From forests to fields: investigating Culicoides (Diptera: Ceratopogonidae) abundance and diversity in cattle pastures and adjacent woodlands.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are hematophagous flies that feed on wild and domestic ruminants. They can transmit arboviruses, such as bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), which circulate in the United States. Larvae occupy a range of aquatic and semiaquatic habitats, and disperse short distances from their development sites. In the southeastern United States, there are limited studies on the abundance and diversity of Culicoides in wooded and adjacent livestock pasture habitats. In this study, we characterized Culicoides diversity and abundance within these distinct habitat types. BG-Sentinel and CDC miniature suction traps baited with CO2 or UV-light were placed in wooded and pasture habitats at 2 locations on a university beef farm in Savoy, Arkansas. Traps were set once per week for 9 wk during August-October of 2021 and 2022. Fifteen species were collected during this study, and the 2 most abundant species were Culicoides haematopotus Malloch and Culicoides stellifer Coquillett. There was a significant effect of site and location on C. haematopotus collections, and a significant effect and interaction of site and trap on C. stellifer collections. In the woods, significantly more C. stellifer were collected from CDC-UV traps, while in the pasture significantly more were collected in CDC-CO2 traps. These data suggest that C. stellifer, a putative vector of BTV and EHDV in the southeast, may be traveling into the pasture to host-seek, while C. haematopotus remains primarily in wooded areas. This study reveals community differences between these habitat types and implications for Culicoides control.

PREVALENCE OF EHRLICHIA AND ANAPLASMA SPP. IN TICKS COLLECTED FROM CATTLE AND DEER IN ARKANSAS, USA.

Bovine anaplasmosis, caused by Anaplasma marginale, is widespread in cattle in the southeast United States. The pathogen is biologically transmitted by Dermacentor spp. ticks, and mechanically transmitted by biting flies and via fomites. Despite high reported regional seroprevalence, Dermacentor spp. are rare on cattle in the southeast. To identify other putative An. marginale vectors, and to characterize cattle exposure to other tick-borne pathogens, we collected ticks from Arkansas cattle herds in 2020-2022. Recognizing that deer are important hosts for some of the same tick species that parasitize cattle, we also collected ticks from hunter-killed deer in the fall and winter of 2021. Ticks were screened for bacteria in the family Anaplasmataceae using qPCR. Positive samples were further amplified using a PCR assay targeting the groEL gene, and the resulting amplicons were sequenced for identification. A total of 3,794 ticks were collected, the majority of which were Amblyomma americanum. Amblyomma americanum was the most common species on cattle, and Ixodes scapularis was most common on deer. No ticks were positive for An. marginale, though Anaplasma phagocytophilum was detected in deer-collected I. scapularis, as well as in a single engorged Am. americanum from cattle. Amblyomma americanum from cattle were infected with Ehrlichia ewingii, Ehrilichia chaffeensis, and Panola Mountain Ehrlichia. Cattle in Arkansas are exposed to several ehrlichial pathogens and may also be exposed to An. phagocytophilum. The importance of these pathogens, particularly Panola Mountain Ehrlichia, in causing cattle disease in Arkansas deserves further study, as does the importance of mechanical transmission of An. marginale in bovine anaplasmosis epidemiology.

Topical, contact, and oral susceptibility of adult Culicoides biting midges (Diptera: Ceratopogonidae) to fluralaner.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are economically important blood-feeding pests closely associated with livestock production. They are the principal vectors of two hemorrhagic disease viruses affecting both wild and domestic ruminants within the US: bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). BTV impacts the US agriculture sector through direct commodity loss and strict international livestock trade restrictions. Yet, despite posing a considerable threat to US livestock, Culicoides are understudied, and management strategies are lacking. Current control tools for Culicoides are limited to synthetic chemicals, predominantly pyrethroids. With limited products available for livestock producers, proper pesticide rotation is difficult. The present study investigates the efficacy of fluralaner, an isoxazoline insecticide, beyond its current labeled use as an ectoparasiticide in anticipation of adding a new class of pesticides into rotation for use against biting midges. METHODS: The efficacy of fluralaner was evaluated by conducting contact, topical, and oral toxicity bioassays on adult female Culicoides sonorensis. Contact toxicity was assessed by using a modified WHO cone assay, which simulates exposure through landing on an insecticide-treated surface. A modified WHO topical toxicity assay, in which fluralaner dilutions were administered to the lateral thorax, was used to assess topical toxicity. For evaluation of oral toxicity, females were offered a blood meal spiked with fluralaner in an artificial membrane feeding system to simulate a systemic insecticide. RESULTS: Contact exposure of fluralaner did not cause extensive or consistent mortality. Even the highest concentration tested (100 mg/ml) resulted in an average of only 24.3% mortality at 24 h, and mortality did not significantly differ between exposed and control midges at any concentration. One hundred percent mortality was consistently achieved at concentrations of 1 mg/ml when fluralaner was applied topically. The LC50 for topical exposure to fluralaner at 24 h was estimated to be 0.011 mg/ml. Oral exposure to fluralaner through ingestion of a spiked blood meal proved to be the most effective exposure method, significantly increasing mortality in a dose-dependent manner at 1 h post-exposure. The LC50 at 24 h following ingestion was 14.42 ng/ml. CONCLUSION: Our results suggest that fluralaner is a viable candidate for use as an insecticide against adult biting midges if exposed orally, such as in a systemic given to livestock. As withdrawal period requirements for meat animals present unique yet definitive challenges, pharmacokinetic studies of isoxazoline drugs need to be pursued and finalized for livestock before fluralaner may be used as a management strategy in this manner. Alternatively, livestock not raised for consumption, such as hair sheep, would directly benefit from administering oral fluralaner as a component of a BTV disease management program.

Co-occurrence of Culicoides guttipennis (Coquillett) with tree hole mosquito species, including Aedes albopictus Skuse, in container habitats in northwest Arkansas, U.S.A.

Natural and artificial water-filled containers serve as development sites for several larval Dipterans, including medically important mosquito species and Culicoides biting midges. Containers are discrete habitats with limited carrying capacity and high levels of both intra- and interspecific competition. While the outcomes of the interactions between mosquito species, particularly native and introduced Aedes, have been well described, competition between mosquitoes and other taxa, including Culicoides, is largely unstudied. To determine the extent of shared resource use between container-inhabiting mosquito and biting midge species, we surveyed water-filled natural and artificial container habitats in Washington County, AR. Larvae were collected from containers in the field and reared in the laboratory until the adult stage, where they were identified to species. Culicoides guttipennis, the only Culicoides species identified, co-occurred with four mosquito species, including the invasive Aedes albopictus. Culicoides larvae were more common in natural than artificial containers. Natural container samples from which Ae. albopictus emerged were significantly less likely to harbor Culicoides larvae than samples where that mosquito species was absent, suggesting the possibility that Ae. albopictus competes either directly or indirectly with other native taxa beyond congeners. Further research is needed on the nature of the interactions between Ae. albopictus and native Culicoides spp.

A Review of the Vector Status of North American Culicoides (Diptera: Ceratopogonidae) for Bluetongue Virus, Epizootic Hemorrhagic Disease Virus, and Other Arboviruses of Concern.

Purpose of Review: Culicoides biting midges transmit several pathogens of veterinary importance in North America, but the vector status of many midge species is unresolved. Additionally, the available evidence of vector competence in these species is scattered and variable. The purpose of this review is to summarize current knowledge on confirmed and putative North American Culicoides arbovirus vectors. Recent Findings: While the vector status of Culicoides sonorensis (EHDV, BTV, VSV) and Culicoides insignis (BTV) are well established, several other potential vector species have been recently identified. Frequently, these species are implicated based primarily on host-feeding, abundance, and/or detection of arboviruses from field-collected insects, and often lack laboratory infection and transmission data necessary to fully confirm their vector status. Recent genetic studies have also indicated that some wide-ranging species likely represent several cryptic species, further complicating our understanding of their vector status. Summary: In most cases, laboratory evidence needed to fully understand the vector status of the putative Culicoides vectors is absent; however, it appears that several species are likely contributing to the transmission of arboviruses in North America.

Aedes aegypti (L.) and Anopheles stephensi Liston (Diptera: Culicidae) Susceptibility and Response to Different Experimental Formulations of a Sodium Ascorbate Toxic Sugar Bait.

Attractive toxic sugar baits (ATSBs) require target insects to locate, orient toward, and feed on an insecticidal sugar solution to control populations. Formulating these baits with different attractants and phagostimulants can increase their efficacy by causing insects to choose the ATSB over competing natural sugar sources, and to ingest more of the bait solution. We tested formulations of a 20% sodium ascorbate (SA) ATSB solution using different sugars, adenosine triphosphate (ATP), gallic acid, and six plant volatile compounds to determine their effect on adult Aedes aegypti (L.) and Anopheles stephensi Liston mortality. Baits formulated with fructose or sucrose had no effect on either species, neither did the addition of ATP. Gallic acid increased the survival of Ae. aegypti. Four of the six volatile compounds increased mortality in at least one species. We also examined An. stephensi response to baits formulated with each of the six volatile compounds. Anisaldehyde significantly increased the number of mosquitoes responding toward the SA-ATSB, but increasing the amount had no effect. Addition of anisaldehyde also significantly increased An. stephensi feeding rates on the SA-ATSB, though mosquitoes will avoid the toxic bait if a nontoxic sugar source is available. Formulation of SA-ATSBs with synthetic blends of attractive compounds can increase bait efficacy and consistency, though further research is needed to assess their performance in the field in the presence of natural sugar sources.

A comparison of BG Sentinel and CDC trap attractants for mosquito surveillance in urban and suburban areas of Montgomery and Prince George's Counties, Maryland, U.S.A.

Monitoring mosquito populations is crucial for vector-borne disease surveillance. Routine mosquito surveillance in many regions of the United States is performed either by vector abatement districts or public health departments. These surveillance programs often use multiple trap types and attractants to target key mosquito species, however setting different traps with varying attractants can be expensive and labor intensive. Because funding for mosquito control is highly variable throughout the U.S., some programs may be limited in their surveillance capabilities. To determine whether a single trap-attractant combination could provide specificity for key vector and nuisance species, as well as sensitivity for rare species, we compared the BG-Sentinel 2 and CDC miniature light traps paired with CO2, UV-LED, BG Lure, BG Sweetscent, octenol, or chicken feathers. Trapping was conducted biweekly from June/July-October 2019 and 2020 in Montgomery and Prince George's County, MD. BG traps collected significantly more Aedes albopictus than CDC traps when paired with BG Lure, Sweetscent, or octenol. BG/CO2 traps collected both the greatest number of total mosquitoes and Culex pipiens. BG/CO2, CDC/CO2, and CDC/UV traps provided the most diverse collections. Trapping with the CO2-baited BG-Sentinel is recommended as an effective strategy for general mosquito surveillance when resources are limited.

Laboratory evaluation of stable isotope labeling of Culicoides (Diptera: Ceratopogonidae) for adult dispersal studies.

BACKGROUND: Stable isotope labeling is a promising method for use in insect mark-capture and dispersal studies. Culicoides biting midges, which transmit several important animal pathogens, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), are small flies that develop in various semi-aquatic habitats. Previous Culicoides dispersal studies have suffered from the limitations of other labeling techniques, and an inability to definitively connect collected adult midges to specific immature development sites. RESULTS: Adult C. sonorensis were successfully labeled with 13C and 15N stable isotopes as larvae developing in a semi-aquatic mud substrate in the laboratory. High and low-dose isotope treatments for both elements significantly enriched midges above the background isotope levels of unenriched controls. Enrichment had no effect on C. sonorensis survival, though a slight (~ 5 day) delay in emergence was observed, and there was no significant effect of pool size on 13C or 15N enrichment levels. CONCLUSIONS: Stable isotope labeling is life-long, and does not interfere with natural insect behaviors. Stable isotope enrichment using 13C or 15N shows promise for Culicoides dispersal studies in the field. This method can be used to identify adult dispersal from larval source habitat where a midge developed. It may be possible to detect a single enriched midge in a pool of unenriched individuals, though further testing is needed to confirm the sensitivity of this method.

Sodium Ascorbate as a Potential Toxicant in Attractive Sugar Baits for Control of Adult Mosquitoes (Diptera: Culicidae) and Sand Flies (Diptera: Psychodidae).

Attractive toxic sugar baits (ATSBs) can be an effective vector control tool, especially in areas where aerial or aquatic applications of pesticides are undesirable or impractical. In general, there is a need to develop novel or alternative insecticides for vector control, and there is a demand from consumers for more 'natural' pest control products. Sodium ascorbate (SA) is a naturally occurring antioxidant compound, found in fruits and vegetables, and is available commercially in the United States as a food additive and supplement. In this study, we continuously exposed groups of adult Aedes aegypti (L.), Anopheles stephensi Liston (Diptera: Culicidae), Phlebotomus papatasi Scopoli, and Lutzomyia longipalpis (Lutz & Neiva; Diptera: Psychodidae) to ATSBs containing SA in concentrations of 6, 8, 10, and 20%, and tracked their mortality over 10 d. We also exposed insects to a 20% SA-ATSB on a single day to determine the effect of a single exposure to the bait on mortality. Concentrations of ≥8% SA significantly reduced survival of both mosquito species over 10 d compared with sugar-fed controls. Sand fly mortality was inconsistent. A single exposure to 20% SA significantly reduced the survival of An. stephensi. Mosquitoes exposed to SA exhibited elevated catalase levels and cell death. The use of SA in ATSBs may be most effective in areas where sugar sources are scarce, and where mosquito species frequently sugar-feed. SA sugar baits may be a particularly attractive option for the general public looking to control mosquito populations using 'natural' alternatives to synthetic insecticides.

The Dark Side of Light Traps.

Light-baited suction traps are one of the most widely used tools for vector surveillance. Their popularity stems from ease of use even in remote locations, range and abundance of species caught, and low cost. The availability of smaller, portable models, like the CDC miniature light trap, have further increased their ubiquity in entomological field studies. However, when researchers have looked, light trap collections are usually biased in ways that may affect data interpretation for epidemiological studies. If used alone, light traps may fail to collect important or infected vectors, and light traps are inefficient or ineffective when competing ambient light is present. In this article, we discuss these biases and limitations in terms of their effect on collection efficiency, population data, and pathogen detection. While light trap data certainly have a purpose, an over-reliance on light trapping risks drawing false conclusions about vector populations and vector-borne disease epidemiology. These concerns are especially troubling when light trap data are used to inform policy decisions meant to protect human and animal health. Particularly when a species' response to light is unknown or poorly characterized, light traps should be used in conjunction with supplemental sampling methods. Researchers conducting vector surveillance field studies should carefully consider their study design and objectives when deciding on a trapping method or methods, and specifically endeavor to understand the limitations of their data. Only then can researchers take advantage of the best attributes of light traps while avoiding their dark side.

Field Distribution and Density of Culicoides sonorensis (Diptera: Ceratopogonidae) Eggs in Dairy Wastewater Habitats.

Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) is a key bluetongue virus vector in the United States. Immatures occur in mud near the edges of wastewater ponds and are understudied targets for control efforts. Eggs of C. sonorensis were collected in the morning from a dairy wastewater pond bank by taking 5-ml surface mud samples along four transects on each of six dates. Surface mud samples parallel to waterline (10-cm long, 1-cm wide, and 0.5-cm deep) were removed at 5-cm increments ranging from 15 cm below waterline up to 25 cm above waterline. Eggs were removed using MgSO4 flotation, held on moist filter paper, and scored for hatching over 3 d. Eggs hatching on days 2 and 3 were assumed to have been laid on the test night. Water levels were stable within a night according to time-lapse camera photos. Most samples from below the waterline had no eggs and were not analyzed statistically. Mean (±SE) sample moisture (25.8 ± 2.1 at 5 cm above waterline and 19.8 ± 2.6% at 25 cm above waterline) did not vary significantly by position above waterline. The highest density of viable eggs (21 eggs/5 ml), proportion of mud samples positive for viable eggs (75%), and proportion of eggs hatching (80%) were found 5 cm above waterline. Oviposition in the few hours after sunset is adaptive, allowing eggs to age, develop the serosal cuticle, and resist later desiccation. As a potential control method, reducing water levels after midnight would encourage young egg desiccation.

Low Temperature Tolerance of Culicoides sonorensis (Diptera: Ceratopogonidae) Eggs, Larvae, and Pupae From Temperate and Subtropical Climates.

Culicoides sonorensis Wirth and Jones biting midges are the primary North American vectors of bluetongue virus (BTV), which infects domestic ruminant livestock and can cause high morbidity and mortality. Both virus and vector exhibit highly seasonal activity patterns, even in subtropical climates like southern California. However, the exact mechanism of BTV and Culicoides overwintering has been debated. In this study, we examined the supercooling point (SCP) and lower lethal temperature of a laboratory colony of C. sonorensis eggs, larvae, and pupae, as well as of field-collected larvae and pupae from subtropical (California) and temperate (Colorado) climates. Larvae and pupae succumbed to temperatures higher than their respective SCPs, indicating death from prefreezing cold injury. Eggs were the most cold-tolerant life stage, and were able to tolerate 1-h exposures to temperatures as low as -20 °C without suffering complete mortality. Larvae were the least cold-tolerant life stage and suffered complete mortality at < -4 °C, while temperatures of -9 to -10 °C were required to kill all pupae. Larvae and embryos suffered chronic cold injury after exposure to subzero temperatures, which increased mortality. Field-collected larvae succumbed to slightly higher temperatures ( > -3 °C) than colony larvae, but did not differ significantly in their survival. Culicoides sonorensis immatures did not cold harden when exposed to 4 °C for 24 h before freezing. Results of this study indicate that C. sonorensis eggs are more tolerant of environmental stress than previously thought. Further work is needed on C. sonorensis winter survival in the field to corroborate laboratory studies.

Progress and knowledge gaps in Culicoides ecology and control.

The Bluetongue and Schmallenberg virus outbreaks in Europe between 1999 and 2013 stimulated progress in Culicoides field ecology and control. In responding to these outbreaks, however, the deficit of broadly trained entomologists became evident. Culicoides research has been uneven, and critical gaps remain in our knowledge of vector ecology. Certain aspects of adult midge biology have gotten more attention (biting rates and relationship to trap collections, seasonal activity and survey, taxonomy of critical species complexes). Some aspects of adult midge biology (survival, resting site selection, or direct dispersal measurements) have been understudied. More work is needed on Culicoides immatures (taxonomy and details of ecology). Control research has centred on insecticide treatments applied to animals. However, our true goal is not vector control, but reducing disease agent transmission. We require field vector control targets (e.g. how far we might need to reduce biting rates) needed to interrupt transmission. The most critical need is for studies incorporating vector control experimentally into epidemiological studies in active arbovirus transmission zones. Such field interdisciplinary studies are absolutely required to understand transmission dynamics. Through the power of experimental field studies, we should be able to develop both theoretical and operational guidelines for disease management.

Bluetongue virus infection creates light averse Culicoides vectors and serious errors in transmission risk estimates.

BACKGROUND: Pathogen manipulation of host behavior can greatly impact vector-borne disease transmission, but almost no attention has been paid to how it affects disease surveillance. Bluetongue virus (BTV), transmitted by Culicoides biting midges, is a serious disease of ruminant livestock that can cause high morbidity and mortality and significant economic losses. Worldwide, the majority of surveillance for Culicoides to assess BTV transmission risk is done using UV-light traps. Here we show that field infection rates of BTV are significantly lower in midge vectors collected using traps baited with UV light versus a host cue (CO2). METHODS: We collected Culicoides sonorensis midges in suction traps baited with CO2, UV-light, or CO2 + UV on three dairies in southern California to assess differences in the resulting estimated infection rates from these collections. Pools of midges were tested for BTV by qRT-PCR, and maximum likelihood estimates of infection rate were calculated by trap. Infection rate estimates were also calculated by trapping site within a dairy. Colonized C. sonorensis were orally infected with BTV, and infection of the structures of the compound eye was examined using structured illumination microscopy. RESULTS: UV traps failed entirely to detect virus both early and late in the transmission season, and underestimated virus prevalence by as much as 8.5-fold. CO2 + UV traps also had significantly lower infection rates than CO2-only traps, suggesting that light may repel infected vectors. We found very high virus levels in the eyes of infected midges, possibly causing altered vision or light perception. Collecting location also greatly impacts our perception of virus activity. CONCLUSIONS: Because the majority of global vector surveillance for bluetongue uses only light-trapping, transmission risk estimates based on these collections are likely severely understated. Where national surveillance programs exist, alternatives to light-trapping should be considered. More broadly, disseminated infections of many arboviruses include infections in vectors' eyes and nervous tissues, and this may be causing unanticipated behavioral effects. Field demonstrations of pathogen-induced changes in vector behavior are quite rare, but should be studied in more systems to accurately predict vector-borne disease transmission.

Desiccation Tolerance in the Eggs of the Primary North American Bluetongue Virus Vector, Culicoides sonorensis (Diptera: Ceratopogonidae), and Implications for Vector Persistence.

Culicoides sonorensis Wirth and Jones transmits bluetongue virus and develops in a variety of polluted mud habitats. Egg desiccation tolerance was tested by obtaining eggs of known age, drying them, and placing them back on wet substrate. Eggs 4-10 h old failed to hatch after 12 h of drying at 75% relative humidity (RH). Older eggs (28-34 h) survived severe desiccation and >50% water weight loss. They regained their water within ≈2 h of rehydration. Relative to control eggs, average egg hatch was reduced by 36% after 12 h of drying, 79% after 24 h, 91% after 36 h, and 97% after 48 h. Some embryos (1%) survived and hatched after 60 h of drying and water losses of nearly 60%. Eggs in specific 25-40 h age categories did not differ in hatch after a 12-h desiccation stress; critical embryo age to survive drying is between 10 and 24 h. Humidity gradients relieved desiccation stress, and eggs appeared to regain water from saturated RH conditions. Individual, gravid C. sonorensis oviposited in 1-liter containers with an artificial mud bank. If they laid eggs, 73% deposited them singly in lines ranging up to 5-6 cm in length (meanderers), while 27% laid eggs in clumps (dumpers). Eggs were positioned an average of 45 ± 12 mm back from waterline. Younger eggs, if laid in early evening, may not experience severe desiccation. Embryo recovery from such severe desiccation could be adaptive in ephemeral habitats where the species may have evolved.