Is Anopheles gambiae a Natural Host ofWolbachia?

Abstract

Wolbachia (Alphaproteobacteria, Rickettsiales) is an intraovarially transmitted symbiont of insects able to exert striking phenotypes, including reproductive manipulations and pathogen blocking. These phenotypes make Wolbachia a promising tool to combat mosquito-borne diseases. Although Wolbachia is present in the majority of terrestrial arthropods, including many disease vectors, it was considered absent from Anopheles gambiae mosquitos, the main vectors of malaria in sub-Saharan Africa. In 2014, Wolbachia sequences were detected in A. gambiae samples collected in Burkina Faso. Subsequently, similar evidence came from collections all over Africa, revealing a high Wolbachia 16S rRNA sequence diversity, low abundance, and a lack of congruence between host and symbiont phylogenies. Here, we reanalyze and discuss recent evidence on the presence of Wolbachia sequences in A. gambiae. We find that although detected at increasing frequencies, the unusual properties of these Wolbachia sequences render them insufficient to diagnose natural infections in A. gambiae. Future studies should focus on uncovering the origin of Wolbachia sequence variants in Anopheles and seeking sequence-independent evidence for this new symbiosis. Understanding the ecology of Anopheles mosquitos and their interactions with Wolbachia will be key in designing successful, integrative approaches to limit malaria spread. Although the prospect of using Wolbachia to fight malaria is intriguing, the newly discovered strains do not bring it closer to realization. IMPORTANCE Anopheles gambiae mosquitos are the main vectors of malaria, threatening around half of the world’s population. The bacterial symbiont Wolbachia can interfere with disease transmission by other important insect vectors, but until recently, it was thought to be absent from natural A. gambiae populations. Here, we critically analyze the genomic, metagenomic, PCR, imaging, and phenotypic data presented in support of the presence of natural Wolbachia infections in A. gambiae. We find that they are insufficient to diagnose Wolbachia infections and argue for the need of obtaining robust data confirming basic Wolbachia characteristics in this system. Determining the Wolbachia infection status of Anopheles is critical due to its potential to influence Anopheles population structure and Plasmodium transmission.

Systematic Review of Wolbachia Symbiont Detection in Mosquitoes: An Entangled Topic about Methodological Power and True Symbiosis

“…Next-generation sequencing (NGS) employing long reads of mosquito and Wolbachia DNA may offer additional data that can help to distinguish between a true Wolbachia infection and integrated bacterium genomic fragments. Long DNA reads allow the detection of Wolbachia DNA integration sites into the mosquito genome or the confirmation of circular Wolbachia genomic DNA that further supports a true Wolbachia infection hypothesis [46]. However, many genome assembly parameters must be considered when analyzing these genomes, including genome coverage and sequencing depth.…”

“…Of the 59 articles analyzed, only one used a method for visualizing the bacterium, which was performed using MET. Although molecular techniques to visualize antigens or cell structures, such as FISH and MET are laborious, require expensive equipment and trained personnel, they can discern the first scenario (integration of Wolbachia gene into the mosquito genome) from a true Wolbachia infection, since the visualization of the bacteria and not just a single structure or gene trace confirm a true infection [46] (Figure 4). An alternative to the high cost required by these cited techniques is to perform a squash of the mosquito's ovaries followed by staining with May-Grunwald-Giemsa method (GIEMSA) or Gimenez staining to visualize pleomorphic structures suggestive of Wolbachia cells, through an optical microscope [61,113,114].…”

“…Wolbachia can be detected through various molecular techniques that show variable sensitivity and specificity, such as polymerase chain reaction (PCR) (with specific and/or degenerate primers), PCR with various markers such as multilocus sequencing Typing (MLST), quantitative PCR (qPCR), microscopy methods, such as Fluorescent In Situ Hybridization (FISH), electron microscopy, and others [42][43][44][45]. The popularization of molecular methods has broadened the capacity of many laboratories to perform Wolbachia DNA detection in several insect species while the different microscopy techniques have been used by a small number of researchers [46]. Each of these techniques has particular advantages and limitations that can influence the detection of different Wolbachia-derived molecules and/or a true Wolbachia infection.…”

Detection of diverse Wolbachia 16S rRNA sequences at low titers from malaria vectors in Kayin state, Myanmar

“…To show an infection, additional experiments are necessary, e.g. showing intracellular localization of the sequences and maternal transmission of the bacteria (see Chrostek and Gerth 2019 2 for further discussion). I suggest changing the wording throughout, from “natural Wolbachia infections” to “Wolbachia 16S/wSpec sequence amplification”.…”

Aquatic Hemiptera in Southwest Cameroon: Biodiversity of Potential Reservoirs of Mycobacterium ulcerans and Multiple Wolbachia Sequence Types Revealed by Metagenomics

“…1 was a more unusual finding, as this clade is less widespread than the A and B supergroups, although it is also the only one known from both arthropod and nematode hosts [120,121]. As the Wolbachia signal in this specimen was relatively low (~6,000 read pairs, or 0.04% of the total), the possibility that the clade F sequences originate from gut contents or an arthropod or nematode parasite or parasitoid cannot be discounted at this stage [122,123]. A priority for future research on the association of aquatic bugs with M. ulcerans will be to determine if Wolbachia influences this relationship; e.g., whether its presence might explain the limited evidence of M. ulcerans infection in our specimens compared with other locations in Cameroon.…”