Mosquitoes of the Anopheles maculipennis Meigen (Diptera: Culicidae) group are of public health concern: five of the 11 morphologically indistinct species have been historically considered as vectors of malaria in Europe. Three members of the An. maculipennis group have been reported in the U.K.: Anopheles atroparvus van Thiel; Anopheles messeae Falleroni, and Anopheles daciae Linton, Nicolescu & Harbach. To study the distribution of the three U.K. species, particularly that of An. daciae, we developed a polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) assay using the nuclear ribosomal internal transcribed spacer 2 (ITS-2) gene. Anopheles daciae was found to be widespread, occurring in four of the five counties surveyed in southern England and on the Welsh island of Anglesey, often in sympatry with the closely related species An. messeae. The host preferences of 237 blood-fed females were determined using either direct sequencing or PCR-based fragment analysis of the mitochondrial cytochrome oxidase b gene with DNA from females' abdomens. All three species were found to be opportunistic, having fed on at least three different hosts. Seventeen individuals contained multiple bloodmeals, including two An. daciae that had fed on humans and birds. Our results show that An. daciae is widespread in England and Wales, occurs in sympatry with other members of the An. maculipennis group, and feeds on humans, which suggests it is a potential vector of disease in the U.K.

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.

The use of invertebrate-derived DNA (iDNA) is a promising non-invasive tool to monitor wildlife. While most studies have been carried out in dense tropical and subtropical forests and have focused on the use of a single category of invertebrates, this study compares the use of flies and mosquitoes-derived DNA to assess vertebrate diversity in semi-urban environments. We conducted our sampling in four different forest plots in Berlin, Germany. Pools of flies and non-bloodfed mosquitoes were metabarcoded using 108-bp vertebrate-specific 12 S rRNA (12 S-V5) and 94-bp mammal-specific 16 S rRNA (16Smam) mitochondrial markers, and individual bloodfed mosquitoes were sequenced using the 340-bp vertebrate-specific 12 S rRNA fragment (Mam-12 S-340). Most sequencing was only successful for mammal species.From the fly pools, we detected 10 mammal species using 16Smam, and six species using 12 S-V5. From the non-bloodfed mosquito pools, we only amplified putative contaminant DNA, indicating that mosquito females without visual signs of a blood meal carry no traces of vertebrate DNA. Finally, in the bloodfed mosquitoes, we identified four mammal species. We did not find significant differences in the proportion of mammal species detected regarding the total available number of species between sampling localities. Fly samples were easier to obtain and more abundant over the sampled localities compared to mosquito samples. We conclude that, while there are a few advantages in using mosquito blood meals, the use of flies in the detection of wildlife in a suburban environment is more effective in terms of collection of samples and detection of vertebrates, although this technique is limited to few mammal species in the urban environment.

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