Fig. 1
Protocol schematic to study arboviral infection in the salivary apparatus of Culicoides spp. 1. Female C. sonorensis Wirth & Jones 1957 (PIRB -s-3 strain), or C. nubeculosus Meigen 1830 (PIRB strain) were infected by either membrane feeding on horse blood containing 7.4 log10 TCID50/mL of BTV-4 MOR2009/07 (BTV-4) or by intrathoracic inoculation with ≤ 0.2 µL at 6.4 log10 TCID50/mL of BTV-4. Female insects were selected and incubated between 5 and 15 days according to experiment (see “Results” section). 2. After incubation, insects were anesthetized with CO2, and heads with the salivary apparatus (SA) still attached were dissected (and any other soft organs of interest). 3. Dissected SA glands were then fixed with 4% paraformaldehyde, permeabilised with 0.5% Triton X-100/PBS and labelled for cellular tubulin (mouse anti-tubulin, from Sigma-Aldrich as primary antibody (ab) and anti-mouse IgG AlexaFluor™ 405 or 568, from Invitrogen as secondary ab), viral structural proteins (guinea pig anti-BTV structural proteins Orab279, from in-house, as primary ab, and anti-guinea pig IgG AlexaFluor™ 488, as secondary ab) and/or viral non-structural protein NS2 (rabbit anti-NS2 Orab1, from in-house, as primary ab, and anti-rabbit IgG AlexaFluor™ 568, as secondary ab), and cell nuclei stained with 6-Diamidino-2-phenylindole (DAPI, from Life Technologies). Labelled and stained salivary apparatus with the head still attached were mounted on a microscope slide within a gene-frame (25 µL, 10 mm x 10 mm; Thermo Scientific™) containing Vectashield® Hardset Mounting Medium (Vector Laboratories). 4. Samples were then imaged using a Leica SP8 CLSM confocal microscope (Leica Microsystems, Wetzlar, Germany), and analyzed and exported using the Leica Application Suite X. 5. Matching bodies to heads and SA, were homogenized using a Tissue Lyzer (Qiagen), and 6, viral RNA was extracted using the MagMAX™ CORE Nucleic Acid purification kit and a KingFisher Flex extraction robot (ThermoFisher Scientific). Bluetongue virus RNA was detected using a Segment-10 BTV serogroup RT-qPCR assay (adapted from [15])