THE ROUTE OF INFECTION INFLUENCES THE CONTRIBUTION OF KEY IMMUNITY GENES TO ANTIBACTERIAL DEFENSE IN ANOPHELES GAMBIAE

Abstract

Pathogens gain access to their hosts through several routes, most of which require contact with barrier epithelia Indeed, studies in insects and mammals suggest that different routes of infection are likely to trigger different physiological responses in the host. However, the nature of these responses and how they impact host resistance and tolerance in different infection routes is not completely understood. Immunity studies in several model insects have focused largely on conducting microbial challenges using microinjections, whereby the microbe is directly injected into the hemolymph, a scenario that is less likely to occur in nature and one that bypasses the multilayered immune response elicited following microbial colonization and invasion of barrier epithelial. Hence, it remains unclear whether and to what extent the contribution of systemic immune defenses to host resistance to infection varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infection of the hemolymph in the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Indeed, we were able to detect Sm in the mosquito hemolymph one day after oral infection, a clear indication that Sm is able to cross the midgut epithelial barrier and gain access to the body cavity. Using functional genetic studies by RNA interference (RNAi), we report that the two humoral immune factors, thioester-containing protein 1 (TEP1) and C-type lectin 4 (CTL4), which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Relish 2 (Rel2)/Immune deficiency (Imd) pathway, indicating that this pathway may either be not activated in response to oral infection or that it is activated but rather non-essential for defense against Sm oral infection. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. A plausible explanation could be that this cellular response is not essential when small numbers of bacteria are present in the hemolymph, as is the case with our infection protocol herein. Transcriptomic analysis of mosquito midguts and abdomens by RNA sequencing (RNA-seq) revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm, despite the fact that injections resulted eventually in higher loads of Sm in the hemolymph. A small overlap was observed when comparing differentially expressed transcripts in midguts and abdomens of mosquitoes injected with Sm to those of mosquitoes fed on Sm, indicating that different physiological responses are triggered in response to the different routes of Sm infection. Functional classification of all differentially expressed transcripts in abdomens and midguts from all treatments revealed that metabolic genes are the most represented class. Surprisingly, oral and septic infections with Sm seem to have little effect on the transcriptome of immunity genes as these were under-represented in both abdomens and midguts from all treatments. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. On the other hand, only four immunity genes were upregulated after Sm oral infections; Galectin 5 and CecA were upregulated in abdomens, whereas, C-type lectin 6 (CTL6) and lysozyme C7 (LYSC7) were upregulated in the midgut. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host anti-microbial defense, but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host.