Development and application of a quantitative real-time PCR assay for the globally invasive tunicate Styela clava

Joanne Gillum

doi:10.3391/mbi.2014.5.2.06

Abstract

Styela clava Herdman, 1881, is a solitary ascidian native to the Northwest Pacific, which has spread globally over the past 90 years, reaching pest levels and causing concern to the aquaculture industry in some regions. It has a relatively short-lived larval stage, spending only limited time in the water column before settling on a desirable substrate. Early detection of this species is an important step in both the prevention of its spread and of successful eradication. Here we report the development of a qPCR based assay, targeted to a region of the mitochondrial cytochrome oxidase I gene, using TaqMan® MGB, for the early identification of S. clava, post-settlement. The assay takes into account the moderately high level of haplotype diversity within this species, while also accommodating the need for species specificity. Using exemplars of closely related ascidian species, we show that our assay is specific for S. clava and is able to detect this species at low concentrations, equivalent to a single propagule. We also demonstrate, through “spike in” tests using environmental extracts, that our assay is robust to the potential inhibitors that might be found in environmental samples, which suggests this assay could have utility in the field, and may be developed further to detect larvae at an earlier stage of introduction.

Highlights

Invasions of non-indigenous species (NIS) are acknowledged as one of the major threats to natural environments - terrestrial, freshwater and marine - having ecological, economic and social consequences
When dealing with the international agreements and measures to prevent introduction of marine Invasive Alien Species (IAS), the following documents are fundamental to the conservation and management of the marine environment: the International Convention for the Control and Management of Ships' Ballast Water and Sediments (IMO 2004), ICES Code of Practice on the Introductions and Transfers of Marine Organisms (ICES 2005), Alien Species in Aquaculture - IUCN considerations for responsible use (Hewitt et al 2006), EC Regulation on concerning use of alien and locally absent species in aquaculture (European Commission 2007) and EC Marine Strategy Framework Directive (European Commission 2008)
A recent study of diatoms and dinoflagellates reported as NIS in European Seas found dubious identifications, and that synonyms were included as separate species (Gómez 2008)

Summary

Introduction

Invasions of non-indigenous species (NIS) are acknowledged as one of the major threats to natural environments - terrestrial, freshwater and marine - having ecological, economic and social consequences. A recently published Proposal for a Regulation of the European Parliament and of the Council on the prevention and management of the introduction and spread of invasive alien species (European Commission 2013) states that “...The impact of IAS on biodiversity is significant ... The EU Biodiversity Strategy (European Commission 2011) aims that "...by 2020, Invasive Alien Species (IAS) and their pathways are identified and prioritised, priority species are controlled or eradicated, and pathways are managed to prevent the introduction and establishment of new IAS...". Stemming from a recent freshwater invasives conference, Caffrey et al (2014) identified ’the top 20 issues that relate to IAS management in Europe“. We agree and support all the issues proposed, and would like to contribute to this important initiative by adding six important topics that relate to the marine environment

Regulatory framework to prevent introduction of IAS

Reliable NIS identifications and loss of taxonomic expertise

Data-gaps impede comprehensive assessments

Need for a unified and appropriate terminology

Standardization of data and information systems

Conclusions