1. Serum and slime vitellogenin in the Atlantic cod, Gadus morhua, and its relation to ovarian development Joe Kunkel, John Bohannon, Rahul Sharma, and Joe Zydlewski UMass Amherst Biology Department

2. Cod Vitellogenesis Purification of Cod Lv Antiserum production and validation New slime and serum collection at sea Estradiol induction in aquarium held cod Serum analysis of NMFS samples Correlation serum Vg with ovarian histology

3. Winter Flounder Lipovitellin Heat stability of Lv! Monomeric. Labile is nicked! Single peptide = stability? Lipoprotein = stability?

4. NOAA/CMER Project Request for Vitellogenin measurement in the Atlantic cod, Gadus morhua NMFS contact: Frank Almeida, NMF, Woods Hole Albatross IV Fall & Spring Bottom Survey

5. ET50 of cod maturation Fall and Spring Bottom Survey Leg IV and V cover Georges Bank and Gulf of Maine Fall categorization of immature vs developing is a problem in establishing ET50 of maturation.

6. Most fish Lipovitellin is heat stable! Heat stable Lv: hagfish, red fish, Atlantic shad, cod, red hake, tautog, plaice, winter flounder, zebra fish. Heat labile Lv: chicken, cockroach, silk moth, gypsy moth, frog, spiny dog fish.

7. Cod Lipovitellin Purification Heat stabile Large Stokes Radius

8. IEP of cod serum and anti-cod-Lvs Both anti-cod-Lvs react with two forms of cod Vg: fast & slow The fast and slow forms of cod Lv are found variably in serum samples

9. anti-cod-Lv reacts with other Gadid Lv! QIEP: Quantitative Immunoelectrophoresis Anti-cod-Lv1 cod Vg = haddock Vg? cod fast Vg = slow Vg?

10. Cod historical average maturation. All ……….. 1305 DEL 94-12... 504 AL 95-02 … 63 Isabel 96-01.. 146 AL 98-02 ….. 75 AL 98-11 ….. 43 AL 99-03 ….. 227 GL 99-01 …... 73 AL 99-10 ….. 174

11. Biggest Gadus morhua in decade? AL 99-03, Leg V. 227 cod sampled by J. Kunkel & J. Burnette. Station 317 cod #1: 141cm 28 kg spent female. sample of slime, blood, otoliths and ovary. No Vg in serum!

12. Atlantic Cod inherited by CMER study All ………… 713 DEL 94-12... 504 AL 95-02 … 63 Isabel 96-01.. 146 Serum and ovary samples from females; plus only a few male sera.

13. Inherited vs new samples All ……….. 1305 DEL 94-12... 504 AL 95-02 … 63 Isabel 96-01.. 146 AL 98-02 ….. 75 AL 98-11 ….. 43 AL 99-03 ….. 227 GL 99-01 ….. 73 AL 99-10 ….. 174

14. Cod in NMF Woods Hole Aquarium Short otter trawl cod. Two weeks acclimation. 10 ug Estradiol injection. Mucous scraped. Tail vein bled. No Vg in mucous!

15. QIEP of Vg in Estradiol injected cod Slime contains no Vg Vg titer in serum starts to decline in 3rd week post injection Sex needs to be determined at end of experiment 5 sequential experiments

16. Kinemage 3-D plot Immature Developing Ripe Spent Resting Males

17. Kinemage 3-D plot Immature Developing Ripe Spent Resting Males

18. Kinemage 3-D plot Immature Developing Ripe Spent Resting Males

19. Kinemage 3-D plot Immature Developing Ripe Spent Resting Males

20. Kinemage 3-D plot Immature Developing Ripe Spent Resting Males (3 of 100)

21. Developing Ovaries, H&E, low X

22. Cod developing follicles, H&E cortical granules, nucleoli, thin chorion small yolk granules, large cortical granules, 2 chorion layers growing yolk granules dominate cortex large yolk granules, growing follicle, thick chorion

23. Odd cod found in Cape Cod Bay 3 similarly deformed cod suggest oddities could be found in the biochemical realm as well.

24. Conclusions anti-cod-Lv is not a litmus for maturation, but could be used to delimit lower reaches of maturation range (eg. > 47 cm.) cod Vg is not secreted in mucous Lv of most fish are heat stable some ‘odd’ cod males may produce Vg

25. The End Thanks to: NMFS partners: Frank Almeida, Jay Burnette. CMER: John Boremann and Kevin Friedland. Shipmates: Nancy McHugh, Linda Despres, John Galbraith, Nina Shepherd, Holly Yachmetz, Jason Link. (+ countless crew) UMass students and colleagues: Ruth Hartling, Ray Moniz, Gerda Kunkel.