STILLMAN, J.H.; TERANISHI, K.S.; Univ. of Hawaii, Manoa; Univ. of Hawaii, Manoa: Hepatopancreas transcriptome profiles during thermal acclimation of the porcelain crab, Petrolisthes cinctipes

We investigated changes in hepatopancreas tissue transcriptome profiles during thermal acclimation of porcelain crabs, Petrolisthes cinctipes, to 8 and 18 �C using a 13,824 spot cDNA microarray. The microarray was constructed from cDNA libraries created from a range of tissue types (including whole crabs, and tissues from gill, heart, muscle, nerve, and hepatopancreas) from crabs exposed to a range of thermal conditions. Crabs were acclimated for four weeks in aquaria held at either 8�C or 18�C and were swapped into the opposite acclimation aquarium at noon on the 29th day of acclimation. Crabs acclimated to 8 and 18�C for four weeks have significantly different cold and warm thermal limits and we hypothesize that crabs at these acclimation temperatures differentially express genes involved in maintenance of thermal limits. During n=10 time points during 48h preceding the swap and n=11 time points during 48h following the swap, n=3 specimens were removed from each acclimation group, and were snap frozen. These samples represent transcriptome states of cold and warm acclimated crabs, as well as the transcriptome responses to acute temperature changes. For each sample, hepatopancreas tissue was thawed in TRIZOL, total RNA was purified, and RNA reverse transcription was used to synthesize amino-allyl cDNA. Amino allyl cDNAs were labeled with Cy3 or Cy5 dye and hybridized on microarrays using a modified loop design. Each sample was hybridized at least once. Fluorescence data were normalized using the Loess function and were log transformed. These data were used in variance-based analyses (SAM in Excel and MAANOVA in R) to determine which of the 13,824 spots had fluorescence intensities that significantly differed among samples with respect to acclimation temperature and acute temperature changes.