ABSTRACT: The rainbow smelt (Osmerus mordax, Mitchill, 1814) is an anadromous teleost that overwinters in the estuaries and inshore waters along the North American Atlantic coastline. In the winter months, smelt avoid freezing in subzero temperatures by the production of antifreeze proteins and high levels of glycerol. Glycerol production (glyceroneogenesis) occurs in the liver via a branch point in glycolysis and gluconeogenesis and is directly activated by low temperature. In these studies, hepatocytes were isolated from the liver of individual warm fish and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature over a 72 h time course. Functional genomic techniques were used to identify and validate hepatic transcripts that were differentially expressed between the warm and cold cells. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for cold-responsive liver transcripts were constructed at the 72 h incubation time. Microarray analyses using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array were performed at the 24, 48 and 72 h incubation times. For quantitative reverse transcription – polymerase chain reaction (QPCR) studies, we focused specifically on the non-colligative [type II antifreeze protein (AFPII)] and colligative (glycerol accumulation) freeze prevention strategies. AFPII (SSH identified) and 21 transcripts (SSH and/or microarray identified or selected by the authors based on a conceptual link to glycerol production) involved in the metabolism of glycolytic (glycogen, glucose) and gluconeogenic (amino acids) sources of glycerol and of lipids with a glycerol backbone (triglyceride, phosphoplipid) were analyzed using QPCR. Rainbow smelt were collected by seine netting from Mount Arlington Heights, Placentia Bay, Newfoundland in late October 2007 and transported to the Ocean Sciences Centre, Memorial University of Newfoundland. The fish were held in a 3000 L indoor free-flowing seawater tank maintained at 8°C to 10°C and followed a natural photoperiod with fluorescent lights set by an outdoor photocell. Hepatocyte isolations were performed for individual male fish (i.e. no pooling of cells from different fish) from Nov. 27th to Dec.17th, 2007. Hepatocytes were not pooled as individual smelt produce different amounts of glycerol in response to cold temperature challenge. Each liver was perfused with medium containing collagenase and yielded approximately 300 million cells per individual. Initial or “pre-incubation” samples were collected and the remaining cell suspension was divided into 20 ml glass scintillation vials (6 x106 cells per vial) containing 2 ml of incubation medium and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature. Duplicate vials from each temperature were sampled at 24, 48 and 72 h incubation times for RNA isolation. RNA isolated from the hepatocytes of the 9 fish with the highest levels of glycerol production at 0.4°C were used to generate 2 mRNA pools (a “cold” pool and a “warm” pool) for each incubation time (24, 48 and 72 h). To summarize, the “cold” pools contained cells from 9 individual fish incubated at 0.4°C for either 24, 48 or 72 h and which exhibited the highest increase in glycerol levels at 72 h relative to pre-incubation levels, and the “warm” pools contained cells from these same 9 individual fish but incubated at 8°C for either 24, 48 or 72 h and therefore exhibited no increase in glycerol levels at 72 h relative to pre-incubation levels. Comparisons were made for the cold compared to warm pools at the 24, 48 and 72 h time points. For each time point, technical quadruplicate slides including two dye swaps were run per comparison. Incubation time: 24 h: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Incubation time: 48 h: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Incubation time: 72 h: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4 Technical replicate: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Technical replicate: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Technical replicate: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4
