Meeting Abstract
The impact of ocean acidification due to increased greenhouse gas emissions on ecosystems and the organisms which they are comprised of can be devastating. At the cellular level, pH changes can be dealt with in a number of ways, one of which is by the use of membrane bound proton pumps. This study focuses on the changes in expression of a V-type proton ATPase found in Ciona intestinalis. This species was chosen because it is widespread in marine habitats globally, and because it has been researched extensively for the effects of other marine pollutants. The objective of this experiment is to investigate how Ciona intestinalis expresses V-type proton ATPase genes to cope with acidic conditions. The C. intestinalis genome contains several proton ATPase subunit isoforms, and two isoforms for subunit A will be tested. The hypothesis is that the two isoforms will be expressed in different situations and different parts of the organism depending on the acidity of the environment. The tunicates will be kept in filtered seawater and fed a combination of dry Spirulina and Phytoplex. To begin, samples of C. intestinalis will be collected, frozen using liquid nitrogen, crushed into powder, and stored at -80o. One set of samples will be collected before the addition of the acid, and several will be collected afterwards at 48, 72, 96, and 120 hours. RNA isolation and RT-PCR methods will be repeated for all samples. The mRNA will be isolated using RNeasy Mini Kit and reverse transcribed using Superscript II RT. The primer sets created for the two genes of interest and one house keeping gene (actin) will be used to run RT-PCR on the cDNA library. After these experiments have been completed, fluorescent ISH will be used to determine if the genes are expressed differently in different parts of the organism. The FISH will be performed on the whole organism without dissecting it in order to visualize the organs better.
