Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax)

Rainbow smelt accumulate high amounts of glycerol in winter. In smelt, there is a predictable profile of plasma glycerol levels that starts to increase in November (<5 μmol/ml), peaks in mid-February (>200 μmol/ml), and thereafter decreases to reach the initial levels in the beginning of May. The aim of this study was to investigate the respective role of the two main mechanisms that might be involved in glycerol clearance from mid-February: 1) breakdown of glycerol to glycerol-3-phosphate through the action of the glycerol kinase (GK) and 2) direct loss toward the environment. Over the entire glycerol cycle, loss to water represents a daily loss of ～10% of the total glycerol content of fish. GK activities were very low in all tissues investigated and likely have a minor quantitative role in the glycerol cycle. These results suggest that glycerol levels are dictated by the rate of glycerol synthesis (accelerated and deactivated during the accumulation and decrease stages, respectively). Although not important in glycerol clearance, GK in liver might have an important metabolic function for other purposes, such as gluconeogenesis, as evidenced by the significant increase of activity at the end of the cycle.     

Response of rainbow smelt, Osmerus mordax (Mitchill), eggs to low pH

The eggs of rainbow smelt were incubated at pH c . 4.5, c . 5.5 and c ontrol ( c . 6.8) for durations of 2–14 days to assess their vulnerability to acid precipitation. These eggs are likely to be exposed to marked pH depressions during their incubation in small streams owing to run-off from snow melt and spring rains. Survival after 48 h exposure was not related to pH, although longer exposure to low pH significantly decreased survival. The developmental stage at initial exposure was also important in determining survival. Overall egg survival varied from 0–87% at pH c . 4.5,0–89% at pH c . 5.5, and 44–100% at control pH levels, (c. 6.8).     

Characterization of di‐ and tetranucleotide microsatellite markers in rainbow smelt (Osmerus mordax)

Twelve tetra‐ and di–tetra compound microsatellite loci are described for the rainbow smelt (Osmerus mordax). These loci were screened among a minimum of 79 individuals and the average number of alleles per locus was 16 with an average heterozygosity of 0.86. These microsatellites are being used to examine population structure and life‐history evolution among natural populations of rainbow smelt.     

The effects of environmental factors on rainbow smelt Osmerus mordax embryos and larvae

Experiments were conducted to identify environmental factors that influence the survival of rainbow smelt Osmerus mordax during their early life stages. Developing rainbow smelt embryos and yolk-sac larvae were cultured under controlled conditions with different dissolved oxygen (DO; 1·09, 2·18, 4·37 and 6·55 mg l⁻¹, pH (4·0, 4·5, 5·0, 5·5, 6·0 and 7·0), nitrate ( 0·7, 3·6, 7·3, 14·6 and 29·2 mg l⁻¹), phosphate (0·04, 0·21, 0·42, 2·08 and 4·17 mg l⁻¹) and salinity (0, 5, 10, 15, 20 and 30) levels. Rainbow smelt embryos were also incubated with simulated tidal salinity fluctuations (2–28), ultraviolet radiation (irradiances of 2·8, 6·2 and 5·1 W m⁻²) and under natural conditions in two rainbow smelt spawning rivers. In the laboratory, hatch was only impaired under the lowest DO and pH conditions (0 and 13% hatch, respectively) and at highest constant salinity levels (0% hatch). Larval survival was only affected by pH levels ≤5·0. The experiment that compared hatch under natural conditions was terminated when embryos became covered with silt and fungus. These results suggest that water acidification, sediment and fungal growth may affect rainbow smelt survival during their early life stages.     

Mortality of young-of-the-year rainbow smelt (Osmerus mordax) in Lake Erie associated with the occurrence of Glugea hertwigi

An extensive mortality of young-of-the-year rainbow smelt (Osmerus mordax) occurred in Lake Erie during the fall of 1969. Dead and dying smelt were observed along the north shore from west of Long Point in the central basin to Port Maitland in the eastern basin. No other fish species was involved. Glugea hertwigi, a microsporidan parasite, was observed in 90% of the distressed smelt examined, and is believed to have been a major contributing factor to the mortality.     

Temporal genetic differentiation: continuous v. discontinuous spawning runs in anadromous rainbow smelt Osmerus mordax (Mitchill)

Genetic differentiation was greater between 'early' and 'late' spawning rainbow smelt Osmerus mordax separated by a hiatus in spawning activity compared to a continuously spawning population over a similar time period. This magnitude of temporal differentiation was comparable to that seen among spatially separated populations.     

Structural and functional characterization of a C-type lectin-like antifreeze protein from rainbow smelt (Osmerus mordax).

Antifreeze proteins (AFPs) are produced by several cold-water fish species. They depress physiological freezing temperatures by inhibiting growth of ice crystals and, in so doing, permit the survival of these fish in seawater cooler than their normal freezing temperatures. The type II AFP from rainbow smelt (Osmerus mordax), which is a member of the C-type lectin superfamily, was characterized in terms of its Ca2+-binding quaternary structure and the role of its single N-linked oligosaccharide. The protein core of the smelt AFP, shown through sequence homology to be a C-type lectin carbohydrate-recognition domain, was found to be protease resistant. Smelt AFP was also shown to bind Ca2+, as determined by ruthenium red staining and a conformational change on Ca2+ binding detected by intrinsic fluorescence. The N-linked oligosaccharide was found to have no effect on protease resistance, dimerization, or antifreeze activity. Thus its role, if any, in the antifreeze function of this protein remains unknown. Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits. This dimerization did not require the presence of N-linked oligosaccharide or bound Ca2+. Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity. This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this AFP.     

Low temperature directly activates the initial glycerol antifreeze response in isolated rainbow smelt (Osmerus mordax) liver cells

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol in winter that serve as an antifreeze. Liver glycogen is a source of glycerol during the early stages of glycerol accumulation, whereas dietary glucose and amino acids are essential to maintain rates of glycerol synthesis. We presently report rates of glycerol and glucose production by isolated hepatocytes. Cells from fish held at 0.4 to -1.5 degrees C and incubated at 0.4 degrees C were metabolically quiescent with negligible rates of glycerol or glucose production. Hepatocytes isolated from fish maintained at 8 degrees C and incubated at 8 degrees C produced glucose but not glycerol. Glycerol production was activated in cells isolated from 8 degrees C fish and incubated at 0.4 degrees C without substrate or when glucose, aspartate, or pyruvate was available in the medium. Incubation at 0.4 degrees C without substrate resulted in similar molar rates of glucose and glycerol production in concert with glycogen mobilization. Glycogenolysis and glycerol production were associated with increases in total in vitro activities of glycogen phosphorylase and glycerol-3-phosphate dehydrogenase. Maximal in vitro activities of hexokinase and glucokinase were not influenced by temperature, but high activities of a low-K(m) hexokinase may serve to redirect glycogen-derived glucose to glycolysis as opposed to releasing it from the cells. Rates of glycerol production were not enhanced in cells from fish held at 8 degrees C and incubated at 0.4 degrees C with adrenergic or glucocorticoid stimulation. As such, low temperature alone is sufficient to activate the glycerol production mechanism and results in a shift from glucose to a mix of glucose and glycerol production.     

A simulation of food-web interactions leading to rainbow smelt Osmerus mordax dominance in Sparkling Lake, Wisconsin

A process-based simulation model was used to examine the nature and intensity of food-web interactions that allow Osmerus mordax to dominate invaded lakes. The model simulates food-web interactions among linked populations of O. mordax, Coregonus artedi and Sander vitreus. Simulations indicated that O. mordax dominate where: (1) adult O. mordax prey on young-of-the-year (YOY) C. artedi, (2) YOY O. mordax negatively affect YOY S. vitreus through competition and (3) adult S. vitreus experience moderate fishing mortality. Osmerus mordax dominated simulations across a broad range of variable values that regulated competition and predation, and displayed threshold responses to increasing angler harvest. Consequently, angler harvest should be carefully managed in lakes susceptible to O. mordax invasions because the alternative could lead to fishery collapse.     

Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production

Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.     

Divergent selection maintains adaptive differentiation despite high gene flow between sympatric rainbow smelt ecotypes (Osmerus mordax Mitchill)

In this study, we investigate the relative role of historical factors and evolutionary forces in promoting population differentiation in a new case of sympatric dwarf and normal ecotypes of the rainbow smelt (Osmerus mordax Mitchill) in Lac Saint-Jean (Québec, Canada). Our first objective was to test the hypothesis that the evolution of sympatric smelt ecotypes in Lac Saint-Jean has been contingent upon the secondary contact between two evolutionary lineages in postglacial times. Secondly, the QST method was applied to test the null hypothesis that the extent of phenotypic differences relative to that of neutral marker variation would be similar in comparisons involving populations within and among ecotypes. Thirdly, we applied a quantitative-genetic method as an exploratory assessment as to whether the amount of gene flow observed between populations could affect divergence in adaptive traits under specific conditions. This study revealed a unique situation of dwarf and normal smelt ecotypes that are, respectively, characterized by selmiparous and iteroparous life histories and the occurrence in each of two genetically distinct populations that synchronously use the same spawning habitat in two tributaries. Historical contingency has apparently played little role in the origin of these populations. In contrast, an important role of divergent natural selection in driving their phenotypic divergence was suggested. While divergent selection has apparently been strong enough to maintain phenotypic differentiation in the face of migration, this study suggests that gene flow has been sufficiently important to modulate the extent of adaptive differentiation being achieved between ecotypes, unless the extent of stabilizing selection acting on smelt ecotypes is much more pronounced than usually reported in natural populations.     

Analysis of the effect of transplantation on morphometric and meristic characters in lake populations of the rainbow smelt,Osmerus mordax (Mitchill)

Synopsis Results of experimental transfer of rainbow smelt into lakes reclaimed by rotenone around 1960 in Maine were originally interpreted to cast doubt on the previously widely accepted hypothesis that there were two hereditarily different forms of rainbow smelt, one large and one small. Study of more recent data from some of the transplanted populations and reanalysis of the original data suggests different conclusions. The initial effect of introductions into a reclaimed lake may be accelerated and/or more prolonged growth which exceeds even interlake differences. This initial phase, however, is followed by a second phase, when the population reaches equilibrium and these effects subside. Data from phase two of the Little Concord-Shagg and Cold Stream-Coleback Lake transfers showed that the growth characteristics of the transplanted populations returned to those of the parental populations. Large differences in growth patterns were thus found only in the initial phase of the introduction. Meristic characters were little affected by transplanting.Analysis of large specimens derived from a postulated second unofficial introduction into Coleback Lake showed that they also differed significantly, having both higher gill raker and vertebral counts than the smaller smelt. This was of interest as smelt vertebral and gill raker counts usually are inversely related; hence we do not equate these for the moment with the large form of smelt known elsewhere.It is concluded that the initial interpretation of transfer experiments be delayed until conditions approaching equilibrium can be expected to exist. Further, our analysis of more recent lake transfer data has shown nothing to refute the hypothesis that there are at least two hereditarily different forms of smelt.     

Control of glycerol production by rainbow smelt (Osmerus mordax) to provide freeze resistance and allow foraging at low winter temperatures

The rainbow smelt (Osmerus mordax) is a small anadromous fish that actively feeds under the ice at temperatures as low as the freeze point of seawater. Freezing is avoided through the production of both non-colligative antifreeze protein (AFP) and glycerol that acts in a colligative manner. Glycerol is constantly lost across the gills and skin, thus glycerol production must continue on a sustained basis at low winter temperatures. AFP begins to accumulate in early fall while water temperatures are still high. Glycerol production is triggered when water temperatures decrease to about 5 degrees C. Glycerol levels rapidly increase with carbon flow from dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) to glycerol. Glucose/glycogen serves as the initial carbon source for glycerol accumulation with amino acids contributing thereafter. The period of glycerol accumulation is associated with increases in GPDH mRNA and PEPCK mRNA followed by elevations in protein synthesis and enzyme activities. Plasma glycerol levels may reach in excess of 500 mM in winter. The high freeze resistance allows rainbow smelt to invade water of low temperature and forage for food. The lower the temperature, the higher the glycerol must be, and the higher the glycerol the greater the loss to the environment through diffusion. During the winter, rainbow smelt feed upon protein rich invertebrates with glycerol production being fueled in part by dietary amino acids via the gluconeogenic pathway. At winter temperatures, glycerol is quantitatively more important than AFP in providing freeze resistance of blood; however, the importance of AFPs to other tissues is yet to be assessed. Glycerol levels rapidly plummet in the spring when water temperature is still close to 0 degrees C. During this period, freeze resistance must be provided by AFP alone. Overall, the phenomenon of glycerol production by rainbow smelt reveals an elegant connection of biochemistry to ecology that allows this species to exploit an otherwise unavailable food resource.     

Genetic structure and relationships among anadromous and landlocked populations of rainbow smelt, Osmerus mordax, Mitchill, as revealed by mtDNA restriction analysis

Larvae of anadromous rainbow smelt originating in various spawning tributaries are retained in the St Lawrence estuary. We proposed that these smelt represent one population genetically differentiated from adjacent populations characterized by geographically distinct larval retention areas. We also analysed four landlocked populations to evaluate the phylogenetic basis of dwarf and normal-sized phenotypes and their relation to an adromous smelt. A phylogenetic distinction was revealed between anadromous and landlocked smelt, with only one of the two mtDNA phylogenetic groups of anadromous fish observed among landlocked smelt. Significant geographical heterogeneity in the distribution of mtDNA genotypes was observed among landlocked smelt, suggesting that dwarfism in smelt may be polyphyletic in origin. St Lawrence smelt were genetically identical but distinct from adjacent populations, supporting the proposition that population genetic structure reflects the number of larval retention zones rather than spawning sites.     

Temperature effects on trimethylamine oxide accumulation and the relationship between plasma concentration and tissue levels in smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) were maintained in a long term acclimation study to elucidate temperature effects on the accumulation of trimethylamine oxide (TMAO) and to determine if the activity of trimethylamine oxidase (TMAoxi) plays a role in modulating the seasonally variable levels of TMAO. In the same experiment, the TMAO content was determined for several tissues at varying plasma TMAO concentrations. TMAO accumulation begins at 5-7 degrees C, well above that which might be normally associated with an antifreeze response. The plasma concentration reached a plateau of 20 mM as temperatures reached 0 degrees C. Plasma TMAO concentration drops to pre-accumulation levels, less than 5 mM, when fish are held at elevated temperature (8-11 degrees C) and increases when fish are chilled below ambient seawater temperatures. However, despite temperatures near or below 0 degrees C, plasma TMAO decreases after the winter season. Changes in TMAoxi activity do not correlate with TMAO levels, suggesting that the activity of this enzyme does not play a key role in regulating TMAO concentrations in smelt. For the first time in any teleost fish, tissue TMAO contents in liver, kidney, brain, and intestine were found to strongly correlate with plasma TMAO concentrations. For these tissues, the intracellular and extracellular concentration of TMAO appears to be approximately equal. Conversely, the heart and white muscle accumulate TMAO, and in the case of white muscle, intracellular concentration is maintained at a constant level of approximately 35 mmol/kg, despite fluctuating plasma concentrations over a range from 0 to over 25 mM.