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

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

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

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

Changes in mosquito species and blood meal composition associated with adulticide applications.

Although adulticide application is a pillar in the integrated management of nuisance and vector mosquitoes, non-target effects of adulticide applications within ecosystems are a substantial concern. However, the impacts of adulticide applications on non-target organisms are not necessarily detrimental, and in some cases, may provide benefits to certain organisms or wildlife. Here, we hypothesized that adulticide applications have beneficial non-target impacts on vertebrate wildlife through reduced biting pressure. To test this, we collected mosquitoes from ultra-low volume Permanone-treated (intervention) and untreated (reference) areas and assessed mosquito abundance and diversity, and abundance of blood-engorged female mosquitoes. We performed DNA barcoding analysis on mosquito blood meals to identify host species. Our results demonstrated a significant reduction in mosquito abundance by 58.9% in the intervention areas, taking into account the reduction in reference areas. Consequently, this decline led to a 64.5% reduction in the abundance of blood-engorged females. We also found a temporal dynamic of mosquito composition driven by mosquito control actions in which different mosquito species became dominant at treated sites while composition at reference areas remained similar during the same period. The present study suggests that the beneficial effects of mosquito control treatments for humans extend to other vertebrates, which represents an unstudied and rarely recognized non-target impact.

Detection of Aedes Scapularis In Southwestern Florida.

Aedes scapularis has recently been detected for the first time in southwestern Florida. During the course of research and surveillance activities by local mosquito control districts in 2020 and 2021, a total of 190 adult females were collected from 14 separate locations in Collier and Lee Counties. To date, Ae. scapularis has been found in 5 counties since its rediscovery in Florida in 2006. Its detection and likely northward expansion into Collier and Lee Counties from the southern Florida Peninsula is in line with ecological niche model predictions that found the environment of the Gulf Coast of southwestern Florida to be highly suitable for the species. Due to its potential ability to transmit both exotic and endemic pathogens such as Everglades virus and Dirofilaria immitis, understanding the range and distribution of Ae. scapularis should be a priority for Florida mosquito control and public health agencies.

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.

Aedes Tortilis, Culex Declarator, and Culex Tarsalis: New County Records for Mosquito Species in Collier County, Florida.

Understanding the distribution of mosquito species is an important element of surveillance. This is especially true in Florida, where detections of nonnative mosquitoes have been increasing. Collier Mosquito Control District performs routine adult mosquito surveillance for operational purposes throughout the year. Here, we report records for 3 species collected in 2021 that had not been documented previously in Collier County, FL: Aedes tortilis, Culex declarator, and Cx. tarsalis. Specimens were initially identified based on morphology, then each species was confirmed by comparing the cytochrome c oxidase subunit I gene sequences to those of other related mosquito species. Although Ae. tortilis and Cx. declarator were collected at multiple sites, Cx. tarsalis was collected only once, making it unclear whether this species has established a permanent population within the county.

A new species of the fish louse genus Dipteropeltis Calman, 1912 (Crustacea: Branchiura) from Peru.

Dipteropeltis is a poorly described genus of fish louse endemic to South America. In a small blackwater region within Loreto, Peru, 13 adult and juvenile specimens of an unidentified species of Dipteropeltis Calman, 1912, as well as one adult specimen of D. hirundo Calman, 1912, were observed and collected. Scanning electron and light micrographs were acquired to examine and measure key features of these specimens. Morphological differences from the two known species of Dipteropeltis, D. hirundo and D. campanaformis Neethling et al., 2014, indicate that the collected specimens represent a new species. Dipteropeltis longicaudatus sp. nov. is diagnosed by elongate abdominal lobes, a chevron-shaped carapace, and uniquely shaped maxillae. One specimen represents the longest branchiuran documented to date at 31.5 mm. Additionally, we provide the first sequence data for this genus using DNA barcoding, which corroborates our designation of a new species. Videos were also captured that document behaviors including host attachment, pulsating abdominal lobes, suction disc "walking", and swimming. Findings have implications for its teleost hosts, Triportheus albus Cope, 1872 and Brycon amazonicus Spix & Agassiz, 1829, the latter being a critical species for aquaculture and commercial fisheries in Amazonia.

Mosquito Blood Meal Analysis.

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

Extracting DNA from Preserved Mosquito Blood Meals.

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

Preservation of Field-Collected Mosquito Blood Meals.

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

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

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

Ticks and Tick-Borne Pathogens from Wild Pigs in Northern and Central Florida.

Invasive wild pigs are distributed across much of the U.S. and are hosts to tick vectors of human disease. Herein, adult ticks were collected from 157 wild pigs in 21 northern and central Florida counties from 2019-2020 during removal efforts by USDA-APHIS Wildlife Services personnel and evaluated for their potential to be used as a method of tick-borne disease surveillance. Collected ticks were identified, screened for pathogens, and the effects of landscape metrics on tick community composition and abundance were investigated. A total of 1415 adult ticks of four species were collected. The diversity of tick species collected from wild pigs was comparable to collections made throughout the state with conventional surveillance methods. All species collected have implications for pathogen transmission to humans and other animals. Ehrlichia, Anaplasma-like, and Rickettsia spp. were detected in ticks collected from wild pigs. These results suggest that tick collection from wild pigs is a suitable means of surveillance for pathogens and vectors. The strongest drivers of variation in tick community composition were the developed open space and mixed forest landcover classes. Fragmented shrub/scrub habitat was associated with increased tick abundance. Similar models may be useful in predicting tick abundance and distribution patterns.

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.

First Records of Aedeomyia squamipennis in Palm Beach County, Florida, USA.

Continuous surveillance, trained personnel, and coordination with other agencies are critical elements of effective nuisance and vector mosquito control. Palm Beach County Mosquito Control, FL, has used routine surveillance to make control decisions and note changes in mosquito populations since the 1940s. In the fall and winter of 2021-2022, Palm Beach County Mosquito Control's surveillance program detected the presence of Aedeomyia squamipennis. This represented the first collection of specimens of Ad. squamipennis in Palm Beach County and the second finding of this species beyond the initial site of detection in Miami-Dade County, FL. Identity was confirmed by morphology and DNA sequencing. Sequenced Ad. squamipennis specimens collected in Palm Beach County were identical (100% sequence similarity) to specimens collected and sequenced from Homestead, Miami-Dade County in 2016. It is suspected that Ad. squamipennis has expanded its range northward from Miami-Dade County into Palm Beach County, a distance of approximately 140 km.

Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator: An Update of Mosquito Species Records for Charlotte County, Florida.

Understanding the geographic occurrence of mosquito species is an important element to addressing public health and nuisance mosquito-related issues, particularly as changing climates and increased global connectivity is likely to facilitate changes in the distribution of mosquitoes and other species. In Charlotte County, FL, routine surveillance of mosquito species for public health in 2019-21 identified 4 mosquito species not previously documented in the county. Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator adults were collected and verified to species level. Aedes pertinax and Cx. declarator and were collected in 2019, whereas An. perplexens and Cx. interrogator were documented from collections in 2021. All 4 species were initially visually identified by external morphology and confirmed by sequencing the DNA barcoding region of the cytochrome c oxidase subunit I gene. Apart from native An. perplexens, in which only 1 specimen has been confirmed to date, the 3 newly documented nonnative species are now recognized throughout the county.

Pollinator biological traits and ecological interactions mediate the impacts of mosquito-targeting malathion application.

Mosquito adulticides are perceived by the public as detrimental to nontarget arthropods, contributing to declines of native and beneficial insects. However, the actual impact of adulticides on nontarget arthropods in nature needs to incorporate biological and ecological elements. Here, we investigated the effect of biological/behavioral traits (butterfly roosting at different heights, roosting in sites underneath foliage, bumblebee hive usage) and interactions (parasitism, predation) of pollinators (butterflies and bumblebees) that could mediate the impacts of malathion application in manipulative semi-field experiments in Florida, USA. Roosting height from the spray route had a significant negative relationship with mortality of butterflies treated with ULV malathion, with high survival at the highest roosting height (7 m), but butterflies roosting among vegetation did not have higher survival. Bumblebees with hive access had significantly higher survival than bumblebees without hive access. Host plants treated with ULV malathion significantly reduced parasitism of monarch eggs by Trichogramma platneri, but increased predation of monarch caterpillars by Polistes paper wasps. These data provide insight into the realistic impacts of adulticide applications on pollinators in nature which will enable mosquito control districts to better limit nontarget effects of adulticide treatments and may help to address concerns related to potential nontarget effects.

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.

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.

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.

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.