Effectiveness of a neutral red viability protocol developed for two colonial tunicate species

In order to evaluate the potential risk posed by transporting invasive tunicates away from infested sites it is important to be able to measure the viability of these organisms. A Neutral Red (NR) viability assay was developed using a colonial invasive tunicate, Botrylloides violaceus (Oka, 1927) that could be applied to other species. Unlike some colonial tunicates B. violaceus' mortality can be determined under light microscopy by observing changes in the physiological parameters of filtration and reaction to tactile stimulus making them a good study organism. A total of 32 B. violaceus segments (3 cm2) were allocated to treatment and control groups. The treatment group was subjected to acetic acid which resulted in 100 % mortality. Viability was then assessed by comparing treatment and control group responses to NR uptake. There was no mortality in the control that demonstrated a response to tactile stimulus and a noticeable amount of stain uptake inside zooids. In contrast, the treatment groups did not respond to tactile stimulus and no stain was taken in by the zooids. In this study the viability of Didemnum vexillum (Kott, 2002) was assessed under controlled experimental conditions by simulating the air exposure encountered during the normal transportation and processing of tunicate fouled Pacific oysters, Crassostrea gigas (Thunberg, 1793). Clusters containing C. gigas and D. vexillum were collected from an aquaculture lease, in Lemmens Inlet, BC, for an air exposure trial comprised of 9 equally divided treatments (0, 0.5, 1.5, 3, 6, 12, 24, 48 and 72 hours). Three segments of D. vexillum on each cluster were removed and evaluated with the NR protocol developed for B. violaceus. Preserved tissues were analyzed and no sign of stain was noticeable, even with the control segments. The NR protocol developed worked efficiently with B. violaceus, but appeared to fail with D. vexillum. The findings are consistent with D. vexillum demonstrating a high tolerance in situations they are not accustomed to reflecting their ability to enter a dormant state during adverse environmental conditions (hence lack of response to clinical