Quantitative PCR assays to detect humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge) in marine water samples

Abstract

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. In order to obtain quantitative datasets for individual species, species-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre to the genus-specific shortbelly rockfish assay, to the humpback whale assay which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Environmental DNA (eDNA) for monitoring marine mammals: Challenges and opportunities

“…Due to potential environmental degradation of DNA over time (Barnes et al, 2014;Collins et al, 2018), studies have focused on amplification of short fragments (< 300bp) to maximize the detection rate of target eDNA. However, shorter amplicons may not contain sufficient nucleotide variation to avoid ambiguous taxonomic assignment, as found in previous studies targeting single species (Foote et al, 2012;Andruszkiewicz et al, 2020) and in metabarcoding studies, in which unequivocal identification of some delphinid species continues to be challenging (Closek et al, 2019;Sigsgaard et al, 2020b;Valsecchi et al, 2021). A recent study using Teleo01 metabarcoding primers (Valentini et al, 2016) demonstrated significant inconsistency in the marine mammals identified, resulting in the erroneous assignment of three non-endemic species to the Black Sea (Zhang et al, 2020b).…”

“…This study of harbor porpoise was the first study to highlight the potential for eDNA isolated from surface seawater samples to detect both detected and undetected marine mammal species at a specific location, and also highlighted the importance of the primer design and optimization to obtain an accurate target species identification. Similarly, primers targeting the D-loop, designed to specifically detect humpback whale (Megaptera novaengliae) eDNA by qPCR assay, showed cross-amplification with minke whale (Balaenoptera acutorostrata) and gray whale (Eschrichtius robustus) (Andruszkiewicz et al, 2020). Both studies provide compelling evidence for the need for careful validation of qPCR assays both in silico and using vouchered DNA samples to ensure assay specificity, capture intraspecific variants and avoid off-target amplification.…”

Capturing marine microbiomes and environmental DNA: A field sampling guide

“…Furthermore, the optimal balance of tradeoffs may depend on the subsequent molecular processing method. For example, the DNA required to capture microbiome diversity (Schmidt et al, 2022) from marker gene amplicon (metabarcoding) or shotgun sequencing likely differs from that needed to successfully conduct a qPCR assay for a specific target (e.g., Andruszkiewicz et al, 2020;Roux et al, 2020;Rourke et al, 2022).…”