| Summary: | Winter flounder (Pseudopleuronectes americanus) survive in icy seawater at temperatures reaching -1.9 °C by producing antifreeze proteins (AFPs). AFPs bind to ice crystals and lower the freezing point of fish blood in a non-colligative manner. Previous studies have shown that AFP levels vary among populations of winter flounder, even when fish are raised in the same location, suggesting a genetic rather than an environmental mechanism. Other studies have shown that AFP gene copy numbers vary among locations and that AFP activity in plasma is proportional to gene copy number. The goal of the current study was to determine the AFP gene copy numbers in winter flounder from several locations along the eastern coast of North America, and to determine whether gene dosage is related to antifreeze activity. We designed a quantitative PCR (qPCR) assay to determine the copy numbers of genes encoding the four major AFPs in winter flounder plasma (wflAFP-6, wflAFP-8, wflAFP-9 and the hyperactive AFP Maxi). AFP gene copy numbers were analyzed in flounder from Newfoundland, St. Lawrence Estuary, Baie des Chaleurs, Passamaquoddy Bay and Georges Bank. Winter flounder from Newfoundland had considerably higher AFP gene copy number for each of the genes tested compared to Georges Bank fish. Flounder from the other locations had intermediate gene dosages, differing from Georges Bank fish for some genes and differing from Newfoundland fish for others. There was a relationship between AFP gene copy numbers and antifreeze activity at the population level. We found the hyperactive gene Maxi to be in considerable lower numbers in comparison to the smaller AFPs wflAFP-6 and 8. We attribute this to the protein size and activity. In order to better understand the gene organization of Maxi, we performed Southern blot analysis on samples from each of the locations studied and found that Maxi is represented by a multi-gene family. The antifreeze activity analysis also showed that some fish which did not have any copies of Maxi still had Maxi-like antifreeze activity. This suggests there might be other genes encoding AFPs with high activity similar to maxi that are contributing to winter flounder freeze resistance.
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