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.

Host associations of biting midges (Diptera: Ceratopogonidae: Culicoides) at deer farms in Florida, USA.

Documenting the host use of vector species is important for understanding the transmission dynamics of vector-borne pathogens. Biting midges (Diptera: Ceratopogonidae: Culicoides) are vectors of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) worldwide. However, relative to mosquitoes and many other vector groups, host associations of this group are poorly documented. In this study, we used PCR-based bloodmeal analysis to determine species-level host associations of 3,603 blood-engorged specimens belonging to 18 Culicoides species at 8 deer farms in Florida, USA. We used a binomial mixed model with a Bayesian framework to compare the effect of host composition on the feeding patterns of Culicoides spp. and employed the Morisita-Horn Index to investigate the similarity of host use between farms for Culicoides stellifer and Culicoides insignis. Results show that the estimated probability of Culicoides spp. feeding upon white-tailed deer depends on the availability of cattle or exotic game and demonstrates differences in host-feeding selection among species. Culicoides insignis had high host similarity across farms suggesting that its host-use patterns are somewhat conserved. Culicoides stellifer had lower host similarity across farms suggesting that it is a more opportunistic feeder. White-tailed deer are fed upon by many Culicoides species on deer farms in Florida, and while most Culicoides species feed on white-tailed deer, the ratio of white-tailed deer bloodmeals to other bloodmeals is likely influenced by host availability. Culicoides spp. taking a majority of their bloodmeals from farmed white-tailed deer should be assessed for their vector competence for EHDV and BTV.

Culex (Phenacomyia) lactator (Diptera: Culicidae) in southern Florida, USA: a new subgenus and species country record.

The Culex subgenus Phenacomyia is a small and poorly studied group of three mosquito species native to the American tropics. Here, we report the first detections of established populations of Culex (Phenacomyia) lactator Dyar & Knab in three counties of southern Florida. Culex lactator was first detected in May 2018 in southern Miami-Dade County, and, at this locality, was collected in subsequent years from 2018 to 2022 as both adults and immatures. Larvae and adults were subsequently collected in 2022, ~175 km northwest of the initial locality at nine sites in Collier and Lee Counties. Identification of specimens collected in these counties as Cx. lactator is supported by molecular analysis and morphological characters of the adult female, male genitalia, and larva. The host associations and vector competence of Cx. lactator have not been extensively studied, and the public health implications, if any, of the addition of this species to Florida's mosquito fauna are unclear. These collections represent the first detections of Cx. lactator, or any Phenacomyia species, in the United States, adding to a trend in which detections of established populations of mosquito species from the American tropics in Florida appear to be increasing.

Evaluating sampling strategies for enzootic Venezuelan equine encephalitis virus vectors in Florida and Panama.

Determining effective sampling methods for mosquitoes are among the first objectives in elucidating transmission cycles of vector-borne zoonotic disease, as the effectiveness of sampling methods can differ based on species, location, and physiological state. The Spissipes section of the subgenus Melanoconion of Culex represents an understudied group of mosquitoes which transmit Venezuelan equine encephalitis viruses (VEEV) in the Western Hemisphere. The objective of this study was to determine effective collection methods that target both blood-engorged and non-engorged females of the Spissipes section of Culex subgenus Melanoconion to test the hypothesis that favorable trapping methods differ between species and by physiological status within a species. Mosquitoes were collected using two commercially available traps, (CDC-light trap and BG-Sentinel trap), two novel passive traps (a novel mosquito drift fence and pop-up resting shelters), and two novel aspirators, (a small-diameter aspirator and a large-diameter aspirator) in Darién, Panama, and Florida, USA. The total number of female mosquitoes collected for each species was compared using rarefaction curves and diversity metrics. We also compared the utility of each trap for collecting total females and blood-engorged females of four Spissipes section mosquito species in Florida and Darién. In Darién, it was found that both blood-engorged and unfed females of Cx. pedroi were most effectively collected using the mosquito drift fence at 57.6% and 61.7% respectively. In contrast, the most unfed Cx. spissipes were collected using the mosquito drift fence (40.7%) while blood-engorged females were collected effectively by pop-up resting shelters (42.3%). In Florida, the best sampling technique for the collection of blood-engorged Cx. panocossa was the large diameter aspirator at 41.9%, while the best trap for collecting Cx. cedecei was the pop-up resting shelter at 45.9%. For unfed female Spissipes section mosquitoes in Florida, the CDC light trap with CO2 collected 84.5% and 98.3% of Cx. cedecei and Cx. panocossa respectively in Florida. Rarefaction analysis, and both the Shannon and Simpsons diversity indices all demonstrated that the mosquito drift fence was capable of collecting the greatest diversity of mosquito species regardless of location. The finding that the proportions of unfed and blood-engorged mosquitoes collected by traps differed both among and between species has implications for how studies of VEEV vectors will be carried out in future investigations. In Florida a combination of pop-up resting shelters and use of a large-diameter aspirator would be optimal for the collection of both VEEV vectors for host-use studies. Results demonstrate that traps can be constructed from common materials to collect mosquitoes for VEEV vector studies and could be assessed for their utilization in vectors of other systems as well. Unfortunately, no single method was effective for capturing all species and physiological states, highlighting a particular need for assessing trap utility for target species of a study.

Predicting the potential distribution of Culex (Melanoconion) cedecei in Florida and the Caribbean using ecological niche models.

Everglades virus (EVEV), an enzootic subtype of Venezuelan equine encephalitis virus, along with its endemic mosquito vector, Culex cedecei, is known only from South Florida. The taxonomy of Cx. cedecei is complex and was once synonymous with Culex opisthopus and Culex taeniopus. We modeled potential distribution of Cx. cedecei in Florida and the Caribbean using an ecological niche model and compared this distribution to the recorded distribution of EVEV in Florida as well as historical records of Cx. opisthopus/Cx. taeniopus. We used recent collections and occurrence data from scientific publications and temperature/precipitation variables and vegetation greenness values to calibrate models. We found mean annual temperature contributed the greatest to model performance. Everglades virus in humans and wildlife corresponded with areas predicted suitable for Cx. cedecei in Florida but not with incidence of antibodies reported in dogs. Most records of Cx. opisthopus/Cx. taeniopus in the Caribbean did not correspond to areas predicted suitable for Cx. cedecei, which may be due to mean annual temperature values in the Caribbean exceeding values within the calibration region, imposing model constraints. Results indicated that this model may adequately predict the distributions of Cx. cedecei within Florida but cannot predict areas suitable in the Caribbean.

Predicting potential transmission risk of Everglades virus in Florida using mosquito blood meal identifications.

The overlap between arbovirus host, arthropod vectors, and pathogen distributions in environmentally suitable habitats represents a nidus where risk for pathogen transmission may occur. Everglades virus (EVEV), subtype II Venezuelan equine encephalitis virus (VEEV), is endemic to southern Florida where it is transmitted by the endemic vector Culex cedecei between muroid rodent hosts. We developed an ecological niche model (ENM) to predict areas in Florida suitable for EVEV transmission based upon georeferenced vector-host interactions from PCR-based blood meal analysis from blood-engorged female Cx. cedecei females. Thirteen environmental variables were used for model calibration, including bioclimatic variables derived from Daymet 1 km daily temperature and precipitation values, and land use and land cover data representing percent land cover derived within a 2.5 km buffer from 2019 National Land Cover Database (NLCD) program. Maximum temperature of the warmest month, minimum temperature of the coldest month, and precipitation of the driest month contributed 31.6%, 28.5% and 19.9% to ENM performance. The land cover types contributing the greatest to the model performance were percent landcover of emergent herbaceous and woody wetlands which contributed 5.2% and 4.3% to model performance, respectively. Results of the model output showed high suitability for Cx. cedecei feeding on rodents throughout the southwestern portion of the state and pockets of high suitability along the northern east coast of Florida, while areas with low suitability included the Miami-Dade metropolitan area and most of northern Florida and the Panhandle. Comparing predicted distributions of Cx. cedecei feeding upon rodent hosts in the present study to historical human cases of EVEV disease, as well as antibodies in wildlife show substantial overlap with areas predicted moderate to highly suitable for these vector/host associations. As such, the findings of this study likely predict the most accurate distribution of the nidus of EVEV to date, indicating that this method allows for better inference of potential transmission areas than models which only consider the vector or vertebrate host species individually. A similar approach using host blood meals of other arboviruses can be used to predict potential areas of virus transmission for other vector-borne diseases.

Potential Distribution of Aedes (Ochlerotatus) scapularis (Diptera: Culicidae): A Vector Mosquito New to the Florida Peninsula.

Aedes scapularis is a neotropical mosquito known to transmit pathogens of medical and veterinary importance. Its recent establishment in southeastern Florida has potential public health implications. We used an ecological niche modeling approach to predict the abiotic environmental suitability for Ae. scapularis across much of the Americas and Caribbean Islands. Georeferenced occurrence data obtained from the Global Biodiversity Inventory Facility and recent collection records of Ae. scapularis from southern Florida served as input for model calibration. Environmental layers included bioclimatic variables provided in 2000 to 2010 average Modern Era Retrospective-analysis for Research and Applications climatic (MERRAclim) data. Models were run in the software program Maxent. Isothermality values often found in costal environments, had the greatest contribution to model performance. Model projections suggested that there are areas predicted to be suitable for Ae. Scapularis across portions of the Amazon Basin, the Yucatán Peninsula, the Florida Peninsula, and multiple Caribbean Islands. Additionally, model predictions suggested connectivity of highly suitable or relatively suitable environments spanning the United States Gulf Coast, which may facilitate the geographic expansion of this species. At least sixteen Florida counties were predicted to be highly suitable for Ae. scapularis, suggesting that vigilance is needed by vector control and public health agencies to recognize the further spread of this vector.

Establishment of Aedes (Ochlerotatus) scapularis (Diptera: Culicidae) in Mainland Florida, With Notes on the Ochlerotatus Group in the United States.

Aedes scapularis (Rondani), a widespread neotropical vector mosquito species, has been included in the mosquito fauna of Florida on the basis of just three larval specimens that were collected in the middle Florida Keys in 1945. Here, we report numerous recent collections of immature and adult Ae. scapularis from multiple locations in two counties of southern Florida. These specimens represent the first records of Ae. scapularis from mainland Florida and the first records of the species in the state since the initial detection of the species 75 yr ago. Collections of both larvae and adults across several years indicate that Ae. scapularis is now established in Broward and Miami-Dade Counties. These contemporary records of this species in Florida may represent novel dispersal and subsequent establishment events from populations outside the United States or a recent reemergence of undetected endemic populations. To confirm morphological identification of Ae. scapularis specimens from Florida, the DNA barcoding region of the cytochrome c oxidase subunit I gene (COI) was sequenced and compared to all other Ochlerotatus Group species from the United States, specifically Aedes condolescens Dyar and Knab (Diptera: Culicidae), Aedes infirmatus Dyar and Knab (Diptera: Culicidae), Aedes thelcter Dyar (Diptera: Culicidae), Aedes tortilis (Theobald) (Diptera: Culicidae), and Aedes trivittatus (Coquillett) (Diptera: Culicidae). Molecular assays and sequencing confirm morphological identification of Ae. scapularis specimens. Maximum likelihood phylogenetic analysis of COI and ITS2 sequences place Florida Ae. scapularis in a distinct clade, but was unable to produce distinct clades for Florida specimens of Ae. condolescens and Ae. tortilis.

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.

Host Associations Of Biting Midges (Diptera: Ceratopogonidae: Culicoides) Near Sentinel Chicken Surveillance Locations In Florida, USA.

Quantifying host use is important for understanding transmission of vector-borne pathogens. Despite the importance of biting midges (Diptera: Ceratopogonidae) in pathogen transmission, the vector-host relationships of most Culicoides species are poorly documented, even in locations where active arbovirus surveillance is conducted. Polymerase chain reaction-based blood-meal analysis was performed on 663 blood-engorged Culicoides females collected by 7 Florida mosquito control districts at 24 sentinel chicken arbovirus surveillance sites in 2017. A total of 638 blood meals were successfully analyzed to determine host species source, representing 11 Culicoides species. The most commonly bitten host was domestic chicken (Gallus gallus) (presumably sentinel chickens), constituting 565 of 638 (88.6%) the total blood meals. Other common hosts included humans (5.8%), white-tailed deer (Odoocoileus virginianus) (2.5%), and brown basilisk (Basiliscus vittatus) (1.6%). Significant differences in distribution of mammal and avian blood meals were found for a number of Culicoides species, and these patterns did not vary across locations. These results indicate that sentinel chickens are exposed to bites by Culicoides, potentially exposing them to Culicoides-borne pathogens. The findings that vertebrate host use was consistent across locations suggests that each Culicoides species has affinity for one or more specific animal groups, and does not feed randomly upon available animals.

Field data implicating Culicoides stellifer and Culicoides venustus (Diptera: Ceratopogonidae) as vectors of epizootic hemorrhagic disease virus.

BACKGROUND: Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus of veterinary importance which is transmitted by biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to ruminants. Culicoides sonorensis Wirth & Jones, the only confirmed vector of EHDV in the USA, is rare in the southeastern states where transmission persists, suggesting that other Culicoides species transmit EHDV in this region. The present study aimed to determine which Culicoides species transmitted EHDV in Florida and Alabama, two states in the southeastern USA. Viral RNA was detected in field-collected midges using molecular methods. These data are presented alongside data on Culicoides blood meal analysis, white-tailed deer (Odocoileus virginianus) aspiration, and seasonality to demonstrate an interaction between potential vector species and EHDV hosts. RESULTS: Out of 661 pools tested, 20 pools were positive for EHDV viral RNA, including six pools from Culicoides stellifer (Coquillett) and 14 pools from Culicoides venustus Hoffman. The overall infection rate was 0.06% for C. stellifer and 2.18% for C. venustus. No positive pools were identified for a further 17 species. Serotypes identified in Culicoides included EHDV-2, EHDV-6, and coinfections of EHDV-2 and EHDV-6 and were identified in similar proportions to serotypes in deer at 3 of 4 deer farms. Viral detections conducted in Alabama also identified one positive pool of C. venustus. Blood meal analysis revealed that both Culicoides species fed on white-tailed deer (verified through aspiration), fallow deer, and elk, species for which EHDV viremia has been documented. Seasonality data indicated that both species were present throughout the period in which viral transmission occurred to EHDV hosts in 2016 in addition to the 2017 epizootic. CONCLUSIONS: Our finding of EHDV positive pools of field-collected C. stellifer and C. venustus and an interaction between these species and EHDV hosts satisfy two of the four criteria for vector incrimination as set by the World Health Organization. Determining the vectors of EHDV is an important step towards developing sound strategies for the control of vector Culicoides and management of EHDV in the southeastern USA.

Ecological niche modeling the potential geographic distribution of four Culicoides species of veterinary significance in Florida, USA.

Epizootic hemorrhagic disease (EHD) is a viral arthropod-borne disease affecting wild and domestic ruminants, caused by infection with epizootic hemorrhagic disease virus (EHDV). EHDV is transmitted to vertebrate animal hosts by biting midges in the genus Culicoides Latreille (Diptera: Ceratopogonidae). Culicoides sonorensis Wirth and Jones is the only confirmed vector of EHDV in the United States but is considered rare in Florida and not sufficiently abundant to support EHDV transmission. This study used ecological niche modeling to map the potential geographical distributions and associated ecological variable space of four Culicoides species suspected of transmitting EHDV in Florida, including Culicoides insignis Lutz, Culicoides stellifer (Coquillett), Culicoides debilipalpis Hoffman and Culicoides venustus Lutz. Models were developed with the Genetic Algorithm for Rule Set Production in DesktopGARP v1.1.3 using species occurrence data from field sampling along with environmental variables from WorldClim and Trypanosomiasis and Land use in Africa. For three Culicoides species (C. insignis, C. stellifer and C. debilipalpis) 96-98% of the presence points were predicted across the Florida landscape (63.8% - 72.5%). For C. venustus, models predicted 98.00% of presence points across 27.4% of Florida. Geographic variations were detected between species. Culicoides insignis was predicted to be restricted to peninsular Florida, and in contrast, C. venustus was predicted to be primarily in north Florida and the panhandle region. Culicoides stellifer and C. debilipalpis were predicted nearly statewide. Environmental conditions also differed by species, with some species' ranges predicted by more narrow ranges of variables than others. The Normalized Difference Vegetation Index (NDVI) was a major predictor of C. venustus and C. insignis presence. For C. stellifer, Land Surface Temperature, Middle Infrared were the most limiting predictors of presence. The limiting variables for C. debilipalpis were NDVI Bi-Annual Amplitude and NDVI Annual Amplitude at 22.5% and 28.1%, respectively. The model outputs, including maps and environmental variable range predictions generated from these experiments provide an important first pass at predicting species of veterinary importance in Florida. Because EHDV cannot exist in the environment without the vector, model outputs can be used to estimate the potential risk of disease for animal hosts across Florida. Results also provide distribution and habitat information useful for integrated pest management practices.

Effects of ultraviolet LED versus incandescent bulb and carbon dioxide for sampling abundance and diversity of Culicoides in Florida.

Biting midges (Ceratopogonidae: Culicoides) are vectors of bluetongue virus and epizootic hemorrhagic disease virus which cause significant morbidity and mortality in ruminants. Recently, ultraviolet light emitting diodes (UV/LEDs) in conjunction with suction traps have been widely utilized for Culicoides spp. collections. Despite the use of these traps, limited work has been done comparing sampling variability associated with these light types with and without CO2. For this objective, mini-CDC light traps with four different attractant combinations were operated at eight sites across Florida between April and October 2017. Trap attractants included white-incandescent lights and UV/LEDs with and without CO2 to determine optimum combinations of light type and attractant for species richness, diversity, and abundance of Culicoides spp. in Florida. The results of the study demonstrate that traps with UV/LED light collect greater richness, diversity, and abundance of Culicoides species than traps with white-incandescent light. Addition of CO2 resulted in greater diversity in traps with UV/LED lights, but lower diversity in traps with white-incandescent light. Therefore, CO2 may be used to increase the abundance of Culicoides spp. collected by traps, regardless of light type, but the ability for CO2 to attract a higher number and diversity of species to traps varies by the light type used. Therefore, we suggest using CO2 primarily in conjunction with UV/LED light. When CO2 is not available, UV/LED light used alone can be substituted without a significant loss in species richness or diversity, although abundance of most Culicoides species will be significantly lower in the absence of CO2.