The Susceptibility of Late Plasmodium Sporogonic Stages to the Anopheles Gambiae Melanization Response, and Functional Characterization of the Toll/Rel1 Pathway in Anopheles Gambiae Immunity

Abstract

The malaria-causing parasites must complete a complex infection cycle in the mosquito vector that also involves attack by the insect’s innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. In the first part of my thesis, I investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 abolished oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization. In the second part of my thesis, I investigated the functional contribution of the Toll/Rel1 pathway to antimicrobial defense in An. gambiae, particularly focusing on bacterial and fungal infections. The Toll and Imd pathways are evolutionary conserved immune signaling pathways, implicated in controlling the expression of hundreds of non immunity and immunity genes, including the antimicrobial peptide genes (AMPs). The downstream transcription factor activated in the An. gambiae Toll pathway is the NF-κβ transcription factor Rel1, orthologue of Drosophila Dorsal. The molecular structure of the An. gambiae Toll pathway remains poorly characterized, since the identity of the Toll receptor and the spätzle ligand that activate the pathway remain largely unknown. Similarly, the functional contribution of this pathway to An. gambiae immunity has not been thoroughly investigated. Here, we investigated the contribution of the Toll pathway to immune defense against fungal and bacterial infections in adult female An. gambiae mosquitoes using Rel1 silencing to block pathway activation or silencing its negative regulator Cactus to over-activate the pathway. Our results suggest that the Toll/Rel1 pathway is not essential for mosquitoes to clear bacterial and fungal infections but can efficiently clear these infections if overactivated by silencing Cactus. The primary roles for clearing fungal and bacterial infections were attributed to the complement-like protein TEP1 and the Imd/Rel2 pathway, respectively. However, we show that Rel1 pathway contributes to the ability of mosquitoes to tolerate infections with the entomopathogenic fungus Beauveria bassiana and with certain virulent bacteria such as Bacillus cereus and Listeria monocytogenes. Collectively, our results suggest that the Toll/Rel1 pathway in An. gambiae contributes mainly to enhancing host fitness in the context of fungal infections and infections with virulent bacteria, through mechanisms that remain to be elucidated. We reason that the Toll/Rel1 pathway in An. gambiae may function as a fail safe immune defense pathway, possibly because the activation of this pathway may incur more fitness costs on the host than other defense mechanisms.