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Bacterial composition; Antibiotic treatment; Salivary gland; Midgut; Susceptibility; Malaria vector
Anopheline gut bacterial community is an important factor against Plasmodium colonization, however there are few observations about the interaction among the microbiota, Plasmodium vivax and Anopheles darlingi. To better understand the interaction between P. vivax-An. darlingi, we proposed to study the composition and diversity of microbiota from midgut and salivary gland of lab-reared and field-caught An. darlingi, and to evaluate the impact of the microbiota on the survival of An. darlingi and artificial infection by P. vivax. Mosquitoes were obtained from An. darlingi colony of the PIVEM/Laboratório de Entomologia of Fiocruz Rondônia and field mosquitoes were collected by protected human landing collection. The V3-V4 and V4 hypervariable region of the 16S rRNA gene were sequenced to profile the microbiota composition of midguts and salivary glands of colonized and field-captured mosquitoes. The effect of microbiota on mosquito survival and in the sporogonic cycle of P. vivax was evaluated from antibiotic-treated and untreated mosquitoes. Mosquitoes infected by P. vivax were dissected at 7 and 14-day post-infection (dpi) for oocyst and sporozoite count, respectively. To analyze alpha-diversity, the Chao1, Shannon and Inverse Simpson indices were estimated, and for beta-diversity the weighted and unweighted Unifrac measures were assessed. Kaplan-Meier survival curves of treated and untreated mosquitoes were constructed and the hazard risk for each group was obtained by Cox Models. The effect of antibiotic treatment on the susceptibility of An. darlingi was evaluated by the Mann-Whitney test for oocyst and sporozoite intensity, and the prevalence determined by Chi-square. Anopheles darlingi microbiota data showed that the midgut and salivary gland had low diversity and dominance of a few bacterial taxa in microbiota communities. The salivary glands showed higher richness and diversity than midguts, regardless of sample origin. However, the salivary glands and midguts only showed dissimilarities in beta diversity between lab-reared mosquitoes. An intra-variability in taxa abundance was observed in the samples, and a high proportion of lab-reared mosquitoes shared a core microbiota, probably due to lab rear conditions. Acinetobacter and Pseudomonas were dominant in the tissues of lab-reared mosquitoes. Sequences of Wolbachia and Asaia were both found in the tissue of lab-reared mosquitoes; however, only Asaia was found in field-captured An. darlingi, but in low abundance. Antibiotic treatment was effective to reduce the bacterial load. The bacterial load reduction resulted an increase on the survival of treated mosquitoes and a protective effect > 63%, regardless the P. vivax infection. In this way, antibiotics may help to control bacterial proliferation and prevent mosquito death before 14 dpi. On the other hand, antibiotic treatment had no effect on the susceptibility of An. darlingi to P. vivax. This raise the question whether the An. darlingi-P.vivax-microbiota interaction could be different from what is known for others Plasmodium spp. and anopheline combinations.