Richard A. Nunamaker

Absence of transovarial transmission of bluetongue virus in Culicoides variipennis: Immunogold labelling of bluetongue virus antigen in developing oocytes from Culicoides variipennis (Coquillett)

1. Culicoides variipennis midges were fed on a blood meal containing bluetongue virus (BTV) serotype 11 (BTV-11) and on four subsequent non-infective blood meals at 4-day intervals. 2. Eggs were collected before each blood-feeding and reared to adults. 3. Progeny from each egg batch were incubated for 14 days (20 degrees C, 40-60% RH) before plaque assay. 4. Oocytes from several parent flies were sectioned for immunoelectron microscopy. 5. Thirty-two percent of the parent females tested by plaque assay were positive for BTV. 6. All 993 progeny flies were negative for BTV. 7. BTV antigen was dense in proteid yolk bodies and in the vitelline membrane of the developing oocytes.

Grasshoppers (Orthoptera: Acrididae) Could Serve as Reservoirs and Vectors of Vesicular Stomatitis Virus

Abstract Vesicular stomatitis (VS) is an economically devastating disease of livestock in the Americas. Despite strong circumstantial evidence for the role of arthropods in epizootics, no hematophagous vector explains the field evidence. Based on the spatiotemporal association of grasshopper outbreaks and VS epizootics, we investigated the potential role of these insects as vectors and reservoirs of the disease. The critical steps in the grasshopper–bovine transmission cycle were demonstrated, including 1) 62% of grasshoppers [Melanoplus sanguinipes (F.)] fed vesicular stomatitis virus (VSV) from cell culture became infected, with titers reaching 40,000 times the inoculative dose; 2) 40% of grasshoppers that cannibalized VSV-infected grasshopper cadavers became infected, amplifying virus up to 1,000-fold; 3) one of three cattle consuming VSV-infected grasshopper cadavers contracted typical VS and shed virus in saliva; and 4) 15% of grasshoppers became infected when fed saliva from this infected cow. The ecological conditions and biological processes necessary for these transmissions to occur are present throughout much of the Americas. Field studies will be required to show these findings are relevant to the natural epidemiology of VSV.

Stress proteins elicited by cold shock in the biting midge, Culicoides variipennis sonorensis wirth and jones

Abstract In vivo protein expression in the abdominal viscera of C. v. sonorensis was examined from adult flies that were cold shocked for various lengths of time at 0, -10, or -15°C and labelled at 25°C with 35 S-methionine at 0, 2, 4 and 6 hr during the recovery period. In vitro labelling showed that seven unique proteins (23, 40, 43, 48, 60, 70 and 92 kDa) were produced in C. v. sonorensis exposed to low temperatures in vivo . In general, the rate of expression and quality of stress proteins were directly proportional to both the severity and duration of the cold shock. A polyclonal antibody to the moth hsp 60/63 crossreacted with antigen from the viscera of the 60 kDa protein that was expressed during recovery from cold shock. This crossreaction with C. v. sonorensis suggests that the 60 kDa protein expressed during recovery from cold shock in the midge is immunologically related to the moth heat shock protein (hsp). Weather records from central Wyoming suggest that if the stress proteins produced by C. v. sonorensis enhance survival of the earliest, normally lethal temperatures (e.g., -5°C), populations of these insects can persist for an additional 20–30 d in an average year and thus extend the time they can transmit bluetongue virus.

Oral Infection of Culicoides sonorensis (Diptera: Ceratopogonidae) by Vesicular Stomatitis Virus

Abstract Vesicular stomatitis virus serotype New Jersey (VSNJV) was mixed with bovine blood or fetal bovine serum (FBS) and fed across silicone membranes to laboratory populations of Culicoides sonorensis Wirth & Jones. In an initial study, virus was detected after 13 d in 21% of the midges that received an FBS/VSNJV mixture. In subsequent time-course experiments, engorged females were collected and maintained at 20.0°C and assayed for VSNJV immediately after feeding and at 1, 3, 7, 10 and 13 d after feeding. Virus was detected after 13 d in 3% of the midges that received a bovine blood/VSNJV mixture and in 9% of the midges that received an FBS/VSNJV mixture. The results indicate that C. sonorensis should be considered as a potential biological vector of VSNJV.

Cryopreservation of embryos of Culicoides sonorensis (Diptera: Ceratopogonidae).

Abstract The successful long-term storage of insects that are used for research purposes can eliminate the need for ongoing colony maintenance on a large scale. In addition, rare and valuable genotypes of insects can be preserved. This study was conducted to determine whether cryopreservation is a suitable means of storing embryos of Culicoides sonorensis Wirth & Jones, an important vector of animal pathogens. We determined that eggs of C. sonorensis can withstand vigorous treatments of dechorionation, permeabilization, and loading with the cryoprotectant, elthylene glycol. Although their viability was reduced, an average of 80.3% of the embryos developed into larvae. Dehydration in vitrification solution caused a much greater reduction in egg viability (42.7% survival), and freezing in liquid propane further reduced the number of eggs that developed into larvae (40.1%), pupae (22.9%) and adults (18.8%). This work demonstrated that this procedure may prove useful for the cryopreservation of standard laboratory colonies and genetic lines of C. sonorensis.

Cuticular Hydrocarbons of Glacially-Preserved Melanoplus (Orthoptera: Acrididae): Identification and Comparison with Hydrocarbons of M. sanguinipes and M. spretus

-The cuticular hydrocarbons of four groups of grasshoppers were identified and quantified by capillary column gas-liquid chromatography (GLC) and GLC-mass spectrometry (GLC-MS), including samples of: 1) a modern species, M. sanguinipes (Fabricius), 2) a morphologically similar extinct species, M. spretus Walsh, 3) 650year old fragments recovered from a receding glacier in southwestern Montana, and 4) 140-year old whole bodies from a glacier in northwestern Wyoming. Quantitative data from 20 n-alkanes and methyland dimethyl-branched alkanes were compared using discriminant analysis, which showed differences in cuticular hydrocarbon patterns between the putative species M. sanguinipes and M. spretus. The resulting discriminant model classified the glacially preserved whole bodies as M. spretus, but the identity of the fragments remains ambiguous [

Identification and characterization of a cDNA clone encoding the heat shock protein (Hsp60) from the biting midge, Culicoides variipennis sonorensis Wirth and Jones.

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67–78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.

Preserved Grasshopper Fauna Of Knife Point Glacier, Fremont County, Wyoming, U.S.A.

In 1987 and 1988, samples of preserved insects were extracted from the ice of Knife Point Glacier, Fremont County, Wyoming. The glacier lies at an altitude of 3500 m a.s.l. in the Shoshone National Forest, Wind River Range, and is known to contain preserved insects. Although the glacier has undergone extensive recession in the last 50 yr, some insect deposits are still embedded at 20 to 25 cm below the surface and perhaps much deeper. The frozen deposits appear to consist entirely of grasshoppers. A few, virtually intact, specimens and body parts were in a state of preservation that allowed their identification as Spharagemon campestris McNeill and Melanoplus spretus (Walsh) or M. sanguinipes (F.). The majority of the deposits consisted of partial bodies and isolated parts, including, in order of frequency: mandibles, tibiae, tentoria, femora, wings (primarily tegmina), and cingulae/epiphalli. Deposits from various depths and locations on the glacier were radiocarbon dated at 205 + 65 to 450 + 80 yr BP. Although access to the glacier is quite difficult, the insects are better preserved than any glacial deposit documented in recent history. Thus, the state of preservation and age of

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a Mr of ∼24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%–74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of ∼28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.