Detecting Natural Wolbachia Infection and Supergroup Identification of Metochus uniguttatus in North India

Abstract

Introduction: Insect endosymbionts have a crucial evolutionary and ecological role. Insects, particularly arthropods, have evolved to tolerate a wide range of endosymbionts that provide them with external biochemical, genetic, and reproductive capacities that improve their fitness. Wolbachia is a widely prevalent bacterial endosymbiont that is gaining popularity in insect research due to its utility in pest and vector management. Although India has a diverse insect population, little research has been undertaken on insect-microbe interactions. Therefore, This study aimed to ascertain the spread of Wolbachia in a randomly sampled set of insects, identify any novel Wolbachia infections, and determine the Wolbachia infection’s supergroup. Materials and methods: Field capture of insects was carried out manually with netting and gloves, and the insects were immediately stored in absolute ethanol to immobilize them. Phenol-chloroform-isoamyl alcohol was the method used to extract the insect DNA. Insect species identification was carried out via the CO1 mitochondrial gene using PCR amplification. PCR was utilized to identify the presence of Wolbachia, and 16S Wspec DNA was amplified to confirm its presence. The Sanger method was used to sequence the amplified CO1 and the positive samples for Wspec. Using NCBI blast, the sequences of the infected insects were compared with the database sequences. The obtained FASTAs were then aligned using Sequencher 5.4.6, and the chromatograms were examined to ensure contig quality and similarity. Results: Among the 21 insects screened, one was found weakly positive (Playpleura octoguttata) and two were strongly positive (Metochus uniguttatus and Velarifictorus micado) with Wolbachia, which represented an infection rate of 14.29%. Although the individual infection rate in this limited sample size falls at the lower end compared to extensive surveys reporting rates between 20% and 76%. This study indicated the dissemination of Wolbachia in randomly screened insects. Moreover, this is one of the first records of Metochus uniguttatus being infected with Wolbachia in North India. Conclusion: This study represents an initial exploration of insects not previously considered hosts of the Wolbachia endosymbiont. The results could be useful for future studies on insect biocontrol and pest management.