Figure 1.
Schematic of cell death and immune signaling pathways in Aedes aegypti.
Panel A. In insects, the primary apoptotic caspase is AeDronc, a caspase-9 homologue with an N-terminal CARD domain for interactions with the caspase adaptor molecule AeARK. AeDronc activation is primarily regulated by the IAP antagonist proteins, Michelob_X and IMP. Together, the IAP antagonists promote cell death by binding to the Inhibitor of Apoptosis Protein, AeIAP1. Once activated, AeDronc will cleave and activate the effector caspase caspase-16. Panel B. AeDredd is a death domain containing caspase that contains two death-inducing domains that interact with the caspase adaptor protein, AeFADD (Aedes Fas Associated Death Domain containing protein). Both Drosophila Dredd and AeDredd were isolated initially as potential inducers of apoptosis. Although apoptotic roles for Dredd have not been ruled out, data suggests that the primary role of Dredd is immune-related.
Figure 2.
Within Strain Comparisons: Relative expression of Argonaute-2, AeIAP1, caspase-16, Aedronc and Aedredd in the midguts of Aedes aegypti in strains that are Refractory (Cali-MIB: Left Panel) or Susceptible (Cali-S: Right Panel) to Dengue virus in the presence (black bars) or absence (white bars) of Dengue virus-2 in the bloodmeal.
The expression levels in the bloodfed samples were arbitrarily set at 1 and the expression levels in the presence of the virus represent fold-differences from the susceptible controls. The bars were geometrically adjusted (2−ΔCT)+SD.-2−ΔCT.
Figure 3.
Between Strain Comparisons: Differential expression of Argonaute-2, AeIAP1, Caspase-16, Aedronc, and Aedredd in the midguts of Resistant Cali-MIB (black bars) and Susceptible Cali-S mosquitoes (white bars) at the same timepoints after exposure to a bloodmeal containing DENv-2 (top panel) or blood alone (bottom panel).
In each pairwise comparison the expression level in the Cali-S strain was arbitrarily set at 1 and the expression levels in Cali-MIB strain represents fold-differences in expression within that pair. The bars are geometric adjusted (2−ΔCT)+SD.
Table 1.
Gene expression ratios comparing midgut gene expression of Argonaute-2, AeIAP1, Caspase-16, Aedronc, and Aedredd over time, compared with time zero (ΔCt), in Cali-S and Cali-MIB strains exposed to blood or blood+DENv-2, as well as between-strain ratios.
Figure 4.
Effects of RNAi knockdown of Aedronc, Caspase-16 or Nautilus on the development of DENv in the refractory Cali-MIB strain of Ae. aegypti.
The expected proportions in the Cali-MIB colony (53% Susceptible: 47% Resistant) were maintained in naïve and Nautilus injected insects. Knockdown of Caspase-16 and Aedronc using RNAi increased the proportion of susceptible mosquitoes to 62% in Caspase 16 Kd insects (Chi2 = = 0.39; p = 0.53) and 78.6% in the Aedronc Kd insects (Chi2 = 3.9; p = 0.03). The numbers in brackets above each pair of bars indicates the # of mosquitoes in 3–5 replicates on which these summaries are based. The * indicates significant difference between Aedronc knockdown and naïve and Nautilus injected controls.