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

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.