Bluetongue virus (BTV) isolate BTV-4 MOR2009/07 (cell passage KC1), was obtained from the Orbivirus Reference Collection (ORC) at The Pirbright Institute, UK (https://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-4.htm#MOR2009/07, accessed on 13th June 2023) and was previously shown to infect Culicoides sonorensis at high rates [36]. Working stocks of the virus were generated by two additional propagations on Culicoides-derived KC cells as previously described [38] and kept at + 4 °C. Infectivity of the viral working stocks was determined by fluorescent Tissue Culture Infective Dose 50% (TCID50) in KC cells as previously described [38].

Adults of both Culicoides sonorensis Wirth & Jones 1957 (PIRB -s-3 strain), and Culicoides nubeculosus Meigen 1830 (PIRB strain) from colonies held at The Pirbright Institute were used. Maintenance was as described previously [2], with the exception that the colonies were sustained by blood feeding through artificial membranes (Parafilm ™) over a heated reservoir (Hemotek, UK) filled with defibrinated horse blood from a commercial supplier (TCS Bioscience, Botolph Claydon, UK).

Both Culicoides species were exposed to BTV by two routes, oral infection via a virus-infected blood meal or intrathoracic inoculation (ITI). For oral infection, approximately 500 adult C. sonorensis at 3 days post eclosion were exposed to a 3:1 horse blood:BTV tissue culture mixture at a calculated titre of 7.4 log10 TCID50/ml using a reservoir and heating unit (Hemotek, UK) at 37 °C and a Parafilm™ membrane. After 30 min exposure to the blood meal, individuals were immobilised under light anaesthesia with CO2. 150 fully engorged, blood-fed females were transferred into a cardboard pillbox (Watkins and Doncaster, Leominster, UK) and incubated in the dark at 25 °C, 80% relative humidity (RH) and fed ad libitum with 10% sucrose on a cotton pad refreshed daily for a period of 8 or 15 days, depending on the experiment. Additionally, 3-day-post eclosion adult female Culicoides were intrathoracically (IT) inoculated with BTV-4. 50 Culicoides were inoculated with ≤ 0.2 µl 6.4 log10 TCID50/ml BTV-4 MOR2009/07 using a pulled glass needle and Nanoject II microinjector (Drummond Scientific, NJ, USA) under light CO2 anesthesia. Inoculation site was either under the dorsal mesonotum or laterally between thoracic plates above the legs depending on individual presentation. Inoculated individuals were transferred to a cardboard pill box and incubated as for membrane-fed Culicoides for 5 or 8 days. For both oral and ITI treatments, post incubation surviving insects were killed by immersion into DPBS (Gibco™, Life Technologies, Inchinnan, UK) + 0.05% Tween® 20 (Sigma-Aldrich®, Gillingham, UK) for 20 min immediately prior to dissection. Mock-infected individuals were treated as above with oral-fed controls receiving a blood meal with no virus via Hemotek and ITI controls inoculated with the same volume of Schneider’s Drosophila media (Gibco™, Life Technologies, Inchinnan, UK) without virus.

Dissection was carried out under a stereomicroscope (Leica MZ60) using sterile 25G hypodermic needles for each individual (Fisher Scientific, Loughborough, UK). Individual Culicoides were placed in single drop of DPBS (Gibco™, Life Technologies, Inchinnan, UK) + 0.05% Tween® 20 (Sigma-Aldrich®, Gillingham, UK). The head was carefully removed, keeping the salivary glands attached, and transferred using the needle to a well in a 96-well flat-bottom microplate with 200 µl DPBS + 0.05% Tween 20. The DPBS was aspirated from the well using a pipette and discarded. 200 µl of 4% paraformaldehyde (PFA) in PBS (Thermo Scientific Chemicals, Inchinnan, UK) was added to each sample and the sample incubated for one hour at room temperature. After fixation, the 4% PFA was removed by aspiration and the samples rinsed with DPBS three times. Samples were stored in DPBS at + 4 °C until immunolabelling. The remaining body of each individual was placed in a sample 96-microtube plate (Qiagen, Manchester, UK) containing 200 µl RPMI (Gibco™, Life Technologies, Inchinnan, UK) + 2% penicillin / streptomycin (Gibco™, Life Technologies, Inchinnan, UK) + 2% amphotericin B (Sigma-Aldrich®, Gillingham, UK) and a 3 mm stainless steel bead (Dejay Distribution Limited Ltd, Launcestron, UK) for homogenisation. Plates were sealed with caps and bodies were homogenised in a Tissue Lyser (Qiagen, Manchester, UK) [41]. Homogenates were then topped up with RPMI + 2% penicillin/ streptomycin + amphotericin B to 1 mL and stored at + 4 °C until RNA extraction.

Dissected and fixed salivary apparatus-head combinations were immunolabelled in 96-well flat-bottom microtiter plates for cellular tubulin, BTV structural proteins (VSPs) and/or the BTV non-structural protein 2 (NS2). Briefly, using a single channel 200 µl pipette, DPBS (Gibco™, Life Technologies, Inchinnan, UK) was removed, and tissue permeabilization was carried out by adding 200 µl of 0.5% Triton X-100 (Sigma-Aldrich®, Gillingham, UK) / DPBS magnesium (Mg) and calcium (Ca) free for 15 min. Triton was then removed as above followed by three washes with 200 µl of DPBS (Mg and Ca free) and a one-hour blocking step with 200 µl of blocking buffer (1% bovine serum albumin (Sigma-Aldrich®, Gillingham, UK) / 0.2% normal goat serum (Sigma-Aldrich®, Gillingham, UK) / DPBS Mg and Ca free. Next, blocking buffer was removed and 200 µl of a cocktail of specific primary antibodies (Table 2) diluted in blocking buffer was added. After 90 min of incubation at room temperature, primary antibodies were removed, organs washed three more times with DPBS as above, and 200 µl of appropriate secondary antibody cocktail (Table 2) diluted in blocking buffer was added and incubated at room temperature in the dark for 90 min. Next, organs were washed three times with DPBS. Where cell nuclei were to be stained, 40, 6-Diamidino-2-phenylindole (DAPI) (Life Technologies Limited, Paisley, UK) was added at this point and incubated for 30 min at the manufacturer’s recommended dilution, followed by three washes with ultra-pure water.

To prevent destruction of organ structures, a gene-frame (25 µl, 10 mm x 10 mm; ThermoFisher Scientific, Loughborough, UK) was placed on glass microscope slides and filled with 30 to 40 µl of Vectashield® Hardset Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Fine forceps were used to place the samples on the microscope slide. Using a stereomicroscope (Leica MZ60), positioning of the sample was assessed and corrected if needed. Finally, a square glass coverslip was glued to the gene-frame and organs were either visualized immediately or stored at + 4 °C for a maximum of three days before being imaged. Organs of mock-infected and dissected C. sonorensis were included as immunofluorescence background controls in all experiments (Fig. 11). Samples were imaged using a Leica SP8 CLSM confocal microscope (Leica Microsystems, Wetzlar, Germany).

Representative images of immunolabelling background controls. A. Salivary apparatus of a C. sonorensis orally fed on BTV-spiked blood, but BTV negative. B. Salivary apparatus of a C. sonorensis fed on mock blood and, C. Salivary apparatus of a C. sonorensis mock infected by intrathoracic inoculation. In A and B cell nuclei are shown in blue (DAPI staining), and cellular tubulin is visualised in red (labelling with mouse anti-tubulin, and anti-mouse IgG AlexaFluor™ 568). Absence of green denotes absence of BTV structural proteins (VSPs) which were labelled with in-house antibody Orab279 and anti-guinea pig IgG AlexaFluor™ 488. In panel C, cellular tubulin is shown in blue (labelling with mouse anti-tubulin, and anti-mouse IgG AlexaFluor™ 405). Absence of red or green denotes absence of BTV non-structural protein 2 (NS2) or BTV VSPs, respectively. Viral protein NS2 was labelled with in-house rabbit antibody Orab1 and anti-rabbit IgG AlexaFluor™ 568; and viral VSPs were labelled with Orab279 followed by anti-guinea pig IgG AlexaFluor™ 488 as in panels A and B. In A, one main salivary gland (SG) with its respective four accessory glands (1 to 4) and the salivary duct (arrow) can be observed. All panels are maximum projections of at least 50 stacked plane images. Microscope magnification is shown on the top right corner and the scale bar represents 20 µm

Total RNA was extracted from 100 µl of each insect homogenate using the KingFisher Flex robotic extraction system (ThermoFisher Scientific, Loughborough, UK) using the MagMAX™ CORE Nucleic Acid purification kit (ThermoFisher Scientific, Loughborough, UK) kit as per manufacturer’s instructions. Six microlitres of each extracted RNA was tested by qRT-PCR targeting Segment 10 (Seg-10) of BTV as described by [15], but adapted for the SuperScript III platinum one-step qRT-PCR (Invitrogen™, Life Technologies, Inchinnan, UK). Viral genome copies were quantified using a 10-fold dilution series of BTV-1 Seg-10 RNA transcript as standard [33]. qRT-PCR results were plotted using GraphPad Prism software version 9.3.1 (GraphPad Software, San Diego, USA).