Fig. 4

Expression of LL3 varies among different molecular forms of An. gambiae. A PCR-RFLP was performed to identify the molecular forms (M and S) that distinguish An. gambiae strains used in this study (X1, TEP1 mutant, G3, and Keele strains) as well as a known M-form strain (Ngousso) of An. coluzzii. DNA was prepared from individual mosquito samples for each strain and displayed consistent RFLP patterns for all mosquitoes analyzed. Individuals from each strain were used to create a representative image of the RFLP patterns for each mosquito strain (a). The X1, TEP1 mutant, and G3 strains display a hybrid pattern (M/S) of three bands (367, 257 and 110 bp), while the Keele and Ngousso strains are M molecular form (367 bp). Relative LL3 expression in the Keele strain is higher than those of the X1 and G3 strains at 24 h P. berghei infection (b), while there is no difference of STAT-A expression among mosquito strains (c). To examine the effects of gene-silencing on hemocyte differentiation, the proportion of granulocytes (out of total cell population) was examined in individual mosquitoes 4 days post-infection with P. berghei. The percentage of granulocytes were measured in dsGFP-, dsLL3-, or dsSTAT-A-treated mosquitoes in both the G3 (d) and Keele (e) strains. STAT-A-silencing abrogated hemocyte differentiation in both G3 and Keele mosquitoes (d) and (e), whereas LL3-silencing only influenced the Keele strain (e). Gene expression data were analyzed with a one-way ANOVA and Tukey post-hoc test, while granulocyte percentages were evaluated by Kruskal-Wallis with a Dunn’s post-hoc test. Asterisks denote significance (*P < 0.05, **P < 0.01, ***P < 0.001); Abbreviation: ns, not significant. Three independent biological experiments were performed for all experiments