First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

Background: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites.

Methods: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing.

Results: was abundantly found in this area in both natural and artificial containers, whereas was absent. Among the breeding sources of molecularly confirmed species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on leaves and were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of and , known JE vectors from this area, and reported JE cases as well. Different vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases.

Conclusions: This study, for the first time, reports the breeding sources of in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.