Status of research during September 1, 1999 through Feb. 29, 2000, on the effect of PUFAs on the growth and development of the Tautog (Tautoga onitis) and planned studies for 2000

I.  Progress

A) Morphometric analysis

1) Dean Perry has announced that due to changes in the budget at Milford, no tautog larvae would be reared this season and thus none would be available for analysis.  This created a problem with respect to the material that we needed to develop the morphometric approach to following growth and development that we had originally proposed.
 
2) We have therefore switched our efforts in developing our morphometric approach to an available species in which a similar problem exists.  The company Bioshelters, of Sunderland MA, rears tilapia and would be interested in a morphometric method for following the rate and uniformity of development.  They are experimenting with transgenic strains transfected with extra growth hormone genes.  These experimental strains are said to show improvements compared to the normal hybrid in that they develop at a rapid and uniform rate.  The breeding of these new strains involves sorting out of individuals which do and do not reliably pass the improved gene set to their offspring.  A simple method of  estimating the uniformity and speed of development would help them immensely.  Bioshelters has agreed to provide us with cohorts of their normal hybrid strain on a weekly basis to allow us to develop the technique.
 
3) We ordered, received and installed a new Dell computer (750 MHz) and camera system (Kodak MDS-120) that provides megapixel images through the Parco stereo microscope which was purchased in year 1 of this project.  We are currently configuring this system with the software which we will use for morphometric analysis.

4) We are learning to take pictures of live larval fish.  This has resulted in large numbers of images of fish larvae that need to be stored for analysis.  We are developing filing systems using zip drives and writable CD files to allow a rapid retrieval of images for analysis.  Video camera input is being used and compared to our new megapixel camera.   Taking several pictures, Fig. 1, with a video camera and averaging them has the potential to provide higher resolution than the megapixel camera.  New image averaging software from James Rohlf allows color averaging of registered images.  This should lead to easier recognition of landmarks and more reliable morphometric analysis.

Fig. 1. Tilapia hybrid reared from yolk sac larva obtained from Bioshelters.

1) Since larval tautog would not be available this year we have modified the lipid analysis of our project in consultation with Dean Perry.   We suggested extending our observations on the tautog larval food sources, particularly the rotifers.  Rotifers are fed on algae (PLY 249) and then after some hours the rotifers are fed to the larval fish.  The feeding would be most effective if the PUFAs of prime interest are still high in concentration in the rotifers at the time of feeding.  We therefore did a timed series of samplings of rotifers after they were fed on algae.  The first 8 hours of sampling were done at Milford and then the cultures were transported to Amherst where we continued sampling at longer intervals up to 4 days.  These cultures had their FAs extracted via our established protocols and the samples were processed and run on gas chromatography.   Problems with the gas chromatography system delayed the FA analysis but it has now been completed and the data analysis phase has begun.
 
2) Wild Tautog up to 5 cm in length were collected by seine from LI Sound and from the Woods Hole area during the summer and early fall.  These fish were frozen and will be photographed for our morphometrics project and subsequently their FAs extracted and PUFAs analyzed to provide a comparison to the few measurements of cultured tautog that we have reported in our earlier reports. 

II.  Antiserum to tautog Lipovitellin.

1) Tautog eggs were collected from spawning females housed at Milford labs.  Lipovitellin was extracted from the eggs and the extract was heat denatured at 85C for 15 minutes according  to our previous experience.   The unprecipitated supernatant was loaded on a BioGel A1.5 column and eluted with 0.15 N KCl buffer.  The protein was aliquoted and frozen and 100 µg aliquots were used for immunization of a New Zealand White rabbit using the University Animal Care Facility to supervise the immunization.  The rabbit was bled after the two month immunization schedule and the resulting antiserum is being characterized.  Since we are not anticipating any larval tautog to use the antiserum on, we will store the antiserum for future use.  The project did establish that tautog Lv is consistent with other fish Lvs in being heat stabile.  This methodology for producing an antiserum has worked successfully on winter flounder, Atlantic cod, American shad and now tautog.

III.  Publications recognizing CMER published during this reporting period.

Kunkel, J.G., J. Bohannon, R. Sharma, and J. Zydlewski. (1999).  Serum and slime vitellogenin in the Atlantic cod, Gadus morhua, and its relation to ovarian development. Abstract 119, American Zoologist SICB Annual Meeting, Atlanta GA.

Hartling RC, Kunkel JG.  (1999). Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.  J Exp Zool 284:686-95.

IV. Active Personnel during this reporting period.

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.
5. Jeff Xu. Graduate student working on image analysis.