Water volume, biological and PCR replicates influence the characterization of deep-sea pelagic fish communities

Abstract

ABSTRACTThe pelagic deep sea is challenging to investigate due to logistical constraints regarding access and collection of samples, however environmental DNA (eDNA) can potentially revolutionize our understanding of this ecosystem. Although advancements are being made regarding technology and eDNA samplers, many countries and research groups still do not have access to modern equipment. At the same time, no investigation has been performed to assess the impact of different experimental designs using common gear found on research vessels (i.e., CTD mounted with Niskin bottles). Here, we investigated the effects of sampled water volume, biological and PCR replicates in characterizing deep-sea pelagic biodiversity. Samples were collected at 450m depth at night in the northern Gulf of Mexico using Niskin bottles, and we targeted the fish community using the MiFish primer (12S rRNA). Our results show that 1L is insufficient to characterize deep-sea pelagic fish communities. The treatments 5L and 10L detected similar communities and numbers of species, but 10L detected more exact sequence variants (ESVs). Five biological replicates can detect up to 80% of the species detected in the water collected in both 5L and 10L treatments. Extrapolation analysis indicates that 10 replicates would also potentially recover 100% of the species. More biological replicates would be necessary to detect all ESVs. PCR replicates also had an important role in taxa and ESV detection, being the best strategy to perform more replicates if you filter less water (optimal number inversely proportional to the water volume filtered). We suggest that future studies collect at least 5L, 5 field replicates, and 5-10 PCR replicates to adequately investigate deep-sea pelagic biodiversity using eDNA, considering the limitations we mention. Our study provides guidance for future eDNA studies and a potential route to democratize eDNA studies at a global scale.