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 taxonomic history of Culex pipiens (1758–present) is reviewed. The central question is whether Cx. pipiens is a single polytypic species or a complex of sibling species? The taxon traditionally known as the Cx. pipiens complex is referred to as the Pipiens Assemblage to avoid difficulties associated with the meaning of the word “complex”. Neotype specimens have been designated to fix the morphological identities of Cx. pipiens, Cx. molestus, and Cx. quinquefasciatus, and Cx. pallens is represented by a holotype, but whether these nominal forms represent one or more biological species remains controversial. Despite extensive morphological and physiological/behavioral variation, there is no indication of subspecific or racial differences in geographically separated populations of Cx. pipiens. Introgression occurs where populations of Cx. pipiens and Cx. quinquefasciatus overlap, but the retention of parental epiphenotypes outside the zone of introgression provides evidence of independent species cohesion. The main conclusions reached are: Cx. pipiens and Cx. quinquefasciatus are separate species which evolved in Africa and hybridize in non-indigenous areas where they were unintentionally introduced by humans; Cx. molestus is nothing more than a phenotypic and physiological variant of Cx. pipiens; and Cx. pallens has no taxonomic status under the provisions of the International Code of Zoological Nomenclature. Based on morphological similarity, the Pipiens Assemblage includes Cx. pipiens, Cx. quinquefasciatus, and possibly Cx. australicus. There is no evidence to suggest that the Pipiens Assemblage includes any other species.

The mosquito known in the northern hemisphere as Culex pipiens (a.k.a. Culex pipiens pipiens or Culex pipiens form pipiens) is not known from Australia. However, there are four species of the Culex pipiens group: two indigenous, Culex australicus and Culex globocoxitus, and two introduced, Culex quinquefasciatus and what is known locally as Culex molestus (?  =  Culex pipiens molestus or Culex pipiens form molestus), all four being members of the pipiens subgroup ( =  ‘pipiens complex’). The species status of the indigenous Cx. australicus and Cx. globocoxitus in Australia appears to be accepted as legitimate; however, the true identity of the mosquito called ‘Cx. molestus’ in Australia remains contentious, even as its local profile has been increasing over the past 40 years. This paper provides an overview of the taxonomic and biologic knowledge of these species, and their public health significance as vectors of arboviruses in Australia.

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.

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.

Until the relatively recent application of molecular identification tools, identification of Culex pipiens f. pipiens and Cx. pipiens f. molestus relied on expressed ecological characteristics, including autogeny, host preference and stenogamy. Herein we test two DNA assays, one based on the microsatellite locus CQ11 and the other on species-diagnostic nucleotide bases in the mtDNA cytochrome oxidase I, on 322 wild-caught Cx. pipiens s.l. collected in above ground habitats from 6 counties across southern England and Wales. Of the 322 Culex pipiens s.l. screened using the CQ11 assay, 205 were identified as Cx. pipiens f. pipiens, 95 as Cx. pipiens f. molestus and 22 were determined as hybrids. Neither above ground Cx. pipiens f. molestus, nor hybrids have previously been reported in UK. However, comparison of COI barcodes (658bp) from 30 individuals from the above defined grouping indicated that inadvertent inclusion of specimens of Cx. torrentium resulted in the expected product sizes purportedly diagnostic for Cx. pipiens f. molestus, Cx. pipiens f. pipiens and hybrids in the CQ11 assay. COI sequences showed Cx. torrentium was misidentified as Cx. pipiens s.l. in more than 50% of cases and that all above ground Cx. pipiens s.l. collected in this study were in fact Cx. pipiens f. pipiens. Thus in regions of the Palearctic where Cx. torrentium and Cx. pipiens s.l. are sympatric, we showed that the CQ11 assay produces misleading results and should not be used.

Portugal is a southern European country that displays favorable ecological conditions for the establishment of West Nile virus (WNV) transmission cycles. Competent mosquito vector species are present throughout the country. Among the species with reported cases of WNV isolation in Portugal, Culex pipiens is the most ubiquitous and abundant mosquito. This species exhibits two biological forms with differences in host preferences. The molestus form has a greater tendency to feed upon humans and other mammals whereas the pipiens form prefers avian hosts. In northern latitudes, both forms are physically separated, with molestus occupying underground habitats and pipiens being found aboveground. However, the warmer climatic conditions of southern regions such as Portugal may favor the sympatric occurrence of both forms hence promoting interform hybridization. Genetic introgression between molestus and pipiens forms may result in a higher propensity for admixed populations to serve as bridge-vectors of WNV between humans and birds. Here we revise our present knowledge on the distribution, role in WNV transmission and genetic structure of the Cx. pipiens complex in continental Portugal. We focus on recent findings of sympatric molestus and pipiens populations that display considerable levels of hybridization and discuss the epidemiological repercussions of this occurrence.

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.

Mosquitoes in the genus Culex are the primary enzootic maintenance and bridge vectors of the North American encephalitides, now including West Nile virus. This review briefly summarizes the biology of three key vector species in western North America, Culex tarsalis, Cx. pipiens complex and Cx. stigmatosoma, focusing on the long history of research done in California. Topics reviewed include population genetic structure, larval ecology, autogeny, mating behavior, host-seeking behavior, host-selection patterns, and overwintering strategies. These attributes collectively have allowed the successful exploitation of anthropogenically altered ecosystems and enabled the role of these species as maintenance and bridge vectors of arboviruses.

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.

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.

Culex pipiens pipiens is comprised of two forms: Cx. pipiens pipiens form pipiens and Culex pipiens pipiens form molestus. One population of Cx. p. pipiens form molestus in the United States, located in the 91^st Street sewer of New York City (NYC), has been relatively well-characterized. The objective of this review is to summarize the results of multiple independent studies on Cx. p. pipiens form molestus in NYC and present the current understanding of the origin of this population. While solid evidence exists supporting the genetic differentiation and population structuring between Cx. p. pipiens form pipiens and form molestus in NYC, the possibility that the 91^st St. sewer population of form molestus was founded by local aboveground Cx. p. pipiens form pipiens cannot be ruled out based on available data to date. Future studies in the US should include sympatric populations of both forms of Cx. p. pipiens from around the world in order to provide the necessary context to resolve these phylogenetic relationships.

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.

Culex pipiens sensu lato populations represent significant nuisance pests and vectors of West Nile virus in California. Despite multiple years of investigation, identifying, controlling and understanding the behaviors and associated “biologies” of the complex members still remain a challenge. Population structure cluster analysis using microsatellite markers revealed extensive population structuring, particularly in the central parts of the State, over and above what can be explained by the presence of Cx. pipiens, Cx. quinquefasciatus and their hybrids. Ace 2 gene sequencing provided evidence for the presence of Cx. p. molestus in multiple locations both above and below ground in California. Lack of congruence of male genitalia morphology (dorsal and ventral arms/dorsal arm of phalosome) and polymerase chain reaction diagnostic assay identifications coupled with considerable heterozygosity of pyrethroid resistance in time and space all suggest complex population structuring not adequately explained using current concepts.

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.

Studies on the vector-host interactions of Culex pipiens complex mosquitoes by sequencing portions of mitochondrial cytochrome b gene indicate that Cx. p. pipiens f. pipiens predominantly feed on avian hosts (93.1%), and focus feeding activity on several key bird species, in particular the American robin, the gray catbird, and the house sparrow in Connecticut. However, Cx. p. quinquefasciatus indiscriminately feed on both birds and mammals. Culex p. quinquefasciatus in Harris County - Texas and southern California acquired 39.1% and 88.2% of bloodmeals from birds, respectively. Mammalian-derived bloodmeals constituted 52.5% and 9.6% in the two regions, respectively. The most frequent avian hosts for this mosquito species in the southwestern U.S. were the mourning dove, the white-winged dove, the house sparrow and the house finch. Humans infrequently served as the source of bloodmeals for Cx. p. pipiens and Cx. p. quinquefasciatus. Microsatellite analysis of mosquitoes from Chicago, Illinois showed that Cx. p. pipiens f. pipiens with mammalian- derived bloodmeals had significantly higher ancestry and proportion of hybrids from Cx. p. pipiens f. molestus than did those with avian-derived bloodmeals.

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.

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.