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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.

Despite the wide variation in mosquito reproductive strategies and the present lack of behavioral and ecological information for the majority of described mosquito species, the predominant egg-laying behaviors observed in well-studied species have led to sweeping generalizations of oviposition patterns across entire mosquito genera. Mosquitoes in the genus Culex , which contains over 800 species across 28 subgenera, are commonly believed to exclusively lay egg masses (“rafts”) that float on the water surface. This oversimplification persists, though we currently know little regarding the reproduction of many Culex species and subgenera. Using field observations, photography, and video recordings, we document the oviposition behavior of a Culex subgenus Micraedes species, Culex antillummagnorum (Dyar), and provide the first evidence, to our knowledge, of a Culex species aerially and skip-ovipositing, rather than laying masses of coniform eggs. Gravid Cx. antillummagnorum females were observed and filmed in the Luquillo Experimental Forest of Puerto Rico for several nights as they performed an elliptic flight pattern while launching single eggs into water-filled bracts of Heliconia caribaea . These findings raise questions regarding the evolution and drivers of such an atypical Culex behavior and indicate that our understanding of Culex ecology and behavior remains incomplete.

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. Despite the significance of mosquitos for human health, little research has focused on their phylogeny. Here, the authors present a resolved phylogenetic history of mosquitoes based on phylogenomics showing that these major disease vectors radiated coincidentally with geologic events and the diversification of their hosts.

Mosquito adulticides are perceived by the public as detrimental to nontarget arthropods, contributing to declines of native and beneficial insects. However, the actual impact of adulticides on nontarget arthropods in nature needs to incorporate biological and ecological elements. Here, we investigated the effect of biological/behavioral traits (butterfly roosting at different heights, roosting in sites underneath foliage, bumblebee hive usage) and interactions (parasitism, predation) of pollinators (butterflies and bumblebees) that could mediate the impacts of malathion application in manipulative semi-field experiments in Florida, USA. Roosting height from the spray route had a significant negative relationship with mortality of butterflies treated with ULV malathion, with high survival at the highest roosting height (7 m), but butterflies roosting among vegetation did not have higher survival. Bumblebees with hive access had significantly higher survival than bumblebees without hive access. Host plants treated with ULV malathion significantly reduced parasitism of monarch eggs by Trichogramma platneri, but increased predation of monarch caterpillars by Polistes paper wasps. These data provide insight into the realistic impacts of adulticide applications on pollinators in nature which will enable mosquito control districts to better limit nontarget effects of adulticide treatments and may help to address concerns related to potential nontarget effects.

Although adulticide application is a pillar in the integrated management of nuisance and vector mosquitoes, non-target effects of adulticide applications within ecosystems are a substantial concern. However, the impacts of adulticide applications on non-target organisms are not necessarily detrimental, and in some cases, may provide benefits to certain organisms or wildlife. Here, we hypothesized that adulticide applications have beneficial non-target impacts on vertebrate wildlife through reduced biting pressure. To test this, we collected mosquitoes from ultra-low volume Permanone-treated (intervention) and untreated (reference) areas and assessed mosquito abundance and diversity, and abundance of blood-engorged female mosquitoes. We performed DNA barcoding analysis on mosquito blood meals to identify host species. Our results demonstrated a significant reduction in mosquito abundance by 58.9% in the intervention areas, taking into account the reduction in reference areas. Consequently, this decline led to a 64.5% reduction in the abundance of blood-engorged females. We also found a temporal dynamic of mosquito composition driven by mosquito control actions in which different mosquito species became dominant at treated sites while composition at reference areas remained similar during the same period. The present study suggests that the beneficial effects of mosquito control treatments for humans extend to other vertebrates, which represents an unstudied and rarely recognized non-target impact.

Biodiversity monitoring is essential to conservation, yet field surveys are expensive, labor-intensive, and require substantial taxonomic expertise. DNA-based approaches are increasingly implemented to indirectly detect the presence of species at diverse types of field sites. In terrestrial ecosystems, invertebrate-derived DNA (iDNA) has potential to contribute to monitoring efforts that aim to characterize the diversity of vertebrate communities or to detect the presence of vertebrate species. We investigated the feasibility of using mosquito blood meals to detect vertebrate animals, focusing on the diversity of species detected by mosquitoes collectively and by individual species. Mosquitoes were collected at the DeLuca Preserve, Osceola County, Florida, USA over an eight-month period using sampling methods known to be effective for blood-fed mosquitoes. Blood-fed mosquitoes were identified and screened for host DNA using DNA barcoding-based blood meal analysis. From a sample of 2,051 identified blood meals, representing 21 mosquito species, mosquitoes collectively detected 86 vertebrate species. This assemblage of vertebrates was taxonomically diverse with species from all four terrestrial vertebrate classes, 22 orders, including large- and small-bodied species, and species that are nocturnal, diurnal, migratory, resident, fossorial, arboreal, and semiaquatic, and those that are imperiled, invasive, and cryptic. The host detection efficiency, a measure of the number of detected vertebrate species relative to the number of identified blood meals, varied among mosquito species, indicating that species do not contribute equally to detecting vertebrate species within a community. Our results demonstrate the feasibility of mosquito-based iDNA as a detection method for vertebrates, capable of detecting diverse vertebrate species with a single sampling technique. Because of variation in mosquito diversity patterns between geographic sites and habitats, and in the host associations and host detection efficiency among mosquito species, preliminary surveys to identify and target mosquito species appropriate to the goals of the monitoring effort would be expected to optimize return on investment in mosquito-based vertebrate surveys.

Global biodiversity is declining at alarming rates, necessitating efficient and scalable biodiversity monitoring methods. This study evaluated the efficacy and efficiency of using mosquito blood meal analysis (MBA) as a tool to survey terrestrial vertebrate assemblages during wet and dry seasons in a subtropical landscape of central Florida. We compared the performance of MBA against that of a suite of conventional sampling methods for birds, mammals, amphibians and reptiles. As a single method, MBA detected the highest number of species. Nonetheless, our results indicated that during the dry season, conventional methods were more effective and efficient than MBA, particularly for detecting small mammals and ectotherms. Similarly, in the wet season, conventional methods outperformed MBA in assessing the diversity of individual guilds. Nonetheless, during this season, MBA performance was comparable to that of the conventional techniques when the goal was to assess the entire vertebrate diversity. This is likely due to the higher abundance of adult mosquitoes in the landscape during the wet season. A community similarity analysis showed that MBA and conventional methods obtained different profiles of the vertebrate assemblages. Each method seemed to perform better for dissimilar sets of species. These findings highlight the potential of MBA as a scalable biodiversity monitoring tool, particularly as complementary to conventional methods, or stand-alone method under some environmental conditions. These results could also aid in identifying vertebrate species suitable to be surveyed using MBA.

Determining effective sampling methods for mosquitoes are among the first objectives in elucidating transmission cycles of vector-borne zoonotic disease, as the effectiveness of sampling methods can differ based on species, location, and physiological state. The Spissipes section of the subgenus Melanoconion of Culex represents an understudied group of mosquitoes which transmit Venezuelan equine encephalitis viruses (VEEV) in the Western Hemisphere. The objective of this study was to determine effective collection methods that target both blood-engorged and non-engorged females of the Spissipes section of Culex subgenus Melanoconion to test the hypothesis that favorable trapping methods differ between species and by physiological status within a species. Mosquitoes were collected using two commercially available traps, (CDC-light trap and BG-Sentinel trap), two novel passive traps (a novel mosquito drift fence and pop-up resting shelters), and two novel aspirators, (a small-diameter aspirator and a large-diameter aspirator) in Darién, Panama, and Florida, USA. The total number of female mosquitoes collected for each species was compared using rarefaction curves and diversity metrics. We also compared the utility of each trap for collecting total females and blood-engorged females of four Spissipes section mosquito species in Florida and Darién. In Darién, it was found that both blood-engorged and unfed females of Cx . pedroi were most effectively collected using the mosquito drift fence at 57.6% and 61.7% respectively. In contrast, the most unfed Cx . spissipes were collected using the mosquito drift fence (40.7%) while blood-engorged females were collected effectively by pop-up resting shelters (42.3%). In Florida, the best sampling technique for the collection of blood-engorged Cx . panocossa was the large diameter aspirator at 41.9%, while the best trap for collecting Cx . cedecei was the pop-up resting shelter at 45.9%. For unfed female Spissipes section mosquitoes in Florida, the CDC light trap with CO 2 collected 84.5% and 98.3% of Cx . cedecei and Cx . panocossa respectively in Florida. Rarefaction analysis, and both the Shannon and Simpsons diversity indices all demonstrated that the mosquito drift fence was capable of collecting the greatest diversity of mosquito species regardless of location. The finding that the proportions of unfed and blood-engorged mosquitoes collected by traps differed both among and between species has implications for how studies of VEEV vectors will be carried out in future investigations. In Florida a combination of pop-up resting shelters and use of a large-diameter aspirator would be optimal for the collection of both VEEV vectors for host-use studies. Results demonstrate that traps can be constructed from common materials to collect mosquitoes for VEEV vector studies and could be assessed for their utilization in vectors of other systems as well. Unfortunately, no single method was effective for capturing all species and physiological states, highlighting a particular need for assessing trap utility for target species of a study.

Background Anopheles squamosus is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with Plasmodium falciparum and Plasmodium vivax . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish An. squamosus from morphologically similar Anopheles species . Described in the following methods is the development and validation of a new PCR assay that will reliably identify An. squamosus . Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. Six sets of primers were evaluated overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens morphologically identified from Zambia and South Africa. Results A new assay consisting of a forward (ITS2-ASQ-R10, 5’-CCC TCG AAG GGT GCT GTG-3’) and reverse (ITS2-ASQ-R10 5’-AAT CCA CGG TGT GAT GGC-3’) primer reliably (> 94.9%) amplified an ITS2 fragment of 301 bp length for An. squamosus . The An. squamosus- specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification. Conclusions The development of this robust PCR assay for An. squamosus is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. To further the validation of this ITS2 PCR assay, more species of Anopheles should be compared in addition to An. squamosus collected in different regions. To refine and optimize the PCR process with these primers, touchdown PCR can be used to increase specificity. Applying genomic tools to correctly identify An. squamosus will allow for a better understanding of their role in malaria transmission and may lead to genomic insights into what influences their behaviour, thus leading to new innovations in malaria elimination.

Vector‐borne diseases (VBD) pose a major concern for public health worldwide. Identifying putative vectors and their potential contribution to transmission is a crucial step in understanding vector‐borne disease hazard. However, existing metrics are limited in their utility to inform transmission hazard in zoonotic multi‐vector, multi‐host VBD systems. We present the Vector Potential Index (VPI), a novel metric for evaluating and comparing the potential of blood‐feeding arthropod vectors to contribute to zoonotic VBD transmission. Taking a meta‐analysis approach, the VPI combines vector competence and host use data obtained from scientific literature to assign relative and absolute VPI ranks across species and transmission cycles. Using West Nile virus (WNV) in the eastern United States as a model system, our results demonstrate the ability of VPI to provide a representative assessment of vector species' potential contribution to transmission hazard in the epizootic and enzootic transmission cycles. Most species had low vector potential, and although Aedes species were the most competent WNV vectors in the laboratory, only Culex species were assigned higher VPI ranks. Additionally, the VPI suggests that the contribution of Culex salinarius to WNV transmission in the U.S. may be greater than previously assumed based on assessments of individual parameters. Relative and absolute VPI ranks assigned to species aligned with recent work reviewing their role as vectors in the transmission cycles, indicating that by jointly considering vector competence and host use, the VPI effectively quantifies the species‐specific potential to contribute to WNV transmission hazard in the natural environment, using existing data. We propose the objective and reproducible VPI as a powerful yet simple tool for scientists and public health practitioners, where this trait‐based approach has considerable potential to provide new insights into disease systems and enhance VBD surveillance and intervention strategies. The Vector Potential Index (VPI) is a novel metric for evaluating and comparing the potential of blood‐feeding arthropod vectors to contribute to zoonotic vector‐borne disease transmission. Taking a meta‐analysis approach, the VPI combines vector competence and host use data obtained from scientific literature to assign relative and absolute VPI ranks across species and transmission cycles. We propose the objective and reproducible VPI as a powerful yet simple tool for scientists and public health practitioners, where it has considerable potential to provide new insights into disease systems and enhance vector‐borne disease surveillance and intervention strategies.