A critical assessment of available molecular identification tools for determining the status of Culex pipiens s.l. in the United Kingdom.

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.