# Effects of Wolbachia removal on microbial composition and diversity in Aedes albopictus: implication of using wAlbB for discriminating irradiation-based sterile and wild males

## TL;DR

This study explores how removing Wolbachia bacteria affects the microbiome of Aedes albopictus mosquitoes and suggests using wAlbB as a reliable marker to distinguish sterile males from wild ones in mosquito control programs.

## Contribution

The study introduces wAlbB as a novel biomarker for differentiating irradiated sterile males from wild males in SIT programs.

## Key findings

- Wolbachia removal significantly alters the microbial composition in male Aedes albopictus mosquitoes.
- wAlbB is a more stable and reliable biomarker for distinguishing sterile males compared to wAlbA or Enterococcus.
- The GT strain of mosquitoes shows no significant fitness costs compared to the wild-type GUA strain.

## Abstract

The sterile insect technique (SIT) requires distinguishing sterile from wild male mosquitoes to evaluate male qualities and maintain an appropriate release ratio for efficient population suppression. Current dye/powder marking methods have limitations and may affect SIT effectiveness, necessitating alternative discrimination strategies. Aedes albopictus naturally harbors two Wolbachia infections (wAlbA/wAlbB), which can be eliminated via tetracycline. Although Wolbachia removal minimally affect host fitness, its impact on microbiota remains unclear. Characterizing post-elimination microbial communities is the first step to identify novel endogenous biomarkers for SIT monitoring.

We analyzed the bacterial diversity and composition of two strains of wild-type GUA (Wolbachia-infected) and GT (Wolbachia-free) mosquitoes using the 16S rRNA V3-V4 region sequencing. qPCR was employed to confirm the relative abundance of four major bacterial genera, while PCR was used to validate selected biomarkers for distinguishing factory-reared sterile males from wild males. Kruskal-Wallis or Mann-Whitney test was used to analyze the comparable parameters between GUA and GT strains.

Five-day-old GUA and GT females showed similar microbial diversity/composition, while young males shared diversity but differed in composition. The core microbiota in both strains consisted of Proteobacteria (64.27%), Firmicutes (16.09%), Actinobacteriota (11.22%), and Bacteroidota (4.96%). Asaia was dominant in both strains (GUA: 47.33%; GT: 32.69%), whereas Enterococcus increased in GT males with aging. Wolbachia was absent in GT mosquitoes, and Elizabethkingia was undetected in GUA males. qPCR further confirmed these trends. PCR analysis revealed that wAlbB exhibited higher stability in differentiating factory-reared GT males from their wild counterparts (96.7% infection in field males, n = 60) compared to wAlbA (61.7%, n = 60) or Enterococcus (65.8%, n = 120). The mark-release-recapture experiment further confirmed the detectability using wAlbB biomarker.

Without obvious fitness costs observed previously in the Ae. albopictus GT strain compared to GUA strain, the removal of Wolbachia significantly changes the microbial composition in male mosquitoes in this study. Wolbachia wAlbB is recommended as a reliable biomarker for distinguishing sterile males from wild males when using GT strain in SIT programs targeting Ae. albopictus.

The online version contains supplementary material available at 10.1186/s40249-025-01343-3.