Our project on the Atlantic cod, Gadus morhua, received funding in the Fall of 1996 and I am reporting the progress made toward its goals in its seventh quarter.

Progress Outline:

1. Anti-Lv Sera Characterization.
2. Antiserum Evaluation Has Continued.
3. Summer Undergraduate Trainee is on board .
4. Lab technician participated in two NOAA Spring Bottom Survey Cruises.
5. New scientist joins the project.
6. Senior personnel participates in  NOAA Spring Bottom Survey Cruise.
7. Estradiol Induction of  Vitellogenin in captive cod.

1. Anti-Lv Sera Characterization: An antiserum against Lipovitellin (Lv) from mature female cod ovary was developed previously but it did not exhibit the high specificity for Lv that was needed for ELISA assays.  There was apparent reactivity of the anti-Lv in ELISA tests to male mucous as well as apparently immature females.   Although this is not the only interpretation of the results, it was thought wise to proceed with production of a second antiserum from more highly purified Lv obtained from cod eggs.  The ovary was thought to have many proteins that might be common to both male and female and might result in the lack of desired specificity.  Therefore an antiserum to Lipovitellin purified from ripe and running ovary eggs (obtained on a Albatross IV Spring Bottom Cruise leg II) has been produced.
2. Antiserum Evaluation Has Continued: The first and second antiserum were further evaluated for their usefulness in Quantitative Immunoelectrophoresis (QIEP) and ELISA.  Both polyclonal antisera contain high titers of antibodies against cod Lv when measured with either QIEP or ELISA and our purified Lv.  In QIEP the antisera also show no reactivity to male serum and high reactivity to reproductive female cod serum.  However in ELISA both antisera show a low but definite background reactivity to both male and female slime irrespective of stage.  We would prefer to use an ELISA assay if one is possible because of the higher sensitivity of the ELISA and because it uses less antiserum.  It is not clear yet if we can tolerate the low background reactivity of samples with no Vg in them.  However we now do have a successful QIEP assay that definitely allows us to detect Lv and Vg and we need to validate that it can work with cod slime.  Because undiluted cod slime comes with a high concentration of salt we can not use it directly in QIEP.  We need to develop a way of removing the salt while not diluting the slime proteins.  A technique that concentrates and desalts the slime proteins is preferable if we will be using QIEP technique for analysis.  This technique will give us a product which will be more tractable than the crude slime and allow us to do a total protein assay and an immune assay to characterize the reproductive state of the female.
3. Summer Undergraduate Trainee is On Board: Ray Moniz a sophomore undergraduate has begun a summer internship for which he will obtain Biology 299 Special Problems credit.  During the summer he is learning to do SDS-PAGE and QIEP in preparation for participating in the Cod-Lv assay development.  He will continue working on the project through the Summer and into the Fall.
4. Lab technician participated in two NOAA Survey Cruises: Lab technician John Bohannon participated in two cruises, starting Feb. 17 with a two week gap between each trip.  He collected cod serum, mucous and traditional size statistics (total weight, GSI) for analysis in the laboratory.  On returning with the samples of slime and cod eggs he used our purification protocol to purify Lv from the eggs and we used this product to immunize a rabbit.  John left the project on June 12 to start graduate school in population biology at Oxford, UK.
5. New scientist joins the project.    John Bohannon's position in the laboratory has been taken over by Joe Zydlewski, of Steve McCormick's Conte Andronomous Fish Lab, who was a finishing graduate student in the UMass OEB program.  His experience with fish physiology, endocrinology and biochemistry will be of great service to the project.
6. Senior personnel participate in  NOAA Bottom Survey Cruise.  PI, Joe Kunkel, participated in leg IV of the NOAA Spring Bottom Surveys, April 13-21.  Four live cod were brought back to Woods Hole and 70 cod were sampled for slime and blood.  The practical problems of sexing cod was experienced first hand in the Spring season when the problem is at a minimum.  We worked out techniques for gathering slime and rapidly bleeding animals directly from the heart.  Several samples of fish roe were obtained from other species to add to our general interest in the heat stability of fish lipovitellins.  We obtained eggs from Atlantic Hagfish, Myxine glutinosa,  Red Fish, Sebastes mentella, Silver Hake, Merluccius bilinearis, Spiny Dogfish, Squalus acanthias.   A digital diary of the trip is found at URL: http://www.bio.umass.edu/biology/kunkel/fish/albatross-iv/.  Joe Kunkel has voluteered for the Albatross IV Fall Bottom Survey Cruise leg III in hopes of collecting samples of slime and serum from more cod and experiencing the difficult problem of determining the reproductive state of females during this season when their ovaries may be fgrowing for the first time.
7. Estradiol Induction of  Vitellogenin in captive cod.   Live cod are being maintained at the aquarium facility and are being used in an experiment in which estrogen is used to induce vitellogenin production.  We will sample fish slime for Vg over long (weekly) and short term (daily) after estradiol injection to see how responsive our slime QIEP assay will be for detecting slime-Vg changes in individuals.  The cod will be sacrificed at the end of the experiments and serum Vg determined.

Respectfully submitted,

Joseph G. Kunkel

jgk/hs