De novo whole genome sequencing and thermal adaptation of new invasive mosquito species Aedes koreicus and Aedes japonicus

Mosquito-borne diseases (MBDs) pose significant risks to public health in tropical and subtropical regions and temperate areas, with potential risks even in regions that are not currently endemic. Invasive mosquitoes play a key role in the spread of MBDs, and climate warming is a key element promoting the diffusion of several mosquito vectors. Consequently, by 2050, an additional billion people are predicted to be exposed to the risk of contracting at least one MBD. Recently, two invasive Aedes mosquito species, the Ae. japonicus and Ae. koreicus, are circulating in several European countries, posing potential health risks to humans and animals. Vector control is the primary option to prevent MBDs. An accurate knowledge of the genomes and transcriptomes of the new invasive mosquitoes is essential to understand their biology and develop effective control strategies. Moreover, considering the increasing impact of climate changes on the transmission of MBDs, a more extensive knowledge of the mechanisms underlying the thermal adaptation of these mosquitoes can translate into more effective and innovative approaches for their control. Methodology: The thesis is divided into two main sections: 1. Whole genome sequencing and transcriptomic analysis of Ae. japonicus and Ae. koreicus. The goal was to assemble the genomes and annotate relevant genes in the two species, with a specific focus on candidate insecticideresistant and thermal adaptation genes. 2. Thermal adaptation studies in Ae. koreicus and other mosquito species. These experiments included analysis of the expression of selected thermal adaptation genes and 16S rRNA metagenomic analysis in response to thermal stress. Comparing mosquitoes of different species exposed to various temperatures, we aimed to identify genes differentially expressed and endosymbiotic bacteria with potential roles in thermal adaptation. Results: 1. The de novo genome assemblies of Ae. japonicus (Ajap1) and Ae. koreicus (Akor1) were produced based on a hybrid approach that combined Oxford Nanopore long-read and Illumina short-read data. The quality of the results was ascertained using various metrics. Masking of repetitive elements, gene prediction, and functional annotation were performed. Sequence analysis showed a high presence of repetitive DNA. The phylogenetic analysis revealed that Ae. koreicus and Ae. japonicus had a common ancestor, recently separated from the other Aedes species. Moreover, we identified various thermal adaptation genes and insecticide-resistance candidate genes in the two species. Exposing larvae and 2 adults of Ae. koreicus and Ae. japonicus to different temperatures, we found several genes showing temperature-dependent differential activation by RNA sequencing analysis. 2. We confirmed the temperature-dependent overexpression of five candidate thermal adaptation genes in Ae. koreicus. We found candidate thermal genes activated during cold stress in Ae. albopictus but not in Cx. pipiens. Interestingly, a few thermal genes were also activated in An. stephensi, meaning that this invasive species could also undergo thermal adaptation. The metagenomics analysis gave insights into the complexity of the mosquito microbiota. Moreover, it highlighted changes in the composition of endosymbiotic bacteria in response to thermal stress potentially relevant to the process of thermal adaptation in these mosquito species.