Artificial membrane system

The established laboratory colony of Cx. pipiens (Buckeye strain) was maintained as previously described [35 (link), 43 (link)]. Mosquitoes in the main colony were kept at 25°C, 75% relative humidity under a long-day photoperiod (16 h light: 8 h darkness). Larvae were provided dried fish food (Tetramin; Blacksburg, VA USA). Adults were provided unlimited access to 10% sucrose solution. Chicken blood (Pel-freez Biologicals; Rogers, AR, USA) was provided using an artificial membrane system (Hemotek; Lancashire, UK) approximately 10 days post-adult emergence, and egg rafts were collected approximately 5 days later. To generate diapausing adults, larvae and pupae were held at 18°C, 75% relative humidity under a short-day photoperiod (8 h light: 16 h darkness). Diapausing adults had access to sugar water for the first 10 days of adult life and then sugar water was removed to simulate the lack of food in their natural environment. Nondiapausing adults used in these studies were generated by rearing all life stages under a diapause-averting, long-day photoperiod (L:D 16:8) with constant access to sugar water. Both diapausing and nondiapausing mosquitoes were reared at the same low temperature (18°C) to ensure that diapause status, and not temperature, was the only factor impacting egg follicle development, lipid accumulation and miRNA expression.

All four insect species were also fed on two ex vivo systems. The first was an artificial membrane system (Hemotek, United Kingdom) containing viremic blood collected from the clinically affected donor calf. The Hemotek was composed of a 3-mL metal reservoir holding the viremic blood and covered with a parafilm membrane which acted as a feeding surface for the insects. A heating unit ensured that the test blood remained at 37.4 to 38.0°C. Venous blood was collected from the jugular vein into tubes containing EDTA on 9 and 10 dpi (during early viremia) and on 11 and 12 dpi (peak viremia) before being loaded into the Hemotek reservoirs. The second ex vivo system incorporated skin lesions sourced from calves with clinical LSD into the Hemotek reservoirs. Samples of cutaneous nodules were collected postmortem from the calves with LSD and stored at −80°C. Thin slices of the cutaneous nodules were then generated using a DermaBlade Shave Biopsy Instrument (Verona, VA) and layered between two sheets of parafilm. This was then placed over a Hemotek containing defibrinated horse blood (TCS Biosciences Ltd.). Once feeding was complete, the skin was collected, cut into pieces, and homogenized in 1 mL high-glucose Dulbecco’s modified Eagle’s medium (DMEM) and LSDV genomic DNA was quantified.

The Cincinnati SRL strain of Cimex lectularius was used in the present study. This strain is derived from a population of individuals collected by technicians from Sierra Research Laboratories, Inc. (Modesto, CA, USA) in 2007 and has been maintained under laboratory conditions since this time. Colonies were maintained at the University of South Dakota in plastic jars containing corrugated cardboard harborages at 28 ± 1 °C and 60–70% relative humidity on a 12:12 photoperiod. The colonies were fed aseptically collected defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA, USA) once per week using an artificial membrane system (Hemotek Ltd., Blackburn, United Kingdom).