Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

Impacts of newly established non-indigenous green crab (Carcinus maenas) on native fauna in Placentia Bay, Newfoundland

The recent invasion of the European green crab (Carcinus maenas) populations in Placentia Bay, Newfoundland and Labrador (NL) raises great concern about potential impacts on local fisheries and native biodiversity. Green crab are highly adaptable and in both native and invaded areas, green crab are well established predators that can outcompete other similarly sized decapods. The main objectives of this thesis were to: 1) identify the native species that green crab compete with for resources; 2) determine the depths and substrate types in which these interactions likely occur; 3) assess the indirect effects of green crab on native crustaceans and their changes in behavior; 4) assess the impacts of green crab on benthic community structure; 5) compare the NL population with other Atlantic Canadian populations in terms of competitive abilities; and 6) compare morphological features of the NL population with other Atlantic Canadian populations. I found that green crab overlap in space and diet with both rock crab (Cancer irroratus) and American lobster (Homarus americanus), potentially leading to a shift in habitat. Laboratory studies on naïve juvenile lobster also suggested shifts in behavior related to green crab, in that lobster decreased foraging activity and increased shelter use in the presence of green crab. Benthic community analyses showed fewer species in mud, sand, and eelgrass sites heavily populated by green crab compared to sites without green crab, although results depended on the taxa involved and I could not eliminate environmental differences through a short term caging study. Foraging ability of green crab varied in intraspecific competition experiments, with populations from NL and Prince Edward Island dominating longer-established populations from Nova Scotia and New Brunswick. Additional studies excluded claw size as a factor driving these results and behavioral differences likely reflected differences in invasion time and population genetics. Overall, green crab in Placentia Bay appear to be altering community structure of benthic invertebrates through predation and they also appear to indirectly impact native crustaceans through competition.