Form Pipiens Research Articles

Insecticide resistance and target site mutations (G119S ace-1 and L1014F kdr) of Culex pipiens in Morocco

BackgroundControl of the mosquito vector Culex pipiens with insecticides is the main way to control arboviruses that the species can transmit such as West Nile virus (WNV) and Rift Valley fever virus (RVFV). However, its efficiency has been hampered by the emergence of insecticide resistance. Little is known about the insecticide-resistance status and underlying resistance mechanisms of field-collected populations of Cx. pipiens in Morocco.MethodsMosquito adults from Mohammadia city in Morocco were reared from immature stages. The level of their susceptibility to insecticides was assessed using standard WHO bioassay. The two forms of the Cx. pipiens complex and their hybrids were identified by a multiplex PCR. Identified mosquitoes were then tested for the presence of the G119S ace-1 and L1014F kdr mutations using PCR-RFLP and PCR assays, respectively.ResultsWHO bioassays indicated that Cx. pipiens was resistant to all tested insecticides: lambda-cyhalothrin (49% mortality), permethrin (63% mortality), DDT (16% mortality), malation (52% mortality) and bendiocarb (39% mortality). The frequency of the 119S allele was almost identical in the pipiens form and hybrids (0.11 and 0.15, respectively) whereas it remained low in the molestus form (0.03). No significant correlation was observed between the G119S allele and the resistance phenotype to two tested insecticides (malathion and bendiocarb). The frequency of the L1014F allele was identical in the pipiens form and hybrids (0.44) whereas it was low in the molestus form (0.36) but no significant difference was detected (χ2 = 1.46, df = 1, P = 0.225). The presence of the L1014F kdr mutation was significantly associated with resistance to three tested insecticides in pipiens form (P = 0.0019, P = 0.0023 and P = 0.023, respectively, to lambda-cyhalothrin, permethrin and DDT) whereas no significant correlation was observed between the L1014F kdr mutation and resistance phenotype in molestus form and hybrids to the three tested insecticides.ConclusionThese findings showed that wild populations of Cx. pipiens have developed resistance against the main insecticide families with different modes of action: organochlorines (DDT), organophosphates (malathion), carbamates (bendiocarb), pyrethroids (lambda-cyhalothrin, permethrin). Therefore, urgent action should be taken to manage the resistance in this species to maintain the effectiveness of arbovirus control.

Assessment of Distribution Patterns of Culex Disease Vectors by Molecular Assays

Study of distribution patterns of Culex pipiensforms and their sibling species Culextorrentium in Berlin and Hannover Germany was done The mosquitoes of the Culex genus have epidemiological significance as vectors of arboviruses and other pathogens of humans and animals Based on molecular genetics methodsit was shown that the park in Berlin in was inhabited by Culexpipiens form pipiens while in the watercontainerin Hannover coexistence of Cx pipiens form pipiens and Cx torrentium was noted All females carrying sucked human blood and caught in the flats belong to Cx pipiens form molestus Molecular differentiation has revealed the coexistence of various Culex taxa in cities which increases the probability of hybridization and the appearance of potential bridge vectors between birds and humans

Ecophysiological characterization and molecular differentiation of Culex pipiens forms (Diptera: Culicidae) in Tunisia

BackgroundThe Culex pipiens complex (Diptera: Culicidae) includes the most widespread mosquito species in the world. Members of this complex are the primary enzootic and epidemic vectors of the West Nile virus (genus Flavivirus) in several countries. The two recognized forms of Cx. pipiens (Linnaeus, 1758) - pipiens and molestus- exhibit behavioral and physiological differences. Natural populations of Cx. pipiens were investigated in several sites in Tunisia to evaluate the ecophysiological and molecular characteristics of their forms.ResultsThe analysis showed the sympatric presence of Cx. pipiens forms and hybrids in all studied sites. Of all the tested larvae of Cx. pipiens, 33.5% were identified as pipiens, 30.8% were identified as molestus, and 35.6% were identified as hybrids. The molestus and hybrid forms were positively correlated with urban habitats and belowground sites while the pipiens form was positively correlated with rural habitats and aboveground sites. Autogeny was expressed in all types of habitats and breeding sites. By contrast with the microsatellite CQ11, the two molecular markers, ace-2 and cytb, did not allow differentiation between the Cx. pipiens forms.ConclusionsOur study shows the ubiquitous distribution and the plasticity of the different forms of Cx. pipiens in a wide range of ecological conditions. It suggests that the behavioral traits assigned to the forms of Cx. pipiens seem to be more flexible than previously assumed. Our analysis also proves that the microsatellite CQ11 remains an efficient tool for distinguishing between Cx. pipiens forms.

Culex pipiens forms and urbanization: effects on blood feeding sources and transmission of avian Plasmodium.

BackgroundThe wide spread mosquito Culex pipiens pipiens have two forms molestus and pipiens which frequently hybridize. The two forms have behavioural and physiological differences affecting habitat requirements and host selection, which may affect the transmission dynamic of Cx. p. pipiens-borne diseases.MethodsDuring 2013, blood engorged Cx. p. pipiens mosquitoes were captured in urban, rural and natural areas from Southern Spain. In 120 mosquitoes, we identified the blood meal origin at vertebrate species/genus level and the mosquito form. The presence and molecular lineage identity of avian malaria parasites in the head-thorax of each mosquito was also analysed.ResultsMosquitoes of the form pipiens were more frequently found in natural than in urban areas. The proportion of Cx. pipiens form molestus and hybrids of the two forms did not differ between habitat categories. Any significant difference in the proportion of blood meals on birds between forms was found. Birds were the most common feeding source for the two forms and their hybrids. Among mammals, dogs and humans were the most common hosts. Two Plasmodium and one Haemoproteus lineages were found in mosquitoes, with non-significant differences between forms.ConclusionThis study supports a differential distribution of Cx. p. pipiens form pipiens between urban and natural areas. Probably due to the similar feeding sources of both mosquito forms and their hybrids here, all of them may frequently interact with avian malaria parasites playing a role in the transmission of Plasmodium.

First report of L1014F-kdr mutation in Culex pipiens complex from Morocco.

BackgroundMosquitoes of the Culex pipiens complex, competent vectors for West Nile virus (WNV) and Rift Valley fever virus (RVFV) are widely targeted by insecticide treatments. The intensive application of chemical insecticides led to the development of resistance in many insects including Culex pipiens mosquitoes. The absence of data on resistance mechanisms in Morocco allow us to assess the levels of lambda-cyhalothrin resistance and the frequency of the mutated gene L1014F kdr in different forms of Cx. pipiens complex from three regions of Morocco.MethodsMosquito adults were reared from immature stages collected in three different regions in Morocco (Tangier, Casablanca and Marrakech). Standard WHO insecticide susceptibility tests were conducted on adults emerged from collected larvae. Specimens were identified as belonging to the Culex pipiens complex using a multiplex PCR assay with diagnostic primers designed from the flanking region of microsatellite CQ11. Identified mosquitoes were then tested for the presence of the L1014F kdr mutation using PCR assay.ResultsOur results showed that 21% of the tested population has a resistance to lambda-cyhalothrin. The molecular identification of survivors shows that 43% belonged to the Cx. pipiens pipiens and only 9.5% to the Cx. pipiens molestus form. On the other hand, 416 specimens were screened for the L1014F kdr mutation. L1014F mutation was detected in different forms of Cx. pipiens in different sites. The frequency of L1014F mutation was similar between the Cx. pipiens pipiens form and hybrid form, while it was lower in the Cx. pipiens molestus form. The presence of the L1014F kdr allele was significantly associated with resistance to lambda-cyhalothrin in Cx. pipiens pipiens (P < 0.0001) and hybrid form (P < 0.0001).ConclusionResistance to lambda-cyhalothrin of Cx. pipiens populations appears to be largely due to the L1014F kdr mutation. To our knowledge, the frequencies of L1014F kdr mutation are examined for the first time in natural populations of the Culex pipiens complex in Morocco. These findings will provide important information to propose more adapted vector control measures towards this mosquito species, potential vector of arboviruses.

Ecological differentiation of members of the Culex pipiens complex, potential vectors of West Nile virus and Rift Valley fever virus in Algeria.

BackgroundWe investigated the ecological differentiation of two members of the Culex pipiens complex, Cx. p. pipiens form pipiens and Cx. p. pipiens form molestus in three sites, El-Kala, M'Sila and Tinerkouk in Algeria. These two forms are the most widespread mosquito vectors in temperate regions exhibiting important behavioural and physiological differences. Nevertheless, this group of potential vectors has been poorly studied, particularly in North Africa.MethodsTen larval populations of Cx. p. pipiens were sampled from various above- and underground habitats in three zones representing the three bioclimatic regions in Algeria. The reproduction characteristics were also investigated in the laboratory to define the rates of autogeny and stenogamy. Identification of Cx. p. pipiens members present in Algeria was achieved using a molecular analysis with the microsatellite CQ11 locus.ResultsWe detected larvae of Cx. p. pipiens in all areas suggesting that the species is a ubiquitous mosquito well adapted to various environments. To our knowledge, this study provides the first molecular evidence of the presence of the Cx. p. pipiens form molestus and hybrids (molestus/pipiens) in Algeria with a high proportion of molestus form (48.3 %) in comparison with hybrids (36.8 %) and pipiens form (14.9 %).ConclusionsSome unexpected correlations between the proportion of forms pipiens, molestus and hybrids, and mosquito biological characteristics were observed suggesting some epigenetic effects controlling Cx. p. pipiens mating and reproduction. Consequences for pathogen transmission are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1725-9) contains supplementary material, which is available to authorized users.

Ecological characterization and molecular differentiation of Culex pipiens complex taxa and Culex torrentium in eastern Austria.

BackgroundCulex pipiens complex taxa differ in behaviour, ecophysiology and epidemiologic importance. Despite their epidemiologic significance, information on genetic diversity, occurrence and seasonal and spatial distribution patterns of the Cx. pipiens complex is still insufficient. Assessment of seasonal and spatial distribution patterns of Culex pipiens forms and their congener Cx. torrentium is crucial for the understanding of their vector–pathogen dynamics.MethodsFemale mosquitoes were trapped from April–October 2014 twice a month for a 24-h time period with BG-sentinel traps at 24 sampling sites in eastern Austria, using carbon dioxide as attractant. Ecological forms of Cx. pipiens s.l. and their hybrids were differentiated using the CQ11 locus, and Cx. pipiens forms and their congener Cx. torrentium using the ACE-2 gene. Differential exploitation of ecological niches by Cx. pipiens forms and Cx. torrentium was analysed using likelihood ratio tests. Possible effects of environmental parameters on these taxa were tested using PERMANOVA based on distance matrices and, if significant, were modelled in nMDS ordination space to estimate non-linear relationships.ResultsFor this study, 1476 Culex spp. were sampled. Culex pipiens f. pipiens representing 87.33 % of the total catch was most abundant, followed by hybrids of both forms (5.62 %), Cx. torrentium (3.79 %) and Cx. pipiens f. molestus (3.25 %). Differences in proportional abundances were found between land cover classes. Ecological parameters affecting seasonal and spatial distribution of these taxa in eastern Austria are precipitation duration, air temperature, sunlight and the interaction term of precipitation amount and the Danube water level, which can be interpreted as a proxy for breeding habitat availability.ConclusionsThe Cx. pipiens complex of eastern Austria comprises both ecologically different forms, the mainly ornithophilic form pipiens and the mainly mammalophilic and anthropophilic form molestus. Heterogeneous agricultural areas as areas of coexistence may serve as hybridization zones, resulting in potential bridge vectors between birds and humans. Occurrence, seasonal and spatial distribution patterns of the Cx. pipiens complex and Cx. torrentium and the presence of hybrids between both forms were quantified for the first time in Austria. These findings will improve the knowledge of their vector–pathogen dynamics in this country.

Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: contribution of Cx. quinquefasciatus in Mediterranean populations.

BackgroundMosquitoes of the Culex pipiens complex are cosmopolitan, and important vectors of neglected tropical diseases, such as arbovirosis and lymphatic filariasis. Among the complex taxa, Cx. pipiens (with two forms pipiens and molestus) and Cx. quinquefasciatus are the most ubiquitous mosquitoes in temperate and tropical regions respectively. Mosquitoes of this taxa lack of morphological differences between females, but have frank behavioral and physiological differences and have different trophic preferences that influence their vectorial status. Hybridization may change the vectorial capacity of these mosquitoes, increasing vector efficiency and medical importance of resulting hybrids.MethodsCulex pipiens s.l. from 35 distinct populations were investigated by the study of mtDNA, symbiotic bacterium Wolbachia pipientis, nuclear DNA and flanking region of microsatellite CQ11 polymorphism using PCR with diagnostic primers, RFLP analysis and sequencing.ResultsSix different mitochondrial haplotypes were revealed by sequencing of the cytochrome oxidase subunit I (COI) gene and three different Wolbachia (wPip) groups were identified. A strong association was observed between COI haplotypes/groups, wPip groups and taxa; haplogroup A and infection with wPipII appear to be typical for Cx. pipiens form pipiens, haplotype D and infection with wPipIV for form molestus, while haplogroup E, characteristic of Cx. quinquefasciatus, were correlated with wPipI and found in Cx. pipiens sl. from coastal regions of Southern Europe and Mediterranean region. Analysis of microsatellite locus and nuclear DNA revealed hybrids between Cx. pipiens form pipiens and form molestus, as well as between Cx. pipiens and Cx. quinquefasciatus, in Mediterranean populations, as opposed to Northern Europe. Phylogenetic analysis of COI sequences yielded a tree topology that supported the RFLP analysis with significant bootstrap values for haplotype D and haplogroup E.ConclusionsMolecular identification provides the first evidence of the presence of hybrids between Cx. quinquefasciatus and Cx. pipiens as well as cytoplasmic introgression of Cx. quinquefasciatus into Cx. pipiens as a result of hybridization events in coastal regions of Southern Europe and Mediterranean region. Together with observed hybrids between pipiens and molestus forms, these findings point to the presence of hybrids in these areas, with consequent higher potential for disease transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1333-8) contains supplementary material, which is available to authorized users.

Genetic divergence between populations of feral and domestic forms of a mosquito disease vector assessed by transcriptomics.

Culex pipiens, an invasive mosquito and vector of West Nile virus in the US, has two morphologically indistinguishable forms that differ dramatically in behavior and physiology. Cx. pipiens form pipiens is primarily a bird-feeding temperate mosquito, while the sub-tropical Cx. pipiens form molestus thrives in sewers and feeds on mammals. Because the feral form can diapause during the cold winters but the domestic form cannot, the two Cx. pipiens forms are allopatric in northern Europe and, although viable, hybrids are rare. Cx. pipiens form molestus has spread across all inhabited continents and hybrids of the two forms are common in the US. Here we elucidate the genes and gene families with the greatest divergence rates between these phenotypically diverged mosquito populations, and discuss them in light of their potential biological and ecological effects. After generating and assembling novel transcriptome data for each population, we performed pairwise tests for nonsynonymous divergence (Ka) of homologous coding sequences and examined gene ontology terms that were statistically over-represented in those sequences with the greatest divergence rates. We identified genes involved in digestion (serine endopeptidases), innate immunity (fibrinogens and α-macroglobulins), hemostasis (D7 salivary proteins), olfaction (odorant binding proteins) and chitin binding (peritrophic matrix proteins). By examining molecular divergence between closely related yet phenotypically divergent forms of the same species, our results provide insights into the identity of rapidly-evolving genes between incipient species. Additionally, we found that families of signal transducers, ATP synthases and transcription regulators remained identical at the amino acid level, thus constituting conserved components of the Cx. pipiens proteome. We provide a reference with which to gauge the divergence reported in this analysis by performing a comparison of transcriptome sequences from conspecific (yet allopatric) populations of another member of the Cx. pipiens complex, Cx. quinquefasciatus.

Divergent host preferences of above- and below-ground Culex pipiens mosquitoes and their hybrid offspring.

Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no-choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above- and below-ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction-based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No-choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian-seeking; an individual's probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F1 and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above- and below-ground populations is an important contributor to epizootic transmission of WNV in North America.

New evidence for the potential role of Culex pipiens mosquitoes in the transmission cycle of West Nile virus in Tunisia.

Physiological and molecular characteristics of natural populations of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) were investigated to elucidate how this species is potentially involved in the transmission of West Nile virus in Tunisia. A total of 215 Cx. pipiens females from 11 breeding habitats were analysed in the laboratory to estimate autogeny and stenogamy rates. They were tested individually for the locus CQ11 to distinguish between the two Cx. pipiens forms, pipiens and molestus. All tested Cx. pipiens populations were stenogamous. Females from underground breeding sites were all autogeneous, whereas females from above-ground habitats were mostly anautogeneous. Of all the females tested, 59.7% were identified as pipiens, 22.4% as molestus, and 17.9% as hybrid pipiens/molestus. Furthermore, both Cx. pipiens forms and their hybrids were found to co-occur in sympatry in all sites. The results of this study represent the first evidence that both Cx. pipiens forms and their hybrids are present in Tunisia. Because hybrids able to act as bridge vectors are present in all studied habitats, Tunisia can be considered to have a high degree of receptivity for the establishment of West Nile virus zoonotic cycles.

Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes

For over 100years studies on avian haemosporidian parasite species have relied on similarities in their morphology to establish a species concept. Some exceptional cases have also included information about the life cycle and sporogonic development. More than 50 avian Plasmodium spp. have now been described. However, PCR-based studies show a much broader diversity of haemosporidian parasites, indicating the possible existence of a diverse group of cryptic species. In the present study, using both similarity and phylogenetic species definition concepts, we believe that we report the first characterised cryptic speciation case of an avian Plasmodium parasite. We used sequence information on the mitochondrial cytochrome b gene and constructed phylogenies of identified Plasmodium spp. to define their position in the phylogenetic tree. After analysis of blood stages, the morphology of the parasite was shown to be identical to Plasmodium circumflexum. However, the geographic distribution of the new parasite, the phylogenetic information, as well as patterns of development of infection, indicate that this parasite differs from P. circumflexum. Plasmodium homocircumflexum n. sp. was described based on information about genetic differences from described lineages, phylogenetic position and biological characters. This parasite develops parasitemia in experimentally infected birds – the domestic canary Serinus canaria domestica, siskin Carduelis spinus and crossbill Loxia curvirostra. Anaemia caused by high parasitemia, as well as cerebral paralysis caused by exoerythrocytic stages in the brain, are the main reasons for mortality. Exoerythrocytic stages also form in other organs (heart, kidneys, liver, lungs, spleen, intestines and pectoral muscles). DNA amplification was unsuccessful from faecal samples of heavily infected birds. The sporogonic development initiates, but is abortive, at the oocyst stage in two common European mosquito species, Culex pipiens pipiens (forms pipiens and molestus) and Aedes vexans. Vectors of this Plasmodium sp. remain unknown.

Population Genetic and Admixture Analyses of Culex pipiens Complex (Diptera: Culicidae) Populations in California, United States

Microsatellite markers were used to genetically characterize 19 Culex pipiens complex populations from California. Two populations showed characteristics of earlier genetic bottlenecks. The overall FST value and a neighbor-joining tree suggested moderate amounts of genetic differentiation. Analyses using Structure indicated K = 4 genetic clusters: Cx. pipiens form pipiens L., Cx. quinquefasciatus Say, Cx. pipiens form molestus Forskäl, and a group of genetically similar individuals of hybrid origin. A Discriminant Analysis of Principal Components indicated that the latter group is a mixture of the other three taxa, with form pipiens and form molestus contributing somewhat more ancestry than Cx. quinquefasciatus. Characterization of 56 morphologically autogenous individuals classified most as Cx. pipiens form molestus, and none as Cx. pipiens form pipiens or Cx. quinquefasciatus. Comparison of California microsatellite data with those of Cx. pipiens pallens Coquillett from Japan indicated the latter does not contribute significantly to genotypes in California.

Feeding patterns of molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in a region of high hybridization

BackgroundTwo biological forms of the mosquito Culex pipiens s.s., denoted pipiens and molestus, display behavioural differences that may affect their role as vectors of arboviruses. In this study, the feeding patterns of molestus and pipiens forms were investigated in Comporta (Portugal), where high levels of inter-form admixture have been recorded.MethodsIndoor and outdoor mosquito collections were performed in the summer of 2010. Collected Cx. pipiens s.l. females were molecularly identified to species and form by PCR and genotyped for six microsatellites. The source of the blood meal in post-fed females was determined by ELISA and mitochondrial DNA sequencing.ResultsThe distribution of the forms differed according to the collection method. The molestus form was present only in indoor collections, whereas pipiens and admixed individuals were sampled both indoors and outdoors. In both forms, over 90% of blood meals were made on avian hosts. These included blood meals taken from Passeriformes (Passer domesticus and Turdus merula) by females caught resting inside domestic shelters.ConclusionGenetic structure and blood meal analyses suggest the presence of a bird biting molestus population in the study area. Both forms were found to rest indoors, mainly in avian shelters, but at least a proportion of females of the pipiens form may bite outdoors in sylvan habitats and then search for anthropogenic resting sites to complete their gonotrophic cycle. This behaviour may potentiate the accidental transmission of arboviruses to humans in the region.

West Nile virus in mosquitoes in Greece

Epidemics of West Nile virus (WNV) occurred for two consecutive years in Greece (in 2010 and 2011). A total of 16,116 adult Culex pipiens mosquitoes collected in two peripheries, Central Macedonia and Thessaly, were tested for WNV infection. WNV lineage 2 was detected in 6/296 mosquito pools, three in each year. The H249P substitution in the NS3 protein, previously associated with increased pathogenicity and thermotolerance, was detected in all six WNV-positive mosquito pools. When 21 individual C. pipiens mosquitoes were tested for the locus CQ11 to distinguish between the two C. pipiens forms, pipiens and molestus, 71.4% were identified as pipiens, 4.7% as molestus, and 19% as hybrid pipiens/molestus, giving the first evidence that both C. pipiens biotypes are present in Greece, with a significant proportion being hybrids. The exact role of the C. pipiens forms and hybrids in the WNV epidemiology, in combination or not with the H249P substitution in the virus genome, remains to be elucidated.

Сравнительная характеристика межгенных спейсеров кластера генов рибосомной РНК комаров родаCulex(Diptera: Culicidae)

Nucleotide sequences of intergenic spacer of ribosomal RNA gene cluster (rIGS) were identified in mosquitoes Culex modestus, Culex torrentium and Culex pipiens pallens. The level of interpopulation variability of the rIGS in the subspecies C. pipiens pipiens (form pipiens--mosquitoes that inhabit the open waters, and form molestus--mosquitoes that inhabit basements) living in Russia was estimated. No extensive repetitive sequences characteristic of the rIGS of all previously described species of mosquitoes were found within the rIGS of Culex mosquitoes. At the same time, evolutionarily conserved motifs and relatively short degenerate sequences of different classes of transposable elements, as well as multiple blocks of variable microsatellite repeats were identified. Our data demonstrated that the rIGS of Culex mosquitoes can be considered as a promising molecular marker for the analysis of population and phylogenetic relationships within this group of insects.

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.

A Comparison of Above-Ground and Below-Ground Populations of Culex pipiens pipiens in Chicago, Illinois, and New York City, New York, Using 2 Microsatellite Assays

Aboveground and belowground populations of the mosquito Culex pipiens pipiens are traditionally classified as form (f.) pipiens and f. molestus, respectively, and gene flow between forms is thought to be limited. Relatively few f. molestus populations have been found in the United States, which has hindered their study in North America. In this investigation, we used microsatellites to characterize a recently discovered population of f. molestus in Chicago, IL, and compared levels of genetic diversity and differentiation in above-ground and below-ground populations from Chicago and New York City, NY. Levels of genetic diversity were markedly lower in both f. molestus populations. Pairwise F(ST) values between populations indicated that f. molestus populations were highly divergent from each other, as well as from their associated aboveground populations. The most likely number of genetic clusters depended on the number of loci used; we began with a set of 8, and reanalyzed the specimens with 17. Using a panel of 17 loci, there were 4 clusters, 1 for each below-ground population, and 1 for each pair of above-ground populations. Our findings are supportive of the hypothesis that f. molestus populations in Chicago and New York City arose from local aboveground populations.

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.

Diversity of Wolbachia pipientis strain wPip in a genetically admixtured, above-ground Culex pipiens (Diptera: Culicidae) population: association with form molestus ancestry and host selection patterns.

Analysis of molecular genetic diversity in nine marker regions of five genes within the bacteriophage WO genomic region revealed high diversity of the Wolbachia pipentis strain wPip in a population of Culex pipiens L. sampled in metropolitan Chicago, IL. From 166 blood fed females, 50 distinct genetic profiles of wPip were identified. Rarefaction analysis suggested a maximum of 110 profiles out of a possible 512 predicted by combinations of the nine markers. A rank-abundance curve showed that few strains were common and most were rare. Multiple regression showed that markers associated with gene Gp2d, encoding a partial putative capsid protein, were significantly associated with ancestry of individuals either to form molestus or form pipiens, as determined by prior microsatellite allele frequency analysis. None of the other eight markers was associated with ancestry to either form, nor to ancestry to Cx. quinquefasciatus Say. Logistic regression of host choice (mammal vs. avian) as determined by bloodmeal analysis revealed that significantly fewer individuals that had fed on mammals had the Gp9a genetic marker (58.5%) compared with avian-fed individuals (88.1%). These data suggest that certain wPip molecular genetic types are associated with genetic admixturing in the Cx. pipiens complex of metropolitan Chicago, IL, and that the association extends to phenotypic variation related to host preference.