BACKGROUND : Mosquitoes 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.
METHODS : Culex 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.
RESULTS : Six 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.
CONCLUSIONS : Molecular 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.
Additional file 1: Discrimination of specific COI alleles. (A) COI
haplotypes after HaeIII digestion of PCR products: 1-type A, 2-type B, 3-
type C, 4-type D, 5-type E, 6-type E1, 7-marker molecular weight M100;
(B) COI haplotypes after AluI digestion: 1-type A, 2-type B, 3-type C, 4-type
D, 5-type E, 6-type E1, 7-marker molecular weight M50. (TIF 141 kb)
Additional file 2: Example of PCR amplification of specific ACE2 (A)
and CQ11 (B) alleles in Tanger, Morocco. 1–13 - samples, samples 7,
10 - Cx. pipiens form pipiens by both assay. Other samples are hybrids by
ACE2 or CQ11 assays; 14 - marker molecular weight; 15 – Cx.
quinquefasciatus; 16 – Cx.pipiens. (TIF 1408 kb)
Additional file 3: Alignments of ace-2 gene sequences for Cx.
pipiens/quinquefasciatus hybrid collected from Kos, Greece.
Sequences are compared with Cx. pipiens (AY196910) and Cx.
quinquefasciatus (AY196911).“*” Indicates the absence of mutation, “.” -
nucleotide substitutions, “-” indels. (DOCX 12 kb)
Additional file 4: Discrimination of specific wPip alleles based on
ank2 and pk1 markers. (A) three alleles: a (313 bp), b (217, 195, 98 bp)
and c (293, 217 bp) after HinfI digestion of the ank2 PCR products; (B)
three alleles: a/e (903, 430 bp), c (851, 498 bp) and d (497, 251, 107 bp)
after TaqI digestion of the pk1 PCR products; (C) allele a (903, 303,
141 bp) after PstI digestion of the pk1 PCR products. (TIF 557 kb)