Advancing DNA-based quantification of Pacific oyster larvae using a HTqPCR multi-marker approach

Abstract

Marine bivalves play important roles in seafood supply/security and ecosystems services, with many aquaculture practices relying on wild stocks as sources of larvae. However, rapid and effective monitoring of larvae occurrence, abundance and distribution in the marine environment can be challenging. DNA-based approaches have shown promising results in providing novel monitoring tools. However, the effect of varying sources of different DNA targets and differential representation at distinct larval stages on genetic signal (copy numbers) are poorly understood. The present study used a High-Throughout real-time quantitative PCR (HTqPCR) platform to screen multiple genetic markers (originating from nuclear, ribosomal and mitochondrial genes) in DNA extracts from known number of larvae at two developmental stages (5-day old D-shape and 20-day old pediveliger), using the Pacific oysters (Magallana gigas) as a model study organism. Findings showed clear trends across sources of DNA, with ribosomal markers showing significantly higher numbers of DNA copies compared to mitochondrial markers, which in turn were significantly higher than nuclear markers. A predictable relationship was found between DNA copies found in five-day old D-shape larvae and 20-day old pediveliger larvae, indicating that developmental stages have significant effects on biomass estimation using DNA-derived data. Nuclear and mitochondrial markers showed higher accuracy for estimating higher amounts of larvae, whereas ribosomal markers showed higher accuracy for estimating lower numbers of larvae, suggesting that a multi-marker approach may be most appropriate. While this study provides empirical evidence on the effect of larval stages and source of DNA in quantifying bivalve larvae, it also provides a framework (enabled by a HTqPCR platform) to assess the suitability of DNA-based approaches for the monitoring of marine larvae occurrence as well as biomass.