicidae) Populations in Colorado, USA Using Microsatellites

Mosquitoes such as those in the Culex pipiens complex are important vectors of disease. This study was conducted to genetically characterize Cx. pipiens complex populations in the state of Colorado, USA, and to determine the number of genetic clusters represented by the data. Thirteen populations located among four major river basins were sampled (n = 597 individuals) using a panel of 14 microsatellites. The lowest-elevation sites had the highest Expected Heterozygosity (HE) values (range 0.54–0.65). AMOVA results indicated the presence of statistically significant amounts of variation within each level when populations were analyzed as one group or when they were grouped either by river basin or by their position on the east or west side of the Rocky Mountains. Most pairwise FST values were significant via permutation test (range 0–0.10), with the highest values from comparisons with Lamar, in southeast CO. A neighbor joining tree based on Cavalli–Sforza and Edwards’s chord distances was consistent with the geographic locations of populations, as well as with the AMOVA results. There was a significant isolation by distance effect, and the cluster analysis resolved five groups. Individuals were also assayed with an additional microsatellite marker, Cxpq78, proposed to be monomorphic in Cx. pipiens but polymorphic in the closely related but biologically distinct species Cx. quinquefasciatus. Low frequencies (≤3%) of Cx. quinquefasciatus alleles for this marker were noted, and mostly confined to populations along the Interstate 25 corridor. Pueblo was distinct in that it had 10% Cx. quinquefasciatus alleles, mostly of one allele size. The degree of population genetic structure observed in this study is in contrast with that of Cx. tarsalis, the other major vector of WNV in the western U.S., and likely reflects the two species’ different dispersal strategies.

Identification of resistant carboxylesterase alleles in Culex pipiens complex via PCR-RFLP

BackgroundCarboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides. As a major transmitter of human diseases, mosquitoes in the Culex pipiens complex have evolved 13 carboxylesterase alleles (Ester) that confer organophosphate resistance. Six alleles, EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11, have been observed in field populations in China, sometimes co-existing in one population. To differentiate the carboxylesterase alleles found in these field populations, PCR-RFLP was designed for use in resistance monitoring.ResultsBased on the DNA sequences of resistant and nonresistant carboxylesterase alleles, Ester B alleles were first amplified with PCR-specific primers and then digested with the restriction enzyme DraI. In this step, Ester2 and Ester11 were differentiated from the other Ester alleles. When the other Ester B alleles were digested with the restriction enzyme XbaI, EsterB1 and the susceptible C. p. pallens Ester were screened out. Ester8 and Ester9 were differentiated from EsterB10 and the susceptible C. p. quinquefasciatus esterase allele, respectively, by amplifying and digesting the Ester A alleles with the restriction enzyme ApaLI. The effectiveness of the custom-designed PCR-RFLP was verified in two field mosquito populations.ConclusionsA PCR-RFLP based approach was developed to differentiate carboxylesterase alleles in Culex pipiens complex mosquitoes. These processes may be useful in monitoring the evolutionary dynamics of known carboxylesterase alleles as well as in the identification of new alleles in field populations.