A pair of intact salivary glands dissected from 10-day old male G. pallidipes exhibiting overt salivary gland hypertrophy symptoms were used to prepare the virus inoculum as previously described (Boucias et al., 2013) with slight modifications including aseptic salivary gland dissections and use of non-filtered virus inoculum.

Virus injection was conducted in the different tsetse species as previously described by Demirbas-Uzel et al. (2018b). In brief, teneral flies were immobilized in the chiller (2–6°C; 5 min) and injected (intra-hemocoelic) with either 2 μl sterile phosphate buffered saline (PBS) or GpSGHV virus suspension. For each species, 160 flies (1:3 male:female) were injected and placed in standard holding cages at a density of 45–80 flies per cage. The experiments were conducted in 2–3 replicates for each species. After the injections, eight flies (six females and two males) were randomly selected and sampled from both virus and PBS injected fly groups at 0-, 1-, 5-, and 9-days post injection (dpi), and subsequently frozen at −20°C until further analysis. Virus density variations were measured separately for both females and males at different dpi.

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