Locating Little Larvae: Environmental DNA Methods Can Detect a Rare Fish Species Under Field Estuarine Conditions

ABSTRACT Rare species are difficult to monitor using traditional methods, making them excellent candidates for environmental DNA (eDNA) monitoring. The larval life stages of Delta Smelt ( Hypomesus transpacificus ) and Longfin Smelt ( Spirinchus thaleichthys ) are potentially impacted by State Water Project operations in the San Francisco Bay Estuary (CA, USA) via entrainment into unfavorable habitat. Despite intensive monitoring efforts, locating these small, rare fishes is difficult as they occur at extremely low densities in a heterogeneous and dynamic estuarine environment. This study presents novel eDNA monitoring efforts for larval Delta Smelt and Longfin Smelt. We first evaluated the limit of detection/limit of quantification (LOD/LOQ) of a novel Quantitative PCR (qPCR) assay using cultured larval Delta Smelt. Next, we confirmed detectable concentrations of eDNA for a range of abundances of larval Delta Smelt through laboratory experiments. The average copy number of eDNA obtained from one larva exceeded the LOD for the qPCR assay with a positive linear relationship between eDNA concentrations and abundance. Finally, we evaluated the feasibility and probability of detection (POD) for three candidate sampling methods (two vessel‐based and a stationary autonomous sampler) under field conditions, using known distances from, and abundances of, an eDNA source (1, 50, or 300 live larvae in submerged cages). We detected all abundances tested from at least one sample replicate and one filter type. We observed high variability between replicates, with greater POD at higher abundances and smaller distances. Under optimal conditions POD approached 100%. Future research with higher replication is needed to reduce standard error and increase confidence. Despite challenges, we successfully detected larval Delta Smelt in controlled and field conditions. These are the first quantitative estimates of POD and the first use of an eDNA autosampler for monitoring this rare Osmerid.