Novel genes in spermatozoa as robust markers for reproductive behavior in fish: Physiological perspectives on environmental RNA sequencing

Environmental RNA (eRNA) is a growing theme that can potentially indicates the functional aspects of the organisms in the wild non-invasively, complementing to the species-identifying application of environmental DNA (eDNA). During fish reproduction, large quantities of sperm and eggs are released into the environment, and our group previously showed that a testis-specific eRNA marker ( klhl10 ) was transiently increased in such scenario. The present study used an eRNA sequencing (eRNAseq) approach, accompanied by differential gene expression analysis and transcript visualization by in situ hybridization chain reaction, to identify and characterize novel and robust eRNA markers for reproductive behavior. In the water where mating behavior was evident, 42 novel gene transcripts, including glutamic-acid rich protein-like ( garp -like) and polyamine modulated factor 1 binding protein 1-like ( pmfbp1 -like), were significantly abundant. The transcript abundance of pmfbp1 -like was a thousand-fold more than that of klhl10 , and the positive correlation between their transcript abundance supports that pmfbp1 -like can become a robust eRNA marker for reproductive behavior. The cellular localizations of garp -like, pmfbp 1-like, and klhl10 in the testis indicated that the stages of spermatozoa expressing these markers are associated with their transcript abundance in the water when spermiation occurs. Technical challenges and potential solutions for eRNAseq were discussed.