Natural Wolbachia infection in field-collected Anopheles and other mosquito species from Malaysia

Abstract

Background The endosymbiont bacterium Wolbachia is maternally inherited and naturally infects some filarial nematodes and a diverse range of arthropods, including mosquito vectors responsible for disease transmission in humans. Previously, it has been found infecting most mosquito species but absent in Anopheles and Aedes aegypti . However, recently these two mosquito species were found to be naturally infected with Wolbachia . We report here the extent of Wolbachia infections in field-collected mosquitoes from Malaysia based on PCR amplification of the Wolbachia wsp and 16S rRNA genes. Methods The prevalence of Wolbachia in Culicinae mosquitoes was assessed via PCR with wsp primers. For some of the mosquitoes, in which the wsp primers failed to amplify a product, Wolbachia screening was performed using nested PCR targeting the 16S rRNA gene. Wolbachia sequences were aligned using Geneious 9.1.6 software, analyzed with BLAST, and the most similar sequences were downloaded. Phylogenetic analyses were carried out with MEGA 7.0 software. Graphs were drawn with GraphPad Prism 8.0 software. Results A total of 217 adult mosquitoes representing 26 mosquito species were screened. Of these, infections with Wolbachia were detected in 4 and 15 mosquito species using wsp and 16S rRNA primers, respectively. To our knowledge, this is the first time Wolbachia was detected using 16S rRNA gene amplification, in some Anopheles species (some infected with Plasmodium ), Culex sinensis , Culex vishnui , Culex pseudovishnui , Mansonia bonneae and Mansonia annulifera . Phylogenetic analysis based on wsp revealed Wolbachia from most of the mosquitoes belonged to Wolbachia Supergroup B. Based on 16S rRNA phylogenetic analysis, the Wolbachia strain from Anopheles mosquitoes were more closely related to Wolbachia infecting Anopheles from Africa than from Myanmar. Conclusions Wolbachia w as found infecting Anopheles and other important disease vectors such as Mansonia ....

Low prevalence of naturalWolbachiain major malarial vectorsAnopheles culicifaciess.l., andAnopheles stephensi:a first report

“…The 16S rDNA phylogeny shows all the Wolbachia isolates from this study belongs to supergroup B and it’s non-monophyletic. Similar to our observation, recent reports (Gomes et al 2017, Sawasdichai et al 2019, Wong et al 2020) show supergroup B is polyphyletic. Though our samples are in separate clades, the genetic diversity within the group (Supplementary Table 3) and clades of supergroup B was less (Supplementary Table 4b).…”

“…introlatus, An. macarthuri (Wong et al 2020) shows lesser natural prevalence rate. Higher prevalence rate has been reported from an unknown Anopheles species from Sub-Saharan Africa (Jeffries et al 2018), An.…”

Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes

“…35 The inability to find Wolbachia reads targeting the 16S rRNA hypervariable V3-V4 region in nested-PCR-positive individuals raises concerns about the validity of nested PCR, which has been commonly used to report the detection of Wolbachia infections in Anopheles. [12][13][14][15][16][17] In comparison, our microbiome analysis shows that, when present, both the wAnM and wAnD strains dominate the microbiome, which would be more consistent with a maternally transmitted endosymbiont. Furthermore, the inability to amplify and sequence the wsp gene from strains detected in the An.…”

“…Although, for many years, Anopheles were thought to be impervious to Wolbachia infection, 9,10 several recent reports detect Wolbachia DNA in a range of species. [11][12][13][14][15][16][17][18] However, the detection of gene sequences does not confirm the presence of endosymbiotic (or even living) bacteria, 19 given the possibility of environmental contamination or integration into the host genome. 20 The majority of these studies are limited to the amplification of only a few genes (particularly 16S rRNA), and these findings have been extrapolated to conclude the presence of genuine Wolbachia infections.…”

Genomic and microscopic evidence of stable high density and maternally inheritedWolbachiainfections inAnophelesmosquitoes

“…In addition, the inability to find Wolbachia reads using microbiome sequencing in nested-PCR positive individuals raises concerns about the validity of this assay, which has been commonly used to report detection of Wolbachia infections in Anopheles mosquitoes [16][17][18][19][20][21] . A recent study using 16S rRNA gene sequencing of nested PCR positive An.…”