Abstract
The recent detection of the invasive colonial tunicate Diplosoma listerianum in Havre- Aubert, Magdalen Islands (Quebec, Canada) in 2008, prompted the development of a molecular assay as a method to detect and monitor for the potential invasion of this species in Prince Edward Island. The aim of this study was to design a highly sensitive, species-specific Polymerase Chain Reaction (PCR) assay capable of detecting D. listerianum with a high efficacy in local water samples. To accomplish this, oligonucleotide primer sets were designed from the 18S rDNA gene of D. listerianum. Primer sets were evaluated for specificity using the GenBank database, followed by a series of spiked water sample trials involving various tunicate species. Assay efficacy was tested and then evaluated by conducting spiked water sample trials using D. listerianum samples from two different geographic locations (Japan and Canada). Primer sets that were shown to be species specific were then tested for their analytical sensitivity and environmental efficacy by spiking local water samples with various amounts of D. listerianum tissue. The primer set DlistF1/DlistR1 was found to be species specific and yielded no false positive results when tested with tissue from the four invasive tunicate species currently present on Prince Edward Island (PEI) (Styela clava, Botryllus schlosseri, Botrylloides violaceus, and Ciona intestinalis). This assay was also capable of detecting D. listerianum DNA from two different populations, demonstrating its potential for use in other geographic locations, which may possess different haplotypes of the species. As the results of this study demonstrate, the DlistF1/DlistR1 assay has a high analytical sensitivity, detecting DNA from as little as 1 zooid in a water sample, and was not inhibited when tested with water samples collected from various bays across both PEI and the Magdalen Islands. The DlistF1/DlistR1 molecular assay provides a monitoring tool for shellfish aquaculture regions and can be used to facilitate early detection of this species. This level of early detection is beneficial to facilitate the implementation of mitigation programs in time to prevent D. listerianum from reaching nuisance levels.
Highlights
Several thousand aquatic invasive species (AIS) are transported globally in ship ballast water, on boat hulls and in sea chests every day (Smith et al 1996; Lambert 2007)
The DlistF1/DlistR1 assay was capable of amplifying target DNA from two different populations of Diplosoma listerianum (Magdalen Islands, Canada, and the Misaki Shipyard, Japan)
This indicates that the assay developed in this study can potentially be used for early detection in the region and has the potential to be used in other geographic locations that may possess different haplotypes, which is an important criterion for invasive species molecular detection tools
Summary
Several thousand aquatic invasive species (AIS) are transported globally in ship ballast water, on boat hulls and in sea chests every day (Smith et al 1996; Lambert 2007). There are currently four invasive tunicate species that are causing challenges in Prince Edward Island (PEI) Canada: Ciona intestinalis Linnaeus, 1767, Styela clava Herdman, 1881, Botrylloides violaceus Oka, 1927, and Botryllus schlosseri Pallas, 1774 (Ramsay et al 2008). These invasive tunicates attach to both natural and artificial substrates and are capable of fouling mussel lines and aquaculture gear including buoys, ropes, cages, and moorings (Astudillo et al 2009; Darbyson et al 2009). Species identity was confirmed using morphological and molecular methods (Nathalie Simard, Sarah Stewart-Clark, and Mary Carman-Personal Communication)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
