Second Semiannual Report on:
Use of Morphometrics and Biochemical Assays to
Study the Development of Larval Tautog.
Proposal response to CMER NOAA/NMFS
RESEARCH TOPICS - 1998:
19. Effect of Dietary Fatty Acid and Amino Acid Composition on
the Growth Rate and Body Composition of Larval Tautog (Tautoga
onitis) and on the Reproductive Success of Adult Tautog
(contact: Dean Perry, 203-579-7030, Milford Laboratory)
Joseph Kunkel
Status of research during June 1, 2000 through October 31, 2000, on
the effect of PUFAs on the growth and development of the larval and juvenile
tautog (Tautoga onitis).
Future of Tautog Lipid Analyses
The methodology for analyzing small samples of lipid was developed on shad
gill samples during the first year of the grant. The results presented
in our last report demonstrated our application of those micro extraction
and analysis methodology to the samples of algae, rotifers, adult food
and tautog eggs and larvae that were provided by the Milford NMF Lab.
The figures provide graphical analysis of the samples but interpretation
of the results will await the final project report. We have requested
an extension of our budget until September 2001 in partial expectation
that some tautog larvae will be reared in the Spring of 2001.
The figures provided with the last report were summaries of much more extensive
data which will be tabulated along with summary figures in our final report.
Antiserum to tautog Lipovitellin.
A rabbit immunized with purified tautog lipovitellin (Lv) produced an
anti-tautog-Lv-serum. This antiserum was characterized and found to be
highly reactive to tautog Lv. Since we are not anticipating
any larval tautog to use the antiserum on until spring 2001, 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 (Hartling et al.,
1997). This methodology for producing an antiserum has worked successfully
in our hands on winter flounder, Atlantic cod, American shad and now tautog.
This antiserum could be used to test for the titer of vitellogenin in tautog
female serum as well as follow the utilization of Lv in tautog larvae (Hartling
and Kunkel, 1999) as we have proposed. Its specificity for Vg in
female serum needs to be tested by demonstrating no reaction to control
male tautog serum. This control testing can be done as soon as we
can obtain serum from male tautog but would ideally be done in the spring
of 2001 when we could also test for the positive reaction with vitellogenic
female serum. In the event that there is some residual reactivity
to male serum components, the antiserum could be made female specific by
adsorbing it with male serum.
Morphometric analysis of Tautog Larval Development
Equipment was purchased and software and protocols developed for recording
and analyzing the homogeneity of larval tautog development. In the
absence of sufficient tautog larvae to develop our protocols we arranged
to use a model system in the methodology development phase. Larval
tilapia were obtained from Bioshelters and photographed at intervals using
a Kodak MDS-120 camera with Parco Stereo Microscope adapter.
This system is superior in resolution to the image averaging of video input
provided by the Rohlf tpsDig software. Although many images
of larvae were collected, it has become obvious that we need to develop
a larval fish holder which will enable us to rotate the larva to an ideal
lateral plane for photography. In particular the eyes of successive
larvae were obviously rotated with different yaw such that in the lateral
aspect the eye looks to be differently situated in otherwise identical
fish. We will continue this approach in the Fall with the help of
a new undergraduate, Jennifer Schnorbus, who is training on the project.
Our current protocol includes brief anaesthesia of the individual larval
tilapia with ms222, photography under water with the megapixel camera through
the disecting microscope. The megapixel images are stored with no
compression and archived onto zip discs. These images are then digitized
for their landmarks. The ms222 anaesthesia provides a neutral identical
posture for different individuals. These results are promising in
their possibility of providing 2-D lateral landmarks if we can develop
a jig for controling the yaw of the larva during photography. The
superposition of the two eyes is being used to aid in rotating the larva
the correct number of degrees to a correct lateral view. A
second approach which we are experimenting with is to take two images at
a fixed angle of rotation and use the two different lateral landmark data
sets to achieve a 3-D placement of landmarks. This will require more
computation but will provide data on the growth in thickness of the larva
which may be valuable in gauging growth. This project will be of
use to Bioshelters, which is providing the embryos from its successful
aquaculture facility, and which is concerned with homogeneity of growth
of their tilapia larvae. We propose to apply this technique to the
tautog culture in the late spring of 2001. Our relatively non-invasive
technology may make it possible to follow growth of aquacultured larvae
in situ at Milford so that we can test actual cohorts of larvae from the
growth tanks enriched with different natural and artificial food choices.
We have money available in the Biology Department MBL Fund to partially
support student participation in travel to research stations for training
or research.
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 immunology assay.
3. Jeff Xu. Graduate student working on image analysis.
4. Jennifer Schnorbus. Undergraduate student. Morphometric
analysis of larvae.
Respectfully submitted,
Joseph G. Kunkel
10/01/2000
Last Updated on 10/01/2000
By Joseph G. Kunkel