The midgut bacterial diversity study of laboratory-reared strains of
An. stephensi identified
Proteobacteria as the dominant phyla. Rani et al. [
9] made similar observation where
Proteobacteria was the dominant phyla in the midgut of field-collected
An. stephensi mosquitoes from Haryana, India.
Proteobacteria was identified as dominant phyla among field-collected
An. stephensi and
An. maculipennis from Iran,
An. gambiae from Cameroon [
11,
24]. Ngo et al. also found
Proteobacteria as the dominant phyla in several of
Anopheles species sampled from two different regions of Vietnam, Dak Nong and Binh Phuoc Provinces [
25,
26]. Most of the dominant genera in the midgut of
An. stephensi identified here, namely
Acinetobacter,
Pseudomonas,
Bacillus,
Prevotella, and
Corynebacterium, have been previously reported in
Anopheles mosquitoes [
8,
9]. Interestingly,
Veillonella genera were found in the midgut of
An. stephensi for the first time, and reported in the present study. However, the presence of
Veillonella sp. has been reported by Fraihi et al. [
27] in the midgut of laboratory-reared sand fly (
Phlebotomus perniciosus, Diptera: Psychodidae). Also, McCarthy et al. [
28] found uncultured
Veillonella sp. in
Lutzomyia longipalpis, a vector of visceral leishmaniasis. In general, the midgut microbial composition in mosquitoes is found to be influenced by various factors, such as species of mosquitoes, life stages, sex, feeding behaviour, geographical locations, seasonality, etc. [
29,
30]. In contrast to other studies, a homogenous microbial population among laboratory-reared stains of
An. stephensi mosquitoes was found in the present study, which may be due to the fact that all the mosquito strains were grown under identical conditions with the same diet for 6–8 generations before sampling them for bacterial diversity studies [
24,
30]. Studying the microbial communities through 16S rRNA gene sequencing is subject to many pitfalls and many sources of bias have been identified with sample processing steps [
31]. Importantly, work up of negative controls alongside samples throughout has been found to be very critical to assess possible contaminants from kits and reagents [
32]. Here, not sequencing the negative controls because of undetectable library yields and excluding the reads from them in downstream analysis might have increased the chances of introducing contaminants thereby misleading the results, so that counteracting bias in metagenomic study is found to be very critical. A study by Boissière et al. [
24] found significant correlation between
Enterobacteriaceae abundance in the mosquito midgut with
Plasmodium infection status. The present study also recorded
Enterobacteriaceae in all field-derived
An. stephensi at an OTU percentage of 1.8% in AS-B, 2.0% in AS-C and 1.4% in AS-M strains. Some species from the genus
Pseudomonas are among
Anopheles gut-derived bacteria with known
Plasmodium-blocking abilities [
14,
33].
Pseudomonas genera in the midgut of all field-derived strains of mosquitoes were detected in the present study. Most of the studies have reported the effect of individual bacteria isolates from the gut of mosquitoes on
Plasmodium development in its host [
12‐
14]. An effort was made to understand the effect of total or entire mosquito midgut microbiota on susceptibility of
An. stephensi originally from 3 different locations to
Plasmodium infections, as this could recognize the influence of entire microbiota on host vector competence [
24]. Although, the lower
Plasmodium infection level among the three tested
An. stephensi strains in the presence of total microbiota was observed, a more comprehensive study is required to draw reliable conclusions on the relationship between the difference in microbiota composition and variation in competence of mosquito for
Plasmodium infections. In recent years, researchers have shown increased emphasis on the use of midgut microbiota of vector mosquitoes in control of vector-borne disease [
34,
35]. In this regard, understanding the diversity, function and dynamics of midgut microflora of
Anopheles would offer newer prospective in control of malaria transmission.