Trap placement and attractant choice affect capture and create sex and parity biases in collections of the biting midge, Culicoides sonorensis.

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.

Lack of Evidence for Laboratory and Natural Vertical Transmission of Bluetongue Virus in Culicoides sonorensis (Diptera: Ceratopogonidae).

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.

Acidification of calf bedding reduces fly development and bacterial abundance.

Environmental stressors, such as high fly density, can affect calf well-being. Sodium bisulfate (SBS) is an acidifier that reduces the pH of flooring and bedding, creating a medium that neither bacteria nor immature flies (also known as larvae or maggots) can thrive in. Two experiments were conducted to investigate the application of SBS to a mixture of rice hull calf bedding and calf slurry (BED) to reduce house fly (Musca domestica L.) larval density and the abundance of bacteria. In experiment 1, dish pans containing 1L of BED and 3,000 house fly eggs were treated with SBS at concentrations of 0, 8.9, 17.7, and 26.5g of SBS/0.05m(2) of BED (CON, LOW, MED, and HIGH, respectively), with each SBS concentration applied to 4 individual pans (16 pans total). Reapplication of the same SBS concentrations in each pan occurred 3 times/wk throughout the 23-d trial. Larval house fly survival was significantly reduced in all pans with SBS relative to CON pans, with lowest survival rates in the MED and HIGH pans (99% and 100% reduction, respectively). The mean pH for each treatment was inversely related to the SBS concentration. In experiment 2, pans containing 1L of BED and 3,000 house fly eggs were treated with either 0g of SBS (CON), 8.9g of SBS/0.05m(2) of BED with reapplication of the acidifier 3 times/wk (SB3x), or 8.9g of SBS/0.05m(2) of BED applied only once at 48h before the end of the 8 d-trial (SB48). Larval house fly survival and bacterial concentrations were reduced (90% larval reduction and 68% bacterial reduction) in the SB3x treatment relative to the CON. Mean pH was also reduced in SB3x pans relative to CON or SB48 pans. Overall, acidification of calf BED using the acidifier SBS resulted in a reduction of bacteria and house fly larval survival. This form of fly control might be expected to reduce adult fly production and, therefore, fly-related stress in calves.

Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies (Diptera: Muscidae).

The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7-specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.

Seasonal abundance and survivorship of Culicoides sonorensis (Diptera: Ceratopogonidae) at a southern California dairy, with reference to potential bluetongue virus transmission and persistence.

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.

Response of male Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) to carbon dioxide and observations of mating behavior on and near cattle.

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.

Comparison of bait cattle and carbon dioxide-baited suction traps for collecting Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) and Culex quinquefasciatus (Diptera: Culicidae).

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.

Failure of a permethrin treatment regime to protect cattle against bluetongue virus.

Holstein heifers in a confined feedlot setting on a southern California dairy were either sprayed individually along the ventral midline using 0.2% permethrin (250 ml/animal) (two pens) or were not treated (two pens). Treatments (n = 6 dates) were applied every 2 wk during the peak fall bluetongue virus transmission season (22 August-29 October). Animals seronegative for bluetongue virus antibodies at the initial bleeding on 15-18 September (n - 106 in the treatment pens and n = 117 in the control pens) were bled again for testing 2 mo later (12-13 November). Seroconversion rates were not significantly different: 56% for the treated animals and 48% for the controls (P > 0.2). The area has many essentially contiguous, confinement dairies with wastewater ponds that produce large numbers of Culicoides sonorensis Wirth & Jones, the primary bluetongue virus vector. Further, these dairies presumably provided a large reservoir of virus-infected cattle to infect vectors in the immediate area. Under these severe virus challenge conditions, permethrin applied at 2-wk intervals failed to reduce exposure to bluetongue virus.

Seasonal transmission of bluetongue virus by Culicoides sonorensis (Diptera: Ceratopogonidae) at a southern California dairy and evaluation of vectorial capacity as a predictor of bluetongue virus transmission.

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.

Environmental effects on vector competence and virogenesis of bluetongue virus in Culicoides: interpreting laboratory data in a field context.

Environmental factors profoundly affect vectorial capacity, governing dynamics and intensity of vector-vertebrate contact in time and space (e.g. seasonal vector population densities, biting rates, and feeding frequencies). Temperature influences vector developmental rates and life history parameters, and may modify vector competence. Studies should move iteratively from field to laboratory, as attempts are made to understand complex epidemiological patterns. Simulation models can be extremely helpful in identifying and predicting geographic and seasonal trends in virus occurrence. Field and laboratory data from the Culicoides sonorensis-bluetongue virus system in North America are incorporated into preliminary estimates of virus prevalence and geographic occurrence along a latitudinal (and temperature) gradient. Geographic information systems technology is likely to be helpful in understanding vector and virus occurrence on a broader scale, especially in temperate latitudes that typify sporadic or emerging transmission zones, areas of particular concern for animal movement.

Feeding and survival of Culicoides sonorensis on cattle treated with permethrin or pirimiphos-methyl.

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.