A Comparative Study of Botryllid Tunicates Regeneration in the San Francisco Bay

Regeneration processes for various botryllid species show some characteristic features in laboratory studies using application of retinoic acid as a developmental initiator. The three botryllid species, Botrylloides violaceus (n = 6), Botrylloides sp. (bicolor)(n = 4), and Botryllus schlosseri (n = 3) were collected from the San Francisco Bay, and settled onto glass plates in the laboratory. Zooids were removed and the remaining vasculature (ampullae and connecting vessels) were cut into fragments (mean = 9/ genotype) and observed for 8 14 days. Retinoic Acid (RA), a natural product of Vitamin A that accelerates regeneration (in botryllids, Rinkevich, 2007) was also used as a variable in this study to test regeneration ability with decreased artificial RA exposure. Regeneration in the field was also tested by deploying vascular fragments at a marina in Fort Baker after 2 day laboratory RA exposure. The purpose of this study is to characterize and compare regeneration variation between botryllid species under standardized conditions and to determine methods for studying regeneration in the field. Tunicate regeneration results in the growth of an entire functioning body from a small sample of the blood. When the zooid, the part of the animal that contains the heart, feeding and excretion system and gonads, are removed, all that remains is the finger-like ampullae which attach the animal to a substrate along with the vasculature that connects them all within a clear tunic. After several days of rearrangement of the ampullae and vasculature, new buds appear which in turn grow into a new, fully functioning zooid. These features include a notochord, hollow dorsal nerve cord, and pharyngeal gill slits, however the first two are reabsorbed upon settlement and metamorphosis into the sessile adult form. At this stage, the tunicate asexually buds numerous identical buds within the same colony, meaning that all parts of a colony are genetically identical. Introduction Methods Regeneration in the laboratory: Samples were taken from tunicates found on various docks of marinas in the San Francisco Bay. Tunicates were settled on glass slides immediately and stored in a tank with bay water circulation. The zooids were cut from the vascular ampullae in fragments which ranged from 2mm – 7mm in length, 2-10 d after settlement within a span of 7 days. Species used in the experiment include B. violaceus (n=6 samples, n = 42 fragments), B. sp. Bicolored (n = 4 samples, n = 52 fragments), and B. schlosseri (n=3 samples, n = 25 fragments). Samples were placed into 10 gallon tanks with bay water which was changed out every other day. Salinity was maintained between 32 33 ppt. A chiller was placed in the water surrounding the tanks maintained the temperature at 18°C. Results