Optimization and Validation of Molecular Assays for Invasive Tunicate Monitoring in Environmental Water Samples

Abstract

There are currently four invasive tunicate species which are causing significant challenges to the aquaculture industry in Atlantic Canada: Ciona intestinalis, Styela clava, Botryllus schlosseri and Botrylloides violaceus. Molecular PCR based assays have been developed in previous studies as high throughput screening tools for invasive tunicate monitoring in Atlantic Canada. In this study, field validation experiments were conducted to ensure that assay efficacy and sensitivity would not be impacted by components in the environment which are known PCR inhibitors. Two sets of protocols were optimized for screening sediment rich mussel processing plant effluent, and sediment light bay water surrounding mussel leases, as DNA extraction method and DNA sample dilution were both shown to impact assay efficacy and sensitivity in different water sample conditions. In addition, the efficacy, sensitivity and specificity of all four assays were compared to the current industry standard method of microscope analysis of water samples for invasive tunicate egg and larvae detection at the bay level. All four assays exhibited high sensitivity 1-5 eggs/larvae in all water conditions and had comparable sensitivity and efficacy with the current industry standard.