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.

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BackgroundDetermination 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.MethodsWe 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.ResultsPreservation 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.ConclusionsOur 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.

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 that Ur. sapphirina feeds on annelid hosts (100% of identified blood meals; n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on Sparganophilus worms 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.

A study was conducted in 4 plots within a newly established olive grove in Florida to assess surveillance methods for insects present around the period of olive bloom. Over 99% of thrips collected were Frankliniella bispinosa (Morgan) (Thysanoptera: Thripidae), with occasional collections of predacious Leptothrips pini (Watson) (Thysanoptera: Phlaeothripidae). Collections of thrips using sticky traps or in tap or brush samples were high at the time of bloom, with low numbers before bloom and very low numbers after bloom. No differences in collections were seen among plots for thrips numbers when sampled using sticky cards. However, one plot had higher thrips numbers when sampled using tap and brush samples. Overall, and especially during bloom, blue sticky traps were most attractive, followed by yellow and then white sticky traps. Clear (color-free) traps collected the fewest thrips. Using tap samples, more thrips were collected on the edges than in the middle of the grove. Highly localized high densities of thrips were detected by the tap samples. Although sticky traps were highly effective for collecting thrips, only tap samples detected the localized hot spots.

The Burmese python, Python bivittatus Kuhl, is a well-established invasive species in the greater Everglades ecosystem of southern Florida, USA. Most research on its ecological impacts focuses on its role as a predator and its trophic interactions with native vertebrate species, particularly mammals. Beyond predation, there is little known about the ecological interactions between P. bivittatus and native faunal communities. It is likely that established populations of P. bivittatus in southern Florida serve as hosts for native mosquito communities. To test this concept, we used mitochondrial cytochrome c oxidase subunit I DNA barcoding to determine the hosts of blood fed mosquitoes collected at a research facility in northern Florida where captive P. bivittatus and Argentine black and white tegu, Salvator merianae (Duméril and Bibron), are maintained in outdoor enclosures, accessible to local mosquitoes. We recovered python DNA from the blood meals of three species of Culex mosquitoes: Culex erraticus (Dyar and Knab), Culex quinquefasciatus Say, and Culex pilosus (Dyar and Knab). Culex erraticus conclusively (P = 0.001; Fisher’s Exact Test) took more blood meals from P. bivittatus than from any other available host. While the majority of mosquito blood meals in our sample were derived from P. bivittatus, only one was derived from S. merianae. These results demonstrate that local mosquitoes will feed on invasive P. bivittatus, a recently introduced host. If these interactions also occur in southern Florida, P. bivittatus may be involved in the transmission networks of mosquito-vectored pathogens. Our results also illustrate the potential of detecting the presence of P. bivittatus in the field through screening mosquito blood meals for their DNA.

Peanut is an important oilseed crop and legume species, with more than 1.9 M tons produced annually in the U.S. Being indeterminate, peanut continually flowers and sets pods throughout the growing season, leading to the potential harvest of both mature and immature pods. To quantify the physiological impacts of peanut seed maturity, a two-year field study was conducted to elucidate the difference in canopy structure and reproductive characteristics, including flower production, yield, and grade between seed obtained from immature and mature seed of two commercial peanut cultivars: TUFRunner™ ‘727’ and FloRun™ ‘107’. Data indicated that seed from the yellow class of pods have lower vigor and overall plant development and performance; further, plants developed from immature seed never achieved a level of performance comparable to that of the mature brown/black pod classes. There were differences between cultivars in the severity of the impact of immaturity, with larger detrimental effects on immature TUFRunner™ ‘727’, which exhibited reduced emergence. Despite these cultivar differences, this study illustrated that mature seed performs better in a field setting than immature seed. These results are critically important to disproving the ‘catch-up' assumption: seed maturity not only has an impact on emergence, but on subsequent life history and performance traits through the remainder of the season.

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