R0561

Culicoides midges were collected from 17 sites across the United States and Canada (Table 1). Specimens were collected either as pupae and reared to adulthood, or as adults using CDC light traps baited with CO₂ and UV light (Bioquip 2836BQ). Individuals morphologically assigned to the C. variipennis complex were sorted out from the by-catch and stored in 95% ethanol at -80 °C. Total DNA was extracted from individuals using a Puregene extraction protocol (Gentra Systems, Inc., D-5500A) with the addition of glycogen (ThermoFisher, R0561) to increase yields. DNA was only extracted only from females as their larger body size (compared to the males) produced sufficient amount of DNA for next-gen sequencing. The DNA quality was checked using gel electrophoresis and DNA concentration was measured using a Qubit 3.0 fluorometer and a Qubit dsDNA HS assay kit (Invitrogen, Q33230). A total of 300–400 ng of DNA per sample was sent to Floragenex, Inc. for library preparation using the protocol from Truong et al. (2012). DNA was digested using the restriction enzymes MseI and PstI. After PCR amplification, the samples in each plate were pooled and sequenced on a lane of single-end 100 bp sequencing on a HiSeq4000 at the University of Oregon Genomics Facility, Eugene, OR.

Conditioned media samples were mixed with twice their volumes of cold methanol and 2 μg of glycogen (molecular biology grade, catalog no. R0561, Thermo Scientific), vortexed, and incubated overnight at 4 °C. Proteins were precipitated by centrifugation at 12000 rpm for 10 min at 4 °C. The supernatant was carefully decanted without dislodging the protein pellet. The pellets were air-dried in a chemical hood for 20–30 min, then mixed with 2X Laemmli sample buffer containing 6 M urea, vortexed, and boiled at 95 °C for 5 min prior to gel loading.