Periodic Reporting for period 2 - RosaLind (Ecogenomics of Mosquito-Microbe Symbiosis for Novel Control Strategies of Infectious Diseases)
Reporting period: 2023-03-01 to 2024-08-31
The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a significant public health threat worldwide. With the rise of insecticide resistance and the absence of an effective vaccine, new strategies are emerging that focus on the mosquito's microbiota. Among these, the endosymbiotic and maternally-inherited bacteria Wolbachia, which can selfishly manipulate the reproduction of their host and interfere with viral pathogens, stands out. However, despite its promise, the genomic variability of Wolbachia and its mobile genetic elements along with its impact on pathogen blocking, in interaction with other symbiotic life, particularly in naturally infected vectors like Culex mosquitoes, remains poorly understood. Leveraging state-of-the-art Ecogenomic tools, the RosaLind project aims to reconstruct bacterial genomes from Culex specimens collected around the world, one mosquito organ at a time. The obtention of novel Wolbachia genomes at the global scale aims to investigate Wolbachia's genomic variability and examine how it contributes to distinct phenotypes of density and protection against viruses. Simultaneously, the project aims to identify and analyse other symbiotic genomes, notably in the midgut of mosquitoes, that could be exploited to fight vector-borne diseases. Moreover, it is expected these newly reconstructed symbiotic genomes serve as reference genomes to study the complex interaction between symbionts and arboviruses through differential expression analyses in infection conditions. Lastly, an important objective of the project is to explore the diversity, biogeography, and transformation capacity of the recently discovered Wolbachia plasmid element (called pWCP for plasmid of Wolbachia in Culex pipiens). While Wolbachia remains not amenable to genetic manipulation, these findings could pave the way for the development of novel vector biocontrol strategies.
The successful sampling of Culex specimen from 10 localities across 4 continents is a major step of the project. It will allow genomic comparison of Wolbachia and its mobile genetic elements together with other symbionts at a scale never done before. Mosquito collection and dissection were done in collaboration with local partners, therefore reinforcing partnership between institutions. Sharing protocols, concepts and entomology skills that have been acquired during the first period of the project with local institutions is an important aspect of the project. The emergence of diseases is a public health issue worldwide and joining forces at the international level appears as key to understand pathogens transmission and to prevent their spread. In parallel to field work, some robust “wet’ and “in silico” developments were set up to investigate the generated data at fine resolution scale. A preliminary metabarcoding study allowed to highlight bacterial microdiversity and niche partitioning within single mosquito individuals. The reconstruction of novel Wolbachia genomes, notably from both ovaries and midguts of single individuals, allowed population genetic analyses that confirmed the absence of strain selection between organs using short reads. Subsequently, developing a hybrid sequencing approach combining short and long-reads (ONT) simultaneously from mosquito ovaries samples allowed obtaining near-complete genomes of Wolbachia. The acquisition of fairly unfragmented Wolbachia genomes represents notably a stepping stone for further investigation of Wolbachia genomic diversity and rearrangement in Culex mosquitoes collected from distinct geographic locations and settings around the globe. In addition, multiple novel genomes belonging to various (cultivable and non-cultivable) bacterial genera were obtained from dissected Culex midguts and are now analyzed. Experimental infection with virus transmitted by Culex (West Nile and Usutu viruses) were successfully performed at the Vectopole in MIVEGEC and newly reconstructed bacterial genomes will be key to explore interactions between symbionts and pathogens. Finally, the large sampled Culex collection allowed investigating the biogeography of pWCP in several islands and continental countries around the world—together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. Data showed that Wolbachia plasmid pWCP is remarkably stable across generations in very different environments and associated selective pressures, confirming a critical role for this mobile element in the endosymbiont biology that warrants further investigation.
Although the architecture, gene content and the very sequence of pWCP is extremely conserved with only minor variations in Culex specimens around the globe, synthesis of the element remains particularly cumbersome. An international and larger group of collaborators is joining forces to attempt developping a much-needed tool for Wolbachia genetics and engineering in different cloning organisms. It is expected the consortium will succeed in delivering a Wolbachia vector shuttle that can be tested and used by a wider Wolbachia community. In addition, given the risk that virus blocking by Wolbachia may evolve through changes in the host or the bacterium in a context of global warming, or in the case of virus epidemics, studying Wolbachia variability at the genomic level at very distinct geographical scales will provide key information on the biology, adaptation and evolution of the bacterium. Studies of the complex interaction between Wolbachia, commensal bacteria and the host in the presence of pathogens in vectors will then allow accumulating knowledge on microbial adaptative molecular mechanisms, and identify some possible other symbionts that may be of use for paratransgenesis approaches. These data are expected to be particularly important for naturally infected Culex populations, which are major vectors of emerging arboviruses such as West Nile virus or Usutu.