Malaria is a devastating parasitic disease transmitted globally by female Anopheles mosquitoes. Different species of Anopheles dominate transmission in various areas of the world, and these mosquitoes show extensive divergence both in their genetics and in their vectorial capacity for Plasmodium falciparum. Previous studies have shown that Anopheles mosquitoes respond to infective pathogens, including Plasmodium, thus establishing a hostile environment for the parasite and resulting in substantial bottleneck in the parasite population. This bottleneck represents a highly attractive target for transmission blocking strategies. Such strategies are considered essential for a comprehensive malaria elimination procedure. However, the current understanding of Anopheles responses to infection have largely been studied in the major African vector species, An. gambiae. The aim of this thesis was to interrogate the responses to early P. falciparum infection in two highly divergent Anopheles mosquitoes, An. gambiae s.s (Kisumu). and An. albimanus (Panama), via bulk RNA sequencing. These species exhibit variable permissibility to P. falciparum infection, but can both be infected the clone P. falciparum X45 which was used in this work. Within this investigation both conserved and single species responses to P. falciparum were profiled, in a tissue resolved manner. To evaluate an antiplasmodial response, parallel infectious (P. falciparum X45) and non infectious (human blood) feeds to mosquitoes of both species were performed, before harvesting the two tissues of interest: midgut and fatbody, at 12- and 24-hour post feed. RNA was extracted, sequenced, and the transcriptional changes analysed. Three analyses were performed in order to analyse the data (orthologue analysis, and two single species studies), this allowed for a comparative cross species analysis alongside the single species investigations. The differential expression (DE) data generated by these three analysis strategies, revealed key differences between the species, both in their baseline physiological responses to blood feeding and in the response to P. falciparum. An. albimanus was found to transcriptionally more enzymatically active than An. gambiae irrespective of infection. These differences between the species, is suggestive of a more hostile baseline environment in An. albimanus which may influence transmission in this species. In each species, a modest number of genes were identified as differentially expressed within the first 24 hours of infection within the midgut. The response to An. albimanus emphasised an upregulation of several immune/defence related genes, while an opposing state was seen in An. gambiae with DE immune related genes being downregulated in response to infection. Overall, An. albimanus mosquitoes show far lower infection intensity and prevalence with various strains of P. falciparum compared to An. gambiae. This infection phenotype is coupled with the differences in transcriptional changes, and its baseline hostile state, establishes An. albimanus as a more responsive vector to infection. This information, when considered alongside the shorter coevolutionary time between An. albimanus and P. falciparum compared to that of An. gambiae with the same parasite species indicates that P. falciparum may be less able to evade the immune response in this species and therefore undergoes far more substantial parasite losses. Interestingly within the time points surveyed, the fatbody showed minimal involvement in an antiplasmodial state, with even fewer genes identified as DE than in the midgut. This appears to contrast with other work that implicate the fatbody as a highly active immune organ in Anopheles. At 12 and 24 hours post infectious feed, parasites have yet to fully traverse the midgut epithelial and form oocysts on the basal lamina, thus, extensive transcriptional fatbody responses appear to not yet be triggered. The differences in sampling between this work and previous studies of this tissue likely explain discrepancies in the role of this tissue in an immune response to P. falciparum. From the transcriptional analysis, several key genes of interest as potential targets for transmission blocking interventions were identified. One such gene SRPN10 was implicated as an important gene in the response to P. falciparum in the midgut samples. The role of this gene through RNAi gene silencing in both species was therefore evaluated. Knockdown of this gene elicited a dramatic reduction in prevalence in both An. gambiae and An. albimanus, as well as moderate reductions in oocyst intensity, but had no effect on oocyst size. Additionally investigated was whether RNAi of SRPN10 affected size of blood meal, but this was not found to occur. Data here implicates SRPN10 as involved in a species conserved parasite clearance process. This coupled with previous studies of SRPN10 upregulation in response to P. berghei are suggestive of this gene playing a role in apoptosis regulation, but further work is needed to confirm this hypothesis. Finally, again utilising the reference RNA seq data set developed, parasite responses to heterogenous host species were studied. P. falciparum X45 parasites were found to shift gene expression pattern in each of the two host Anopheles species, implicating 83 genes as differentially expressed between the hosts. These DE genes included some known invasion related genes, (SOPT, TRAP like) as well as many genes with unknown function, potentially showing novel activity in these highly divergent hosts. This provides further evidence of the phenomenon of transmission stage parasites varying their transcriptional trajectories in response to different hosts they encounter. Overall, this thesis has revealed the concise nature of the response to early P. falciparum in two extensively divergent species of Anopheles mosquito; with An. albimanus showing a more drastic response to infection which potentially explains the lower susceptibility of this species. This work has also evidenced the limited overlap in response to infection between these species, likely a result of their extensive evolutionary divergence. In addition, this work has identified SRPN10 as a conserved P. falciparum parasite responsive gene and indicated that it may play a negative immunoregulatory role over a parasite elimination process from the midgut. This establishes this gene as a potent target for transmission blocking strategies in multiple important P. falciparum vector species.
Amelia Jane Cox (Thu,) studied this question.
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