The Holobiont in hybrid zones: Interplay between microbiota and host during the hybridization process

Hybridization is an important evolutionary process capable of generating novel genetic and phenotypic combinations, thereby contributing to adaptation and biological diversification. At the same time, the holobiont concept has highlighted that organisms should be considered together with their associated microbial communities, which play a fundamental role in their biology and evolution. However, the role of hybridization in shaping holobiont structure remains poorly understood. This thesis uses the coastal mosquito complex Aedes mariae as a study system to investigate the interactions among host, microbiota, and environment in the context of hybridization. In particular, the study evaluates the relative contributions of environmental factors and host genetic background to the assembly of host-associated microbial communities. Analysis of the bacterial microbiota revealed that hybrid individuals harbor microbial communities distinct from those of the parental species. Application of the 4H (Hybridization–Holobiont–Hologenome–Hierarchy) theoretical framework showed that hybrids predominantly lose rare or low-abundance parental taxa while acquiring novel taxa, whereas taxa shared between the parental species and occurring at higher abundance are preferentially retained. These findings suggest that hybridization promotes the reorganization of the associated microbiota, contributing to the emergence of novel holobiont configurations. Because knowledge of the viral component associated with the Aedes mariae complex was extremely limited, this thesis also provides an exploratory characterization of the Aedes mariae virome, revealing remarkable viral diversity and a substantial degree of viral community sharing between mosquitoes and their aquatic habitat. Although the effects of hybridization on the virome were not directly investigated, these findings provide a valuable reference for future studies on the viral component of the holobiont. Finally, the application of highly accurate long-read sequencing technologies (PacBio HiFi) demonstrated the potential of these approaches to improve the characterization of mosquito-associated viral communities and enhance the detection of viral diversity. Overall, the findings demonstrate that holobiont structure emerges from the interplay between environmental factors and host genotype, highlighting the role of hybridization in reshaping the associated bacterial microbiota. In parallel, the thesis expands current knowledge of the viral diversity associated with Aedes mariae and its natural habitat and shows that highly accurate long-read sequencing technologies represent a promising approach for virome characterization. Taken together, the three studies provide new insights into the processes underlying holobiont assembly and establish a conceptual and methodological foundation for future research on host–microbiota–environment interactions.