Viral Metagenomic Analysis of Bat Fly Pupae

Bats are important reservoirs of zoonotic viruses, yet the role of their obligate ectoparasites, particularly bat flies, in viral maintenance and transmission remains poorly understood. Although viral sequences have been detected in adult bat flies, vertical transmission to their offspring has not been investigated. Here, we present the first metagenomic analysis of bat fly pupae to assess transgenerational viral persistence. Eighty pupae associated with a large Miniopterus schreibersii colony in Hungary were analyzed by next-generation sequencing. We generated more than 69 million reads and over 28,000 assembled contigs. Although initial classification suggested the presence of viral sequences, none were confirmed following sequence-level validation. These findings highlight the substantial potential for false-positive viral assignments in metagenomic datasets and the importance of rigorous post-classification validation. While our results provide no evidence that vertical transmission is a major mechanism of viral maintenance in bat–bat fly systems, this conclusion is limited by the relatively small sample size and single sampling occasion. Highly divergent or low-abundance viruses may also have remained undetected. Broader sampling across seasons and locations is therefore needed to assess the generality of these findings. Nevertheless, this study provides a valuable first step toward understanding potential transgenerational viral persistence in bat flies and highlights the need for complementary approaches to clarify their role in viral transmission.