Our project on the Tautog, (Tautoga onitis), received funding in the Fall of 1998 and I am reporting the progress made toward its goals in its first quarter, Oct. through Dec. 1998.
Progress Outline:
The success of this project was contingent on identifying the necessary
personnel interested in this type of research. I had previously identified the
lab of Eric Decker, UMass Food Sciences Department, as a key resource which
could provide the PUFA analysis expertise. I was able to interest a, then
graduate student, Joseph Zydlewski, in the PUFA analysis project because of his
continuing interest in the PUFA content of gill tissue from migrating shad. I
invited Joe Zydlewski to be the point person in the PUFA aspects of the tautog
project. We met Sept. 16 with Eric Decker to plan the tentative protocols of
the proposed analysis. By the time that the project was funded, Oct.
1998, Joe had
defended and officially deposited his dissertation with the grad school and
was postdoctoral in status. In addition, I officially assigned Joe as my
laboratory manager. We further interested Raymond Moniz,
a biochemistry undergraduate,
to work with us as a technical assistant on this PUFA project.
I further identified Mike Pelak, a Biology (major) and Wildlife (minor) to
become involved in aspects of the biochemistry we wanted to pursue, the
vitellogenin and calmodulin content of embryos.
After an initial planning and sampling visit to Milford on June 17, 1998,
Joe Zydlewski, Ray Moniz and I visited NMF, Milford in late August 1998. We
met with Dean Perry and Laurel Ramseyer and obtained additional samples
of tautog
embryos for morphometric analysis as well as tautog fry for preliminary lipid
analysis. We also obtained samples of food (algae strains and rotifers) fed to
the larvae and fry as well as rock crab fed to the adult females. On the
earlier visit we had also obtained some unfertilized eggs from a tautog female
which we could use for purification of lipovitellin and calmodulin.
The most critical protocols to develop were those that were
novel to our laboratory. Micro-methods were already available for protein and
DNA but we needed to familiarize ourselves with micro lipid analysis.
While we were familiar with extraction of lipid
from small protein samples, we did not have any experience in how small
a sample one could work with. Our objective was to be able to extract
lipid from single embryos. Our fallback would be to determine what small
number of embryos needed to be pooled to provide an adequate sample for
PUFA analysis.
Joe Zydlewski pursued the lipid extraction protocol development in the first
quarter of this grant cycle.
Joe Zydlewski along with Ray Moniz set to work learning the routines of gas
chromatography in Eric Decker's Laboratory with the instruction of Eric
Decker's lab personnel.
As part of this procedure, we purchased internal standard material and PUFA
standard mixtures that we could use in the identification and quantification
of the PUFAs detected. By December 1998 we had successfully run some
standard solutions as well as some practice extracts through the gas
chromatograph. Analysis of these preliminary results and
subsequent planning for analytical runs of important samples would start in
January 1999. Part of the analysis protocol involved transcription of the
dot matrix tape output from the gas chromatography equipment.
Joe Kunkel pursued the protein and morphometric analysis
protocols. We used alcohol fixed embryos and larvae obtained from Milford
as well as live Zebrafish cultures established in our lab animal room.
Joe Kunkel practiced with the morphometric analysis of growth using the
fixed embryos. Images were grabbed with available equipment to establish
requirements for the projected study.
Mike Pelak started practicing extraction of calmodulin from the
Zebrafish larvae. He succeeded in the Fall 1998 term of being able to
visualize the calmodulin fraction of a one larva equivalent on an SDS-PAGE
apparatus. The next step for the spring semester will be to develop
a western blotting technique for quantifying the calmodulin content using
anti-CaM-serum.
We obtained a small grant from the Biology Department
Woods Hole Field Study Fund ($800) which was partially spent on purchasing
a refractometer to be used in determining refractive indices of solutions.
We deferred acquisition of the proposed microscope until we ascertained all
the size classes of embryos and fry that we would need to cover with the
microscopes available objectives as seen by the CCD camera to be mounted at
the trinocular.
Dean Perry has been as cooperative as possible in light of their low yield of tautog larvae and fry from the 1998 growing season. We have proceeded with the specimens provided and look forward to a stream of samples from the first official season covered by this grant.
In conclusion, we feel we are in most respects on task in our analysis of dietary effects on tautog growth parameters. We are ready for the samples to be provided in the first season of tautog culturing during which we will be prepared to analyze PUFAs, protein and DNA content and be able to secure samples for CaM and Vg estimation in embryos and morphometrics of larvae.