Phylogenomics reveals the history of host use in mosquitoes.

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.

Diel activity patterns of vector mosquito species in the urban environment: Implications for vector control strategies.

Mathematical models have been widely used to study the population dynamics of mosquitoes as well as to test and validate the effectiveness of arbovirus outbreak responses and mosquito control strategies. The objective of this study is to assess the diel activity of mosquitoes in Miami-Dade, Florida, and Brownsville, Texas, the most affected areas during the Zika outbreak in 2016-2017, and to evaluate the effectiveness of simulated adulticide treatments on local mosquito populations. To assess variations in the diel activity patterns, mosquitoes were collected hourly for 96 hours once a month from May through November 2019 in Miami-Dade County, Florida, and Brownsville, Texas. We then performed a PERMANOVA followed by a SIMPER analysis to assess whether the abundance and species richness significantly varies at different hours of the day. Finally, we used a mathematical model to simulate the population dynamics of 5 mosquito vector species and evaluate the effectiveness of the simulated adulticide applications. A total of 14,502 mosquitoes comprising 17 species were collected in Brownsville and 10,948 mosquitoes comprising 19 species were collected in Miami-Dade County. Aedes aegypti was the most common mosquito species collected every hour in both cities and peaking in abundance in the morning and the evening. Our modeling results indicate that the effectiveness of adulticide applications varied greatly depending on the hour of the treatment. In both study locations, 9 PM was the best time for adulticide applications targeting all mosquito vector species; mornings/afternoons (9 AM- 5 PM) yielded low effectiveness, especially for Culex species, while at night (12 AM- 6 AM) the effectiveness was particularly low for Aedes species. Our results indicate that the timing of adulticide spraying interventions should be carefully considered by local authorities based on the ecology of the target mosquito species in the focus area.

Diel activity patterns of two distinct populations of Aedes aegypti in Miami, FL and Brownsville, TX.

The diel biting activity of Aedes (Stegomyia) aegypti (L) populations was extensively investigated in the early 1900s to gain more information on the biology of Ae. aegypti, and this information was used to devise effective approaches to controlling populations of this species and protect the human population from widespread arbovirus outbreaks. However, few contemporary studies are available regarding the diel activity patterns of Ae. aegypti. To assess the diel activity patterns of Ae. aegypti in southern Florida and Texas, we conducted 96-h uninterrupted mosquito collections once each month from May through November 2019 in Miami, Florida, and Brownsville, Texas, using BG-Sentinel 2 Traps. The overall diel activity pattern in both cities was bimodal with morning and evening peak activity between 7:00 and 8:00 and between 19:00 and 20:00. There were significant daily, monthly, seasonal, and site-specific differences in activity patterns, but these differences did not affect the overall peak activity times. These differences suggest daily, monthly, seasonal, and site-specific variations in human exposure to Ae. aegypti. Our observations can be used in planning and executing Ae. aegypti vector control activities in southern Florida and southern Texas, specifically those targeting the adult mosquito populations.

Insecticide Resistance in Aedes aegypti Varies Seasonally and Geographically in Texas/Mexico Border Cities.

Insecticide use is the primary method of attempting to reduce or control the spread of mosquito-borne diseases. Insecticide resistance is a major concern as resistance will limit the efficacy of vector-control efforts. The lower Rio Grande Valley region of South Texas has had autochthonous transmission of multiple mosquito-borne diseases including those caused by dengue virus, chikungunya virus, and Zika virus. However, the current status of mosquito resistance to commonly used pesticides in this region is unknown. In this study, we collected field samples from multiple municipalities in South Texas and assessed resistance using the Centers for Disease Control and Prevention bottle bioassay. All populations exhibited characteristics of resistance, and permethrin was the most effective insecticide with an average mortality rate of 44.78%. Deltamethrin and sumethrin had significantly lower mortality rates of 20.31% and 32.16%, respectively, although neither of these insecticides are commonly used for vector-control activities in this region. Depending on which insecticide was used, there was little significance between each of the 7 cities. Seasonal variation in resistance was observed among the collection sites. Both deltamethrin and sumethrin exhibited an increase in susceptibility over the course of 10 months, while permethrin exhibited a decrease in susceptibility. These data highlight the need for further studies to determine if variations in resistance observed are repeated. The data and future findings may be useful in determining the most effective strategies for pesticide use and rotation.

Assessment of Two Novel Host-Derived Beauveria bassiana (Hypocreales: Cordycipitaceae) Isolates Against the Citrus Pest, Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), vectors 'Candidatus Liberibacter spp.', the causative agent of Citrus Greening Disease (CGD) or Huanglongbing (HLB). Managing populations of psyllids in the Lower Rio Grande Valley (LRGV), TX, United States is imperative given a continuous increase in HLB-positive trees. A component of integrated pest management (IPM) program is the use of strains of entomopathogenic fungi for the biological control of D. citri. In an attempt to find endemic strains of entomopathogenic fungi that grow favorably under LRGV environmental conditions and naturally infect D. citri, psyllids were collected from local residential areas, surface sterilized, and plated on a semi-selective agar medium. Collection of over 9,300 samples from 278 sites throughout the LRGV led to the positive identification of two Beauveria bassiana (Balsamo-Crivellii) Vuillemin (Hypocreales: Cordycipitaceae) isolates, ACP18001 and ACP18002. Chi-square analysis of primary and secondary acquisition bioassays revealed that both field isolated strains outperformed Cordyceps (Isaria) fumosorosea (Wize) (Hypocreales: Cordycipitaceae) Apopka97 under both primary (direct spray) and secondary acquisition (adult exposure to sprayed foliage) bioassays with ACP18002 marginally outperforming ACP18001 under secondary acquisition. Slopes of the dose response regression lines for the three fungi were not significantly different. In addition, the thermal profiles for vegetative growth of each isolate indicated that the field isolates grew at higher rates than the standard at higher temperatures. The new isolates may prove to be good candidates for the management of D. citri populations in the LRGV.

Temperatures Influence Susceptibility to Insecticides in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Mosquitoes.

Aedes aegypti and Aedes albopictus (Diptera: Culicidae) are vectors for several arboviruses, including dengue, Zika virus and chikungunya virus. The primary method of controlling these diseases is controlling the vector population, often with insecticides. Insecticide resistance may impact the success of these efforts. We tested the effect of variable temperature exposures on susceptibility to insecticides by exposing adult A.aegypti and A. albopictus to different temperatures and tested their susceptibility to insecticides. We hypothesized that adults maintained at high temperatures would show increased susceptibility to insecticides relative to lower temperatures. Colony mosquitoes were hatched, reared to adulthood and then maintained in three temperature regimes that reflect average seasonal temperatures in the Rio Grande Valley, TX. Susceptibility to permethrin and deltamethrin was assessed using the CDC bottle bioassay method. Overall Aedes albopictus had higher susceptibility to all insecticides than Aedes aegypti. Mosquitoes kept at different temperatures exhibited differential susceptibility to insecticides. Low temperature exposed mosquitoes had decreased susceptibility while high temperature conditions resulted in increased mortality. Our results suggest public health officials must consider temperature effects when controlling mosquitoes with insecticides.

Increasing Public Health Mosquito Surveillance in Hidalgo County, Texas to Monitor Vector and Arboviral Presence.

From 2016 to 2018, Hidalgo County observed the emergence of Zika virus (ZIKV) infections along with sporadic cases of Dengue virus (DENV) and West Nile virus (WNV). Due to the emergence of ZIKV and the historical presence of other mosquito-borne illnesses, Hidalgo County obtained funding to enhance mosquito surveillance and educate residents on arboviruses and travel risks. During this time period, Hidalgo County mosquito surveillance efforts increased by 1.275%. This increase resulted in >8000 mosquitoes collected, and 28 mosquito species identified. Aedes aegypti, Ae albopictus and Culex quinquefasciatus made up approximately two-thirds of the mosquitoes collected in 2018 (4122/6171). Spatiotemporal shifts in vector species composition were observed as the collection period progressed. Significantly, temperature variations (p < 0.05) accounted for associated variations in vector abundance, whereas all other climate variables were not significant.

A Method for Repeated, Longitudinal Sampling of Individual Aedes aegypti for Transmission Potential of Arboviruses.

Mosquito-borne viruses are the cause of significant morbidity and mortality worldwide, especially in low- and middle-income countries. Assessing risk for viral transmission often involves characterization of the vector competence of vector-virus pairings. The most common determination of vector competence uses discreet, terminal time points, which cannot be used to investigate variation in transmission aspects, such as biting behavior, over time. Here, we present a novel method to longitudinally measure individual biting behavior and Zika virus (ZIKV) transmission. Individual mosquitoes were exposed to ZIKV, and from 9 to 24 days post-exposure, individuals were each offered a 180 µL bloodmeal every other day. Biting behavior was observed and characterized as either active probing, feeding, or no bite. The bloodmeal was then collected, spun down, serum collected, and tested for ZIKV RNA via qRT-PCR to determine individuals' vector competence over time. This included whether transmission to the bloodmeal was successful and the titer of expectorated virus. Additionally, serum was inoculated onto Vero cells in order to determine infectiousness of positive recovered sera. Results demonstrate heterogeneity in not only biting patterns but expectorated viral titers among individual mosquitoes over time. These findings demonstrate that the act of transmission is a complex process governed by mosquito behavior and mosquito-virus interaction, and herein we offer a method to investigate this phenomenon.

Use of the sea hare (Aplysia fasciata) in marine pollution biomonitoring of harbors and bays.

Our study evaluated heavy metal concentrations in soft tissues of sea hare, Aplysia fasciata, from the Lower Laguna Madre, Texas. Heavy metals in tissues followed Se>As>Pb>Cd. Concentrations ranged As (BDL-28.08), Cd (BDL-5.50), Pb (BDL-12.85) and Se (4.25-93.43ppm). Median As, Cd, Pb, and Se tissue levels exceeded exposure levels. Significant relationships occurred in metal-metal (AsCd, AsPb, CdPb, CdSe, and PbSe), metal-tissue (significant Se uptake by inhalant and exhalant siphons and As in the hepatopancreas), and metal-metal within tissue (AsPb in the hepatopancreas and CdPb in the digestive cecum) analyses (p<0.05). Bioaccumulation factors (BAF) suggested the inhalant siphon, hepatopancreas, and digestive cecum function as macroconcentrators of Cd, hepatopancreas and digestive cecum as macroconcentrators of Pb, and all tissues were deconcentrators for As and Se. As a bioaccumulator of heavy metals, Aplysia was evaluated as a bioindicator of marine pollution in harbors and bays.

Differential Vector Competency of Aedes albopictus Populations from the Americas for Zika Virus.

To evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain.

Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission.

To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log 10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquitoes appear to be competent vectors of Zika virus in several regions of the Americas.

Seasonal detection of atrazine and atzA in man-made waterways receiving agricultural runoff in a subtropical, semi-arid environment (Hidalgo County, Texas, USA).

Atrazine is a widely-used herbicide that can impact non-target organisms in the environment but can be biologically degraded by several types of microorganisms. In this study, the gene atzA, which encodes for the initial step in bacterially-mediated atrazine degradation, was used as an indicator of atrazine pollution in agricultural canals located in Hidalgo County, Texas, USA. The concentration of atrazine and atzA were monitored once per month for 12 months during 2010-2011. Atrazine was measured using an enzyme-linked immunosorbent assay; atzA abundance was monitored using Quantitative Polymerase Chain Reaction (Q-PCR) analyses. Abundance of atrazine and atzA were compared with rainy versus dry months and during planting versus non-planting months. Results showed that atrazine levels varied from below detection to 0.43 ppb and were not influenced by precipitation or planting season. Concentrations of the gene atzA were significantly different in rainy versus dry months; during planting versus non-planting times of the year; and in the interaction of precipitation and planting season. The highest concentration of atzA, approx. 4.57 × 108 gene copies ml-1, was detected in July 2010-a rainy, planting month in Hidalgo County, South Texas. However, atrazine was below detection during that month. We conclude that Q-PCR using atzA as an indicator gene is a potential method for monitoring low levels of atrazine pollution in environmental samples.

Aedes aegypti and Aedes albopictus Habitat Preferences in South Texas, USA.

The South Texas region has a historical record of occasional dengue outbreaks. The recent introduction of chikungunya virus to the Caribbean suggests that this disease may be a concern as well. Six different cities and three field habitat types (residential, tire shops, and cemeteries) were examined for evidence of habitat and longitudinal preference of two vector species, Aedes aegypti and Aedes albopictus. A. aegypti was more prevalent in tire shop sites, while A. albopictus was more prevalent in cemetery sites. In residential sites, the relative abundance of the two species varied with longitude, with A. albopictus being more abundant near the coast, and A. aegypti being more abundant inland. There was also a temporal variation, with A. aegypti declining in frequency over time in residential sites. These results have implications for control strategies and disease risk and suggest a greater need for increased surveillance and research in the region.

Dengue vectors, human activity, and dengue virus transmission potential in the lower Rio Grande Valley, Texas, United States.

Dengue virus is an emerging disease of concern in the Americas. Recent outbreaks in Florida highlight the potential for the virus to return to the United States. The Lower Rio Grande Valley region of Texas directly borders Mexico, and has experienced dengue transmission in the past concurrent with outbreaks in Mexico along the border region. We examined the potential for dengue virus transmission by examining the vectors in the region, as well as assessing human behavior. We further hypothesized that dengue vector abundance would influence human behavior. Two dengue vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), were found in the region in high abundance. More mosquitoes were collected in rural sites and sites with high vegetation. Of the two species, only Ae. albopictus showed any significant habitat preferences, being more common in rural site. While there was no correlation between human behavior and mosquito abundance, the results support a significant correlation between knowledge of mosquitoes and dengue virus and behavioral practices that might reduce risk of disease transmission. Dengue risk may be higher in certain regions of the Lower Rio Grande Valley based on socioeconomic conditions, specifically in economically poor regions such as the undeveloped colonias found in the region. Because of the proximity of this region to an area with endemic dengue, continued surveillance and risk assessment is suggested.

Effects of forced egg retention on the temporal progression of West Nile virus infection in Culex pipiens quinquefasciatus (Diptera: Culicidae).

Environmental factors that impact the biology of mosquito vectors can have epidemiological implications. Lack of oviposition sites facilitated by environmental factors such as temperature and drought can often force Culex spp. mosquitoes to retain their eggs. Culex pipiens quinquefasciatus Say were fed blood meals containing West Nile virus (WNV; family Flaviviridae, genus Flavivirus) and either allowed to oviposit or forced to retain their eggs through different time points postinfection (9, 13, 20, 27 d) at 28 degrees C. Oviposition status did not significantly affect rates of WNV infection (% with virus-positive bodies), dissemination (% with virus-positive legs), or transmission (% with virus-positive saliva) for any of the tested time points. As expected, WNV titers in bodies and legs were significantly (P < 0.05) higher at late time points compared with early time points. No significant differences were observed in WNV titers in saliva between time points. There were no significant effects of oviposition status on virus titers of bodies, legs, or saliva. However, we found that egg retention may increase vector competence at early and late time points after infection and that a single oviposition event may decrease vector competence, possibly by activating an immune response against the virus. Environmental changes that influence mosquito biology are important determinants of virus transmission, and further studies are needed to assess the effects of drought on virus transmission risk and how these interactions affect our interpretation of field data.

A model to assess the accuracy of detecting arboviruses in mosquito pools.

Vigilant surveillance of virus prevalence in mosquitoes is essential for risk assessment and outbreak prediction. Accurate virus detection methods are essential for arbovirus surveillance. We have developed a model to estimate the probability of accurately detecting a virus-positive mosquito from pooled field collections using standard molecular techniques. We discuss several factors influencing the probability of virus detection, including the number of virions in the sample, the total sample volume, and the portion of the sample volume that is being tested. Our model determines the probability of obtaining at least 1 virion in the sample that is tested. The model also determines the optimal sample volume that is required in any test to ensure a desired probability of virus detection is achieved, and can be used to support the accuracy of current tests or to optimize existing techniques.

Hatch plasticity in response to varied inundation frequency in Aedes albopictus.

Eggs of container-breeding mosquitoes are able to withstand drought conditions as an egg and hatch when submerged. Frequent rainfall can be simulated by frequent submersion, and drought conditions can be simulated by infrequent submersion. We examined the hatch response of Aedes albopictus (Skuse) eggs to simulated drought conditions. Ae. albopictus eggs from a strain originating outside Kobe, Japan, were subjected to one of three treatments; high-frequency hatch stimulation consisting of submerging the eggs in a nutrient broth mixture every 3 d, low-frequency hatch stimulation consisting of submerging the eggs every 7 d, and delayed high-frequency hatch stimulation. Eggs that were subjected to lower-frequency stimulation showed a significant decrease in hatch delay, which was the opposite of the predicted response. This decrease in hatch delay may be an example of hatch plasticity in response to drought conditions. This response could not be explained as a result of the difference in the ages of the eggs on any given stimulus. A decreased hatch delay response to potential drought conditions combined with rapid larval development may enable Ae. albopictus, whose eggs are not as desiccation resistant as some other container-breeding mosquitoes, to survive extended drought.

Climate and geographic trends in hatch delay of the treehole mosquito, Aedes triseriatus Say (Diptera: Culicidae).

Eggs of Aedes triseriatus mosquitoes are stimulated to hatch when inundated with water, but only a small fraction of eggs from the same batch will hatch for any given stimulus. Similar hatching or germination patterns are observed in desert plants, copepods, rotifers, insects, and many other species. Bet hedging theory suggests that parents stagger offspring emergence into vulnerable life history stages in order to avoid catastrophic reproductive failures. For Ae. triseriatus, a treehole breeding mosquito, immediate hatching of an entire clutch leaves all of the parent's progeny vulnerable to extinction in the event of a severe drought. Natural selection has likely favored parents that pursued a bet hedging strategy where the risk of reproductive failure is distributed over time. Considering treehole mosquitoes, bet hedging theory could be used to predict that hatch delay would be positively correlated with the likelihood of drought. To test this prediction, we collected Ae. triseriatus from habitats that varied widely in mean annual precipitation and exposed them to several hatch stimuli in the laboratory. Here we report that, as predicted, Ae. triseriatus eggs from high precipitation regions showed less hatch delay than areas of low precipitation. This strategy probably allows Ae. triseriatus to cope with the wide variety of climatic conditions that it faces in its extensive geographical range.

A method to increase efficiency in testing pooled field-collected mosquitoes.

Testing field-caught mosquito collections can result in thousands of pools, and testing pools of 50 mosquitoes each can be both time consuming and cost prohibitive. Consequently, we have developed an alternative approach to testing mosquito pools for arboviruses, utilizing a superpool strategy. When mosquito samples are processed for extraction of viral RNA and subsequent virus testing via quantitative real-time polymerase chain reaction, each pool is tested individually. Using the method described here, 0.025 ml from each of 10 pools is combined into a superpool for RNA extraction and testing. When a virus-positive superpool sample is found, each of the original 10 pools that constitute this sample is tested individually in order to find the specific positive sample. By retesting the original samples after the initial superpool screen, we are still able to obtain reliable estimates for minimum infection rates or maximum likelihood estimations. To test this principle, we created controlled mosquito pools of known titer and subjected them to our superpool process. We were able to detect our entire range of laboratory-created pools as being West Nile virus (WNV) positive. In 2005, field surveillance efforts from our laboratory resulted in over 4,000 mosquito pools tested, with 8 resulting WNV-positive samples. We found that all of these field samples were detected as WNV positive using the superpool method and contained calculated virus titers from < 0.1 to 4.1 log10 plaque-forming units/ml WNV, indicating that the limit of superpool detection of WNV is below this point. These results reveal that the superpool method could be accurately used to detect WNV in field-collected specimens.