Some background first. Wolbachia, the alpha-proteobacterial queen of insect reproductive manipulations, is an obligately intracellular and maternally transmitted bacterium. Various folks have been investigating it for its ability to prevent mosquitoes (and other insects) from harboring or transmitting viruses (see earlier post on this here). Dr. Rasgon, and his lab, have been trying to infect the Malaria vector, Anopheles, with Wolbachia in order to take advantage of this sweet side-effect...it's been rough. Anopheles seem inordinately recalcitrant to Wolbachia infection -- very few infections found in the wild, difficulty establishing maternal transmission etc. However, it turns out, Wolbachia can infect Anopheles tissues ex vivo as well as cell lines, suggesting that there is something found in the whole organism that is preventing infection. Let's see if it's the microbiome!
The authors focus on using the Wolbachia strain wAlbB in this manuscript (from Aedes albopictus), since there is no native infection for Anopheles, this makes perfect sense. Normally, when Anopheles is infected with Wolbachia, they show severe defects in transmission and/or fecundity. In A. gambiae, the mosquitoes transmit the bacterium at very low titer and in A. stephensi, no progeny are obtained when mosquitoes are injected with Wolbachia.
In this work, the authors first show that antibiotic treatment actually allows Wolbachia to infect two different Anopheles species (gamiae (A) and stephensi (B) below), without the fecundity defects observed previously. They use qPCR (looking at the ratio between a Wolbachia gene - wsp - and a host gene s7).
Neat! Looks like antibiotic treatment allows Wolbachia to colonize. Now, most folks would say "job done!" at this point and publish the work as quickly as possible but Hughes et al. go further -- they try to figure out which microbiome member could be contributing to this difference and if that microbiome member is necessary and sufficient for the Wolbachia transmission defects and host induced phenotypes.
Next they do some basic amplicon work to identify microbial community members in these mosquitoes before and after antibiotic treatment. Interestingly, they find an increase in the prevalence of many major taxonomic groups after antibiotic treatment and a reduction in one major genus: Asaia.
Shamefully, I had never heard of this genus before reading it in this paper. It's a fascinating member of the Acetobacteraceae (or Acetic-acid bacteria) that associates with many different insects. It turns out that this bacterium is maternally transmitted and also culturable - leading many folks to think of it as a potential paratransgenesis tool for mosquitoes. But I digress. In the next set of truly elegant experiments, Hughes et al culture Asaia and generate antibiotic resistant mutants. They then add these back to the previously gnotobiotic insects and voila! Now these mosquitoes are unable to maintain a Wolbachia infection and they die after a blood meal!
Above you see that supplementation with antibiotic-resistant Asaia plus a Wolbachia infection recapitulates the phenotypes originally observed in the mosquito pre antibiotic treatment. That is to say, that Asaia at least partially explains why Wolbachia has such a severe effect on the host. Ah, you ask, but the title of the paper is: "Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes" not "Asaia plus Wolbachia kill mosquitoes." True dat. The authors do count Wolbachia titers after the addition of this interesting Acetic-acid bacterium and they found that these mosquitoes, when supplemented with Asaia, cannot transmit Wolbachia (qPCR results below showing infected offspring).
Brilliant! Clearly this second maternally transmitted symbiont protects its niche from Wolbachia and the collateral damage is the host. Although a tidy story, this study really provokes more questions and potential lines of inquiry into this system. For example, diversity actually increases when Asaia is removed from mosquitoes. Do any of these bacteria contribute to Wolbachia maintenance? What is the mechanism behind the host phenotypes induced by the Asaia-Wolbachia conflict? and Are Asaia-free mosquitoes more fit than those with Asaia? Looking forward to hearing more from this group!