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.

Predicting temperature-dependent transmission suitability of bluetongue virus in livestock.

The transmission of vector-borne diseases is governed by complex factors including pathogen characteristics, vector-host interactions, and environmental conditions. Temperature is a major driver for many vector-borne diseases including Bluetongue viral (BTV) disease, a midge-borne febrile disease of ruminants, notably livestock, whose etiology ranges from mild or asymptomatic to rapidly fatal, thus threatening animal agriculture and the economy of affected countries. Using modeling tools, we seek to predict where the transmission can occur based on suitable temperatures for BTV. We fit thermal performance curves to temperature-sensitive midge life-history traits, using a Bayesian approach. We incorporate these curves into S(T), a transmission suitability metric derived from the disease's basic reproductive number, [Formula: see text] This suitability metric encompasses all components that are known to be temperature-dependent. We use trait responses for two species of key midge vectors, Culicoides sonorensis and Culicoides variipennis present in North America. Our results show that outbreaks of BTV are more likely between 15[Formula: see text] C and [Formula: see text], with predicted peak transmission risk at 26 [Formula: see text] C. The greatest uncertainty in S(T) is associated with the following: the uncertainty in mortality and fecundity of midges near optimal temperature for transmission; midges' probability of becoming infectious post-infection at the lower edge of the thermal range; and the biting rate together with vector competence at the higher edge of the thermal range. We compare three model formulations and show that incorporating thermal curves into all three leads to similar BTV risk predictions. To demonstrate the utility of this modeling approach, we created global suitability maps indicating the areas at high and long-term risk of BTV transmission, to assess risk and to anticipate potential locations of disease establishment.

Temperature and food sources influence subadult development and blood-feeding response of Culicoides obsoletus (sensu lato) under laboratory conditions.

BACKGROUND: Culicoides obsoletus (s.l.) is the most abundant Culicoides species in northern Europe and an important vector of bluetongue virus and Schmallenberg virus. Nevertheless, information on its subadult life stages remains scarce and no laboratory-reared colony exists. METHODS: C. obsoletus (s.l.) adults were collected in Belgium and transferred to the laboratory in an attempt to establish a laboratory-reared colony. C. obsoletus (s.l.) were reared from eggs to adults at different temperatures (28 °C, 24 °C, 20/16 °C) and under different food regimes. RESULTS: The most suitable temperature for rearing seemed to be 24 °C for most developmental parameters, but resulted in a biased 3:1 male/female sex ratio. The latter could be optimized to a 1:1 sex ratio when a 20/16 °C day/night temperature gradient was applied, but rearing at these low temperature conditions resulted in significantly lower egg hatching and pupation rates and a longer subadult development time. Independent of the rearing temperature, adding dung as an additional food source during larval development resulted in a significantly higher adult emergence rate and a decrease in subadult development time. Furthermore, blood-feeding rates of field-collected C. obsoletus (s.l.) were compared for different blood sources and feeding systems. The overall blood-feeding success was low and only successful with cotton pledgets (2.7% blood-fed midges) and through a membrane system with chicken skin (3.5% blood-fed midges). Higher feeding rates were obtained on cattle blood compared to sheep blood. CONCLUSIONS: These results will help us to determine the necessary conditions to rear a viable laboratory colony of this important vector species, although further optimization is still required.

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.

Physicochemical factors affecting the diversity and abundance of Afrotropical Culicoides species in larval habitats in Senegal.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are the biological vectors of arboviruses of global importance in animal health. We characterized the physicochemical parameters that determine the density and composition of the main Culicoides species of veterinary interest in larval habitats of the Niayes region of Senegal. For this purpose, we combined larval and substrate sampling in the field in different habitat types with adult emergence and physicochemical analyses in the laboratory. Three major habitat types were identified, conditioning the predominant species of Culicoides and pH and the amount of organic matter were positively correlated with the abundance of larvae and emerging Culicoides, as opposed to salinity. The diversity of emerging Culicoides was positively correlated with pH while it was negatively correlated with salinity. Culicoides distinctipennis was the predominant species in the larval habitat group of freshwater lake edges. In the larval habitat group of pond and puddle edges, C. oxystoma and C. nivosus were predominant; both species were again most abundant in the larval habitat group of saltwater lake edges. These variabilities in physicochemical parameters support the distribution of different Culicoides species in different habitat groups. These results make it possible to implement effective, selective and environmental-friendly control measures but also to improve current models for estimating the abundance of adult vector populations at a local scale.

Assessment of the Hemotek® system for the in vitro feeding of field-collected Culicoides imicola (Diptera: Ceratopogonidae) in South Africa.

The optimising and standardisation of in vitro blood feeding protocols for field-collected Culicoides species (Diptera: Ceratopogonidae) will be of essence for the comparison of the vector competencies of various populations of viruses of veterinary importance and the establishment of laboratory colonies of putative vector species. A custom-made feeding chamber to accommodate the small size of Culicoides imicola Kieffer was designed for the commercially available Hemotek® system and compared to existing membrane and cotton pledge feeding methods. High feeding rates coupled to higher mean blood meal volume than that of the existing OVI device indicated that the Hemotek system will be suitable for the feeding of field-collected Culicoides. The Hemotek system was subsequently used to identify factors that may affect feeding success in the laboratory. Evaluated factors were the source (host) and temperature of the blood meal, time of the day of feeding, the position of the blood reservoir in relation to the midges and exposure time to the blood. While only feeding orientation and the temperature of the blood source seems to significantly affect the feeding rate, all the factors did influence the volume of blood consumed.

Development and lifespan of Culicoides obsoletus s.s. (Meigen) and other livestock-associated species reared at different temperatures under laboratory conditions.

Culicoides Latreille (Diptera: Ceratopogonidae) transmit arboviruses affecting wild and domestic ruminants such as bluetongue (BTV) and Schmallenberg virus (SBV). The sub-adult development and lifespan of Culicoides obsoletus s.s. (Meigen), Culicoides circumscriptus Kieffer and Culicoides paolae Boorman were examined at three different temperatures under laboratory conditions. Insects were collected from field between spring and autumn 2015 in two livestock farms located in Majorca (Spain). Gravid females were held individually at 18, 25 or 30 °C. Low temperatures increased the adult lifespan, time to oviposit and rate of development, whereas high temperatures increased the number of eggs, successful pupation and adult emergence as well as the larvae growth rate. The results showed that C. obsoletus s.s. have optimum development at 18 °C, whereas the optimal rearing temperature for C. circumscriptus and C. paolae was under warmer conditions of 25-30 °C. Variations in temperature/humidity and assays with different materials and substrates for oviposition should be considered in future studies. Understanding the requirements of the different species of Culicoides optimizing the results should be of special interest for predicting environmental change effects on these species, in addition to determining the rearing conditions for candidate Culicoides vectors.

Sampling Considerations for Adult and Immature Culicoides (Diptera: Ceratopogonidae).

Developing sampling programs for Culicoides can be challenging due to variation in ecology and behavior of the numerous species as well as their broad distributions and habitats. In this paper, we emphasize the need to clearly define research goals to select appropriate sampling methods. This includes not just the choice of sampling device, but also choice of attractant, site, number of traps per site, the duration and frequency of sampling, and the number of traps per unit area. Animal-baited trapping using enclosure traps and direct animal aspiration is more labor-intensive but yields information on species attracted to specific hosts as well as their biting rates. Sampling immatures is discussed with respect to choosing collection sites in semiaquatic mud, soil, and rich organic habitats. Sorting and extracting larvae using emergence traps, flotation, and Berlese funnels is also discussed.

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.

Identification of a cyanidin-3-O-ß-galactoside in gall tissue induced by midges on Japanese beech (Fagus crenata Blume).

Insect gall structures have many characteristic forms and colors, which are distinguishable from host plants. In this study, we identified an anthocyanin from red color insect galls and revealed that the anthocyanin biosynthesis of plants was induced by the gall extracts. The galling insects presumably regulate the anthocyanin biosynthesis of host plants to protect their larvae from environmental stresses.

A new methodology for sporogony research of avian haemoproteids in laboratory-reared Culicoides spp., with a description of the complete sporogonic development of Haemoproteus pastoris.

BACKGROUND: Haemosporidian parasites of the genus Haemoproteus (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of Culicoides (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus Parahaemoproteus. However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared Culicoides nubeculosus. This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of Haemoproteus (Parahaemoproteus) pastoris, a widespread parasite of the common starling Sturnus vulgaris. METHODS: The methodology consists of the following main stages: (i) laboratory rearing of C. nubeculosus from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to H. pastoris (cytochrome b lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively. RESULTS: Culicoides nubeculosus females were successfully reared and exposed to H. pastoris, which completed sporogonic development 7-9 days post-infection when sporozoites were observed in the salivary glands. CONCLUSIONS: The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of Parahaemoproteus parasites, which might be used in a broad range of parasitology and genetic studies. Culicoides nubeculosus is an excellent experimental vector of subgenus Parahaemoproteus and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

Update of the species checklist of Culicoides Latreille, 1809 biting midges (Diptera: Ceratopogonidae) of Morocco.

BACKGROUND: Investigations of Culicoides fauna, including inventories, were carried out in Morocco at different periods after the country had faced major bluetongue and African horse sickness outbreaks. However, no comprehensive reference publication has provided a clear overview of the Culicoides species diversity. This study reviewed available data on Culicoides biting midge species in Morocco from 1968 to 2015 (published and grey literature in French and English) in order to revise the current checklist, in light of state of the art taxonomic and systematic knowledge, and confirmed the checklist with morphological and molecular identifications of specimens collected from the region of Rabat. METHODS: Literature related to Culicoides collections in Morocco was collated. Authors were contacted to obtain raw data and additional information for the collections. Fresh Culicoides material was collected and examined from two sites around Rabat, the capital of Morocco. Each collected individual was examined and morphologically identified, if possible, to the species level. In addition, molecular identification was performed to separate closely related species, to confirm difficult morphological identifications and to confirm new species records. RESULTS: A total of 6121 individuals of Culicoides spp. were collected and analyzed and at least 17 species were identified: C. cataneii/C. gejgelensis, C. circumscriptus, C. fagineus, C. festivipennis, C. imicola, C. jumineri, C. kingi, C. longipennis, C. montanus, C. newsteadi, C. obsoletus, C. paolae, C. parotti, C. puncticollis, C. sahariensis, C. scoticus and C. subfagineus. Seven species were confirmed using phylogenetic analyses. Two new species records for Morocco are reported: C. paolae and C. subfagineus. CONCLUSIONS: The Moroccan fauna of Culicoides now includes 54 valid species. Further work would certainly increase this total, as one of the clades we identified was not affiliated to any described and valid species.

Latitudinal diversity of biting midge species within the Obsoletus group across three habitats in Europe.

Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country-habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm-to-farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission.

Combined larvicidal and adulticidal treatments to control Culicoides biting midges (Diptera: Ceratopogonidae): Results of a pilot study.

Culicoides biting midges can transmit viruses such as bluetongue virus and Schmallenberg virus to ruminants and African horse sickness virus to equines. The control of Culicoides population can be based on physical and/or chemical methods. This study aims to evaluate the efficacy of larvicides and adulticides combined treatments to control the vector Culicoides populations. Field trials have been conducted during 2014 on seven farms in northern Sardinia (Italy). Three insecticide treatments (larvicides and adulticides) have been carried out on three farms, whereas four farms remained unsprayed as untreated controls. For the larval control two commercial formulations, based on diflubenzuron and Bacillus thuringiensis var. israelensis (H-14), have been used. Against adults a deltamethrin based product has been sprayed on the walls and roofs of animal shelters. Adult Culicoides have been collected on each farm twice a week with an Onderstepoort blacklight suction trap. After each treatment a significant reduction in the abundance of total Culicoides, C. imicola and C. newsteadi s.l., has been observed. The Obsoletus complex density has been affected by the first two treatments. Our results suggest the possibility to control the Culicoides populations with a combination of adulticide and larvicide treatments.

Community analysis of the abundance and diversity of biting midge species (Diptera: Ceratopogonidae) in three European countries at different latitudes.

BACKGROUND: The outbreaks of bluetongue and Schmallenberg disease in Europe have increased efforts to understand the ecology of Culicoides biting midges and their role in pathogen transmission. However, most studies have focused on a specific habitat, region, or country. To facilitate wider comparisons, and to obtain a better understanding of the spread of disease through Europe, the present study focused on monitoring biting midge species diversity in three different habitat types and three countries across Europe. METHODS: Biting midges were trapped using Onderstepoort Veterinary Institute light traps at a total of 27 locations in Sweden, the Netherlands and Italy, comprising farm, peri-urban and wetland habitats. From July 2014 to June 2015 all locations were sampled monthly, except for during the winter months. Trapped midges were counted and identified morphologically. Indices on species richness, evenness and diversity were calculated. Community compositions were analysed using non-metric multidimensional scaling (NMDS) techniques. RESULTS: A total of 50,085 female midges were trapped during 442 collection nights. More than 88% of these belonged to the Obsoletus group. The highest midge diversity was found in Sweden, while species richness was highest in the Netherlands, and most specimens were trapped in Italy. For habitats within countries, diversity of the trapped midges was lowest for farms in all countries. Differences in biting midge species communities were more distinct across the three countries than the three habitat types. CONCLUSIONS: A core midge community could be identified, in which the Obsoletus group was the most abundant. Variations in vector communities across countries imply different patterns of disease spread throughout Europe. How specific species and their associated communities affect disease risk is still unclear. Our results emphasize the importance of midge diversity data at community level, how this differs across large geographic range within Europe, and its implications on assessing risks of midge-borne disease outbreaks.

The Immatures of Culicoides trilineatus (Diptera: Ceratopogonidae) Potential Vector of the Bluetongue Virus.

The fourth instar larva and pupa of Culicoides trilineatus Fox (Diptera, Ceratopogonidae), a species considered as potential vector of the bluetongue virus in Central and South America, are described, illustrated, and photomicrographed for the first time by using binocular, phase-contrast, and scanning electron microscopy. The immatures were collected by using a siphon bottle in tree holes in Salta Province, Argentina, transported to the laboratory, and there reared to the adult's emergence. They are compared with the immatures of Culicoides debilipalpis Lutz (Diptera, Ceratopogonidae), another Neotropical species that develops in tree holes. Details on larval biology and habitat are given.

Geo-statistical analysis of Culicoides spp. distribution and abundance in Sicily, Italy.

BACKGROUND: Biting midges belonging to Culicoides imicola, Culicoides obsoletus complex and Culicoides pulicaris complex (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palaearctic regions. Culicoides obsoletus complex includes C. obsoletus (sensu stricto), C. scoticus, C. dewulfi and C. chiopterus. Culicoides pulicaris and C. lupicaris belong to the Culicoides pulicaris complex. The aim of this study was a geo-statistical analysis of the abundance and spatial distribution of Culicoides spp. involved in bluetongue virus transmission. As part of the national bluetongue surveillance plan 7081 catches were collected in 897 Sicilian farms from 2000 to 2013. METHODS: Onderstepoort-type blacklight traps were used for sample collection and each catch was analysed for the presence of Culicoides spp. and for the presence and abundance of Culicoides vector species (C. imicola, C. pulicaris / C. obsoletus complexes). A geo-statistical analysis was carried out monthly via the interpolation of measured values based on the Inverse Distance Weighted method, using a GIS tool. Raster maps were reclassified into seven classes according to the presence and abundance of Culicoides, in order to obtain suitable maps for Map Algebra operations. RESULTS: Sicilian provinces showing a very high abundance of Culicoides vector species were Messina (80% of the whole area), Palermo (20%) and Catania (12%). A total of 5654 farms fell within the very high risk area for bluetongue (21% of the 26,676 farms active in Sicily); of these, 3483 farms were in Messina, 1567 in Palermo and 604 in Catania. Culicoides imicola was prevalent in Palermo, C. pulicaris in Messina and C. obsoletus complex was very abundant over the whole island with the highest abundance value in Messina. CONCLUSIONS: Our study reports the results of a geo-statistical analysis concerning the abundance and spatial distribution of Culicoides spp. in Sicily throughout the fourteen year study. It provides useful decision support in the field of epidemiology, allowing the identification of areas to be monitored as bases for improved surveillance plans. Moreover, this knowledge can become a tool for the evaluation of virus transmission risks, especially if related to vector competence.

Overview and features of larval developmental sites of biting midges species associated with livestock in Israel with implications to their control.

The larval developmental sites of Culicoides species were assessed from 1155 samples collected during the years 1969-2003. Culicoides circumscriptus was most prolific in mud with medium organic matter with proportional representation of 55.8%. Culicoides imicola breeds mainly in damp mixture of rotten, decomposed animal dung with or without some mud, with proportional representation of 97.2%. Culicoides obsoletus was found mostly in rotten banana stumps, with proportional representation of almost 100%. Culicoides puncticollis was found in mud with a medium amount of organic matter with proportional representation of 35.6%. Culicoides distinctipennis showed 22.2% proportional representation for mud poor in organic matter. Culicoides schultzei gr. was most prolifically in mud rich in organic matter with proportional representation of 6.2%. Culicoides cataneii was most prolifically in mud with a medium amount of organic matter with proportional representation of 1.8%. Maximum duration time to emergence from the larval developmental sites ranged from seven weeks in C. obsoletus to fourteen weeks in C. circumscriptus and nine weeks in C. imicola. All the studied species breed throughout the year. Significant innate differences in the proportions of the sexes were found in the emergence of six species.

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.

Egg and Larval Morphology of Culicoides sonorensis (Diptera: Ceratopogonidae).

The egg and larval instars (L1-L4) of Culicoides sonorensis Wirth and Jones are described. Average (range) of body lengths of a laboratory colony (Van Ryn) were 0.45 (0.43-0.48) mm for eggs, 0.72 (0.39-0.92) mm for L1, 1.8 (1.3-2.2) mm for L2, 3.6 (2.2-5.0) mm for L3, and 5.9 (5.0-6.9) mm for L4. Head capsule lengths were 0.067 (0.064-0.070) mm for L1, 0.100 (0.094-0.110) for L2, 0.157 (0.144-0.168) for L3, and 0.316 (0.300-0.324) mm for L4. Wild and colony L4 larvae differed in many standard metrics such as head length or width, but head ratios and pharyngeal armature measurements were comparable. Standard external and internal features of the head capsule are described. Scanning electron microscope photos revealed pharyngeal armature structure was similar for L2-L4. The pharyngeal armature and piercing mandibles suggest a generalist feeder and opportunistic predator. Egg ansullae were randomly distributed (as opposed to rows in most other Culicoides spp.) and averaged 1.98 ansullae per 25 µm2 of egg surface. Ansullae were widened and porous apically, averaging 1.29 microns long, 0.65 µm at the base, and 1.06 µm at the apex. Only the L1 had a proleg, structurally complex, and featuring five rows of posteriorily directed, shorter spines (1-3 µm long) and 8-10 setae 18 µm long with hooked tips. On terrestrial mud, this proleg was used for surface crawling, and the L1 navigated dry conditions better than the more aquatic L2-L4 stages. Eggs are laid above waterline, and the L1 proleg is probably an advantage in terrestrial surface movement.