Evidence of natural Wolbachia infections in field populations of Anopheles gambiae

Abstract

Wolbachia are maternally transmitted intracellular bacteria that invade insect populations by manipulating their reproduction and immunity and thus limiting the spread of numerous human pathogens. Experimental Wolbachia infections can reduce Plasmodium numbers in Anopheles mosquitoes in the laboratory, however, natural Wolbachia infections in field anophelines have never been reported. Here we show evidence of Wolbachia infections in Anopheles gambiae in Burkina Faso, West Africa. Sequencing of the 16S rRNA gene identified Wolbachia sequences in both female and male germlines across two seasons, and determined that these sequences are vertically transmitted from mother to offspring. Whole-genome sequencing of positive samples suggests that the genetic material identified in An. gambiae belongs to a novel Wolbachia strain, related to but distinct from strains infecting other arthropods. The evidence of Wolbachia infections in natural Anopheles populations promotes further investigations on the possible use of natural Wolbachia-Anopheles associations to limit malaria transmission.

Figure 1. Mosquito collection sites and distribution of Wolbachia-infected individuals.

The maps describe the three villages (Soumousso, VK5 and VK7) where An. gambiae couples from different mating swarms (indicated by circles) and larvae from different breeding sites (indicated by squares) were collected. Swarm sites and larval breeding grounds are identified by numbers and letters, respectively (see Supplementary Table 1 and 2). Sites where Wolbachia-positive mosquitoes were found are highlighted in red. Maps were adapted with permission from originals courtesy of A. A. Millogo, IRSS/Centre Muraz, Bobo-Dioulasso, Burkina Faso.

Figure 2. Phylogenetic tree for Wolbachia 16S rRNA sequences.

The tree was built using V4 16S rRNA fragments from Wolbachia reference sequences available in public repositories (NCBI and SILVA) and from the sequences obtained in this study. The sequences are clustered at 99% identity and the cardinality of each OTU obtained is reported in a logarithmic scale as a bar chart external to the tree (green for reference sequences and red for the new sequences). Shadings highlight subtrees with high bootstrapping support (>90%) and host organisms are reported for those OTUs in which at least one-third of the sequences are consistently associated to the same host.

Figure 3. Whole-genome shotgun sequencing of a Wolbachia-positive sample identifies a new Wolbachia strain in An. gambiae.

Ovaries from a Wolbachia-positive female (sample S1) were sequenced using WGS. (a) Percentage (Perc.) identity of 395 short sequences uniquely attributable to Wolbachia from sample S1 versus eight sequenced strains. The Wolbachia reference strains and the supergroups are indicated with the corresponding percentage of assigned sequences. (b) Distribution of phylogenetic distances between different Wolbachia supergroups, from phylogenies reconstructed separately on each of the short sequences universally conserved within Wolbachia genomes (light blue inset in a). (c) Functional classification of Wolbachia loci identified by alignment to read sequences, based on NCBI annotated gene functions. (d) Wolbachia phylogeny, comprising the new Wolbachia strain wAnga isolated in An. gambiae, reconstructed from the concatenated sequences of b. These analyses show that wAnga is different from all other strains sequenced so far. The same analyses for another PCR-positive Wolbachia sample are available in Supplementary Fig. 3.

Figure 4. Vertical transmission of Wolbachia from mother to offspring.

A total of 14 Wolbachia-positive blood-fed An. gambiae females collected from houses in VK5 were allowed to lay eggs individually. The progeny of the five Wolbachia-positive females (W+) that laid eggs was screened for the presence of Wolbachia at the fourth larval developmental stage (L4). Red indicates Wolbachia-positive larvae and adults. The number of larvae screened is shown for each female (n).