Sympatric occurrence of Culex pipiens (Diptera,Culicidae) biotypes pipiens,molestus and their hybrids in Portugal,Western Europe: feeding patterns and habitat determinants

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host‐feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri‐urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri‐urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.     

Chromosome-level assembly of Culex pipiens molestus and improved reference genome of Culex pipiens pallens (Culicidae,Diptera)

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.     

The haemagglutination activity in the different developmental stages and changes of this activity caused by analogues of insect hormones in two members of the Culex pipiens complex of mosquitoes

Abstract. Haemagglutination activity was studied in last larval instars, pupae and adults of both sexes of the Culex pipiens mosquito complex. In females of Culex pipiens quinquefasciatus Say, an anautogenous member of C. pipiens complex, activity was detected in the homogenate of the head‐thorax complex, gut and remaining abdomen (after removing the gut), before and after sucking water, glucose solution or blood. Similar investigations were made in females of autogenous Culex pipiens molestus Forskal, a member of the same mosquito complex. The results were compared with data obtained for males of these subspecies, plus the larvae and pupae. The results show that haemagglutination activity in adult females depends not on the sucking of blood, but on the age of the females. In newly emerged females, the level of activity was very low, but after 24 h or 48 h of activity was highly enhanced, even in females that had no possibility to suck water or other liquids including blood. Furthermore, in C. p. molestus, the haemagglutination activity does not change in hungry or engorged females, and a high level of activity was found in the gut of last larval instars, a developmental stage that never takes blood. Treatment with methoprene reduces haemagglutination activity in both sexes, but 20‐hydroxycdysone application decreased activity in the gut of females. It is suggested that haemagglutination activity may be regulated indirectly by the endocrine system. The possibility that haemagglutination activity may depend on some aspect of the digestive system that has no direct connection to blood uptake is discussed. In addition, the activity may be important not only for the elimination of infections, but also for the processing of food and the utilization and transport of nutrients.     

Electrophysiological and behavioural responses of mosquitoes to volatiles of Silene otites (Caryophyllaceae)

In order to understand the biological significance of flower odour for attraction of mosquitoes, electrophysiological responses to headspace flower odour samples of Silene otites (L.) Wibel were investigated on Culex pipiens pipiens biotype molestus Forskal 1775 and Aedes aegypti L. using coupled gas chromatographic-electroantennographic detection (GC-EAD). No remarkable differences in antennal responses to the odour compounds have been found between these two mosquito species. Further, the behavioural attractiveness of the electrophysiologically active compounds, singly or as multiple odour mixtures, was evaluated with bioassay experiments with C. pipiens molestus. In bioassays, C. pipiens responded to 14 electrophysiologically active compounds in different magnitudes (65–20%) and acetophenone, linalool oxide (pyranoid), phenyl acetaldehyde and phenylethyl alcohol were found as more attractive in comparison to the least attractive compound, hexanol. In two-stimulus choice test, mosquitoes were significantly more attracted to the mixture of the four most attractive compounds compared to the mixture of all 14 compounds. The results of present study confirm that floral odours are attractive cues for mosquitoes.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.     

Comparative characteristics of the intergenic spacer of the ribosomal RNA gene cluster in mosquitoes of the genus Culex (Diptera: Culicidae)

The intergenic spacer of the ribosomal RNA gene cluster (IGS) was sequenced for mosquitoes Culex modestus, C. torrentium, and C. pipiens pallens. The interpopulation variation of the IGS was estimated for the C. pipiens pipiens forms pipiens, which inhabits open waters, and molestus, which inhabits basements, of Russia. The IGSs of the Culex mosquitoes were not found to contain extended repetitive sequences characteristic of all known mosquito IGSs. At the same time, evolutionarily conserved motifs, relatively short degenerate sequences belonging to various classes of transposable elements, and multiple blocks of variable microsatellite repeats were identified. Based on the results, the IGS of the Culex mosquitoes was assumed to provide a promising molecular marker for population and phylogenetic analyses of the group.     

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.     

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.     

Variability of quantitative morphological traits of mosquito larvae in some species of the <Emphasis Type="Italic">Culex pipiens</Emphasis> complex (Diptera,Culicidae)

The variability of 13 larval morphological characteristics in the urban mosquito, Culex pipiens f. molestus and Culex torrentium, was investigated. These species, belonging to the Culex pipiens complex, are characterized by rapid ecological and evolutionary processes following the human-induced environmental changes. A comparison of 4 geographical larval populations of C. pipiens f. molestus and Culex torrentium revealed different rates of divergence of different morphological traits. In the older form in terms of evolutionary development, C. torrentium, the two local populations differed in the structures of the trophic morphological complex characterized by a higher functional trophic load, while the structures with a lower functional load did not differ between the populations. On the contrary, the populations of the younger form, C. pipiens f. molestus, which had recently colonized new urban sites, differed in the structures with a low functional trophic load (some structures of siphon and abdominal segment IX), whereas structures of the trophic morphological complex were similar in different populations. The possible mechanisms of these morphological transformations and their role in the evolution of mosquito larvae are discussed.     

Hemolymph pH of the larvae of three species of mosquitoes,and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.     

The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

A preliminary simulation model of movement of larvae ofCulex pipiens molestus (Diptera: Culicidae) (Experimental studies on the dispersal of insects,II)

Experiments for analyzing the movement and dispersal of larvae of C. pipiens molestus were made and preliminaly simulation models were constructed. The models were based on two distributions, the distance and direction of movements between 1 minute intervals. The distribution of distance is represented by a unimodal curve, and that of angle by a bimodal one. The results of simulation reasonably depicted the actual process of movements. Nevertheless, the simulated time-dispersal curves overestimated the number of dispersed individuals. This might be due to mutual attraction among the larvae.     

The arbovirus vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe

Two species of arbovirus vector, Culex torrentium and Culex pipiens (Diptera: Culicidae), occur in several European countries, but difficulties in their accurate identification and discrimination have hampered both detailed and large‐scale distribution and abundance studies. Using a molecular identification method, we identified to species 2559 larvae of Cx. pipiens/torrentium collected from 138 sites in 13 European countries ranging from Scandinavia to the Mediterranean coast. In addition, samples of 1712 males of Cx. pipiens/torrentium collected at several sites in the Czech Republic were identified to species based on the morphology of their hypopygia. We found that the two species occur together in large areas of Europe, and that Cx. torrentium dominates in northern Europe and Cx. pipiens dominates south of the Alps. The transition in dominance occurs in central Europe, where both species are roughly equally common. There was a strong correlation between the length of the growing season at different sites and occurrences of the two species. As the growing season increases, the proportion and detection of Cx. torrentium decrease, whereas those of Cx. pipiens increase. The present findings have important consequences for the interpretation of the results of studies on major enzootic and link‐vectors of mosquito‐borne bird‐associated viruses (i.e. Sindbis, West Nile and Usutu viruses), especially in central Europe and Scandinavia.     

A study of the mosquito Culex pipiens (Diptera, Culicidae) population structure in the transcaucasia using molecular identification methods

The mosquito species Culex pipiens consists of two forms, or ecotypes: the typical pipiens form and the molestus form. These forms are similar morphologically but have significant ecophysiological differences. The Cx. pipiens population structure was studied in open and underground habitats (house basements) in several localities in Abkhazia, Georgia, and Azerbaijan. The pipiens and molestus forms were identified by a molecular method, namely by restriction analysis of the amplification products with HaeIII endonuclease. Altogether, 90 individuals from 10 local populations were studied. The pipiens form was found only in surface water bodies (8 cases); in two cases both forms were found in one habitat. Homogeneous molestus populations were recorded in two underground habitats. Analysis of the Cx. pipiens population structure by the molecular method agrees with the results of biological and ecological studies of this mosquito in Georgia by Sh. Sichinava (1974, 1978, 1989). Thus, molecular diagnostics of intraspecific forms in Cx. pipiens populations is doubtlessly reliable.     

Contributions to the mosquito fauna (Diptera, Culicidae) of Belgorod Province

The results of long-term studies of mosquitoes in Belgorod Province are reported. The material was collected at 54 sites situated in 15 districts of the province. Different biotopes were investigated during the spring-autumn period. Larvae and adult mosquitoes were used for identification. A total of 27 mosquito species were found in the region examined. The following species were recorded for the first time: Ae. (Och.) communis. Ae. (Och.) diantaeus. Ae. (Och.) pulchritarsis. Ae. (Och.) sticticus, and Cx. (Bar.) modestus; and also the form Cx. (Cux.) pipiens pipiens biotype molestus. The frequency of occurrence of each species in different biotopes and different localities is given. The most widespread and common species in Belgorod Province include Ae. (Och.) cantans (Mg.), Ae. (Och.) cataphylla Dyar, Ae. (Fin.) geniculatus (Ol.), Ae. (Adm.) vexans (Mg.), Cx. (Cux.) pipiens, and Cx. (Cux.) pipiens pipiens biotype molestus.     

Activité comparée de 22 variétés deBacillus thuringiensis sur 3 espèces deCulicidae

Résumé Le pouvoir larvicide des cultures totales de 22 variétés deBacillus thuringiensis Berliner représentant 15 sérotypes H a été testé sur larves L4 d'Aedes aegypti (L.)Culex pipiens pipiens (L.) etAnopheles stephensi (Liston). Seul le sérotype H 14, variétéisraelensis, est réellement actif, provoquant 100% de mortalité à la dilution 10⁻⁵. Avec des doses beaucoup plus fortes, 10⁻², une certaine toxicité peut être manifestée par les variétésentomocidus, galleriae etkyushuensis en ce qui concerneAe. aegypti etC. pipiens pipiens, ou par les variétésentomocidus, tolworthi, kyushuensis etaizawai, pourAn. stephensi. Cependant cette activité n'a rien de comparable avec celle de la variétéisraelensis.

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Summary We have studied the 15 H serotypes ofBacillus thuringiensis Berliner including 22 varieties. The larvicidal potency of the whole cultures of these varieties is evaluated on 4th instar larvae ofAedes aegypti (L.),Culex pipiens pipiens (L.) andAnopheles stephensi (Liston). The H-14 serotype, varietyisraelensis is the only one to show a true toxicity at 10⁻⁵ dilution on larvae of the 3 mosquito species. A low mortality at 10⁻² dilution is observed onAe. aegypti andCx. pipiens pipiens larvae withentomocidus, galleriae andkyushuensis varieties; onAn. stephensi withentomocidus, tolworthi, kyushuensis andaizawa? varieties. Nevertheless, this activity cannot be compared to the extremely high toxicity of theisraelensis variety.

     

A rural–urban latitudinal study of the distributions of Culex quinquefasciatus and Culex pipiens bioforms in their southernmost sympatric fringe

Mosquitoes grouped in the complex Culex pipiens L. (Diptera: Culicidae) are important vectors of medical and veterinary diseases. In the South American sympatric region, Cx. pipiens and Culex quinquefasciatus Say coexist and potentially hybridize. To identify key drivers of their geographical distribution, mosquito immatures were collected from flower vases of eight urban/rural cemetery pairs within a 5° latitudinal transect along Buenos Aires Province, Argentina. The specimens were identified by molecular methods and their relative proportion modelled as a function of environmental variables. At the beginning of the warm season, northern and southern cemeteries presented exclusively Cx. quinquefasciatus and Cx. pipiens, respectively, with different proportions of both at mid latitudes. By the end of the summer, Cx. quinquefasciatus was present throughout the study area, exclusively in 11 of the 16 cemeteries both rural and urban, whereas Cx. pipiens was predominant only in the southernmost pair. Mean annual temperature, photoperiod variability and time of the season were key drivers of their distributions. All specimens of Cx. pipiens were identified as form molestus and no hybrids were recognized. The reported distribution patterns and the potential absence of Cx. pipiens f. pipiens and hybrids are discussed, along with their implications in disease transmission.     

Effect of light and temperature on the daily rhythm of adult eclosion in the blood-sucking mosquitoes <Emphasis Type="Italic">Aedes communis</Emphasis> De Geer and <Emphasis Type="Italic">Culex pipiens</Emphasis> L. (Diptera,Culicidae)

A comparative study on the effect of light and temperature on the daily rhythm of adult eclosion was carried out on the blood-sucking mosquitoes Aedes (Ochlerotatus) communis De Geer and Culex pipiens pipiens L. Two forms of C. p. pipiens were investigated: the autogenous form molestus occurring in the underground habitats (the urban mosquito), and the anautogenous form pipiens inhabiting mostly the above-ground biotopes. The study included both field observations and laboratory experiments. Under natural conditions (southern Karelia, 62°N) at optimal temperatures A. communis and C. p. p. f. pipiens demonstrated a bimodal eclosion rhythm with morning and evening peaks, whereas in C. p. p. f. molestus only an evening maximum was observed. The fraction of adults eclosing in the middle of the day increased at lower temperatures. In all the mosquitoes studied, the daily eclosion dynamics in the nature did not differ significantly between the variants with different levels of illumination, suggesting that temperature was the main factor regulating the eclosion rhythm. However, experiments with constant temperature showed that light also could influence the timing of eclosion. The responsiveness to light was different in two studied forms of C. p. pipiens. Under a constant temperature and light-dark cycles the rhythm became weak or disappeared completely in the mosquitoes from above-ground biotopes (A. communis and C. p. p. f. pipiens) but persisted in the urban mosquito. The effect of gradual twilight transitions was more prominent than that of switching the light on or off abruptly. The observed differences in the light and temperature dependence of the eclosion rhythm are discussed in relation to the ecological conditions in different habitats.     

Predation differentially structures immature mosquito populations in stormwater ponds

1. Understanding the factors underlying the abundance and distribution of species requires the consideration of a complex suite of interacting biotic and abiotic factors operating on multiple spatial and temporal scales. Larval mosquitoes inhabiting small human-constructed ponds represent a unique opportunity to investigate the relative importance of these structuring mechanisms while simultaneously generating applied knowledge on mosquito control. 2. A multi-year field survey of 32 stormwater ponds was conducted in central Illinois (Champaign County). From each pond, data were collected on pond structure type and hydroperiod, the presence/absence of cattails (Typha spp.), and measures of total nitrogen, phosphorus, and organic carbon, and chlorophyll a. The communities of crustacean zooplankton and aquatic insects were characterised, and these taxa were assigned into two main groups: predators and competitors of larval mosquitoes. Structural equation modelling was used to explore the direct and indirect effects of these biotic and abiotic factors on larval density for three species of culicine mosquitoes (Culex pipiens, Culex restuans, and Aedes vexans). 3. Hydroperiod had an indirect negative effect on C. pipiens density. However, this effect was mediated by predator density; more permanent ponds had more predators, which therefore reduced the density of C. pipiens larvae. Aedes vexans density was positively correlated with predator density. No predictor variables were found that explained variation in C. restuans density. 4. This study show that the relative importance of these biotic and abiotic factors varies among species of culicine mosquitoes inhabiting stormwater ponds.     

The sex structure of the larval populations of the urban mosquito <Emphasis Type="Italic">Culex pipiens pipiens</Emphasis> f. <Emphasis Type="Italic">molestus</Emphasis> Forskal (Diptera,Culicidae) in St. Petersburg

The study of sex ratio in larval populations in the urban mosquito Culex pipiens pipiens f. molestus inhabiting underground sites in St. Petersburg was carried out for the first time in 1997–2001, as part of monitoring of local populations of this mosquito in order to estimate its reproductive potential. Samples of the immature stages were regularly collected in the underground breeding places flooded with water with a varying degree of pollution in five city districts (altogether 27 samples, 14 251 larvae). Pupation was observed under laboratory conditions at 20°C for 6–46 days. The sex was determined at the pupal stage. In 19 samples collected in basements of dwelling houses the incidence of males widely varied from 40.2 to 65.6%, and the sex ratio (males: females), from 0.7: 1 to 1.9: 1. Medians defining the percentage of males in the populations in different basements during a certain period (from 3 to 11 months) were 57.5%, 47.0%, and 50.2%. The greatest incidence of males (the median being 62.2%) and sex ratio from 1.1: 1 to 3: 1 was observed in autumn in 1999–2001 in populations inhabiting the sewer pipe. Analysis of all the 27 samples showed that the males constituted on the average 54.5%, i.e., that is the sex ratio was close to the normal value of 1: 1. Such important population characteristics as normal sex ratio at the pupal stage and the high autogeny rate are indicative of high reproductive potential of the urban mosquito in St. Petersburg. The hydrotechnical (mostly drainage of basements) and special mosquito control measures are needed to decrease mosquito abundance and restrict their further dispersal within the city.