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.

Trait- and density-mediated effects of predation on fecundity and fertility of Aedes aegypti (Diptera: Culicidae) mosquitoes.

Prey populations that encounter predators experience density-mediated effects through lethality and associated numerical changes in the population. Prey also exhibit trait-mediated effects through nonlethal alterations in phenotypic traits associated with exposure to predators. Immature stages of mosquitoes commonly co-occur in habitats along with predators, a natural source of mortality and potential biocontrol agent. Toxorhynchites rutilus Coquillett 1896 is a natural source of mortality with potential as a biological control agent. Previous studies have shown that predation and the mere presence of Tx. rutilus (predator cues) can alter the life-history traits of Aedes aegypti (L. 1762). In addition to observed reductions in recruitment of adults (lethality), exposure to Tx. rutilus without consumption resulted in adult Ae. aegypti females with altered growth and reduced lifespan. To determine the influence of predation on the reproductive biology of Ae. aegypti, we tested the hypothesis that predation, or exposure to predator cues, will compromise the reproduction of adult survivors through reductions in fecundity (egg batch size) and fertility (hatch rate). We observed that for both female and male Ae. aegypti, survival to adulthood was the lowest and development time the shortest in treatments containing prey removal effects, attributable to predation and treatment manipulations of density reduction. There were effects of Ae. aegypti weight, but not predation treatments, on fecundity and fertility. Results suggest that predator-mediated effects on Ae. aegypti derive from lethal effects due to consumption and alterations in other phenotypic traits of survivors, including development, weight, lifespan of adults, and population growth, but not reproductive parameters measured here.

Does prior exposure to larvicides influence dengue virus susceptibility in Aedes aegypti (Diptera: Culicidae)?

Control of mosquito vector populations is primarily intended to reduce the transmission of pathogens they transmit. Use of chemical controls, such as larvicides, can have unforeseen consequences on adult traits if not applied properly. The consequences of under application of larvicides are little studied, specifically the impacts on pathogen infection and transmission by the vectors that survive exposure to larvicides. We compared vector susceptibility of Aedes aegypti (L.) for dengue virus, serotype 1 (DENV-1) previously exposed as larvae to an LC50 of different classes of insecticides as formulated larvicides. Larval exposure to insect growth regulators (methoprene and pyriproxyfen) significantly increased susceptibility to infection of DENV-1 in Ae. aegypti adults but did not alter disseminated infection or transmission. Larval exposure to temephos, spinosad, and Bti did not increase infection, disseminated infection, or transmission of DENV-1. Our findings describe a previously under observed phenomenon, the latent effects of select larvicides on mosquito vector susceptibility for arboviruses. These data suggest that there are unintended consequences of sublethal exposure to select larvicides that can influence susceptibility of Ae. aegypti to DENV infection, and indicates the need for further investigation of sublethal effects of insecticides on other aspects of mosquito biology, especially those parameters relevant to a mosquitoes ability to transmit arboviruses (life span, biting behavior, extrinsic incubation period).

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.

Assessment of insecticide resistance of Aedes aegypti (Diptera: Culicidae) populations to insect growth regulator pyriproxyfen, in the northeast region of Brazil.

Vector control has been an essential strategy in Brazil to manage vector-borne diseases, and the use of insecticides plays an important role in this effort. Pyriproxyfen (PPF) has become a common insect growth regulator used to control juvenile stages of mosquitoes by disturbing their growth and development. This study assesses the susceptibility and resistance status of Brazilian Ae. aegypti populations that previously showed low resistance levels to PPF. Eggs of Ae. aegypti were collected from six cities located in the northeast states of Ceará (Quixadá, Icó, and Juazeiro do Norte), and Bahia (Itabuna, Brumado, and Serrinha). We used the Ae. aegypti Rockefeller strain as an experimental control and a strain known to be susceptible to insecticides. Inhibition of emergence rates by 50% of Ae. aegypti populations varied from 0.0098-0.046 µg/L. Mosquitoes from Icó, Serrinha, and Brumado showed low resistance levels [resistance ratio (RR50) = 2.33, 4.52, and 4.83, respectively], whereas moderate levels of resistance were detected in populations from Juazeiro do Norte (RR50=5.83) and Itabuna (RR50=7.88). Aedes aegypti collected from the Quixadá population showed a high resistance level to pyriproxyfen (RR50=11). The evolution of resistance in Brazilian Ae. aegypti populations to PPF can compromise vector control efforts. Continuous monitoring of insecticide resistance in Ae. aegypti is essential for making timely management decisions for effective vector control and management.

Native Wolbachia infection and larval competition stress shape fitness and West Nile virus infection in Culex quinquefasciatus mosquitoes.

Introduction: Wolbachia transinfections established in key mosquito vectors, including Aedes aegypti are typically associated with pathogen blocking-reduced susceptibility to infection with key pathogens and reduced likelihood those pathogens are transmitted to new hosts. Host-symbiont-virus interactions are less well understood in mosquitoes like Culex quinquefasciatus, which naturally harbor Wolbachia, with pathogen blocking observed in some populations but not others, potentially due to innate differences in their Wolbachia load. In nature, mosquito larvae are often subject to developmental stresses associated with larval competition, which can lead to reduced body size and differential susceptibility to arbovirus infection. Methods: In this study, we sought to understand whether competition stress and Wolbachia infection in Cx. quinquefasciatus combine to impact host fitness and susceptibility to infection with West Nile virus. We reared Wolbachia-infected and uninfected Cx. quinquefasciatus larvae under three competition stress levels, increasing larval density without increasing the amount of food supplied. We then monitored larval development and survival, measured wing length and quantified Wolbachia density in adults, and then challenged mosquitoes from each treatment group orally with West Nile virus. Results and Discussion: We observed that high competition stress extended development time, decreased the likelihood of eclosion, decreased body size, and increased susceptibility to West Nile virus (WNV) infection. We also observed that Wolbachia infection reduced WNV load under low competition stress, and significantly improved the rate of survival for larval reared under higher competition stress. Consequently, our data suggest that native Wolbachia infection in Cx. quinquefasciatus has differential consequences for host fitness and susceptibility to WNV infection depending on competition stress.

Vertical Transmission of Zika Virus by Florida Aedes aegypti and Ae. albopictus.

The Zika virus pandemic of 2015, with mosquitoes Aedes aegypti and Ae. albopictus as the putative vectors, prompted public health concerns and the need to improve our understanding of both the horizontal and vertical transmission of Zika virus. Local transmission is especially concerning for Florida, where these two mosquito species are abundant and widespread throughout much of the year. Here, we evaluate the relative vertical transmission and filial infection rate of progeny of Florida Ae. aegypti and Ae. albopictus following ingestion of infected blood by parental mosquitoes at either 6 or 7 log10 plaque forming units/mL of Zika virus. Florida Ae. aegypti exhibited higher rates of disseminated infection than Ae. albopictus, consistent with other studies indicating greater permissibility of Zika virus in Ae. aegypti. We observed low vertical transmission in both Ae. aegypti (1.1-3.2%) and Ae. albopictus (0-0.3%) mosquitoes, despite imbibing infected blood at titers that yielded high susceptibility to infection and modest horizontal transmission rates. Filial infection rates, testing individual mosquitoes for Ae. aegypti and Ae. albopictus, were 6-10% and 0-6.4%, respectively. Both these invasive Stegomyia mosquitoes were capable of vertically transmitting Zika virus under laboratory conditions, and approximately 5% of female progeny of Ae. aegypti were capable of transmitting Zika virus upon first bite.

Transmission of Asian Zika Lineage by Aedes aegypti and Ae. albopictus Mosquitoes in Florida.

The Asian lineage of Zika virus (ZIKV), a mosquito-borne pathogen originally from Africa, caused an epidemic into Brazil in 2015 and subsequently spread throughout the Americas. Local transmission in the U.S. is a public health concern, especially for Florida where the mosquito vectors Aedes aegypti and Ae. albopictus are widespread, abundant, and there is a high potential for virus introduction due to imported cases. Here we evaluate relative susceptibility to infection and transmission of Zika virus among geographic populations of Ae. aegypti and Ae. albopictus in Florida. Both species have been implicated as ZIKV vectors elsewhere, but both virus and vector genotype are known to influence transmission capacities and, hence, the risk of outbreaks. We test the hypothesis that Ae. aegypti and Ae. albopictus show geographic differences in midgut and salivary gland barriers that limit ZIKV transmission, using local populations of the two vector species recently colonized from three regions of Florida to compare their susceptibility to ZIKV infection, disseminated infection, and transmission potential. Susceptibility to infection was higher in Ae. aegypti (range 76-92%) than Ae. albopictus (range 47-54%). Aedes aegypti exhibited 33-44% higher susceptibility to infection than Ae. albopictus, with Ae. aegypti from Okeechobee, FL having 17% higher susceptibility to infection than Ae. aegypti from Miami, FL. Similarly, disseminated infection was higher in Ae. aegypti (range 87-89%) than Ae. albopictus (range 31-39%), although did not vary by region. Enhanced infection and disseminated infection in Ae. aegypti were associated with higher viral loads in mosquito samples than in Ae. albopictus. Transmission rates did not vary by species or region (range 26-47%). The results support the hypothesis that Ae. aegypti, but not Ae. albopictus, exhibited regional differences in midgut infection barriers. Our observation of higher vector competence for Ae. aegypti than Ae. albopictus, together with this species greater propensity to feed on humans, lends support to the notion that Ae. aegypti is regarded as the primary vector for ZIKV and public health concern in continental U.S.

Lethal and Sublethal Concentrations of Formulated Larvicides Against Susceptible Aedes aegypti.

Chemical control of vectors depends on the effective application of formulated insecticides. In this study we evaluated formulated larvicides using a larval bioassay against susceptible Aedes aegypti. The estimated larvicide lethal concentrations for 50% mortality (LC50s) were 25.7 µg/liter (Natular 2EC), 3.13 µg/liter (Abate 4E), 0.43 µg/liter (Altosid), 0.03 µg/liter (Nyguard), and 500.6 ITU/liter (VectoBac12AS containing Bacillus thuringiensis israelensis). Sublethal effects were identified and documented from adults that survived exposure to these estimated LC50s (body size and sex proportion). We observed changes in net growth as measured by adult wing lengths. For those larvae exposed to estimated LC50s, the average size of adults was between 0.1% and 10.6% smaller for males and between 1.1% and 13.6% smaller for females compared to controls. Sex proportions varied between larvicides, but some were significantly different from the control, favoring greater survival of females than males.

Brazilian Populations of Aedes aegypti Resistant to Pyriproxyfen Exhibit Lower Susceptibility to Infection with Zika Virus.

Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from populations with moderate to high levels of IR exhibited similar or lower susceptibility to ZIKV infection than those from populations with low IR or the susceptible strain. These observations suggest an association between IR and arbovirus infection, which may be attributable to genetic hitchhiking. The use of PPF to control Brazilian Ae. aegypti may be associated with an indirect benefit of reduced susceptibility to infection, but no changes in disseminated infection and transmission of ZIKV among PPF-resistant phenotypes.

Ingestion of spinosad-containing toxic sugar bait alters Aedes albopictus vector competence and vectorial capacity for dengue virus.

Dengue virus (DENV) is a highly prevalent vector-borne virus that causes life-threatening illnesses to humans worldwide. The development of a tool to control vector populations has the potential to reduce the burden of DENV. Toxic sugar bait (TSB) provides a form of vector control that takes advantage of the sugar-feeding behavior of adult mosquitoes. However, studies on the effect of ingestion of toxins in TSB on vector competence and vectorial capacity for viruses are lacking. This study evaluated vector competence for DENV serotype-1 of Aedes albopictus at 7 and 14 days post-ingestion of TSB formulated with spinosad (of bacteria origin) as an oral toxin. Our results and others were modeled to estimate effects on Ae. albopictus vectorial capacity for DENV. Ingestion of TSB caused a reduction in survival of females, but increased mosquito susceptibility to DENV infection, disseminated infection, and transmission. However, this increase in vector competence was obviated by the reduction in survival, leading to a lower predicted vectorial capacity. The findings of this study highlight the importance of evaluating the net impact of TSB ingestion on epidemiological parameters of vectorial capacity in the context of vector control efforts to reduce the risk of transmission of vector-borne viruses.

Temperature-Mediated Effects on Mayaro Virus Vector Competency of Florida Aedes aegypti Mosquito Vectors.

Mayaro virus (MAYV) is an emerging mosquito-borne arbovirus and public health concern. We evaluated the influence of temperature on Aedes aegypti responses to MAYV oral infection and transmission at two constant temperatures (20 °C and 30 °C). Infection of mosquito tissues (bodies and legs) and salivary secretions with MAYV was determined at 3, 9, 15, 21, and 27 days post ingestion. At both temperatures, we observed a trend of increase in progression of MAYV infection and replication kinetics over time, followed by a decline during later periods. Peaks of MAYV infection, titer, and dissemination from the midgut were detected at 15 and 21 days post ingestion at 30 °C and 20 °C, respectively. Mosquitoes were able to transmit MAYV as early as day 3 at 30 °C, but MAYV was not detectable in salivary secretions until day 15 at 20 °C. Low rates of MAYV in salivary secretions collected from infected mosquitoes provided evidence supporting the notion that a substantial salivary gland barrier(s) in Florida Ae. aegypti can limit the risk of MAYV transmission. Our results provide insights into the effects of temperature and time on the progression of infection and replication of MAYV in Ae. aegypti vectors.

Spinosyns Delivered in Sugar Meals to Aedes aegypti and Aedes albopictus (Diptera: Culicidae): Acute Toxicity and Subacute Effects on Survival, Fecundity, and Fertility.

Sugar is an essential source of nutrition for adult mosquitoes to acquire energy. Toxic sugar bait (TSB) provides a promising method for mosquito control by incorporating toxins into artificial sources of sugar (i.e., toxic baits) presented to wild populations. Spinosyns comprise a family of bacterial secondary metabolites with a unique mode of action against the insect nervous system, an appealing environmental safety profile, and potential for incorporation into sugar baits. This research evaluated acute and subacute effects of spinosad (spinosyns A and D) and spinetoram (spinosyns J and L) in sugar meals on survival, fecundity, and fertility of Aedes aegypti and Aedes albopictus. Acute toxicity of spinosyns doubled from 24 to 48 h of assessment, revealing a relatively slow and cumulative action of the formulated spinosyns. Median lethal concentrations at 48 h were lower for spinetoram than for spinosad, lower for Ae. albopictus than Ae. aegypti, and lower for males than females. When exposed to subacute LC50 concentrations of spinosad and spinetoram for 24 h, survival of males and females of both species was diminished compared with controls, fecundity of females was increased, but fertility as measured by hatch rate of eggs was decreased. The formulations may have increased the nutritive value of the sugar meals thereby boosting fecundity, while toxifying embryos, reducing fertility. The inclusion of subacute effects of spinosyns allows assessment of the broader consequences of TSB for adult mosquito control.

Population genetics of an invasive mosquito vector, Aedes albopictus in the Northeastern USA.

The Asian tiger mosquito (Aedes albopictus) arrived in the USA in the 1980's and rapidly spread throughout eastern USA within a decade. The predicted northern edge of its overwintering distribution on the East Coast of the USA roughly falls across New York, Connecticut, and Massachusetts, where the species has been recorded as early as 2000. It is unclear whether Ae. albopictus populations have become established and survive the cold winters in these areas or are recolonized every year. We genotyped and analyzed populations of Ae. albopictus from the northeast USA using 15 microsatellite markers and compared them with other populations across the country and to representatives of the major global genetic clades to investigate their connectivity and stability. Founder effects or bottlenecks were rare at the northern range of the Ae. albopictus distribution in the northeastern USA, with populations displaying high levels of genetic diversity and connectivity along the East Coast. There is no evidence of population turnover in Connecticut during the course of three consecutive years, with consistent genetic structure throughout this period. Overall, these results support the presence of established populations of Ae. albopictus in New York, Connecticut, and Massachusetts, successfully overwintering and migrating in large numbers. Given the stability and interconnectedness of these populations, Ae. albopictus has the potential to continue to proliferate and expand its range northward under mean warming conditions of climate change. Efforts to control Ae. albopictus in these areas should thus focus on vector suppression rather than eradication strategies, as local populations have become firmly established and are expected to reemerge every summer.

Evaluation of Pyriproxyfen Effects on Aedes aegypti and Predatory Mosquito Toxorhynchites rutilus (Diptera: Culicidae).

Control of mosquito vectors of pathogens remains heavily dependent on the application of conventional insecticides. Pyriproxyfen (PPF) is a novel insecticide that has been proposed for use in autodissemination techniques to control mosquito vectors. The use of PPF can inhibit adult emergence but does not inhibit larval development. This feature is favorable for controlling Aedes aegypti because PPF has the potential to work in combination with natural sources of mortality (competition, predation) during the immature stages, and other control methods, including biocontrol agents that further suppress recruitment of adult mosquitoes. However, the PPF effects on life-history traits of Ae. aegypti in comparison to predatory mosquito Toxorhynchites rutilus, a source of mortality, are not fully understood. Here, we show that larval exposure to PPF concentrations that inhibit 50-90% of adult emergence in Ae. aegypti had a negligible effect on adult emergence and lifespan of Tx. rutilus. Weights of adult Ae. aegypti and Tx. rutilus were not influenced by PPF. These findings suggest that the use of PPF to control mosquito vectors may have low effects on mosquito biocontrol agents. Our results extend and confirm earlier data showing that PPF has potential to implement with Tx. rutilus to suppress Ae. aegypti and provide an additional advantage of PPF use in autodissemination control strategies.

The Extrinsic Incubation Period of Zika Virus in Florida Mosquitoes Aedes aegypti and Ae. albopictus.

The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread throughout the Americas. In July 2016, Florida experienced its first locally acquired ZIKV infection in the continental U.S. Concerns about health risks from ZIKV infection have increased the need to investigate the interactions between potential mosquito vectors and ZIKV. The time it takes for an arbovirus to propagate within a mosquito, and become transmissible, is the extrinsic incubation period (EIP). The EIP for potential mosquito vectors in Florida is unknown. To address this gap in the understanding of ZIKV epidemiology, Florida Aedes aegypti (L.) and Ae. albopictus (Skuse) were orally exposed to ZIKV infected blood meals and fully engorged mosquitoes were held at a constant temperature of 28 °C through the duration of the experiment. Saliva expectorates were collected from cohorts of mosquitoes and tested for the presence of ZIKV at three-day intervals over a period of 24 days to allow for an evaluation of the EIP of the emergent Asian lineage of ZIKV. High rates of infected bodies in Ae. albopictus (75-94%) and Ae. aegypti (68-86%) were observed throughout the incubation period, which did not differ by species. Higher rates of disseminated infection were observed later during the incubation period but did not differ between species. We calculated the 50% EIP to be shorter in Ae. albopictus than Ae. aegypti (16.2 and 18.2 days post infection, respectively). The competence for ZIKV observed in both species may contribute to high rates of ZIKV transmission in Florida populations.

Juvenile hormone analog enhances Zika virus infection in Aedes aegypti.

In recent years, there has been a rise in the emergence of arboviruses of public health importance, including Zika, chikungunya, dengue, and yellow fever viruses. Insecticide-based mosquito control has been the primary method for mitigating transmission of arboviruses. The consequences for the application of insecticides include both lethal and sublethal effects, and associated development of insecticide resistance. However, little is known about the influence on arboviral transmission. Mosquitoes with phenotypes that exhibit insecticide resistance or experience sublethal effects may be associated with altered susceptibility to arbovirus infection and transmission. Juvenile hormone analogs (JHAs) are insecticides that prevent pupa to adult molting of mosquitoes by mimicking the action of their natural juvenile hormone. Here, we examined whether the JHA pyriproxyfen interacts with ambient temperature (20 °C and 30 °C) during juvenile stages to influence life-history traits, population growth (λ'), and Zika virus (ZIKV) infection in Aedes aegypti. Development time of females was lengthened at 20 °C and in the presence of JHA. Prevention of pupa to adult molting by JHA was differentially higher at elevated temperature than low temperature. Size of females was larger at 20 °C and smaller at 30 °C. Infection, disseminated infection, and transmission of ZIKV in females were enhanced by JHA at both 20 °C and 30 °C relative to the controls. These results demonstrate that mosquito life-history and vector competence parameters are strongly influenced by interactive effects of JHA and temperature. The JHA-induced enhancement of ZIKV infection in females should be a consideration when implementing JHA in vector control strategies.

Vector Competence of Florida Culicoides insignis (Diptera: Ceratopogonidae) for Epizootic Hemorrhagic Disease Virus Serotype-2.

Epizootic hemorrhagic disease virus (EHDV; family Reoviridae, genus Orbivirus) is an arthropod-borne virus of ungulates, primarily white-tailed deer in North America. Culicoides sonorensis, the only confirmed North American vector of EHDV, is rarely collected from Florida despite annual virus outbreaks. Culicoides insignis is an abundant species in Florida and is also a confirmed vector of the closely related Bluetongue virus. In this study, oral challenge of C. insignis was performed to determine vector competence for EHDV serotype-2. Field-collected female midges were provided bovine blood spiked with three different titers of EHDV-2 (5.05, 4.00, or 2.94 log10PFUe/mL). After an incubation period of 10 days or after death, bodies and legs were collected. Saliva was collected daily from all females from 3 days post feeding until their death using honey card assays. All samples were tested for EHDV RNA using RT-qPCR. Our results suggest that C. insignis is a weakly competent vector of EHDV-2 that can support a transmissible infection when it ingests a high virus titer (29% of midges had virus positive saliva when infected at 5.05 log10PFUe/mL), but not lower virus titers. Nevertheless, due to the high density of this species, particularly in peninsular Florida, it is likely that C. insignis plays a role in the transmission of EHDV-2.

Paternal and maternal effects in a mosquito: A bridge for life history transition.

Parental (transgenerational) effects occur when the conditions experienced by a mother or father contribute to offspring phenotype. Here we show that parental larval diet in mosquitoes, Aedes aegypti, results in differential allocation of resources in offspring of parents depending on the nutritional condition (quality) of their mate. Maternal effects influenced the number of eggs produced by females as well as their lipid investment. Low nutrient females mated with high nutrient males laid eggs with significantly higher lipid content than those laid by high nutrient females. Paternal effects showed that when high nutrient males mated with low nutrient females, resulting eggs had higher lipid content than when low nutrient males mated with low nutrient females. Overall, our results are consistent with a pattern predicted by the differential allocation of resources hypothesis, when females experience nutritional deprivation, which asserts that mate quality directly influences reproductive allocation.

Modelling distributions of Aedes aegypti and Aedes albopictus using climate, host density and interspecies competition.

Florida faces the challenge of repeated introduction and autochthonous transmission of arboviruses transmitted by Aedes aegypti and Aedes albopictus. Empirically-based predictive models of the spatial distribution of these species would aid surveillance and vector control efforts. To predict the occurrence and abundance of these species, we fit a mixed-effects zero-inflated negative binomial regression to a mosquito surveillance dataset with records from more than 200,000 trap days, representative of 53% of the land area and ranging from 2004 to 2018 in Florida. We found an asymmetrical competitive interaction between adult populations of Aedes aegypti and Aedes albopictus for the sampled sites. Wind speed was negatively associated with the occurrence and abundance of both vectors. Our model predictions show high accuracy (72.9% to 94.5%) in validation tests leaving out a random 10% subset of sites and data since 2017, suggesting a potential for predicting the distribution of the two Aedes vectors.