Oviposition of Culicoides insignis (Diptera: Ceratopogonidae) under laboratory conditions with notes on the developmental life history traits of its immature stages.

BACKGROUND: Culicoides insignis is a confirmed vector of bluetongue virus (BTV) throughout the American tropics and a possible vector of epizootic hemorrhagic disease virus (EHDV) in Florida. Despite its importance, fundamental information on the biology and ecology of this vector species is lacking. In this study, we examined the oviposition of C. insignis under laboratory conditions, monitored the development of immature stages and attempted colonization of this species. METHODS: Live C. insignis females were collected from the field using CDC-UV-LED traps, allowed to blood-feed on live chicken and given various natural substrates for oviposition in two-choice assays. The eggs deposited were transferred to 0.3% agar slants, and the hatched larvae were provided a diet of Panagrellus redivivus Linnaeus nematodes and the development of all immature stages was monitored. RESULTS: Culicoides insignis females exhibited an overall oviposition preference for dishes containing mud from their larval habitat as gravid females deposited a significantly higher number of eggs on these dishes (35.3 eggs/female) than on controls (17.7 eggs/female). The ovipositing females also deposited a higher percentage of eggs on substrates with habitat mud and other organically enriched muds (≥ 75.2%) compared to controls (31.0%). The larvae developed successfully to adulthood on the nematode diet, exhibiting high overall larval survival rates (85.0%). Sex ratios of the F1 generation were male biased, approximately 3:1 (male:female). Captive mating could not be induced in the F1 adults. CONCLUSIONS: Mud from the larval habitat and other organically enriched muds provide strong oviposition cues to C. insignis under laboratory conditions. Further studies will be needed to identify the key biotic/abiotic factors influencing midge oviposition in the field. The agar/nematode method is effective for the rearing of C. insignis larvae. However, further studies will be needed to address the issue of male-biased sex ratios in the progeny and to examine the mating habits/cues of C. insignis in nature, which may provide clues towards inducing captive mating in the F1 adults.

Host Bloodmeal Source Has No Significant Effect on the Fecundity and Subsequent Larval Development Traits of the Progeny in Culicoides furens Poey (Diptera: Ceratopogonidae).

Culicoides Latreille species (Diptera: Ceratopogonidae) are insects of significant medical and veterinary importance worldwide because their bites can cause major annoyance, allergic reactions, and/or pathogen transmission to vertebrates, including humans. In this study, we examined the effect of host bloodmeal source on the fecundity and subsequent larval development traits of the progeny of Culicoides furens Poey, a nuisance species in coastal Florida. Field-collected females were fed on two different classes of hosts: birds (chicken) or mammals (human). Fecundity outcomes of the females and larval developmental traits of the progeny were recorded and compared between the two groups. The percentage of females that developed eggs, percentage of gravid females that deposited eggs, number of eggs produced per female, number of eggs deposited per female, egg hatch rates, larval survival rates, time to pupation, pupal sex-ratios, and adult eclosion rates were not significantly different between the two groups. Our results demonstrate that the host bloodmeal source has no significant effect on the fecundity and subsequent larval developmental life-history traits of the progeny in C. furens. Previous studies reported C. furens to be a generalist feeder that shows no host preference for birds or mammals. Collectively, these findings suggest that C. furens is well adapted, not just behaviorally, but also physiologically to efficiently utilize blood meals from avian and mammalian hosts, a quality that offers a major evolutionary advantage to the success of this midge species as a generalist feeder.

Vector Competence of Florida Culicoides insignis (Diptera: Ceratopogonidae) for Epizootic Hemorrhagic Disease Virus Serotype-2.

Epizootic hemorrhagic disease virus (EHDV; family Reoviridae, genus Orbivirus) is an arthropod-borne virus of ungulates, primarily white-tailed deer in North America. Culicoides sonorensis, the only confirmed North American vector of EHDV, is rarely collected from Florida despite annual virus outbreaks. Culicoides insignis is an abundant species in Florida and is also a confirmed vector of the closely related Bluetongue virus. In this study, oral challenge of C. insignis was performed to determine vector competence for EHDV serotype-2. Field-collected female midges were provided bovine blood spiked with three different titers of EHDV-2 (5.05, 4.00, or 2.94 log10PFUe/mL). After an incubation period of 10 days or after death, bodies and legs were collected. Saliva was collected daily from all females from 3 days post feeding until their death using honey card assays. All samples were tested for EHDV RNA using RT-qPCR. Our results suggest that C. insignis is a weakly competent vector of EHDV-2 that can support a transmissible infection when it ingests a high virus titer (29% of midges had virus positive saliva when infected at 5.05 log10PFUe/mL), but not lower virus titers. Nevertheless, due to the high density of this species, particularly in peninsular Florida, it is likely that C. insignis plays a role in the transmission of EHDV-2.

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.

Laboratory Rearing of Culicoides stellifer (Diptera: Ceratopogonidae), a Suspected Vector of Orbiviruses in the United States.

Laboratory rearing procedures of Culicoides stellifer Coquillett (Diptera: Ceratopogonidae) were evaluated with an aim towards colonization of this species. Eggs collected from field-collected gravid females were placed on 0.25% agar slants and given a diet of 1) nematodes (Panagrellus redivivus Linnaeus), 2) nematodes + lactalbumin and yeast (LY), 3) microbes from nematode medium, and 4) tap water (autoclaved). Complete larval development to adult stage occurred only in two treatments: 1) nematodes and 2) nematodes + LY. Culicoides stellifer larvae could not survive beyond 1 wk on a diet of microbes alone or in the sterile water treatment. Larval survival rates were high using nematode diet (79.2 ± 11.3% [mean ± SE]) but were slightly lower in the nematode + LY group (66.5 ± 19.6%). Larval stage lasted ~21 d in both treatments. Sex ratio of F1 adults was ~1:1 (M:F) using nematode diet but was male biased (~2:1) with nematode + LY diet. These findings collectively suggest that a microbial community is required for midge larvae, either to support invertebrate prey base or as a potential food source. But in the present study, the supplied microbes alone were not sufficient to support midge survival/development. It appears that other nutritional components may also be essential to support the larval survival/development of C. stellifer. Overall, a simple diet of bacterial feeding nematodes and their associated microorganisms can be used to rear C. stellifer larvae under laboratory conditions. However, captive mating in F1 adults poses a major obstacle for successful colonization of this species currently.

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.

Vector Competence of Culicoides sonorensis (Diptera: Ceratopogonidae) for Epizootic Hemorrhagic Disease Virus Serotype 2 Strains from Canada and Florida.

Epizootic hemorrhagic disease virus (EHDV), an Orbivirus transmitted by Culicoides spp. vectors, is represented by seven serotypes and numerous strains worldwide. While studies comparing vector competence between serotypes exist, studies between viral strains are lacking. In this study, we examined the rates of infection, dissemination, and transmission of two strains of EHDV-2 orally fed to the known vector, Culicoides sonorensis Wirth & Jones. Culicoides sonorensis cohorts were fed an infectious blood meal containing EHDV-2 strains from either Alberta, Canada (Can-Alberta) or Florida (5.5 log10 PFUe/mL) and tested for the vector's susceptibility to infection and dissemination. In addition, transmission rates of the virus were assessed and compared using capillary tube and honey card methods. Our results show that the Florida strain had higher infection and dissemination rates than the Can-Alberta strain in spite of the Florida strain having significantly lower viral titers in C. sonorensis bodies, legs, and saliva than the Can-Alberta strain. Overall transmission rates were not significantly different between the two strains but varied significantly between the methods used. These findings suggest that the consequences of EHDV infection in C. sonorensis vary between virus strains and have huge implications in future vector competence studies involving Culicoides species and Orbiviruses.

Laboratory studies on the oviposition stimuli of Culicoides stellifer (Diptera: Ceratopogonidae), a suspected vector of Orbiviruses in the United States.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) exert a significant impact on animal agriculture worldwide because they transmit bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) to ruminants. Without effective vaccines, BTV/EHDV vector management strategies are needed, particularly in commercial white-tailed deer (WTD) facilities. However, detailed information on the ecology of midge immatures in/around cervid operations is currently lacking. Towards filling this knowledge gap, we conducted two-choice oviposition experiments with field-collected Culicoides stellifer Coquillett (a suspected vector of BTV/EHDV in the USA) under laboratory conditions to examine which natural source from the larval habitat is relatively more attractive for midge oviposition. METHODS: Field-collected C. stellifer females (CDC-UV light traps) were given a blood meal from live chicken and examined for their oviposition preferences for individual (or mixed) potential larval habitat oviposition stimuli in two-choice bioassays. Substrates included mud from C. stellifer habitat, mud from allopatric site, vegetation (Sphagnum spp. mosses), field water, WTD manure and de-ionized water (control). RESULTS: The majority of midges (91%) oviposited in only one dish, with few females (9%) ovipositing in both the dishes. Gravid females demonstrated an overall oviposition preference for substrates with mud and vegetation from the larval habitat, depositing a significantly higher proportion of eggs on mud (52.3%) and vegetation (81.8%) than on controls (≤ 18.2%) (P ≤ 0.0320). Moreover, greater number of eggs per female were deposited on mud (29.5-40.7 depending on trial) and vegetation (38.2) than on controls (≤ 5.8). WTD manure, field water and mud from allopatric site were not found to be more attractive than controls for oviposition. Combining individual substrates (mud + WTD manure; mud + moss + WTD manure + field water) did not elicit greater oviposition responses than mud or moss alone. CONCLUSIONS: Management strategies to discourage C. stellifer oviposition in/around commercial cervid facilities should likely focus on mud and/or vegetation, rather than WTD manure. However, further studies are needed to examine whether the spatial distributions of C. stellifer and Sphagnum spp. moss are correlated, and to determine whether targeting vegetation in/around cervid facilities can contribute to reductions in local midge densities.

Larval Development of Culicoides sonorensis (Diptera: Ceratopogonidae) in Mud Supplemented With Manure of Various Farm Animals.

Understanding factors that affect Culicoides larval development is critical in suppressing adult midge populations that transmit economically important pathogens to ruminants such as bluetongue virus and epizootic hemorrhagic disease virus. In this study, development of Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) was investigated in sterilized mud mixed with varying concentrations (3.2, 6.4, 12.6, 25.0, 50.0, and 100.0%) of dairy cattle and white tailed deer manures. In addition, C. sonorensis development was also evaluated in manure (25.0% concentration) of six other farm animals (beef cattle, sheep, goat, pig, horse, and chicken). First instar larvae (~100/treatment) were added to each substrate, and adult emergence and development times were monitored for 90 d. In substrates with dairy cattle manure, significantly more adults emerged (≥76.7%) and development time was shorter (≤25.5 d) from 25.0% manure substrate than from lower or higher manure concentrations (≤41.3% emerged; ≥31.2 d). Comparatively, white-tailed deer and chicken manures supported C. sonorensis development poorly with low emergence rates (deer ≤ 13.0%; chicken = 0%) and longer development time (deer ≥ 29.0 d). Mud enriched with manure of beef cattle, sheep, goat, pig, and horse generally supported C. sonorensis development, although adult emergence and development times varied widely between species. These results suggest that manure of several farm animals, except for white-tailed deer and chicken, can contribute substantially to C. sonorensis development in the field. Therefore, the potential of animals other than cattle in sustaining local populations of C. sonorensis cannot be overlooked when designing management strategies.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

Impact of the blood meal on humoral immunity and microbiota in the gut of female Culicoides sonorensis.

Although Culicoides sonorensis is an important vector of orbiviruses causing significant disease in domestic and wild ruminants in the USA, little is known about factors contributing to midge vector competence. In other vectors such as mosquitoes, interactions among the humoral immune response, microbiota, and ingested pathogens within the vector gut directly impact pathogen survival and therefore vectoring potential. We recently described components of the humoral immune response in the reference transcriptome for adult female C. sonorensis and analysed their temporal expression profiles across several dietary states (unfed, blood, or sugar fed). Blood feeding altered the transcription of several humoral immune components of the Immune deficiency (Imd), dual­oxidase (DUOX), and Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) pathways. Genes for immune effectors, such as antimicrobial peptides, were in particular highly induced. Since blood feeding also stimulated proliferation and diversification of bacterial populations colonising the gut of female midges, we infer that changes in immune gene expression were a result of fluctuations in gut microbiota. Thus, diet can indirectly (via microbiota) impact gut immune status and therefore should be carefully considered in subsequent studies assessing vector competence in biting midges.