Jason R. Treburg, Connie E. Wilson, Robert C. Richards, K. Vanya Ewart, and William R. Driedzic

1. The freeze-avoidance response of smelt Omerus mordax: initiation and subsequent suppression of glycerol, trimethylamine oxide and urea accumulation. Jason R. Treburg, Connie E. Wilson, Robert C. Richards, K. Vanya Ewart, and William R. Driedzic The Journal of Experimental Biology 205, 1419- 1427 (2002)

2. Freeze avoidance • Migration • Supercooling • Physiological freeze-resistance mechanisms. 1. colligative 2. non-colligative

3. Rainbow Smelt • Osmerus mordax • Phylum: Chordata • Class: Osteichthyes (bony fishes) • Order: Salmoniformes • Family: Osmeridae (smelts) • Habitat: live in offshore schools in the pelagic zone; undergo seasonal migration.

4. Smelt • Produce antifreeze proteins (AFPs) in low quantities. • Raise their plasma & bodily fluid osmolarity to near iso-osmotic levels. • Do this by: 1. Accumulation of small organic molecules (a) Glycerol (b) TMAO (c) Urea 2. Accumulate inorganic ions

5. Organic Solutes • Must be obtained from food, or synthesized in the body. • Levels of non-organic solutes were higher in the winter in wild-caught smelt . • Glycerol, TMAO and Urea levels can be changed experimentally through acclimation.

6. Experiment Purpose To examine the freeze response in smelt, caught from the same geographic location, over an entire winter season. Primary Method Measure the accumulation of glycerol, TMAO, urea, AFP activity, and AFP mRNA expression in order to determine the temporal sequence of the antifreeze response. Hypothesis Smelt suppress their antifreeze response during the winter only when temps are above freezing in order not to build up organic osmolytes or AFPs.

7. Figure 1 • Smelt were kept in two groups: 1. ambient 2. approx. 5º C • Samples were taken monthly (indicated by the arrows).

8. *Plasma Osmolarity tends to increase with decreasing habitat temperature Figure Two Fig. 2. Plasma osmolality, in warm and ambient smelt. Values are means ± S.E.M. (N=2-5); asignificant difference from initial value, *significant difference between warm and ambient fish (P<0.05).

9. Figure Three Fig. 3. Glycerol levels in warm and ambient smelt. (A) Plasma; (B) muscle; (C) liver. Values are means ± S.E.M. (N=6 initially; N=5 for all other points). aSignificant difference from initial value; *significant difference between warm and ambient fish (P<0.05). Plasma Muscle *Glycerol concentration increases in smelt living at colder temperatures Liver

10. Figure Four *TMAO concentration in plasma and liver decreases at higher habitat temperature. Plasma Muscle Fig. 4. TMAO levels in warm and ambient smelt. (A) Plasma; (B) muscle; (C) liver. Values are means ± S.E.M. (N=6 initially; N=5 for all other points). aSignificant difference from initial value; *significant difference between warm and ambient fish (P<0.05). Liver

11. Figure Five *Urea concentration increases with decreasing habitat temperature. Plasma Muscle Fig. 5. Urea levels, in warm and ambient smelt. (A) Plasma; (B) muscle; (C) liver. Values are means ± S.E.M. (N=6 initially; N=5 for all other points). aSignificant difference from initial value; *significant difference between warm and ambient fish (P<0.05). Liver

12. Figure Six *No significant difference in liver enzyme activity was observed Aspertate Aminotransferase Fig. 6. Liver activities of aspartate aminotransferase (AspAT), alanine aminotransferase (AlaAT) and glycerol-3-phosphate de- hydrogenase (GPDH) from warm and ambient smelt. Values are means ± S.E.M. (N=6 initially; N=5 for all other points). aSignificant difference from initial value. Alanine Aminotransferase Glycerol-3-phosphate Dehydrogenase

13. Figure Seven *No significant difference in GPDH mRNA in liver at varying temperatures. Fig. 7. Liver glycerol-3-phosphate dehydrogenase (GPDH) mRNA expression in smelt. The blot was probed as described in the Materials and methods. (A) Densitometric analysis of the blots showing mean values ± S.E.M. (B) Blot images for three males sampled on January 11.

14. Figure Eight *Warm smelt had significantly higher glycogen levels than ambient smelt in the liver. Fig. 8. Liver glycogen levels in warm and ambient smelt. Values are means ± S.E.M. (N=6 initially; N=5 for all other points). aSignificant difference from initial value; *significant difference between warm and ambient fish (P<0.05).

15. Figure Nine *Thermal hysteresis (AFP production) increased with time, but was not significantly different for each group. Fig. 9. Plasma thermal hysteresis in warm- and ambient-held smelt. Values are means ± S.D. (N=2-5). aSignificantly different (P<0.05) from initial value; *significantly different (P<0.05) between warm and ambient fish.

16. Figure Ten *AFP mRNA expression in January is significantly higher in ambient fish, despite no difference in activity. Fig. 10. Liver AFP mRNA expression in smelt. The blot was probed as described in Materials and methods. (A) Densitometric analysis of the blots; values are means ± S.E.M. (N=3). (B) Blot images for three males sampled on January 11. *Significant difference (P<0.05).

17. Figure Eleven Fig. 11. Proposed pathways for the production of glycerol in smelt. FBP, fructose- 1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3- phosphate; PEP, phosphoe nolpyruvate; OAA, oxaloacetate; KG, alpha- ketoglutarate. Enzymes are (1) aspartate aminotransferase (2) alanine aminotransferase; (3) glutamate dehydrogenase; (4) pyruvate carboxylase; (5) phophoenolpyruvate carboxykinase; (6) aldolase; (7) triose isomerase; (8) glycerol-3-phosphate dehydrogenase; (9) glycerol-3-phosphatase. Dotted arrows indicate multiple conversion steps.

18. DISCUSSION The results indicate that the organic osmolytes studied have a particular order of accumulation. TMAO seems to be expressed first, because TMAO expression of warm fish decreased over time, suggesting that TMAO levels were high when the fish were collected. 2. Urea and Glycerol accumulation appeared to begin later. Because osmolyte levels in the warm fish decreased over a period of time, the mechanisms regulating freeze- avoidance appear to bedownregulated when when not needed.

19. Glycerol, TMAO and urea made up approximately one third of the total osmolality of the smelt plasma in both the initial samples and those collected from both groups on February 29th, suggesting the involvement of other organic ions or solutes in freeze- avoidance. Antifreeze Protein Thermal Hysteresis indicated an increase in AFP over time in both groups, but no significant difference was found between groups. AFP tended to be greater in ambient fish, except for the January date. However, AFP mRNA was found in significantly greater quantities in ambient fish from the January 11th collection date.

20. Antifreeze Strategy Short Term: Regulation of Osmolytes (colligative) 1. Easily Controlled 2. Metabolically Costly Long Term: AFP Expression (non- colligative) 1. Metabolically more efficient

21. Decrease in Glycogen in Liver The decrease in glycogen in the liver (Fig 8) is attributed, in part, to its use in the synthesis of glycerol (Fig 3). Liver Enzymes -Enzyme concentration for AspAT, AlaAT and GPDH did not vary between groups, only with time. -Concentration decreased with time, indicating that expression had been stimulated previously and was returning to normal levels. -There was no correlation between GPDH and glycerol levels.

22. Future Experiments The data indicate that the antifreeze response had already begun when researchers began the experiment. In the follow- up experiment, collection dates should be scheduled during warmer weather. Conduct this experiment with a different population of smelt to limit sampling error.