Detection of Wolbachia sp. in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) populations collected from some districts of Antalya using real-time PCR

The tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), commonly known as the tomato leafminer, is an economically important pest of tomato. This pest harbors cytoplasmically transmitted Wolbachia bacteria, which is found in the arthropods. In this study, we developed a quantitative real-time PCR (qRT-PCR) assay to screen the dynamics of Wolbachia infections across different developmental stages of tomato pinworm. A FAM-labeled TaqMan probe targeting the Wolbachia ftsZ gene was used for qRT-PCR, while a HEX-labeled TaqMan probe targeting the Tuta absoluta, Aktin gene was used for the host gene quantity. Larval samples were collected from tomato grown greenhouses in Antalya, during the 2021 summer season. Some larval samples were reared to specific age under the laboratory condition. Total nucleic acid was extracted from age-specific whole specimens and subjected to optimized qRT-PCR assay. Each age-specific stage was represented with at least 20 biological samples. Though quantitatively variable, all tested Tuta absoluta samples showed 100% infected with Wolbachia. Using TaqMan Prob assay, a significant difference in Wolbachia densities was detected among the developmental stages of the pest. Overall, the first larval stage showed the lowest Wolbachia signal level, with an increasing amount towards the fourth larval developmental stage. The amount of Wolbachia was found to be higher in the pupal stage compared to the larval stage, but slightly lower than in the adult stage. No differences were observed among the 1-, 3-, 6- and 9-day old pupae. Similarly, the Wolbachia amount seemed not to differ in 1-, 3-, 6- and 9-day old moths. The Wolbachia densities in fourth-instar larvae collected from different districts of Antalya were analyzed using qRT-PCR. As a result, the highest Wolbachia density was detected in the Kepez district, while the lowest was observed in Finike district. Upon examining different body regions of the larvae, it was found that Wolbachia density was highest in abdominal segments 3-6, followed by the thoracic segments, the head, and the last segment of the abdomen. In the adult stage, the highest Wolbachia density was found in the head, followed by the thoracic segments and abdomen. The lowest density was observed in the wings and legs. The results indicated significant differences in Wolbachia density between developmental stages and body regions of the pest. The qRT-PCR analysis was evaluated for its efficiency and applicability in studying the host-endosymbiont interaction dynamics.