Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

A network analysis framework to improve the delivery of mosquito abatement services in Machala, Ecuador.

BACKGROUND: Vector-borne disease places a high health and economic burden in the American tropics. Comprehensive vector control programs remain the primary method of containing local outbreaks. With limited resources, many vector control operations struggle to serve all affected communities within their districts. In the coastal city of Machala, Ecuador, vector control services, such as application of larvicides and truck-mounted fogging, are delivered through two deployment facilities managed by the Ecuadorian Ministry of Health. Public health professionals in Machala face several logistical issues when delivering mosquito abatement services, namely applying limited resources in ways that will most effectively suppress vectors of malaria, dengue, and encephalitis viruses. METHODS: Using a transportation network analysis framework, we built models of service areas and optimized delivery routes based on distance costs associated with accessing neighborhoods throughout the city. Optimized routes were used to estimate the relative cost of accessing neighborhoods for mosquito control services in Machala, creating a visual tool to guide decision makers and maximize mosquito control program efficiency. Location-allocation analyses were performed to evaluate efficiency gains of moving service deployment to other available locations with respect to distance to service hub, neighborhood population, dengue incidence, and housing condition. RESULTS: Using this framework, we identified different locations for targeting mosquito control efforts, dependent upon management goals and specified risk factors of interest, including human population, housing condition, and reported dengue incidence. Our models indicate that neighborhoods on the periphery of Machala with the poorest housing conditions are the most costly to access. Optimal locations of facilities for deployment of control services change depending on pre-determined management priorities, increasing the population served via inexpensive routes up to 34.9%, and reducing overall cost of accessing neighborhoods up to 12.7%. CONCLUSIONS: Our transportation network models indicate that current locations of mosquito control facilities in Machala are not ideal for minimizing driving distances or maximizing populations served. Services may be optimized by moving vector control operations to other existing public health facilities in Machala. This work represents a first step in creating a spatial tool for planning and critically evaluating the systematic delivery of mosquito control services in Machala and elsewhere.

Patterns of Abundance, Host Use, and Everglades Virus Infection in Culex (Melanoconion) cedecei Mosquitoes, Florida, USA.

Everglades virus (EVEV), subtype II within the Venezuelan equine encephalitis (VEE) virus complex, is a mosquitoborne zoonotic pathogen endemic to south Florida, USA. EVEV infection in humans is considered rare, probably because of the sylvatic nature of the vector, the Culex (Melanoconion) cedecei mosquito. The introduction of Cx. panocossa, a tropical vector mosquito of VEE virus subtypes that inhabits urban areas, may increase human EVEV exposure. Field studies investigating spatial and temporal patterns of abundance, host use, and EVEV infection of Cx. cedecei mosquitoes in Everglades National Park found that vector abundance was dynamic across season and region. Rodents, particularly Sigmodon hispidus rats, were primary vertebrate hosts, constituting 77%-100% of Cx. cedecei blood meals. Humans were fed upon at several locations. We detected EVEV infection in Cx. cedecei mosquitoes in lower and upper regions of Everglades National Park only during the wet season, despite an abundance of Cx. cedecei mosquitoes at other sampling times.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

BACKGROUND: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. METHODS: We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). RESULTS: The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. CONCLUSIONS: Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

Reemergence of St. Louis Encephalitis Virus in the Americas.

We summarize and analyze historical and current data regarding the reemergence of St. Louis encephalitis virus (SLEV; genus Flavivirus) in the Americas. Historically, SLEV caused encephalitis outbreaks in the United States; however, it was not considered a public health concern in the rest of the Americas. After the introduction of West Nile virus in 1999, activity of SLEV decreased considerably in the United States. During 2014-2015, SLEV caused a human outbreak in Arizona and caused isolated human cases in California in 2016 and 2017. Phylogenetic analyses indicate that the emerging SLEV in the western United States is related to the epidemic strains isolated during a human encephalitis outbreak in Córdoba, Argentina, in 2005. Ecoepidemiologic studies suggest that the emergence of SLEV in Argentina was caused by the introduction of a more pathogenic strain and increasing populations of the eared dove (amplifying host).

Deforestation and vector-borne disease: Forest conversion favors important mosquito vectors of human pathogens.

The global burden of vector-borne diseases accounts for more than 17% of infectious diseases in humans. Rapid global expansion of previously obscure pathogens, such as Zika and chikungunya viruses in recent years highlights the importance of understanding how anthropogenic changes influence emergence and spillover of vector-borne diseases. Deforestation has been identified as one anthropogenic change that influences vector-borne disease prevalence, although contrasting pictures of the effects of deforestation on vector-borne disease transmission have been reported. These conflicting findings are likely attributable to the inherent complexity of vector-borne disease systems, which involve diverse groups of vectors, hosts and pathogens, depending on geography. The current study represents a quantitative exploration of the link between deforestation and mosquitoes, the most important common constituents of vector-borne disease systems. Analysis of data compiled from published field studies for 87 mosquito species from 12 countries revealed that about half of the species (52.9%) were associated with deforested habitats. Of these species that are favored by deforestation, a much larger percentage (56.5%) are confirmed vectors of human pathogens, compared to those negatively impacted by deforestation (27.5%). Moreover, species that serve as vectors of multiple human pathogens were all favored by deforestation, including Anopheles bancroftii, Anopheles darlingi, Anopheles farauti, Anopheles funestus s.l., Anopheles gambiae s.l., Anopheles subpictus, Aedes aegypti, Aedes vigilax, Culex annulirostris, and Culex quinquefasciatus. Our quantitative analysis of vector and non-vector species, demonstrates that the net effect of deforestation favors mosquitoes that serve as vectors of human disease, while the obverse holds true for non-vectors species. These results begin to unify our understanding of the relationship between deforestation and vector mosquitoes, an important step in quantifying how land use change, specifically deforestation, affects human risk of vector-borne disease.

Mammal decline, linked to invasive Burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease.

Invasive apex predators have profound impacts on natural communities, yet the consequences of these impacts on the transmission of zoonotic pathogens are unexplored. Collapse of large- and medium-sized mammal populations in the Florida Everglades has been linked to the invasive Burmese python, Python bivittatus Kuhl. We used historic and current data to investigate potential impacts of these community effects on contact between the reservoir hosts (certain rodents) and vectors of Everglades virus, a zoonotic mosquito-borne pathogen that circulates in southern Florida. The percentage of blood meals taken from the primary reservoir host, the hispid cotton rat, Sigmodon hispidus Say and Ord, increased dramatically (422.2%) from 1979 (14.7%) to 2016 (76.8%), while blood meals from deer, raccoons and opossums decreased by 98.2%, reflecting precipitous declines in relative abundance of these larger mammals, attributed to python predation. Overall species diversity of hosts detected in Culex cedecei blood meals from the Everglades declined by 40.2% over the same period (H(1979) = 1.68, H(2016) = 1.01). Predictions based upon the dilution effect theory suggest that increased relative feedings upon reservoir hosts translate into increased abundance of infectious vectors, and a corresponding upsurge of Everglades virus occurrence and risk of human exposure, although this was not tested in the current study. This work constitutes the first indication that an invasive predator can increase contact between vectors and reservoirs of a human pathogen and highlights unrecognized indirect impacts of invasive predators.

Host stress hormones alter vector feeding preferences, success, and productivity.

Stress hormones might represent a key link between individual-level infection outcome, population-level parasite transmission, and zoonotic disease risk. Although the effects of stress on immunity are well known, stress hormones could also affect host-vector interactions via modification of host behaviours or vector-feeding patterns and subsequent reproductive success. Here, we experimentally manipulated songbird stress hormones and examined subsequent feeding preferences, feeding success, and productivity of mosquito vectors in addition to defensive behaviours of hosts. Despite being more defensive, birds with elevated stress hormone concentrations were approximately twice as likely to be fed on by mosquitoes compared to control birds. Moreover, stress hormones altered the relationship between the timing of laying and clutch size in blood-fed mosquitoes. Our results suggest that host stress could affect the transmission dynamics of vector-borne parasites via multiple pathways.

Vector Competence and Capacity of Culex erraticus (Diptera: Culicidae) for Eastern Equine Encephalitis Virus in the Southeastern United States.

Field studies of the ecology of eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) in the southeastern United States have demonstrated that Culex erraticus (Dyar and Knab) is the most common mosquito at many enzootic sites and is often infected with the virus. However, the competence of Cx. erraticus for EEEV has not been explored in detail. Culex erraticus females were collected from the field and fed upon EEEV-infected chicks. The infected mosquitoes were provided honey for nutrition and to monitor for time to infectiveness. Of the mosquitoes that survived the 14-d postfeeding period, 89% were infected and 84% had evidence of a disseminated infection, though titers were generally low. EEEV was first detected in honey 6 d postinfection and was detected in samples collected from 94% of the mosquitoes with a disseminated infection overall. These data and others were then employed to estimate the relative vectorial capacity of Cx. erraticus at an EEEV enzootic site in Alabama. The vectorial capacity of Cx. erraticus at this site was 44% of Culiseta melanura (Coquillett), the accepted enzootic vector, suggesting Cx. erraticus may play a role in transmitting EEEV in areas where it is abundant and Cs. melanura rare.

Testing of Visual and Chemical Attractants in Correlation with the Development and Field Evaluation of an Autodissemination Station for the Suppression of Aedes aegypti and Aedes albopictus in Florida.

Three exotic mosquito-borne pathogens-dengue, chikungunya, and Zika viruses-transmitted by Aedes albopictus and Ae. aegypti have undergone dramatic global expansion in recent years. The control of vector populations and minimizing bites from these vectors are the primary methods of reducing risk of transmission of these viruses to humans. However, Ae. albopictus and Ae. aegypti are notoriously challenging to control through conventional chemical means, due primarily to difficulties in applying pesticides to their cryptic larval habitats. A novel strategy for suppressing populations of these species is the autodissemination of insect growth regulators (IGRs), in which adult female mosquitoes are attracted to a treatment station where they are tainted with small amounts of potent IGR. When the adult females subsequently visit oviposition sites, they inadvertently disseminate the IGR to larval development sites, suppressing their own population. Implementing this technology to control natural vector populations presents substantial logistical challenges. The current manuscript describes laboratory bioassays and field evaluations to design a novel autodissemination station (ADS) and test the methodology at field locations in Florida where Ae. aegypti and Ae. albopictus are abundant and pose a risk for transmission of emerging pathogens. The prototype ADS is intended to attract host-seeking, resting site-seeking, and oviposition site-seeking females through a combination of visual and olfactory cues. The efficacy of this strategy was assessed through the use of sentinel ovicups at field locations in Indian River County and Martin County, FL. Greatest efficacy (45.3 ± 7.7% mortality in treatment sentinel ovicups) was achieved at a field site with few competing natural ovisites, while much lower efficacy was observed in locations with numerous competing ovisites (0.0 to 29.0 ± 8.2% mortality). The efficacy of the ADS is likely to be strongly affected by the abundance of competing ovisites, the population dynamics, and climatic conditions.

Ecology of Culiseta Melanura and Other Mosquitoes (Diptera: Culicidae) from Walton County, FL, During Winter Period 2013-2014.

Winter ecology of putative vectors of eastern equine encephalomyelitis virus (EEEV) in northern Florida was investigated at field locations with evidence of historic EEEV winter transmission. Light traps and resting shelters were used to sample the mosquito community in the vicinity of eight sentinel flocks throughout the winter period (November-April) of 2013 and 2014 in Walton County, FL. Overall mosquito activity was relatively low, although mosquitoes were captured during each week of the study period. Mosquito activity was linked to morning temperature, and females were captured when ambient morning temperatures were quite low (1-5°C). Anopheles crucians Wiedemann, Culex erraticus (Dyar and Knab), Culex territans Walker, and Culiseta melanura (Coquillett) were the most commonly collected mosquito species (of 20 total species). Analysis of blood-engorged mosquitoes revealed a number of mosquito species feeding upon chickens, other birds, amphibians, and domestic and wild mammals. Cs. melanura fed primarily upon chickens and songbirds (Passeriformes), suggesting that this mosquito species is the likely winter vector of EEEV to sentinel chickens in northern Florida. Both resident and nonresident songbird species were fed upon, constituting 63.9 and 36.1% of total songbird meals, respectively. Our results suggest important roles for Cs. melanura and songbird hosts for the winter transmission of EEEV in northern Florida.

LA CROSSE VIRUS VECTOR RESTING BEHAVIORS - FIELD STUDIES WITH PROKOPAK AND RESTING SHELTER COLLECTIONS PROVIDE LOW YIELD.

Resting adult mosquito collections provide opportunities to sample broad physiological conditions (e.g., blood-engorged, gravid, nectar-engorged, and/or parous) that yield important biological information necessary to understand vector and pathogen transmission ecology. In this study, we evaluated Prokopak aspirations of Rhododendron spp. and human-powered pop-up resting shelter collections at 4 residences with historical evidence of proximal La Crosse virus (LACV) transmission from May through September 2022. The goal of this study was to investigate these sampling methods in the context of LACV vector biology-focused principally on Aedes triseriatus (primary LACV vector) and 2 invasive species (Ae. albopictus and Ae. japonicus) that likely serve as secondary LACV vectors. Overall, 304 resting shelters and 80 Prokopak collections yielded a grand total of 33 mosquitoes, of which a third were LACV vectors (Ae. triseriatus [n = 1, 3.0%], Ae. albopictus [n = 4, 12.1%], and Ae. japonicus [n = 6, 18.2%]). Anopheles punctipennis (n = 9, 27.2%) was the most frequently collected species followed by Culex erraticus (n = 7, 21.2%), whereas the least frequently collected species were Ae. triseriatus and Cx. pipiens (n = 1, 3.0%). Despite substantial collection efforts, and concurrent gravid-trap evidence of LACV vectors at the collection sites, Prokopak aspiration of Rhododendron spp. and human-powered pop-up resting shelters did not yield a meaningful number of LACV vectors and thus, as described within, may not be useful adjuncts for the evaluation of LACV ecology and disease risk. Additional approaches to evaluate the resting behavior of these vectors in LACV endemic areas are needed.

Host associations of Culex panocossa (Diptera: Culicidae) in southern Florida and its implications for arbovirus transmission.

Culex panocossa, Dyar and Knab, an important enzootic vector of Venezuelan equine encephalitis virus subtype ID in Central and South America, was found to have invaded and become established in southern Florida in 2016. No information is currently available regarding the ecology of this invasive mosquito in the United States. Here, we use PCR-based blood meal analysis to investigate vertebrate host associations of Cx. panocossa from Florida to provide information necessary for determining the potential importance of this mosquito for arbovirus transmission in the United States. Culex panocossa fed mainly upon birds (49.5%) but took a substantial fraction of blood meals from mammals (33.3%) and reptiles (17.1%). By feeding upon amplifying hosts of Everglades virus (hispid cotton rat) and eastern equine encephalitis virus (wading birds) and humans, Cx. panocossa could act as a bridge vector for these pathogenic Alphaviruses in Florida, potentially resulting in increased human disease.

Culex erraticus (Diptera: Culicidae) utilizes gopher tortoise burrows for overwintering in North Central Florida.

Mosquito-borne diseases represent a significant threat to human and animal health in the United States. Several viruses, including West Nile, Saint Louis encephalitis, and Eastern equine encephalitis are endemic. In humans, the disease is typically detected during the summer months, but not during the winter months. The ability of these viruses to reemerge year after year is still not fully understood, but typically involves persistence in a reservoir host or vector during periods of low transmission. Mosquito species are known to overwinter at different life stages (adults, larvae, or eggs) in manufactured or natural sites. Gopher tortoise burrows are known to serve as refuge for many vertebrate and invertebrate species in pine savannas. In this study, we surveyed the interior of gopher tortoise burrows for overwintering mosquitoes. We identified 4 species (Anopheles crucians s.l., Culex erraticus, Mansonia dyari, and Uranotaenia sapphirina). Cx. erraticus was the most abundant, and its presence and abundance increased in winter months, implying that this species utilized gopher tortoise burrows for overwintering. Bloodfed Cx. erraticus and An. crucians s.l. females were detected. While An. crucians s.l. fed exclusively on the white-tailed deer, Cx. erraticus had a more diverse host range but fed primarily on the gopher tortoise. Tortoises and other long-lived reptiles like the American alligator have been shown to sustain high viremia following West Nile virus (WNV) and Eastern equine encephalitis virus (EEEV) infection and therefore could play a role in the maintenance of these viruses. In addition, Cx. erraticus is naturally infected with WNV and is a known bridge vector for EEEV. As such, these overwintering sites may play a role in perpetuating over-winter arboviral activity in Florida.

Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Culicoides Midge Abundance across Years: Modeling Inter-Annual Variation for an Avian Feeder and a Candidate Vector of Hemorrhagic Diseases in Farmed Wildlife.

(1) Background: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are orbiviruses that cause hemorrhagic disease (HD) with significant economic and population health impacts on domestic livestock and wildlife. In the United States, white-tailed deer (Odocoileus virginianus) are particularly susceptible to these viruses and are a frequent blood meal host for various species of Culicoides biting midges (Diptera: Ceratopogonidae) that transmit orbiviruses. The species of Culicoides that transmit EHDV and BTV vary between regions, and larval habitats can differ widely between vector species. Understanding how midges are distributed across landscapes can inform HD virus transmission risk on a local scale, allowing for improved animal management plans to avoid suspected high-risk areas or target these areas for insecticide control. (2) Methods: We used occupancy modeling to estimate the abundance of gravid (egg-laden) and parous (most likely to transmit the virus) females of two putative vector species, C. stellifer and C. venustus, and one species, C. haematopotus, that was not considered a putative vector. We developed a universal model to determine habitat preferences, then mapped a predicted weekly midge abundance during the HD transmission seasons in 2015 (July-October) and 2016 (May-October) in Florida. (3) Results: We found differences in habitat preferences and spatial distribution between the parous and gravid states for C. haematopotus and C. stellifer. Gravid midges preferred areas close to water on the border of well and poorly drained soil. They also preferred mixed bottomland hardwood habitats, whereas parous midges appeared less selective of habitat. (4) Conclusions: If C. stellifer is confirmed as an EHDV vector in this region, the distinct spatial and abundance patterns between species and physiological states suggest that the HD risk is non-random across the study area.

Mosquito Blood Meal Analysis.

The host associations of mosquitoes vary by species, with some species being relative generalists, whereas others specialize, to varying extents, on a particular subset of the available host community. These host associations are driving factors in transmission dynamics of mosquito-vectored pathogens. For this reason, characterizing the host associations of mosquito species is critical for understanding the epidemiology of mosquito-vectored pathogens. Diverse methods have been used to associate mosquito species with their hosts. These typically include collecting mosquitoes that bite a restrained host (bait) or collecting wild blood-engorged mosquitoes and matching their blood meal to reference samples (blood meal analysis). Blood meal analysis refers to a collection of molecular techniques for determining the taxonomic identity of the source of a mosquito blood meal using cytological, serological, or DNA-based characteristics of the blood meal. Blood meal analyses that are based on DNA markers have advantages over cytological and serological methods and are effective for determining species-level identities of hosts from a broad range of potential host taxa. Here, we discuss effective techniques for analyzing blood meals.

Extracting DNA from Preserved Mosquito Blood Meals.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

Preservation of Field-Collected Mosquito Blood Meals.

All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.

Amplification and Identification of Vertebrate Host Cytochrome c Oxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals.

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.