Impact of thermal variation on the transcriptome of Plasmodium falciparum infected Anopheles stephensi

Temperature is a key determinant of malaria transmission, influencing both parasite development and mosquito physiology, yet the underlying mechanisms remain poorly understood. Here, we examined how temperature and time modulate gene expression in Anopheles stephensi infected with Plasmodium falciparum. Using RNA-sequencing over 1-19 days post-blood meal and three temperature regimes (20, 24, and 28{degrees}C with diurnal fluctuations of 9{degrees}C), we characterize transcriptome responses to infection with P. falciparum at the site of infection in the midgut, and systemically, in the carcasses. Oocyst prevalence and density declined over the thermal gradient, albeit with distinct, non-linear temporal dynamics in parasite development rates. Although infection contributed minimally to global variation in gene expression relative to temperature and time, infection-associated genes in the midgut showed coordinated transcriptional responses enriched in canonical Plasmodium associated extracellular, proteolytic, immune, and metabolic functions; notably, decline in oocyst infections in the midguts over the thermal gradient was reflected in reduced expression of immune genes known to regulate P. falciparum. Network analysis demonstrated that these genes participate in a significantly interconnected protein-protein interaction network, within which a small number of high betweenness centrality proteins act as bottlenecks linking immune, metabolic, reproductive and behavioral processes. Our results suggest responses to infection may be mediated through coordinated physiological networks rather than large-scale transcriptional changes. Our findings also indicate that differences in thermal conditions may be an important factor when comparing mechanisms of vector- parasite interactions between Plasmodium species. Together, our results highlight the importance of integrating thermal context into mechanistic studies of vector-parasite interactions.