Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

Key to the Adult Females of Species of Culex Subgenus Melanoconion in Florida, USA.

The subgenus Melanoconion of Culex is a diverse and taxonomically challenging group of mosquitoes (Diptera: Culicidae) endemic to the American tropics, with a few species occurring in temperate regions. With the introduction and establishment of Culex (Melanoconion) panocossa in southern Florida, the existing taxonomic resources for identifying species of Melanoconion in Florida need updating. Here we provide an up-to-date photographic key for identifying females of Culex (Melanoconion) spp. known to occur in Florida, USA, and summarize relevant literature on the biology of each species. Given the challenge of identifying members of subgenus Melanoconion, updated resources for identifying females of this medically important group are warranted. The keys, photographs, and summarized biological information in this report should facilitate research, surveillance, and decisions regarding control.

Modeling Abundance of Culicoides stellifer, a Candidate Orbivirus Vector, Indicates Nonrandom Hemorrhagic Disease Risk for White-Tailed Deer (Odocoileus virginianus).

(1) Background: Hemorrhagic diseases in white-tailed deer (Odocoileus virginianus) are caused by orbiviruses and have significant economic impact on the deer ranching industry in the United States. Culicoides stellifer is a suspected vector of epizootic hemorrhagic disease virus (EHDV), with recent field evidence from Florida, but its natural history is poorly understood. Studying the distribution and abundance of C. stellifer across the landscape can inform our knowledge of how virus transmission can occur locally. We may then target vector management strategies in areas where viral transmission can occur. (2) Methods: Here, we used an occupancy modeling approach to estimate abundance of adult C. stellifer females at various physiological states to determine habitat preferences. We then mapped midge abundance during the orbiviral disease transmission period (May-October) in Florida. (3) Results: We found that overall, midge abundance was positively associated with sites in closer proximity to large-animal feeders. Additionally, midges generally preferred mixed bottomland hardwood and agricultural/sand/water habitats. Female C. stellifer with different physiological states preferred different habitats. (4) Conclusions: The differences in habitat preferences between midges across states indicate that disease risk for deer is heterogeneous across this landscape. This can inform how effective vector management strategies should be implemented.

Invasive Burmese pythons alter host use and virus infection in the vector of a zoonotic virus.

The composition of wildlife communities can have strong effects on transmission of zoonotic vector-borne pathogens, with more diverse communities often supporting lower infection prevalence in vectors (dilution effect). The introduced Burmese python, Python bivittatus, is eliminating large and medium-sized mammals throughout southern Florida, USA, impacting local communities and the ecology of zoonotic pathogens. We investigated invasive predator-mediated impacts on ecology of Everglades virus (EVEV), a zoonotic pathogen endemic to Florida that circulates in mosquito-rodent cycle. Using binomial generalized linear mixed effects models of field data at areas of high and low python densities, we show that increasing diversity of dilution host (non-rodent mammals) is associated with decreasing blood meals on amplifying hosts (cotton rats), and that increasing cotton rat host use is associated with increasing EVEV infection in vector mosquitoes. The Burmese python has caused a dramatic decrease in mammal diversity in southern Florida, which has shifted vector host use towards EVEV amplifying hosts (rodents), resulting in an indirect increase in EVEV infection prevalence in vector mosquitoes, putatively elevating human transmission risk. Our results indicate that an invasive predator can impact wildlife communities in ways that indirectly affect human health, highlighting the need for conserving biological diversity and natural communities.

Tracking Community Timing: Pattern and Determinants of Seasonality in Culicoides (Diptera: Ceratopogonidae) in Northern Florida.

Community dynamics are embedded in hierarchical spatial-temporal scales that connect environmental drivers with species assembly processes. Culicoides species are hematophagous arthropod vectors of orbiviruses that impact wild and domestic ruminants. A better sense of Culicoides dynamics over time is important because sympatric species can lengthen the seasonality of virus transmission. We tested a putative departure from the four seasons calendar in the phenology of Culicoides and the vector subassemblage in the Florida panhandle. Two years of weekly abundance data, temporal scales, persistence and environmental thresholds were analyzed using a tripartite Culicoides ß-diversity based modeling approach. Culicoides phenology followed a two-season regime and was explained by stream flow and temperature, but not rainfall. Species richness fit a nested pattern where the species recruitment was maximized during spring months. Midges were active year-round, and two suspected vectors species, Culicoides venustus and Culicoides stellifer, were able to sustain and connect the seasonal modules. Persistence suggests that Orbivirus maintenance does not rely on overwintering and that viruses are maintained year-round, with the seasonal dynamics resembling subtropical Culicoides communities with temporal-overlapping between multivoltine species. Viewing Culicoides-borne orbiviruses as a time-sensitive community-based issue, our results help to recommend when management operations should be delivered.

Habitat associations of Culicoides species (Diptera: Ceratopogonidae) abundant on a commercial cervid farm in Florida, USA.

BACKGROUND: Biting midges in the genus Culicoides (Diptera: Ceratopogonidae) transmit bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) to ruminants, thus exerting a significant economic impact on animal agriculture worldwide. However, very little is known about the larval habitat characteristics of Culicoides species associated with BTV/EHDV transmission, particularly in southeastern USA, limiting the establishment of effective midge control strategies. In this study, we examined the habitat associations of Culicoides species abundant on a commercial cervid farm in Florida, USA and quantified several environmental variables of their habitat to identify the key variables associated with midge abundance. METHODS: Mud/substrate samples from three potential larval habitats on the farm (edges of streams, puddles and seepages) were brought to the laboratory and incubated for adult emergence, and the percentage organic matter, macronutrients, micronutrients, pH, electrical conductivity, moisture and microbial concentrations of the substrate were quantified. RESULTS: Strong habitat associations were observed for Culicoides haematopotus (Malloch) (stream edge), Culicoides stellifer (Coquillett) (puddles) and Culicoides loisae (Jamnback) (stream edge), the most commonly emerging midge species from the samples. Suspected vector species of BTV/EHDV on the property, C. stellifer and Culicoides venustus (Hoffman), emerged mainly from habitats with moderate-high levels of pollution (edges of puddles and seepages) as indicated by the relatively higher concentrations/levels of organic matter, nutrients and other environmental variables in these samples. The emergence of C. insignis was too low to form any meaningful conclusions. For each Culicoides species, only weak positive or negative associations were detected between midge abundance and the various environmental variables quantified. CONCLUSIONS: Habitat associations of Culicoides species abundant on a local cervid/animal farm vary, most likely as a function of certain biotic/abiotic characteristics of the habitat. Further studies across a larger spatial and temporal scale will be needed to experimentally evaluate/identify the key factors more strongly associated with the abundance of target Culicoides species. This information, in the long term, can be potentially exploited to render local habitats unsuitable for midge oviposition/larval development.

Rapid Loss of Resistance to Satyrization in Invasive Mosquitoes and the Effects of Age on Interspecific Mating Frequency.

In several areas where Aedes aegypti (L.) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) have come in contact following successful invasions, Ae. aegypti have been rapidly displaced by Ae. albopictus. Recent work has confirmed that mating interference, in the form of satyrization, is likely a driving factor in these competitive displacements. However, in sites of sympatry, Ae. aegypti females evolve resistance to satyrization, and in the laboratory, satyrization-susceptible Ae. aegypti can evolve resistance within a few generations of cage exposure to Ae. albopictus. The resistance trait may be costly to maintain, as satyrization-resistant females have been shown to invest more time in mate selection. Here, we show that increased satyrization-resistance does not persist in the absence of satyrization pressure, confirming a cost to maintaining this trait. Ae. aegypti lines that had been previously selected for increased satyrization-resistance demonstrated rapid decreases in this trait over eight generations. Support for the hypothesis that condition-dependent mate preferences lead to variation in mating choice among individuals of differing quality within a population has been accumulating. Here we examine how age-related changes in reproductive effort or reproductive value may result in age-dependent mate choice. We hypothesize that older, lower quality individuals may be more likely to accept a subpar mating, in this case an interspecific mating. Our results demonstrate that mosquito age significantly affects interspecific mating rates with older mosquitoes (male and female) engaging in interspecific mating more frequently than younger counterparts (26.32 vs 9.41%). We discuss the possibility of age-related signal variation causing the breakdown of mating barriers with age.

Identification of Uranotaenia sapphirina as a specialist of annelids broadens known mosquito host use patterns.

Feeding upon vertebrate blood by mosquitoes permits transmission of diverse pathogens, including viruses, protozoa, and nematodes. Despite over a century of intensive study, no mosquito species is known to specialize on non-vertebrate hosts. Using molecular analyses and field observations, we provide the first evidence, to our knowledge, that a mosquito, Uranotaenia sapphirina, specializes on annelid hosts (earthworms and leeches) while its sympatric congener, Uranotaenia lowii, feeds only on anurans (frogs and toads). Our results demonstrate that Ur. sapphirina feeds on annelid hosts (100% of identified blood meals; n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on Sparganophilus worms and freshwater leeches. These findings indicate that adult mosquitoes utilize a much broader range of host taxa than previously recognized, with implications for epidemiology and the evolution of host use patterns in mosquitoes.

Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol.

The future of infectious disease surveillance and outbreak response is trending towards smaller hand-held solutions for point-of-need pathogen detection. Here, samples of Culex cedecei mosquitoes collected in Southern Florida, USA were tested for Venezuelan Equine Encephalitis Virus (VEEV), a previously-weaponized arthropod-borne RNA-virus capable of causing acute and fatal encephalitis in animal and human hosts. A single 20-mosquito pool tested positive for VEEV by quantitative reverse transcription polymerase chain reaction (RT-qPCR) on the Biomeme two3. The virus-positive sample was subjected to unbiased metatranscriptome sequencing on the Oxford Nanopore MinION and shown to contain Everglades Virus (EVEV), an alphavirus in the VEEV serocomplex. Our results demonstrate, for the first time, the use of unbiased sequence-based detection and subtyping of a high-consequence biothreat pathogen directly from an environmental sample using field-forward protocols. The development and validation of methods designed for field-based diagnostic metagenomics and pathogen discovery, such as those suitable for use in mobile "pocket laboratories", will address a growing demand for public health teams to carry out their mission where it is most urgent: at the point-of-need.

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.

Environmental Drivers of Seasonal Patterns of Host Utilization by Culiseta melanura (Diptera: Culicidae) in Florida.

Field studies were carried out in four Florida counties to investigate winter and spring ecology of host use by Culiseta melanura (Coquillet), the primary vector of eastern equine encephalomyelitis virus (EEEV) in North America. Bloodmeal analysis by PCR was used to identify 233 host bloodmeals, which mainly originated from birds (78.5%) and reptiles (17.2%), primarily Anolis spp. lizards. Across counties, the percentage of bloodmeals from reptiles (7-37% depending upon county) increased with increasing day length and temperature in the spring. Multiple logistic regression revealed that differences in reptile host use across collection sites were largely explained by differences in average day length and temperature on the day of collection, and is probably owing to environment-driven behavioral patterns of ectothermic animals. Although past studies have demonstrated reptile biting by epizootic vectors of EEEV, including Culex (Melanoconion) spp., this is the first study to demonstrate widespread and common feeding upon ectothermic hosts by Cs. melanura. This work suggests that reptiles, particularly anole lizards, play a role in the ecology of EEEV in Florida either as amplifying hosts or as noncompetent hosts which dilute vector feedings thereby suppressing transmission. Detailed laboratory studies investigating impacts of environmental variables (temperature and photoperiod) on EEEV competence of anoles are needed to assess whether these animals support virus amplification.

Aedeomyia squamipennis (Diptera: Culicidae) in Florida, USA, a New State and Country Record.

Aedeomyia squamipennis (Lynch Arribalzaga) is a tropical mosquito (Diptera: Culicidae) found throughout most of the American Tropics, from eastern Mexico through Argentina, including several Caribbean islands. Larvae are typically associated with bodies of water with dense growths of aquatic vegetation, particularly Pistia stratiotes L., water lettuce. Adult females feed predominantly on the blood of birds and seek hosts in forest canopies. Aedeomyia squamipennis is considered an important vector of Gamboa virus and avian malaria, and is also suspected of transmitting Venezuelan equine encephalitis virus. Collections from Florida City, FL, near the southern tip of the Florida peninsula, yielded larvae, adult males, and females of Ad. squamipennis, constituting a new genus and species record for Florida and the United States. The widespread availability of larval habitat and suitable hosts in Florida will likely lead to expansion of Ad. squamipennis in Florida, and perhaps into neighboring states. In South America, Ad. squamipennis is found as far south as Buenos Aires, Argentina, at ∼35° S latitude, which is equivalent in N latitude to coastal North Carolina. The northern limit of expansion of Ad. squamipennis in North America will likely be limited by winter temperatures and availability of larval habitat.

Oviposition Strategies of Florida Culex (Melanoconion) Mosquitoes.

In the genus Culex, oviposition is often generalized as laying floating egg rafts directly on the surface of the water, as reported for arbovirus vectors Culex pipiens L. and Culex tarsalis Coquillett, both members of subgenus Culex. The current work reports on investigations of exceptions to this strategy by species of Melanoconion, a diverse and mostly tropical subgenus of Culex, with many arbovirus vectors. Wild gravid females from Vero Beach, FL, were introduced into outdoor cages with open water, partially covered water, and emergent solid structures for oviposition. Three species, Culex cedecei Stone and Hair, Culex iolambdis Dyar, and Culex pilosus Dyar and Knab laid vast majorities (80-100%) of egg clusters attached to solid surfaces, although at different heights and angles, while Culex atratus Theobald laid floating egg clusters (100%) directly on the water. When offered mud, open water, and woody material, Cx. iolambdis laid 86% of clusters on nearly vertical surfaces of mangrove rootlets. Culex iolambdis eggs laid on solid surfaces hatched within 3-4 d or dried up without hatching. Results suggest that Melanoconion species utilize a variety of oviposition strategies, yet most species studied oviposit out of water. For some species, such as Cx. pilosus, this is a strategy for diapause and desiccation resistance, while for others, such as Cx. iolambdis, ovipositing above water may protect eggs from predators or being flushed from larval habitat. A summary of oviposition strategies in genus Culex shows that above-water oviposition is widespread and that the Culex egg raft paradigm needs reassessment.

Culex (Melanoconion) panocossa from peninsular Florida, USA.

Culex (Melanoconion) panocossa is a suspected vector of Venezuelan equine encephalitis virus in Central America. Prior to this report, Cx. panocossa was known from Central America (Belize, Costa Rica, El Salvador, Guatemala, southern Mexico, Panama), northern South America (Colombia, Venezuela) and the Greater Antilles (Cuba and Jamaica). Larvae (n=5) and adults (n=4286) of Cx. panocossa were collected at two locations near Homestead, FL, which indicates substantial established populations of this probable vector species in the continental US. Since larvae of Cx. panocossa are associated with Pistia spp. (water lettuce), the distribution of this mosquito is likely to expand in Florida, where water lettuce is a major invasive plant in freshwater ecosystems. The putative establishment of Cx. panocossa in Florida is of significant concern from a public health perspective, as its proliferation in developed areas could link historically sylvatic transmission foci of Everglades virus with populated centers such as the greater Miami Metropolitan area.

Host use and seasonality of Culex (Melanoconion) iolambdis (Diptera: Culicidae) from eastern Florida, USA.

Culex (Melanoconion) iolambdis (Dyar, 1918) is a mosquito (Diptera: Culicidae) species found throughout much of tropical America, including southern Florida. Relatively few reports are available regarding the ecology of Cx. iolambdis, despite its widespread distribution and putative involvement in transmission of Venezuelan equine encephalitis virus. To quantify habitat and host utilization, adults of Cx. iolambdis were sampled from resting shelters at a field site in Vero Beach, Florida, over a 12-month period. Culex iolambdis (1109 males, 3072 females) constituted more than half (56.76%) of all mosquitoes sampled (24 species) and was active year-round. Unfed females and gravid females of Cx. iolambdis were significantly more abundant in mangrove habitat, while males and blood-fed females were not. PCR-based bloodmeal analysis of 305 females revealed that Cx. iolambdis has very wide host breadth, feeding on birds (37.0% overall), reptiles (26.6%), amphibians (23.3%) and mammals (13.1%). Green heron (Butorides virescens), Southern leopard frog (Lithobates sphenocephala) and American alligator (Alligator mississippiensis) were the most commonly fed upon hosts. Bloodmeals from different host classes varied significantly with season, suggesting that Cx. iolambdis may play a role in the amplification and epidemic transmission of zoonotic arboviruses affecting human health.

Carbon dioxide generated from carbonates and acids for sampling blood-feeding arthropods.

Carbon dioxide (CO2) is utilized to attract mosquitoes and other blood-feeding arthropods to traps around the world. Commercial forms of CO2 (e.g., dry ice and compressed gas) are often unavailable or extremely expensive in developing nations, where vector surveillance is essential to make life-saving decisions. We developed and tested inexpensive and reproducible methods of CO2 production from the combination of acids and carbonates, ranging from very basic (crushed seashells and vinegar) to relatively elaborate (a device that controls the timing of the acid-carbonate reaction and extends the reaction over several hours). When utilized with mosquito traps in Florida, USA and black fly traps in Region des Cascades, Burkina Faso, these carbonate-acid CO2 sources attracted significantly greater numbers of both vector groups, than did unbaited traps. CO2 was generated for more than four hours at levels sufficient to attract vectors over the entire period. The utility of this simple methodology in developing nations should be further evaluated.

Competitive reduction by satyrization? Evidence for interspecific mating in nature and asymmetric reproductive competition between invasive mosquito vectors.

Abstract. Upon mating, male mosquitoes transfer accessory gland proteins (Acps) that induce refractoriness to further mating in females. This can also occur because of cross-insemination by males of related species, a process known as mating interference (satyrization). This mechanism could explain the competitive displacement of resident Aedes aegypti by the invasive Aedes albopictus where they co-occur. We tested this hypothesis in mosquito populations in Florida. A new polymerase chain reaction species diagnostic applied to sperm dissected from 304 field-collected females revealed bidirectional cross-mating in five (1.6%) individuals. Cross-injections of females with Acps showed that Ae. albopictus males induced monogamy in heterospecific females but not Ae. aegypti males. Despite its low frequency in the areas under study, the first evidence of cross-mating in nature and the asymmetric effect of Acps on mating suggest that satyrization may have initially contributed to the observed competitive reduction of Ae. aegypti by invasive Ae. albopictus in many areas.