The use of environmental RNA for inferring fish spawning behavior.

Environmental DNA (eDNA) is a popular method to study species composition, community structure, and invasive species presence but this method is limited for inferring dynamic physiological activities of the identified organisms. Recently, environmental RNA (eRNA) collection and analysis has emerged as a useful tool to shed light on the functions and status of organisms, transcending the eDNA limitation by capturing dynamic biological activities, and shifting the paradigm of field monitoring. During fish spawning, gametes released to the environment can markedly increase gamete-specific eRNA. Using literature- and transcriptome-based information, testis-specific klhl10 and epithelium-specific muc5ac were shortlisted as potential eRNA markers for gametes and epithelia. Medaka mating behavior was observed, tank water was filtered for eRNA extraction, and selected markers were quantified by real time PCR. Early in the daily light cycle, mating attempt duration in medaka was positively correlated to the increase of klhl10 transcripts in the water, supporting that gamete release was the cause of increased klhl10 levels in water. Our results pave the way for the use of eRNA markers to monitor physiological activities of fish in the field, the application of which will benefit aquaculture, environmental assessment, and conservation of species diversities.