Evaluation of Enrichment Approaches for the Study of the Viromes in Mollusk Species

Invasive alien species such as freshwater snails have significantly affected the food, environment, and the health of humans and animals, which have unfortunately received insufficient attention. To facilitate the study of viromes in snail species, we compared the enrichment effect of cesium chloride (CsCl) and sucrose density gradient ultracentrifugations in the recovery of diverse viruses in Pomacea canaliculata and Achatina fulica. First, we showed that CsCl-based ultracentrifugation enriched more virus contigs and reduced the nucleic acid background of the Pomacea canaliculata and was thus beneficial for virus recovery. Further studies comparing CsCl- and sucrose-based density gradient ultracentrifugations revealed that the former enriched more viral contigs and viral families of RNA viruses, while the latter yielded more DNA viruses from both Pomacea canaliculata and Achatina fulica. Certain RNA virus families, such as Rhabdoviridae, Arenaviridae, Hepeviridae, Astroviridae, and Alphatetraviridae, were exclusively enriched by CsCl-based ultracentrifugation. Conversely, several DNA virus families including Bacilladnaviridae, Nudiviridae, Malacoherpesviridae, and Adintoviridae were solely identified using the sucrose-based method. Therefore, the selection of viral enrichment technique (either CsCl or sucrose density gradient ultracentrifugation) should be carefully considered based on the specific virome (DNA or RNA viruses) being studied in mollusk species.