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In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.

Lepeophtheirus salmonis is an ectoparasitic copepod that can cause serious disease outbreaks in salmonids. Previous work has shown that Atlantic salmon Salmo salar show very little, if any, tissue response to infection, whereas less-susceptible host species exhibit strong inflammatory responses. The objective of this study was to examine whether a low-level L. salmonis infection in the absence of a stress response would have an effect on expression of Atlantic salmon immune-related genes over time. The effects of low-level infection (approximately 8-11 lice/fish) on kidney tissue and head kidney leukocyte immune-related gene expression were studied at two postinfection time points. At this level of infection, there was no evidence of a cortisol stress response. By use of real-time polymerase chain reaction, constitutive expression of the major histocompatibility (MH) class I gene was shown to be 2-10-fold lower in infected Atlantic salmon head kidneys by 21 d postinfection (dpi) than in head kidneys of uninfected fish held under the same conditions. Conversely, by 14 and 21 dpi, constitutive MH class II expression was significantly increased (.10-fold) in infected fish. Constitutive expression of interleukin (IL) 1b also increased threefold in head kidneys of infected fish by 21 dpi; however, no differences were observed in cyclooxygenase (COX)-2 expression over the course of the infection. Adherent head kidney leukocytes from control Atlantic salmon showed an increase in MH class I and COX-2 expression after 3 h of lipopolysaccharide (LPS) stimulation for all three time points collected. The use of LPS stimulation on similar leukocytes from infected fish did not further increase levels of MH class I and COX-2 expression. This is the first report on host gene expression during an ectoparasitic copepod infection. The implications of these expression changes will be discussed with respect to the hostparasite relationship.

BackgroundThraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars.ResultsCharacterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid.ConclusionsThe presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1246-1) contains supplementary material, which is available to authorized users.