From rivers to the sea: a single gene assay for New Zealand freshwater eel eDNA monitoring

Use of environmental DNA and RNA (eDNA and eRNA) is rapidly expanding as an unintrusive and timely method of studying rare or cryptic organisms. These methods have been proposed for studying the unknown migration routes and spawning sites of Aotearoa New Zealand longfin ( Anguilla dieffenbachii ) and shortfin ( Anguilla australis ) eels. However, these areas may be in sympatry with other members of the genus, necessitating species-specific eDNA and eRNA assays. Accurately comparing eDNA and eRNA quantity and degradation rates of two different species can only be done when assessed on the same gene. In this study, species-specific droplet digital polymerase chain reaction (ddPCR) primers and probes were developed for two closely-related species of catadromous eels, A. dieffenbachii and A. australis . Primer and probe assays were created using phylogenetic inference and novel software (assayID) to identify regions of the mitochondrial genome that remain conserved within species but are sufficiently differentiated between target species and other congenerics. In consensus trees, non-anguillid outgroups clustered separately from the Pacific anguillid taxa for the marker of interest, indicating that there is considerable divergence between the assay region in anguillids compared to other taxa. Assays were designed targeting the same region of the nicotinamide adenine dinucleotide hydride 5 (NADH5) mitochondrial gene. The targeted regions span 174 bp for A. dieffenbachii and 177 bp for A. australis . Primers were tested against other species of Pacific Anguilla in end-point PCR, and probes were evaluated in ddPCR. Although faint bands of non-target amplification were observed in end-point polymerase chain reaction (PCR), ddPCR showed minimal cross-reactivity. A ten-fold dilution series of target species’ genomic DNA was used in a multiplex reaction with these primers and probes to assess limits of detection and quantification (LOD and LOQ). Both assays achieved high sensitivy with LOD and LOQ values of 0.20–0.51 copies/µL. Degradation rates of A. dieffenbachii and A. australis eDNA and eRNA were quantified by applying the developed ddPCR assays to a previously generated set of samples from a controlled in-tank degradation experiment conducted over 168 hours at nine time points. The eDNA half-life for both species ranged from 5 to 23 hours, and the eRNA half-life ranged from 4.5 to 16 hours. For both species, greater amounts of eDNA were detected than eRNA. For both assays, strong associations were found between the concentration of loaded DNA and ddPCR copy numbers. Future use of these assays on collections from the wild will further validate their utility in diverse environmental and sympatric contexts. These assays establish a reliable framework for species-specific detection of A. dieffenbachii and A. australis where multiple Anguilla species may be present and hold potential to unveil their migration routes and cryptic spawning areas.