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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 13 C and 15 N 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 13 C or 15 N enrichment levels., Conclusions: Stable isotope labeling is life-long, and does not interfere with natural insect behaviors. Stable isotope enrichment using 13 C or 15 N 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.

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.

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.

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., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) is the primary North American vector of bluetongue virus (BTV), which can cause high morbidity and mortality in ruminant livestock or wildlife. Worldwide, most Culicoides surveillance relies on light (usually UV) traps typically placed near animals or larval development sites. However, the trapping method can cause sex, species and parity biases in collections. We collected C. sonorensis from three dairies in California using suction traps baited with CO2 , UV light or CO2  + UV placed near animals, wastewater ponds, or in fields. Higher numbers of parous females were collected using CO2  + UV traps, although this difference was only significant on one dairy. UV traps were poor at collecting nulliparous females, but the addition of UV to a trap increased the abundance of males in a collection. Traps set in open fields collected significantly higher numbers of males and females than in either of the other two locations. In some cases, there was a significant interaction between the trap type and site. We discuss the limitations of traditional trapping methodologies for C. sonorensis and make suggestions for vector surveillance., (© 2016 The Royal Entomological Society.)

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.

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.

Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of two important viruses impacting North American ruminants--bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses have been identified for over 60 years in North America, but we still lack an adequate understanding of the basic biology and ecology of the confirmed vector, Culicoides sonorensis, and know even less about other putative Culicoides vector species. The major gaps in our knowledge of the biology of Culicoides midges are broad and include an understanding of the ecology of juveniles, the identity of potential alternate vector species, interactions of midges with both pathogens and vertebrates, and the effectiveness of potential control measures. Due to these broad and numerous fundamental knowledge gaps, vector biologists and livestock producers are left with few options to respond to or understand outbreaks of EHD or BT in North America, or respond to emerging or exotic Culicoides-transmitted pathogens. Here we outline current knowledge of vector ecology and control tactics for North American Culicoides species, and delineate research recommendations aimed to fill knowledge gaps.

Culicoides sonorensis (Wirth & Jones) is the principal North American vector of bluetongue virus (BTV). BTV infection of livestock is distinctly seasonal (late summer and fall) in temperate regions of the world such as California, which has led to speculation regarding vertical transmission of the virus within the midge vector as a potential mechanism for interseasonal maintenance ("overwintering") of the virus. To evaluate potential vertical transmission of BTV in its midge vector, we fed adult midges BTV-spiked blood and used a BTV-specific quantitative reverse transcriptase polymerase chain reaction assay to evaluate parent, egg, and progeny stages of laboratory-reared C. sonorensis for the presence of viral nucleic acid. Whereas BTV nucleic acid was weakly detected in egg batches of virus-fed female midges, virus was never detected in subsequent progeny stages (larvae, pupae, and F1 generation adults). Similarly, BTV was not detected in pools of larvae collected from the waste-water lagoon of a BTV-endemic dairy farm in northern California during the seasonal period of virus transmission. Collectively, these results indicate that BTV is not readily transmitted vertically in C. sonorensis, and that persistence of the virus in long-lived parous female midges is a more likely mechanism for overwintering of BTV in temperate regions., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.)

Culicoides midges are abundant hematophagous flies that vector arboviruses of veterinary and medical importance. Dramatic changes in the epidemiology of Culicoides-borne arboviruses have occurred since 1998, including the emergence of exotic viruses in northern temperate regions, increases in global disease incidence, and enhanced virus diversity in tropical zones. Drivers may include changes in climate, land use, trade, and animal husbandry. New Culicoides species and new wild reservoir hosts have been implicated in transmission, highlighting the dynamic nature of pathogen-vector-host interactions. Focusing on potential vector species worldwide and key elements of vectorial capacity, we review the sensitivity of Culicoides life cycles to abiotic and biotic factors. We consider implications for designing control measures and understanding impacts of environmental change in different ecological contexts. Critical geographical, biological, and taxonomic knowledge gaps are prioritized. Recent developments in genomics and mathematical modeling may enhance ecological understanding of these complex arbovirus systems.

ImageJ is an open-source software tool used for a variety of scientific objectives including cell counting, shape analysis and image correction. This technology has previously been used to estimate mosquito abundance in surveillance efforts. However, the utility of this application for estimating abundance or parity in the surveillance of Culicoides spp. (Diptera: Ceratopogonidae) has not yet been tested. Culicoides sonorensis (Wirth and Jones), a biting midge often measuring 2.0-2.5 mm in length, is an economically important vector of ruminant arboviruses in California. Current surveillance methods use visual sorting for the characteristics of midges and are very time-intensive for large studies. This project tested the utility of ImageJ as a tool to assist in gross trap enumeration as well as in parity analysis of C. sonorensis in comparison with traditional visual methods of enumeration using a dissecting microscope. Results confirmed that automated counting of midges is a reliable means of approximating midge numbers under certain conditions. Further evaluation confirmed accurate and time-efficient parity analysis in comparison with hand sorting. The ImageJ software shows promise as a tool that can assist and expedite C. sonorensis surveillance. Further, these methods may be useful in other insect surveillance activities., (© 2014 The Royal Entomological Society.)

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., (© 2014 Entomological Society of America.)

The Sacramento (northern Central) Valley of California (CA) has a hot Mediterranean climate and a diverse ecological landscape that is impacted extensively by human activities, which include the intensive farming of crops and livestock. Waste-water ponds, marshes, and irrigated fields associated with these agricultural activities provide abundant larval habitats for C. sonorensis midges, in addition to those sites that exist in the natural environment. Within this region, C. sonorensis is an important vector of bluetongue (BTV) and related viruses that adversely affect the international trade and movement of livestock, the economics of livestock production, and animal welfare. To characterize the seasonal dynamics of immature and adult C. sonorensis populations, abundance was monitored intensively on two dairy farms in the Sacramento Valley from August 2012- to July 2013. Adults were sampled every two weeks for 52 weeks by trapping (CDC style traps without light and baited with dry-ice) along N-S and E-W transects on each farm. One farm had large operational waste-water lagoons, whereas the lagoon on the other farm was drained and remained dry during the study. Spring emergence and seasonal abundance of adult C. sonorensis on both farms coincided with rising vernal temperature. Paradoxically, the abundance of midges on the farm without a functioning waste-water lagoon was increased as compared to abundance on the farm with a waste-water lagoon system, indicating that this infrastructure may not serve as the sole, or even the primary larval habitat. Adult midges disappeared from both farms from late November until May; however, low numbers of parous female midges were detected in traps set during daylight in the inter-seasonal winter period. This latter finding is especially critical as it provides a potential mechanism for the "overwintering" of BTV in temperate regions such as northern CA. Precise documentation of temporal changes in the annual abundance and dispersal of Culicoides midges is essential for the creation of models to predict BTV infection of livestock and to develop sound abatement strategies.

"Candidatus Cardinium hertigii" (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of "Candidatus Cardinium" infection was documented as moderate, with no detectable sex bias. We therefore investigated whether "Candidatus Cardinium" affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account "Candidatus Cardinium" infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with "Candidatus Cardinium" infection data. The findings revealed no difference in survival of Culicoides infected with "Candidatus Cardinium" and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for "Candidatus Cardinium"-infected versus uninfected midges. In aggregate, these findings support our conclusion that "Candidatus Cardinium" does not have an overt effect on the survival and size of adult C. imicola midges. "Candidatus Cardinium" may affect immature stages or may alter adult reproductive performance.

Culicoides species (Diptera: Ceratopogonidae) belonging to the Obsoletus and Pulicaris groups are considered to be the main vectors of bluetongue virus (BTV) in non Mediterranean Europe. Selected terrestrial microhabitats (n=17) on a farm in northern Spain were sampled repeatedly over a year-long period and characterized for use by Culicoides species for immature development. Concurrent use of CDC light traps showed the presence of 37 species and 66,575 specimens of adult Culicoides. A total of 28 species and 11,396 individuals emerged from laboratory-maintained soil samples. Culicoides obsoletus and Culicoides scoticus (pooled as Obsoletus complex) were particularly abundant (comprising 58.6% and 74.5% of the total collections in light traps and emergence traps respectively). Potential key vectors of animal viruses (such as BTV) were found in two main terrestrial types of microhabitats. In the case of C. obsoletus, different types of manure (old and composted manure, manure mixed with organic matter, and fresh manure) produced most of the specimens. In contrast, larvae of C. scoticus and Culicoides lupicaris were associated with soil substantially comprised of rotting leaf litter that included the parasitic plant Lathraea clandestina. Several species, Culicoides festivipennis, Culicoides punctatus and Culicoides brunnicans, were very common in mud at pond margins. Indeed, pond microhabitats and runoff below barn rooflines supported the greatest species richness. In the pond habitat, 49.4% of Culicoides specimens emerged from mud at the water edge, as opposed to 50 cm above (32.4%) and 1 meter above waterline (18%). Similar species richness, but statistically significant differences in abundance, were observed among the four pond microhabitats. Overall, the majority of the specimens were found in the upper layer (0-3 cm), except in manure, where they preferred deeper layers (>6 cm). Maximum peaks of abundance occurred in both light traps and soil samples in summer months, whereas increased captures in autumn were noticed only in light traps. Both trapping systems failed to collect adult Culicoides midges in the coldest months of December, January and February. The literature on immature habitats of species suspected in BTV transmission in Europe, the Pulicaris group and particularly the Obsoletus group, is briefly reviewed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bluetongue (BT) is an important viral disease of ruminants that is transmitted by hematophagous Culicoides midges. We examined the seasonal patterns of abundance and infection of Culicoides sonorensis at four dairy farms in the northern Central Valley of California to develop estimates of risk for bluetongue virus (BTV) transmission to cattle at each farm. These four farms were selected because of their similar meteorological conditions but varying levels of vector abundance and BTV infection of cattle. C. sonorensis midges were collected weekly at each farm during the seasonal transmission period, using three different trapping methods: traps baited with either carbon dioxide (CO(2)) alone or traps with CO(2) and UV light, and by direct aspiration of midges from sentinel cattle. Analysis of BTV-infected midges using group and serotype-specific quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) assays confirmed that BTV serotypes 10, 11, 13 and 17 are all present in the region, but that midge infection rates and the number of BTV serotypes circulating differed markedly among the individual farms. Furthermore, more serotypes of BTV were present in midges than in sentinel cattle at individual farms where BTV circulated, and the virus was detected at each farm in midges prior to detection in cattle. BTV infection rates were remarkably lower among female C. sonorensis midges collected by CO(2) traps with UV light than among midges collected by either animal-baited aspirations or in CO(2) traps without light. A subsample of female midges examined from each collection method showed no overall differences in the proportion of female midges that had previously fed on a host. Findings from this study confirm the importance of using sensitive surveillance methods for both midge collection and virus detection in epidemiological studies of BTV infection, which is especially critical if the data are to be used for development of mathematical models to predict the occurrence of BTV infection of livestock., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bluetongue is an economically important arboviral disease of ruminants that is transmitted by hematophagous Culicoides midges. In light of dramatic recent changes in the global distribution of bluetongue virus (BTV), the goals of this study were to re-evaluate the prevalence of BTV infection of cattle and abundance of Culicoides midges on individual dairy farms in California. A serosurvey of adult dairy cattle confirmed that BTV infection is prevalent throughout much of the state, although the coastal northwestern region remains free of infection and prevalence varies markedly among farms in the remainder of the state. Intensive sampling for one year of 4 farms in the northern Central Valley of California showed that the abundance of Culicoides midges was markedly different and coincided with the prevalence of BTV infection of sentinel cattle on each farm. Mean maximum and minimum temperatures and other meteorological parameters were similar on all 4 farms, thus we speculate that particular management practices were responsible for both the increased midge abundance and prevalence of BTV infection of cattle at individual farms. Specifically, it is concluded that variation in vector abundance at individual farms most likely is the result of waste-water lagoon and irrigation management practices, leading to higher BTV infection rates among livestock held on farms with more waste-water lagoons and greater acreage of land for waste-water irrigation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An enclosure trapping experiment compared numbers and engorgement of Culicoides spp. taken from treated sheep (7.5% deltamethrin) to Culicoides from untreated sheep. Attack rates were low (0.2/min), but 58% of Culicoides obsoletus s.l. and 67% of Culicoides parroti Kieffer engorged on untreated sheep, and no engorgement occurred on treated sheep on 0 and 4 d posttreatment. A UV light trap in a livestock barn collected eight Culicoides spp. (510 individuals), dominated by C. obsoletus (Meigen) (68%), Culicoides imicola Kieffer (19%), Culicoides circumscriptus Kieffer (8%), and Culicoides alazanicus Dzhafarov (4%). A more powerful but nonattractive fan trap collected five species (121 individuals) dominated by C. obsoletus (48%), C. imicola (36%), C. alazanicus (8%), and C. circumscriptus (7%). Parity of C. obsoletus and C. imicola did not vary between the light and fan traps. Engorged Culicoides in the barn (33 C. obsoletus and three C. imicola) had fed on sheep or goats (precipitin test).

Biting midges in the genus Culicoides (Diptera: Ceratopogonidae) were collected near sunset by direct aspiration from sheep in northeastern Spain to determine species-specific biting rates and crepuscular activity. Midges were also collected by UV-baited light traps and CO2-baited traps over the same period to compare species diversity and abundance using these common surveillance methods to actual sheep attack rates. Culicoides aspirated from sheep included C. obsoletus, C. parroti, C. scoticus, C. punctatus, and C. imicola. Peak host-seeking activity during the time period examined for the two most commonly collected species (C. obsoletus and C. parroti) occurred just before sunset and activity ceased within 1 h after sunset. Host attack rates near sunset averaged 0.9 midges/min for both species with maximum attack rates of 3/min for C. obsoletus and 4/min for C. parroti. For both species, approximately 35% of midges collected from the sheep were engorged, giving a maximum biting rate of 1.1/min for C. obsoletus and 1.5/min for C. parroti. Traps baited with CO2 collected fewer midges of each species relative to other collection methods. Traps baited with UV light provided a good indication of species richness but significantly underestimated the host attack rate of C. obsoletus and C. parroti while overestimating the host attack rate of C. imicola. Animal-baited collecting is critical to interpret the epidemiological significance of light trap collections used for surveillance of the midge vectors of bluetongue virus and African horse sickness virus.

Arthropod sampling via periodic direct bird examination and regular light trapping was conducted between June 2000 and October 2002 to survey for potential vectors of Haemoproteus lophortyx to nonnative, captive-raised bobwhite quail (Colinus virginianus) in northern California, USA. Examination of individual bobwhite quail (from 5 weeks of age through adult, total n=76) was conducted on several dates during the transmission period (June-October). No ectoparasites, including hippoboscid flies (reported as Haemoproteus vectors to wild quail in early literature), were collected from the birds. Trapping with ultraviolet light suction traps near the quail revealed nine species of biting midges (Culicoides spp.). Of these, 94% were C. bottimeri, which was abundant near the birds, and 65% of collected C. bottimeri were engorged with blood. C. bottimeri adult activity began in late-April, slightly before the onset of disease in the quail. Activity peaked between July and late-September, coincident with maximum reported transmission, and adult activity ceased by early-November. Nonengorged C. bottimeri had a parity rate of 43.6% overall, suggesting excellent survival for biological transmission of a pathogen like H. lophortyx. A controlled study was done injecting a macerated slurry of pooled, nonengorged, wild-caught C. bottimeri into the peritoneum of 1-day-old bobwhite quail hatchlings held in insect-proof containers. Blood smears 13-19 days later confirmed H. lophortyx infection in zero controls but all insect-injected quail. Biting midges, especially C. bottimeri, transmit H. lophortyx to captive quail and probably are the dominant vector to native California quail (Callipepla californica) as well.

Biting insects were sampled to investigate the cause(s) of dermatitis (putative Culicoides hypersensitivity [CHS]) on horses on the Palos Verdes Peninsula, Los Angeles County, California. Suction traps baited with CO2 were operated at 5 sites from April 2002 to August 2003, supplemented by sampling from horses. Six species of Culicoides, 7 species of mosquitoes, and occasional Simulium and Stomoxys were collected in traps. Culicoides sonorensis was the most common midge trapped, although C. obsoletus and C. freeborni also were collected repeatedly. Insects from the belly region of horses in spring (peak dermatitis period) were mostly C. obsoletus, which were far more abundant than indicated by CO2-baited suction traps. The predominant mosquito was Culiseta incidens; Culex quinquefasciatus and Cx. tarsalis also could be common, depending on the site. Mosquitoes, particularly Cs. incidens, were more abundant the 2nd year (normal rainfall), whereas C. sonorensis and C. freeborni were more abundant the 1st year (drought period). Culicoides obsoletus, supplemented by C. sonorensis, is regarded as a primary suspect causing CHS in this area.

Adult female Culicoides occidentalis Wirth and Jones were collected in a coastal salt marsh habitat in southern California over a period of 15 mo using CO2-baited suction traps. Adults were active year-round. Adult females overall were large based on wing length (1.86 mm). Because of coastal influences, daily and seasonal changes in air temperatures were greatly buffered, and mean temperatures ranged from 10 to 15 degrees C in winter to 19-21 degrees C in summer. Wing lengths varied inversely with air temperatures, with an average of 2.13 mm (February) to 1.6 mm (July). Parity was consistently low (3.6% overall) and may reflect poor host availability in this isolated salt marsh habitat.

The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)-nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111-222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTV as determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RT-nested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV.

To determine the variability of the NS3/NS3A gene of field strains of BTV contained in Culicoides sonorensis collected from a single site in California (CA), the NS3/NS3A gene was directly amplified and sequenced from 22 pools of C. sonorensis and compared with those of previously characterized field isolates from CA, as well as to viruses that caused recent outbreaks of bluetongue disease in ruminants in CA. Phylogenetic analysis established that the NS3/NS3A gene of strains of BTV contained in C. sonorensis collected from the site exists as a heterogeneous population. The two most divergent nucleotide sequences of the NS3/NS3A genes of these viruses differed by 2.5% (18 nucleotides). Comparison with the NS3/NS3A gene sequences from viruses that caused recent instances of bluetongue disease in ruminants in CA indicated that BTV strains from different geographic regions can exhibit a higher degree of genetic heterogeneity (up to 6.6%; 0-48 nucleotide differences) than those contained in C. sonorensis collected from a single site.

Bluetongue virus (BTV) is the cause of an insect-transmitted virus infection of ruminants that occurs throughout much of the world. Individual gene segments differ between field strains of BTV; thus, we hypothesized that key viral genes undergo genetic drift during alternating passage of BTV in its ruminant and insect hosts. To test this hypothesis, variation in the consensus sequence and quasispecies heterogeneity of the VP2 and NS3/NS3A genes of a plaque-purified strain of BTV serotype 10 was determined during alternating infection of vector Culicoides sonorensis and a sheep and calf. Consensus sequences were determined after reverse transcriptase-nested PCR amplification of viral RNA directly from ruminant blood and homogenized insects, and quasispecies heterogeneity was determined by the sequencing of clones derived from directly amplified viral RNA. Comparison of these sequences to those of the original BTV inoculum used to initiate the cycle of BTV infection demonstrated, for the first time, that individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Furthermore, a unique viral variant was randomly ingested by C. sonorensis insects that fed on a sheep with low-titer viremia, thereby fixing a novel genotype by founder effect. Thus, we conclude that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV.

Vectorial capacity of Culicoides sonorensis Wirth & Jones for the transmission of bluetongue (BLU) virus was examined at a southern California dairy from January 1995 to December 1997. Insects were collected one to two times per week in five CDC-type suction traps (without light) baited with CO2 at a constant release rate of 1,000 ml/min. BLU virus was detected in midges collected from May through December with an estimated overall infection rate of 0.08%. The BLU virus infection rate of field-captured midges was not correlated with sentinel calf seroconversions to BLU virus. Sentinel calf seroconversions were highly seasonal, occurring from August through November with most calves seroconverting during September and October. Vector competence of field-collected nulliparous flies fed a locally acquired serotype of BLU virus in the laboratory was stable among years (17-23%). Vectorial capacity was strongly correlated with BLU virus transmission (measured by sentinel calf seroconversions) during 1995 and 1996, but not during 1997. Host biting rate estimated for traps nearest to the sentinel calves was the index best correlated with BLU virus transmission for all study years and was most highly correlated with sentinel seroconversions 4 wk later. The utility of vectorial capacity and its component variables is discussed for this system.

Seasonal abundance and survivorship of Culicoides sonorensis Wirth & Jones were examined at a dairy in southern California from January 1995 to December 1997. Insects were collected one to two times per week using five CDC-type suction traps (without light) baited with CO2 at a constant release rate of 1,000 ml/min. Female and male abundance was greatest during late summer and early fall and was directly correlated with mean monthly air temperature. Parity of females was lowest during late summer and early fall. The gonotrophic cycle was estimated to require 3-4 during hot summer months and up to 14 d during cool winter months. Estimated extrinsic incubation of bluetongue virus (BLU) was 9-10 d during August and September. The estimated daily survival ranged from < 60% in the summer to > 95% in the winter, resulting in an expectation of life of only 2-3 d in summer and > 10 d in winter. The probability of females surviving the extrinsic incubation period for BLU virus, and the expectation of infective life were both lowest during late summer and early fall. During 1997, midge abundance during late summer was not high enough to overcome very low survivorship, and the absolute number of females expected to survive the extrinsic incubation period was relatively low. However, in 1995 and 1996, very high midge abundance compensated for low survivorship during summer and the number of females expected to survive the extrinsic incubation period was relatively high. Although abundance was generally very low during the cool winter and spring, host-seeking females were captured throughout the year, and their winter survival was high. Overwintering of BLU virus by continued transmission of the virus by active midges appears possible.

The persistence of permethrin (5% a.i.) and pirimiphos-methyl (27% a.i.), applied to the dorsum of calves in the field against Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), was estimated using a hair-blood-feeding bioassay in the laboratory. Hair clippings were taken before treatment and 3, 7, 14, 21, 28, 42 and 56 days after treatment from the dorsum, side and belly of treated and control calves. Laboratory-reared insects were allowed to feed through thin hair layers and a parafilm membrane on sheep blood warmed using a water-jacketed feeder. Some intoxication after exposure to hair was noted up to 28 days after treatment with permethrin and up to 14 days after treatment with pirimiphos-methyl. Hair from the dorsum caused more intoxication for a longer period than hair from other body regions. Permethrin and pirimiphos-methyl applied to the back did not significantly reduce overall engorgement (body regions pooled) after treatment. Permethrin residues on hair remained far higher on the back than other body regions and were related to insect intoxication and reduction in engorgement in the laboratory. Residues on belly hair never exceeded 12p.p.m. and did not result in significantly reduced feeding at any time. Engorged insects that exhibited sublethal intoxication from feeding through permethrin-treated hair did recover and matured numbers of eggs comparable to controls. Field trials using treated and control calves and enclosure nets showed that dorsal applications of 5% permethrin were not effective in reducing engorgement, despite some intoxication. Vacuum samples from a calf showed that C. sonorensis fed primarily on the belly. A 0.2% permethrin application on the belly (250 ml) did result in > 80% reduction of C. sonorensis in the enclosure nets at 3 and 7 days after treatment, but activity had subsided by 10 days after treatment. The utility of insecticidal treatments for suppression of this vector is discussed.

A plant-derived mosquito repellent, based on the oil of Eucalyptus maculata var. citriodora Hook, was evaluated against the biting midge Culicoides imicola Kieffer. Suction black light-traps covered with repellent-impregnated polyester mesh and deployed near horses attracted large numbers of C. imicola, which were seen near the treated net within a few minutes of the start of the experiment. Initial collections in the traps were approximately 3 times as large as those in control traps with untreated mesh. Numbers collected in treated traps were similar to untreated control traps after 4 h. Traps with mesh treated with DEET or another plant-derived (Meliaceae) proprietary product, AG1000, acted as repellents relative to the control. The differential activity of repellents against blood-feeding Diptera is discussed.

Carbon dioxide-baited suction trap collections were related to simultaneous bait cattle collections of Culicoides variipennis sonorensis Wirth & Jones. Trap collections varied directly with numbers taken from each of 3 calves. More (3.4x) C. v. sonorensis were taken in a trap with 1,000 ml CO2/min versus a trap baited with approximately 300 ml/min (matched to calves by weight). Numbers of insects taken from three individual calves relative to their equivalent CO2 output did not differ, but engorgement of C. v. sonorensis was higher on 1 calf compared with the other 2 calves. Up to 281 C. v. sonorensis fed on a calf exposed for 10 min. More male (7.7x) and female (6.1x) C. v. sonorensis were collected from the calf relative to its equivalent amount of CO2. Under conditions described here, female C. v. sonorensis caught by a suction trap multiplied by 7.2 approximates numbers expected to be caught near a calf. Engorgement of C. v. sonorensis on individual calves declined as abundance increased. Culex quinquefasciatus Say was collected in equal numbers from the calves and their equivalent CO2 output, and none fed on the cattle. Most (85%) C. v. sonorensis in the vicinity of the CO2-baited suction trap were collected in the catch bag, compared with 26% of Cx. quinquefasciatus.

The response of male Culicoides variipennis sonorensis Wirth & Jones to carbon dioxide (CO2) was examined at a dairy in the Chino Basin of southern California. Males were collected using CDC-type suction traps (no light) during 6 evenings in September and October 1995 when C. v. sonorensis were abundant. Traps were baited with 1.8 kg of dry ice, 200-350 nulliparous females, or nothing. Greater than 8 times as many males were captured in CO2-baited suction traps than in either female-baited or unbaited traps. Female-baited and unbaited traps did not differ in the number of males captured. Observations of mating behavior near and on a host calf were made at a nearby dairy. Males swarmed 1-2 m downwind (east) of a restrained calf and 0.3-1.0 m above ground level. Males were also observed coupled with blood-feeding females on the calf venter (especially umbilicus and teats). Virgin female C. v. sonorensis were captured in CO2-baited suction traps and by aspiration while they were engorging on the venter of a tethered calf. There appear to be at least two mating strategies in this species: mating presumably may occur near hosts in male swarms as well as stenogamously on the venter of the host. These mating strategies may serve as prezygotic reproductive isolating mechanisms between closely related and sometimes sympatric members of the C. variipennis complex.

Infectivity of the mermithid nematode Heleidomermis magnapapula, a parasite of Culicoides variipennis variipennis and C. v. sonorensis in dairy wastewater pond habitats, was tested against larvae of aquatic Diptera in the laboratory. Observations were made on preparasite penetration. If the host species could be reared after parasitization, further observations determined nematode development and emergence. Genera in the families Syrphidae (Eristalis), Psychodidae (Psychoda), and Chironomidae (Tanypus), common in wastewater pond habitats, were not attacked. Larvae of Bezzia and Dasyhelea (Ceratopogonidae) also were not attacked. Larvae of Chironomus (Chironomidae) were penetrated, but rapidly encapsulated the nematodes. All Culicoides spp. exposed were readily penetrated by H. magnapapula preparasites. The nematodes successfully emerged from C. v. occidentalis. Some nematode maturation was seen in C. lahontan, and limited adult nematode emergence was seen in C. boydi and C. cacticola. No nematode development was observed in 5 other Culicoides spp. penetrated.

Adult females of Leptoconops (Brachyconops) californiensis Wirth & Atchley and 1 female of Leptoconops (Holoconops) sp. near knowltoni were collected from 5 species of lizards in desert sand dune habitats in San Bernardino and Riverside counties, California, and northern Sonora, Mexico. Feeding by L. californiensis was observed on the dorsum (usually the heads) of the lizards, with up to 6 L. californiensis on a host at 1 time. The L. sp. near knowltoni appeared to be trying to feed but did not contain blood. Midges fed during calm, warm daylight periods from March through October. Reptile feeding by Ceratopogonidae is reviewed.

Improvements in rearing techniques have decreased maintenance needs and have increased the parasitism rate of larval Culicoides variipennis sonorensis Wirth & Jones by the mermithid nematode, Heleidomermis magnapapula Poinar & Mullens. In colony pans with 2,500-3,000 larval C. v. sonorensis, H. magnapapula adults emerged from 22.5% of hosts, with an average yield of 159 female H. magnapapula per pan. H. magnapapula females refrigerated for 1 wk showed no decline in preparasite infectivity compared with nonrefrigerated females. Female insemination did not differ with various mating combinations of 1 or 2 females with 1 or 2 males; 37-47% of these pairs failed to produce fertilized eggs. In 9-cm petri dishes, the optimal preparasite/host ratio was 900 infective preparasites (1 female's entire clutch) to 650-700 C. v. sonorensis larvae. Overall, parasite production was more time- and labor-efficient when larvae were parasitized in regular colony pans compared with petri dishes.

Two field populations of Culicoides variipennis (Coquillett) from southern California, C. v. occidentalis Wirth & Jones from the Salton Sea and C. v. sonorensis Wirth & Jones from a dairy wastewater pond in the Chino Basin, were sampled monthly from February to July (6-7 mo). Morphometric analyses of slide-mounted adults reared from field-collected larvae and pupae indicated that females of the 2 forms were indistinguishable. Two of the standard characters, wing length and mandibular teeth, were correlated with seasonal temperature changes. Males of C. v. sonorensis were distinguishable by the presence of spicules on the aedeagus, which were entirely lacking in C. v. occidentalis. Two populations of C. v. occidentalis (Salton Sea and Bolsa Chica Marsh) and a laboratory strain of C. v. sonorensis hybridized successfully in the laboratory and were maintained for 6 generations. Differential hybrid viability (F1) was observed in reciprocal crosses. Males of C. v. occidentalis mated with females of C. v. sonorensis resulted in a lower egg hatch (7.4%) than did the reciprocal cross (75.6%). Hybrid males displayed spicules on the aedeagus (a character of C. v. sonorensis), but the number of spicules was sometimes reduced compared with parental C. v. sonorensis (AA strain). Spicules in a field population of C. v. sonorensis were similar in number to the laboratory C. v. sonorensis-C. v. occidentalis hybrids. Based on successful hybridization, the 2 forms should be considered closely related. The 2 forms are separated ecologically by the nature and distribution of their larval habitats.

Adult culicoides variipennis sonorensis Wirth & Jones were tested using fluorescence spectrophotometry to determine whether age or sex influenced fluorescence. Males and nulliparous females 0.5, 2.5, and 5.5 d after eclosion were processed, as were uniparous and biparous females 14 d after eclosion. Variability among insects within a sex and age group was moderately high. Males and nulliparous females showed significantly greater fluorescence when very young (0.5 d old). Parous females had significantly lower levels of fluorescence compared with younger nulliparous females and males, but uniparous females could not be differentiated from biparous females of the same absolute age. Fluorescence as applied here appears to have limited utility as a tool for age determination in this vector species.

Adult Culicoides variipennis sonorensis Wirth & Jones were collected using miniature suction traps during 12 nights in October and November at a southern California dairy. Traps were baited with carbon dioxide at a constant release rate of 300, 1,000, or 3,000 ml/min or with 1.3 kg of dry ice. The release rate of CO2 from the dry ice in the laboratory declined from 1,486 ml/min during the first 2 h to 303 ml/min after 10-12 h. Nulliparous and parous females responded in a similar and positive manner to increasing CO2 release rates. An automatic interval suction trap baited with CO2 was used to assess diel host-seeking periodicity. Peak host-seeking activity occurred 0-3 h after sunset, when the trap collected up to 170 females or 103 males per hour. Average peak time of collection during the dusk period was 45 min after sunset. Although numbers were lower, parity was higher in the middle of the night. A smaller, secondary peak of activity occurred near sunrise.

Culicoides variipennis sonorensis females were fed bluetongue virus serotype 11 mixed in sheep blood and were held at constant temperatures of 32, 27, 21 and 15 degrees C. Virogenesis, as measured by enzyme-linked immunosorbent assay (ELISA), proceeded significantly faster at higher temperatures. Based on ELISA absorbance > or = 0.2, some flies first were categorized as infected after 1 day, 2 days and 4 days at 32, 27 and 21 degrees C, respectively. Peak levels of virus antigen were seen after 5-7, 7-13 and 18-22 days for flies held at 32, 27 and 21 degrees C, respectively. There was no significant virus replication in flies held at 15 degrees C for 22 days, but latent virus replicated and was detected easily (44% infection) 4-10 days after these flies were transferred to 27 degrees C. The implications for temperature effects on bluetongue epizootiology are discussed.

Colonized female Culicoides variipennis were fed through an artificial membrane on sheep blood meals containing 0-1.0 microgram/ml of ivermectin. Significant mortalities were present at 48 h postfeeding, with probit analysis yielding an LC50 of 0.35 microgram/ml and a slope of 4.12. All dosages caused significant decreases (P < 0.01) in egg production and sporadic decreases in egg hatch, with no eggs produced at 1.0 microgram/ml. There was a negative linear correlation (r2 = 0.91) between dose rate and number of eggs produced per living female. An inoculation of ivermectin at the recommended dosage of 200 micrograms/kg of body weight would not produce the serum concentration that could be expected to have noticeable effects on bloodfeeding C. variipennis.

Culicoides variipennis sonorensis Wirth & Jones was reared from egg to adult in petri dishes on a 0.5 or 1% agar substrate with bacterial-feeding nematodes (Pelodera, Panagrellus) as food. All larval stages except the first instar were observed attacking and feeding on nematodes. Developmental rate on agar was approximately 25% slower than that of larvae reared in pans of water with bacteria, fungi, and algae as food. Pupation success was approximately 75% from dishes (3.5 or 9 cm diameter) with 30 and 100 eggs, respectively. The technique has significant advantages for maintainance of small laboratory colonies and allows continuous observation of developing immatures for experimental studies.

Permethrin (65% AI) was applied to the dorsum of 3 goats (3-4 ml/animal). Hair clippings were taken before treatment and 4-6, 18-20, 39-41 and 67-69 days after treatment. Culicoides variipennis fed upward through a thin layer of hair and through a membrane on an artificial feeder containing cattle blood. Midge knockdown was usually complete after the 45 min exposure period, but many bloodfed first. Belly hair residues did not statistically reduce engorgement beyond 4-6 days. Head hair residues reduced engorgement by up to 98% at 18-20 days, but did not significantly reduce engorgement at 67-69 days. Back hair residues significantly reduced engorgement for the entire period (99-100% through day 39-41 and 66% by day 67-69). Partial engorgement was higher in the treatments.

Culicoides boydi new species is described from Riverside County, CA. A taxonomic key, table of numerical characters, and female wing photographs are presented to distinguish it from the three other species of the Culicoides pusillus species group of the subgenus Avaritia. Brief notes are presented on its biology and distribution and suspected vector potential in the transmission of bluetongue virus to desert bighorn sheep in California.

The ability of third and fourth instars of Culicoides variipennis (Coquillett) to survive in drying sandy loam soil was tested under simulated field conditions. Two hundred larvae were added to each of a number of soil-filled, plastic tubes, which were buried in the field and retrieved after 2, 5, and 7 d. Of 306 pupae or pupal exuviae recovered, 98.1% were in the top 2 cm of mud. Estimated larval mortality on day two ranged from 25.6 to 87.1% among three trials, and 92.3% of live larvae were in the top 2 cm. Estimated larval mortality on day 5 ranged from 81.4 to 97.2%, and 75.5% of live larvae were in the top 2 cm. On day 7 mortality was 95.2-100%, and 63% of surviving larvae were in the top 2 cm. Some larvae dispersed as deep as 7-10 cm (the maximum depth in the tests). Larval tolerance of unfavorable drying conditions may allow relic populations to persist in some situations, but probably this is of little concern in control of this species through water management in most settings.

Blood-feeding midges (Culicoides sp. and Leptoconops sp.) were sampled in the Santa Rosa Mountains, Riverside County, California (USA), to determine which species might be involved in the transmission of bluetongue and epizootic hemorrhagic disease viruses to peninsular bighorn sheep (Ovis canadensis cremnobates). Host-seeking midges were sampled with CO2-baited suction traps over a period of 30 mo. Nineteen species of Culicoides and seven of Leptoconops were collected. Five of the Culicoides sp. recovered are previously undescribed. The most abundant and widely distributed Culicoides sp. during spring (presumed virus transmission period to lambs) were C. (Selfia) brookmani, C. variipennis, C. (Avaritia) sp. (a new species near C. pusillus), and C. lahontan. Of these, C. brookmani (all elevations) and C. (Avaritia) sp. (elevations greater than 750 m) were common in the mountainous terrain inhabited by bighorn sheep. Culicoides variipennis, a vector of bluetongue virus in agricultural settings, and C. lahontan were numerous in sandy washes but were much less common in the mountains themselves. Leptoconops belkini and L. foulki were occasionally common in upper Deep Canyon in spring (April-June), while L. torrens was very abundant in the same area for 2 wk following heavy summer rains. Parity (an indicator of longevity and success in finding hosts and oviposition sites) in mountain areas was very low in C. variipennis (5%), low-moderate in C. (Avaritia) sp. (13%) and C. lahontan (21%), and relatively high in C. brookmani (40%). Vectorial capacity of Culicoides spp. for these hemorrhagic disease viruses is discussed, and it is suggested that species in addition to C. variipennis be considered as potential vectors of hemorrhagic disease viruses to desert bighorn sheep.

The duration of oogenesis and time to oviposition after blood feeding was studied in Culicoides variipennis held at constant laboratory temperatures. As assessed by daily dissection of ovaries, the time required for greater than or equal to 80% of females to complete egg development at 13, 17, 21, 27, 30 and 34 degrees C was 10, 5, 4, 3, 2 and 2 days, respectively. Mean times to oviposition at these temperatures were 13.8, 8.2, 4.9, 3.6, 3.0, and 2.6 days, while modal values were 13, 6, 4, 3, 3 and 3 days, respectively. Allowing 7 days after onset of oviposition in groups of 100-150 females, cumulative eggs laid/live female were 4.4, 62.6, 68.9, 63.5, 66.5 and 62.4, respectively. These data should prove helpful in estimating the duration of the gonotrophic cycle and deriving daily survivorship estimates from seasonal parity studies in the field.

The use of a time series analysis to estimate the survival rate and gonotrophic cycle length of Culicoides variipennis at 2 California sites is described. Collections were made daily for 28 days in Yolo County (northern California) and for 25 days in Riverside County (southern California) in July and August of 1989, respectively, using CO2-baited suction traps. The time series analysis of these collections yielded a gonotrophic cycle length estimate of 3 days. Stage-specific and daily survivorship estimates 0.242 and 0.623, respectively, were determined for the northern California site. The time series method was found unsuitable for estimating the gonotrophic cycle length or daily survivorship at the southern California site.