Genome-wide detection of Wolbachia in natural Aedes aegypti populations using ddRAD-Seq

Abstract

Background: Wolbachia, an endosymbiotic bacterium, is globally used to control arboviruses because of its ability to block arboviral replication and manipulate the reproduction of Wolbachia host, Aedes aegypti. Polymerase chain reaction (PCR)-based Wolbachia detection has been recently reported from natural Ae. aegypti populations. However, due to the technical limitations of PCR, such as primer incompatibility, PCR-based assays are not sufficiently reliable or accurate. In this study, we examined double digestion restriction site-associated DNA sequencing (ddRAD-Seq) efficiency and limitations in Wolbachia detection and quantification in field-collected Ae. aegypti natural populations in Metro Manila, the Philippines, compared with PCR-based assays.

Methods: A total of 217 individuals Ae. aegypti were collected from Metropolitan Manila, Philippines. We separated it into 14 populations consisting of 7 female and male populations. We constructed a library for pool ddRAD-Seq per population and also screened for Wolbachia by PCR assays using wsp and 16S rRNA. Wolbachia density per population were measured using RPS17 as the housekeeping gene.

Results: From 146,239,637 sequence reads obtained, 26,299 and 43,778 reads were mapped across the entire Wolbachia genome (with the wAlbA and wAlbB strains, respectively), suggesting that ddRAD-Seq complements PCR assays and supports more reliable Wolbachia detection from a genome-wide perspective. The number of reads mapped to the Wolbachia genome per population positively correlated with the number of Wolbachia-infected individuals per population based on PCR assays and the relative density of Wolbachia in the Ae. aegypti populations based on qPCR, suggesting ddRAD-Seq-based semi-quantification of Wolbachia by ddRAD-Seq. Male Ae. aegypti exhibited more reads mapped to the Wolbachia genome than females, suggesting higher Wolbachia prevalence rates in their case. We detected 150 single nucleotide polymorphism loci across the Wolbachia genome, allowing for more accurate the detection of four strains: wPip, wRi, TRS of Brugia malayi, and wMel.

Conclusions: Taken together, our results demonstrate the feasibility of ddRAD-Seq-based Wolbachia detection from field-collected Ae. aegypti mosquitoes.

Keywords: Aedes aegypti; Philippines; Wolbachia; ddRAD-seq; genome-wide.

Figure 1

Sampling site locations of Ae. aegypti in Manila City, Metropolitan Manila, Philippines. Big circles in red, yellow, dark green, light green, gray, dark blue, and purple indicate the geographical midpoints of Ae. aegypti populations per location; small circles near each big circle indicate the households in the sampling locations. F and M indicate the total number of female and male individuals per population, respectively (Muharromah et al., 2023).

Figure 2

Correlation plots between the percentage of mapped reads to the Wolbachia genome and that of Wolbachia-positive mosquitoes with 16S rRNA gene (A), wsp gene (B), both 16S rRNA and wsp genes (C), and the relative Wolbachia density in the pooled data (D). Percentage of reads mapped in the Wolbachia genome = total number of reads mapped in the Wolbachia genome divided by the total number of reads after trimming and filtering, percentage of Wolbachia-positive mosquitoes = total number of Wolbachia-positive individuals divided by the total number of individuals per population.

Figure 3

The visualization of sequence reads generated based on the ddRAD-Seq approach (green) and gap regions (white) on the Wolbachia pipientis wAlbB complete genome. The green arrow indicates the region of single nucleotide polymorphisms (SNPs) located across the Wolbachia pipientis wAlbB complete genome. Supplementary Table 2 includes detailed information about the 150 SNPs.