Temporal and form-dependent dynamics of culturable midgut bacteria in Anopheles stephensi during Plasmodium berghei infection

The gut microbiota of Anopheles mosquitoes modulates vector competence for Plasmodium through immune activation, metabolic competition, and antimicrobial metabolite production. Anopheles stephensi , a major malaria vector in Asia and an emerging threat in Africa, comprises distinct biological forms with divergent transmission capacities. However, infection-responsive dynamics of its culturable midgut bacteria, particularly in the mysorensis and intermediate forms, remain poorly understood. We tracked aerobic culturable midgut bacteria in An. stephensi from Chabahar ( mysorensis ) and Bandar Abbas ( intermediate ) over 20 days, spanning three gonotrophic cycles. Mosquitoes were fed on Plasmodium berghei -infected or uninfected mice, and midguts (n = 5/day/group) were dissected daily. Bacterial isolates were identified via 16S rRNA gene sequencing and analyzed for relative recovery, diversity, and infection association. Among 298 isolates, we identified 17 bacterial species, dominated by Proteus mirabilis (44.3%), Serratia marcescens (27.2%), and Providencia vermicola (13.8%). P. mirabilis and P. vermicola were significantly enriched in infected mosquitoes ( P < 0.05), whereas S. marcescens , a known prodigiosin-producing antiplasmodial bacterium, showed lower abundance in infected mosquitoes. Infected mosquitoes exhibited reduced diversity within the culturable midgut bacterial fraction (Shannon index; P < 0.05), indicating a simplification of the culturable bacterial community. Despite sharing a similar core microbiota, vector competence differed drastically: 77% of mysorensis mosquitoes developed salivary gland sporozoites versus only 20% of intermediate specimens. Major microbial shifts occurred during the first 10 days post-infection, coinciding with ookinete invasion and oocyst formation. P. berghei infection was associated with temporal shifts in the diversity and relative abundance of the culturable midgut bacterial community in An. stephensi . The enrichment of P. mirabilis and P. vermicola and the reduced recovery of S. marcescens in infected mosquitoes highlight taxa warranting further functional investigation. These findings provide a longitudinal, culture-based view of mosquito–microbiota– Plasmodium interactions and establish a basis for future mechanistic studies of microbiome-associated variation in malaria transmission.