Development of 18S rDNA and COI gene primers for the identification of invasive tunicate species in water samples

Abstract

Invasive tunicates are creating costly fouling problems to the mussel aquaculture industry in many regions including Prince Edward Island, Canada. These invasive tunicates are also posing a threat to the ecosystem integrity of native species. There are currently four invasive tunicate species present in waters surrounding Prince Edward Island including: Ciona intestinalis, Styela clava, Botryllus schlosseri, and Botrylloides violaceus . Current monitoring practices for the presence of these species in PEI are conducted by costly and time consuming recruitment plates or via microscopy surveys for egg and larval stages using dissecting microscopes. However, these methods are inadequate for early detection. Recruitment plates only allow for the detection of tunicates once they have already established, and microscopic methods can be inaccurate since it is difficult to distinguish between eggs and larvae of different species of tunicate. In this study, we propose polymerase chain reaction (PCR) based detection as a means to overcome the limitations of current monitoring practices in water samples. For this purpose, oligonucleotide primers were generated to enable the rapid analysis of samples for the presence of invasive tunicates by PCR. Primers were designed from 18S rDNA and COI gene sequences specific to each species and were evaluated for efficacy, specificity, and sensitivity in detecting target tunicate species. Through efficient detection methods and careful monitoring it is hoped that further invasions of these tunicate species throughout Prince Edward Island waters can be prevented or managed in their early stage.