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 eigth quarter.

Progress Outline:

1. 2nd Anti-Lv Sera Characterization.
2. Antiserum Evaluation Has Continued.
3. Summer Undergraduate Trainee activity.
4. Joe Zydlewski adds fish experience to project.
5. Senior personnel to participates in  NOAA Fall Bottom Survey Cruise.
6. Estradiol Induction of  Vitellogenin in captive cod.
7. Analysis of serum and slime sampled fish.

1. 2nd Anti-Lv Sera Characterization: An antiserum to Lipovitellin purified from ripe and running ovary eggs (obtained on a Albatross IV Spring Bottom Cruise leg II) has been produced and tested.
2. Antiserum Evaluation Has Continued: We have questioned the use of an ELISA assay for cod LV and VG because of a background reaction to slime components.  Quantitative Immunoelectrophoresis (QIEP) as reported before remains specific for females and Vg and LV specificly.  Because undiluted cod slime comes with a high concentration of salt we can not use it directly in QIEP.  We developed a way this past quarter of removing the salt while not diluting the slime proteins.  This technique gives 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 activity: Ray Moniz a sophomore undergraduate did a summer internship for which he obtained Biology 299 Special Problems credit.  During the summer he learned SDS-PAGE and QIEP.  He participated in the Cod-Lv QIEP assay development.
4. Joe Zydlewski adds fish experience to project.    Joe Zydlewski, with experience in Andromodous fish and fish in general, was able to provide us with valuable samples of shad and lamprey eggs which are being used to expand our understanding of the heat denaturation approach to purifying fish LVs.
5. Senior personnel to participate in  NOAA Fall Bottom Survey Cruise.  PI, Joe Kunkel, has signed on to participate in leg III of the NOAA Fall Bottom Surveys.  This survay will provide us a new opportunity to sample cod slime and serum during the time when surveyors find it difficult to determine the reproductive status of young female cod.
6. Estradiol Induction of  Vitellogenin in captive cod.   Three of five original live cod were able to be maintained at the NMF aquarium facility at Woods Hole and were used in an experiment in which estrogen induced vitellogenin production.  We sampled fish slime for Vg over long (weekly) and short term (daily) schedules after estradiol injection to see how responsive our slime QIEP assay will be for detecting slime-Vg changes in individuals.  The cod were sacrificed at the end of the experiments and serum Vg determined.  Despite the fact that high titers of Vg were found in the cod serum at the end of the experiment, no VG was detected using our QIEP assay secreted in the slime.   This has several possible interpretations: (1) the measured Vg in cod slime is derived from trauma induced secretion of plasma components into slime only in trawl stressed individuals.  (2) Vg secretion into slime is a natural phenomenon but controlled by other hormones or conditions in the wild populations of fish which were not operant in our captive fish during the summer.
7. Analysis of serum and slime sampled fish. We plan to analyse the samples obtained from our spring and fall cruise with this possibility in mind.  We will concentrate on whether serum contamination exists in the slime.  To this end we will attempt to test for other blood components in the slime, such as hemoglobin from lysed RBCs and fish serum albumin.

Respectfully submitted,

Joseph G. Kunkel

jgk/hs