Altered gut microbiota and immunity defines Plasmodium vivax survival in Anopheles stephensi

Abstract

<jats:title>Abstract</jats:title><jats:p>Blood feeding-enriched gut-microbiota boosts mosquitoes’ anti-<jats:italic>Plasmodium</jats:italic> immunity. Here, we ask how <jats:italic>Plasmodium vivax</jats:italic> alters microbiota, anti-<jats:italic>Plasmodial</jats:italic> immunity and impact tripartite <jats:italic>Plasmodium</jats:italic>-mosquito-microbiota interactions in the gut lumen. Using a metagenomics analysis, we predominantly detect <jats:italic>Elizabethkingia meningitis</jats:italic> and <jats:italic>Pseudomonas sps.</jats:italic> in naïve mosquitoes. Naïve blood fed gut shows a heightened presence of <jats:italic>Elizabethkingia</jats:italic>, <jats:italic>Pseudomonas</jats:italic> and <jats:italic>Serratia</jats:italic>. A parallel RNAseq analysis of blood-fed midguts identify <jats:italic>Elizabethkingia</jats:italic>-transcripts, which may have role in iron metabolism. Post, a <jats:italic>Plasmodium vivax</jats:italic> infected blood-meal, however, we do not detect bacterial until circa 36 hours. Intriguingly, transcriptional expression of a selected array of antimicrobial arsenal cecropins 1-2, defensin-1 and gambicin remains low during the first 36 hours–a time frame when ookinietes/early oocysts invade gut. We conclude during the preinvasive phase, <jats:italic>Plasmodium vivax</jats:italic> outcompetes midgut-microbiota. Suppression of important immune factors, likely due to altered microbiota, may enhance <jats:italic>Plasmodium vivax</jats:italic> survival. Additional finding of a novel <jats:italic>Wolbachia</jats:italic> association warrants further research to design ‘paratransgenesis’ tools for malaria control.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>Successful malaria transmission relies on the competitive interactions of <jats:italic>Plasmodium</jats:italic> and mosquito’s tissue specific immune potential. Within 24hrs of blood meal gut-microbiota grows exponentially and lead to robust enhancement of mosquito immune response, which is detrimental to parasite survival and development. But the mechanism how <jats:italic>Plasmodium</jats:italic> manages to evade this pre-invasive immune barrier is not well known. We investigated the influence of tripartite gut-microbiome-parasite interaction on human malaria parasite <jats:italic>Plasmodium vivax</jats:italic> in its natural/native vector <jats:italic>Anopheles stephensi</jats:italic>. Surprisingly we found that infectious blood meal lead to dramatic suppression in gut-bacteria population, a plausible strategy of <jats:italic>P. vivax</jats:italic> ookinetes to avoid immune responses. Our study suggests that for its own survival <jats:italic>Plasmodium vivax</jats:italic> causes early suppression of bacterial population, possibly by scavenging Fe from the blood meal which is indispensable for bacterial growth. Disruption and manipulation of this gut-microbe-interaction may help to design new ‘paratransgenesis’ molecular tool for malaria control.</jats:p></jats:sec>

Hemocyte‐specific FREP13 abrogates the exogenous bacterial population in the hemolymph and promotes midgut endosymbionts in Anopheles stephensi

“…gambiae 42 . Our recent study suggests that P. vivax infection restricts gut‐flora proliferation, which consequently facilitates Plasmodium development 34 . In this study, we observed that P. vivax infection not only caused a limited change in the expression of hemocyte FREP13 (Figure 6a) during the late phase but also caused significant downregulation in the midgut during the early phase of infection (Figure 6b).…”

“…To test this hypothesis, we examined the effect of FREP13 silencing on gut microbiota proliferation following blood‐feeding. Although blood‐meal causes a multifold expansion of gut symbionts within 24 h of blood‐feeding in naïve mosquitoes, 34,48 surprisingly, depletion of FREP13 messenger RNA in the hemocytes causes an initial increase of gut bacteria within 12‐h after blood‐meal, followed by rapid declination and re‐enrichment of the total bacterial population in the gut after 24 or 48 h of blood‐feeding, respectively (Figure 6c).…”

Bacterial Microbiome in Wild-Caught Anopheles Mosquitoes in Western Thailand

“…This genus appears to have a role in increasing iron metabolism necessary for bacterial growth and Plasmodium development (Clark et al, 2014; FIGURE 4 | Bacterial genera identified in Anopheles species by PCR-TTGE. Sharma et al, 2019). Bacterial microbiota in Anopheles and their effects on the development of Plasmodium parasites as a potential means of controlling malaria transmission have been reported in various Anopheles species, but the precise mechanisms of activity remain poorly understood (Dong et al, 2009;Meister et al, 2009;Abdul-Ghani et al, 2012;Gendrin and Christophides, 2013;Minard et al, 2013;Hughes et al, 2014;Sharma et al, 2014;Villegas and Pimenta, 2014;Romoli and Gendrin, 2018).…”

“…gambiae (Boissière et al, 2012;Ngwa et al, 2013). Sharma et al (2019) reported an interesting microbial interaction and immune modulation, whereby the presence of P. vivax in An. stephensi provoked a metabolic alteration of the availability of iron and nutritional physiology required for ideal bacterial growth resulting in a reduction of the microbiota present in the midgut, particularly the predominant bacteria Elizabethkingia and Pseudomonas.…”

SalivaryAsHPX12influence pre-blood meal associated behavioral properties in the mosquitoAnopheles stephensi

“…stephensi mosquitoes were fed on the P. vivax infected patient's blood (~2% gametocytemia) through preoptimized artificial membrane feeding assay (34). The confirmation of the P. vivax infection was done by staining the midgut with 5% mercurochrome to visualize the oocysts after 4 days postinfection (DPI), as described earlier (34). After confirmation, ~20-25 mosquitos' Salivary gland dissected at 9-12DPI and 12-14DPI, respectively.…”