Status of research during July 1, 1999 through September 30, 1999, on the effect of PUFAs on the growth and development of the Tautog (Tautoga onitis) and proposed studies for remainder of 1999

I.  Progress

A)  Larval and Juvenile Tautog PUFA Analysis

Based on the decision to reduce the effort of the Milford lab on tautog aquaculture for the growing season of summer 1999 we needed to change our plans for lipid analysis which were going to concentrate on sampling the larvae and juveniles.  We visited the Milford Lab and collected small samples of tautog larvae that represented the maximum number that could be spared of the small cohorts being reared.   Plans were made for the Milford staff to freeze small numbers of tautog larvae and juveniles to allow some minimal measurements of PUFA content to be established.  We visited a local Milford beach on LI Sound where we seined for and collected  tautog which were frozen and stored for later analysis.  We picked up the Milford tautog samples at a later visit to the lab in late August.  In addition we visited Woods Hole and seined for wild tautog in the Woods Hole Marina area and the Nobska Beach area.  Samples of juvenile tautog were frozen on dry ice in the field and stored in a -20 C freezer in Amherst for potential use in lipid analysis or morphometrics.

B)  Rotifer Enrichment Experiment

In addition we planned with Dean Perry experiments on the rotifer enrichment process which could aid the aquaculture methodology if the tautog aquaculture program were resumed.  The plan of the proposed experiment was to maximize the FA content and PUFA fraction of the FA content in algal enriched rotifers.  Since rotifer cultures were being maintained for the summer, we could arrange to come down for a day to collect samples from time zero, when algae were added to a rotifer culture previously maintained on yeast, and sample until the algae were exhausted.

C)  Antiserum to tautog Lipovitellin.

Progress was made in making an antiserum against tautog lipovitellin (Lv).  Rahul Sharma was enlisted to work on the column chromatography of the egg LV extracted from tautog eggs which we had obtained from Dean Perry.  The eggs had been stripped from tautog adults maintained at the Milford NMF Lab.  The starting material were eggs which had been frozen.  Eggs were homogenized in distilled H₂O to break any yolk granule membranes.  The homogenate was adjusted to 0.15 N KCl and centrifuged in a high speed refrigerated centrifuge at 10,000 rpm for 15 min.  The supernatant was heated at 85C for 15 minutes producing substantial heat denaturation which formed a coagulum.  The coagulum was removed by centrifugation.  The supernatant was saved and dialyzed against 0.2 N KCl and 10 ml of it was layered onto a 25 mm by 1 m column of BioRad A1.5 beaded agarose.  The high molecular weight penetrating peak of protein was collected and proved to be a relatively homogeneous high molecular weight protein by SDS PAGE analysis.  This protein was stored in aliquots of 100 ug per ml for use in immunization of a rabbit this Fall.

III.  Personnel:

1. Joe Kunkel, PI.  Morphometric analysis and protein purification and analysis.
2. Joe Zydlewski, Postdoctoral Associate.  Lipid analysis and immunologic assay.
3. Ray Moniz, Undergraduate.  Lab technician projects assisting Joe Zydlewski.
4. Rahul Sharma, Undergraduate.  Lab technician projects assisting Joe Kunkel.