The transmission dynamics of mosquito-vectored pathogens are, in part, mediated by mosquito host-feeding patterns. These patterns are elucidated using blood meal analysis, a collection of serological and molecular techniques that determine the taxonomic identities of the host animals from which blood meals are derived. Modern blood meal analyses rely on polymerase chain reaction (PCR), DNA sequencing, and bioinformatic comparisons of blood meal DNA sequences to reference databases. Ideally, primers used in blood meal analysis PCRs amplify templates from a taxonomically diverse range of vertebrates, produce a short amplicon, and avoid co-amplification of non-target templates. Few primer sets that fit these requirements are available for the cytochrome c oxidase subunit I (COI) gene, the species identification marker with the highest taxonomic coverage in reference databases. Here, we present new primer sets designed to amplify fragments of the DNA barcoding region of the vertebrate COI gene, while avoiding co-amplification of mosquito templates, without multiplexed or nested PCR. Primers were validated using host vertebrate DNA templates from mosquito blood meals of known origin, representing all terrestrial vertebrate classes, and field-collected mosquito blood meals of unknown origin. We found that the primers were generally effective in amplifying vertebrate host, but not mosquito DNA templates. Applied to the sample of unknown mosquito blood meals, > 98% (60/61) of blood meals samples were reliably identified, demonstrating the feasibility of identifying mosquito hosts with the new primers. These primers are beneficial in that they can be used to amplify COI templates from a diverse range of vertebrate hosts using standard PCR, thereby streamlining the process of identifying the hosts of mosquitoes, and could be applied to next generation DNA sequencing and metabarcoding approaches. Female mosquitoes take blood meals from diverse vertebrate hosts, including amphibians, birds, mammals, reptiles, and fishes. Mosquito species vary in their host-use patterns. Identifying which mosquitoes feed from which hosts is critical to understanding how mosquitoes transmit disease-causing pathogens between vertebrates. Host-use patterns are determined by blood meal analysis. In modern blood meal analysis, host DNA is extracted from a mosquito, amplified in a polymerase chain reaction (PCR), sequenced, and identified. We designed new primers that selectively amplify a fragment of the DNA barcoding gene, cytochrome c oxidase subunit I (COI), of vertebrates, while avoiding that of mosquitoes. We validated the new primers in experiments using previously identified mosquito blood meals and mosquito DNA, and demonstrated their efficacy in identifying unknown mosquito blood meals. We found that the primers are well-suited to mosquito blood meal analysis applications. They are universal across vertebrates, target the COI DNA barcoding gene, and are effective in standard amplification reactions.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188–250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Feeding upon vertebrate blood by mosquitoes permits transmission of diverse pathogens, including viruses, protozoa, and nematodes. Despite over a century of intensive study, no mosquito species is known to specialize on non-vertebrate hosts. Using molecular analyses and field observations, we provide the first evidence, to our knowledge, that a mosquito,Uranotaenia sapphirina, specializes on annelid hosts (earthworms and leeches) while its sympatric congener,Uranotaenia lowii, feeds only on anurans (frogs and toads). Our results demonstrate thatUr. sapphirinafeeds on annelid hosts (100% of identified blood meals;n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding onSparganophilusworms and freshwater leeches. These findings indicate that adult mosquitoes utilize a much broader range of host taxa than previously recognized, with implications for epidemiology and the evolution of host use patterns in mosquitoes.

Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

The composition of wildlife communities can have strong effects on transmission of zoonotic vector-borne pathogens, with more diverse communities often supporting lower infection prevalence in vectors (dilution effect). The introduced Burmese python, Python bivittatus, is eliminating large and medium-sized mammals throughout southern Florida, USA, impacting local communities and the ecology of zoonotic pathogens. We investigated invasive predator-mediated impacts on ecology of Everglades virus (EVEV), a zoonotic pathogen endemic to Florida that circulates in mosquito-rodent cycle. Using binomial generalized linear mixed effects models of field data at areas of high and low python densities, we show that increasing diversity of dilution host (non-rodent mammals) is associated with decreasing blood meals on amplifying hosts (cotton rats), and that increasing cotton rat host use is associated with increasing EVEV infection in vector mosquitoes. The Burmese python has caused a dramatic decrease in mammal diversity in southern Florida, which has shifted vector host use towards EVEV amplifying hosts (rodents), resulting in an indirect increase in EVEV infection prevalence in vector mosquitoes, putatively elevating human transmission risk. Our results indicate that an invasive predator can impact wildlife communities in ways that indirectly affect human health, highlighting the need for conserving biological diversity and natural communities.