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.

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.

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Abstract. Polymorphism of the mtDNA gene COI encoding cytochrome C oxidase subunit I was studied in the mosquitoes Culex pipiens Linnaeus, 1758 and C. torrentium Martini, 1925 from sixteen locations in Russia and in three laboratory strains of subtropical subspecies of the C. pipiens complex. Representatives of this complex are characterized by a high ecological plasticity and there are signifi cant ecophysiological differences between its morphologically similar members. The full-size DNA sequence of the gene COI spans 1548 bp and has a total A+T content of 70.2 %. The TAA is a terminating codon in all studied representatives of the C. pipiens complex and C. torrentium. 64 variable nucleotide sites (4 %) were found, fi fteen haplotypes were detected, and two heteroplasmic specimens of C. torrentium were recorded. COI haplotype diversity was low in Wolbachia-infected populations of the C. pipiens complex. Monomorphic haplotypes were found in C. p. quinquefasciatus and C. p. pipiens f. molestus. Three haplotypes were detected for the C. p. pipiens, but these haplotypes were not population-specifi c. On the other hand, each of the ten studied Wolbachia-uninfected C. torrentium individuals from three different populations had unique mitochondrial haplotypes. Polymorphism of the 478-bp ITS2 nucleotide sequences was similar in infected C. p. pipiens and C. p. pipiens f. molestus and uninfected C. torrentium specimens. The ITS2 genetic distance between C. p. pipiens and C. torrentium reached 12.5 %. Possible effects of Wolbachia invasion on C. pipiens populations are discussed.

Dirofilariasis is endemic in Russia, as well as in many other European countries. The aim of this study was to assess the ability of mosquitoes to transfer Dirofilaria immitis and Dirofilaria repens in regions with temperate and subtropical climates. The possible impact of the symbiotic bacterium Wolbachia on Dirofilaria transmission was also investigated. 5333 female mosquitoes were collected at 11 points in central European Russia and on the Black Sea coast during the period 2013–2017. Out of 20 mosquito species examined, 14 were infected with D. repens and 13 with D. immitis. Both species of Dirofilaria were found in different climatic regions. The total Dirofilaria spp. estimated infection rate (EIR) in the central part of Russia varied from 3.1% to 3.7% and, in the southern region, from 1.1% to 3.0%. The highest estimated infection rate was found in Anopheles messeae, the lowest in Culex pipiens. The greatest epidemiological danger was represented by Aedes aegypti, Ae. geniculatus, An. messeae and Ae. communis. Six out of 20 mosquito species were infected with Wolbachia. Pools of Aedes albopictus, Cx. pipiens and Coquillettidia richiardii were simultaneously infected with Dirofilaria and Wolbachia. After checking mosquitoes individually, it was found that there was no development of Dirofilaria to the infective larval stage in specimens infected with Wolbachia. Twenty-two Dirofilaria-infective pools were Wolbachia-free and only two mosquito pools were Wolbachia-infected. The potential for transmission of Dirofilaria in mosquito species naturally uninfected with the symbiotic bacterium Wolbachia is higher than in species infected with the bacterium.

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Polymorphism of the gene COI encoding cytochrome C oxidase subunit I and of the rRNA second internal transcribed spacer (ITS2) was measured in Aedes albopictus collected in three locations in Piedmont region and one location in Liguria region in Italy. According to our data, besides the middle part of the COI gene usually used for analysis, there are also SNPs in the 5 half of the gene, which can provide additional information about the polymorphism of A. albopictus populations. Based on COI gene, three various mitochondrial haplotypes were identified in A. albopictus from four north Italian sampling sites. Different sources of introductions can be considered. The variability between COI sequences of the north Italian A. albopictus is 0.001 while the variability between them based on the pairwise analysis of ITS2 sequences is 10 time more-0.011. For the first time, a 4% divergence between the populations of A. albopictus of tropical and subtropical origins was found on the basis of analyzing the ITS2. A relatively high level of variability of ITS2 between tropical and subtropical populations can be used to develop new markers for phylogenetic studies. No individuals with mitochondrial and nuclear haplotypes specific to tropical populations were found in the four north Italian sampling sites.