Figure 5.
Highly acidic and highly alkaline conditions found in the midgut can affect DCV infectivity: (A) upon dissection of L1, L2, and L3 larvae, the m-cresol purple ingested shifts to a red/orange hue in the middle midgut indicating a pH between 1.2 and 2.5 (pointer a), and to a brown/purple hue towards the end of the posterior midgut, close to the Malpighian tubules, indicating a pH > 9.0 (pointer b). These colours fade towards yellow rapidly after dissection, as the gut is neutralised when exposed to the glycerol-PBS. Hence, colours depicted do not accurately reflect those observed in vivo or during dissection; saturation was increased for illustrative purposes. In contrast, the guts of L0 did not display the red or purple colouration of m-cresol purple at acid (pH < 2.8) or alkaline (pH > 8.5) conditions, respectively, either before or after dissection (data not shown [48]). (B) Aliquots of purified DCV (stock at 2.5 × 109 = 109.4 IU/mL) were incubated under acidic-to-neutral (citrate; left red circles), neutral (PB control; centre grey circles), or neutral-to-alkaline (bicarbonate; right blue circles) buffers for 15 min at room temperature and used to infect Drosophila S2 cells. DCV titre was calculated by TCID50, and normalised using log-transformation. Each condition was compared to the PB-treated control (in which the purified DCV aliquots were maintained) using Šídák’s multiple comparisons test. Compared to the PB pH 7.2 treatment, highly acidic conditions (Citrate, pH 1.0) decreased the viral titre by 2 orders of magnitude (mean, 107.37 vs. 109.39 IU/mL), whereas highly alkaline conditions (bicarbonate, pH = 12.0) decreased the viral titre by 3 orders of magnitude (mean, 106.30 vs. 109.39 IU/mL) (p < 0.0001 for both comparisons). Acidic conditions found within the middle midgut (pH 1–2) may significantly decrease the DCV infectivity, whereas alkaline conditions similar to those found in the posterior midgut (pH 7.5–10.5) are likely not to affect the stability or the remaining infectious viruses.