Evaluating the Effect of Sampling Scale on Mosquito Virome Characterization Using PacBio HiFi Long-Read Metagenomics

Characterizing the mosquito virome is essential for understanding host–microbiota interactions and vector competence, but it can be influenced by sample scale, sequencing strategy, and host depletion. This study evaluated the effect of sampling scale on mosquito virome characterization using a third-generation sequencing (TGS) metagenomics approach based on PacBio HiFi long reads, applied to L4 larvae and adults of Aedes mariae, analyzing single individuals and pools of increasing size before and after host genome removal. The results showed that sequencing yield did not increase with pool size, indicating that the total number of reads is not proportional to the number of individuals. Host genome removal reduced the overall number of reads but altered their composition, increasing the relative proportion of assigned viral reads and reducing unclassified sequences. Despite a similar total read output, virome diversity increased with pool size, with larger pools showing greater taxonomic richness driven by the contribution of each individual. However, the high proportion of unassigned reads suggests the presence of uncharacterized viruses. This methodological workflow was technically feasible for both single-individual and pooled samples. Single-individual analyses may provide complementary information on individual-level virome composition and on low-abundance viral taxa that could be less apparent in pooled samples, whereas pooled samples may facilitate the detection of a broader range of viral taxa and may better capture the shared component of viral diversity within the analyzed population.