Environmental DNA Based Surveillance for the Highly Invasive Carpet Sea Squirt Didemnum vexillum: A Targeted Single-Species Approach

Iveta Matejusova,Fiona Bland,John D Bishop,Jenni E Kakkonen,Kirsty F Smith,Eric Dalgarno,Lyndsay Brown,Guillaume Herman,Jennifer Graham,Alex Douglas,Jean-Pierre Lacaze

doi:10.3389/fmars.2021.728456

Abstract

The presence and diversity of marine non-native species, the number of new invasions, and the impact on native communities and habitats are important metrics used to assess the health of marine ecosystems. Monitoring for marine non-native species, using traditional approaches such as rapid assessment surveys (RASs), requires taxonomic expertise and may still fail to detect rare or inconspicuous species. This study reports a validation process for a quantitative PCR (qPCR) assay based on the cytochrome oxidase 1 gene, designed to detect highly invasive tunicate Didemnum vexillum by targeting environmental DNA (eDNA) present in water samples. The D. vexillum qPCR assay showed high sensitivity, with the threshold limit of detection (LOD) and modeled LOD3 (based on triplicate qPCR reactions) estimated as 9.187 and 1.117 copies reaction–1, respectively and the limit of quantification (LOQ) was calculated as 18 copies reaction–1. Analyses of water samples collected from selected Pacific oyster farms and recreational marinas in Scotland showed 100% concordance between the historical data on presence of D. vexillum from RASs and detection of D. vexillum eDNA. Consistency of detection of D. vexillum eDNA among different sampling points within each infected sampling site varied, ranging between 100% positive throughout the site to some sampling points testing “negative” or only as “suspected” for D. vexillum. Sites with lower within-site detection consistency included sites with a low density of D. vexillum as reported by RASs or were sites undergoing D. vexillum management. The present pilot monitoring program demonstrates the potential to generate important data on presence of D. vexillum. This program will be scaled up across large geographic regions and used in the first instance to focus and target the traditional RASs to D. vexillum eDNA-positive sites in a cost-effective way, with an aim to verify the species presence by visual observation and direct Sanger sequencing of positive qPCR products.

Highlights

The carpet sea squirt Didemnum vexillum Kott, 2002 is a colonial tunicate native to the Northwest Pacific Ocean including Japan (Stefaniak et al, 2012)
This study presents the results of a D. vexillum monitoring program carried out between 2017 and 2019 in selected recreational marinas and shellfish aquaculture farms in Scotland based on detection of target species environmental DNA (eDNA) by quantitative PCR (qPCR) and eDNA Surveillance for Didemnum vexillum describes the assay validation process for the use of this assay in the Northern Hemisphere
Stefaniak et al (2009) (Accession number EU472661) for this species from Panama being identical to D. vexillum in the assay probe region, whereas the entry published by Da Silva Oliveira et al (2017) (Accession number KU221189) from Brazil showed three nucleotide differences

Summary

Introduction

The carpet sea squirt Didemnum vexillum Kott, 2002 is a colonial tunicate native to the Northwest Pacific Ocean including Japan (Stefaniak et al, 2012). A wide variety of substrates, including both natural substrates [e.g., gravel, boulders, subtidal rocks (Bullard et al, 2007; Valentine et al, 2007b), bryozoans, eelgrass, macroalgae, sponges, tubeworms (Valentine et al, 2007b; Hitchin, 2012; Carman et al, 2014; Vercaemer et al, 2015), and bivalve species (Fletcher et al, 2013a; Vercaemer et al, 2015; Forrest and Atalah, 2017)] and artificial man-made structures [e.g., harbor, marina, and aquaculture installations (Pederson et al, 2005; Coutts and Forrest, 2007; Hitchin, 2012; Bishop et al, 2015b; Cottier-Cook et al, 2019)] have been reported as suitable for the attachment and successful establishment of D. vexillum colonies. The report highlighted the difficulty of eradication and the numerous lessons learnt by unsuccessful attempts in New Zealand and Wales (Coutts and Forrest, 2007; Forrest and Hopkins, 2013; Sambrook et al, 2014)

Methods

Results

Conclusion