Use of Morphometrics and Biochemical Assays to Study the Development of  Larval Tautog.
Progress and year two budget for CMER NOAA/NMFS Project- 1998:
by Joseph G. Kunkel
Biology Department
University of Massachusetts at Amherst
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, 203-783-4230, and Laurel Ramseyer, 203-783-4222,  Milford Laboratory)

"For tautog aqua culture to advance, the effect of diet quality on egg production, larval survival and larval growth must be quantified" (Perry and Ramseyer, 1997). This request for analytical assistance requires a special laboratory that is familiar with dealing with both biochemical analyses and the concepts of morphological growth and development of embryos. The maternal effects of the female diet and larval diet on the success of the offspring can be transmitted in several ways. First, the health of the mother may be affected and her general health may determine egg quantity and quality; therefore we are examining the females diet. Second, the maternal effect on egg quality may have its effects on embryonic processes that precede the utilization of stored egg reserves (lipid, yolk protein) or the ability to access those reserves; therefore we are examining larval development from hatching through yolk utilization.  Finally, the maternal effect may be exhibited in the reduction or modification of the reserves that are stored in the egg; therefore we are measuring amounts of stored components. In addition the quality of the larval and juvenile diets may directly affect their development; therefore we are examining both the larval diet and its effects on early fish development.


Our intent is to show that a battery of basic biochemical measurements combined with simple landmark measurements and modern morphometric analysis can assist the assignment of a quantitative index of developmental state to embryonic and larval stages of fish. This battery of measurements will use modern inexpensive technology which can be applied to individual embryos. This approach would allow factorial experiments to be designed to evaluate various aspects of aquaculture methods to the rate and uniformity of larval growth.   Factorial experiments are being designed by our NMF partners, Dean Perry and Laurel Ramseyer, to test the effectiveness of defined applied nutritional regimes. The dietary factors to be tested include various live algal species cultured at the Milford NMF Laboratory which will be analyzed with respect to their spectrum of polyunsaturated fatty acid and amino acid contents.  The timing of conversion to a less expensive commercial fish diet will be a factor in the experimental design. The contrasting diets will be built from analyzed components with the C3 and C6 polyunsaturated fatty acids (PUFAs) as major variables.


My laboratory is prepared to continue developing all the methodology required to satisfy the Perry and Ramseyer request for technical assistance in studying dietary fatty acid and amino acid composition on the growth rate and body composition of larval tautog and reproductive success of the adults.  Briefly we have made substantial methodological progress since obtaining funding in Oct 1998.  We have to this date developed the micro extraction methods needed to obtain the PUFA fraction and to transform it into the state necessary for gas chromatography.  Further we have obtained standards for the typical fish PUFAs and have used fish tissue samples to simulate the types and amounts of tissue expected from NMF Milford.  We have trained two individuals Joe Zydlewski and Raymond Moniz in the extraction procedures and the gas chromatography procedures in Eric Decker's laboratory.  Furthermore we have established a data collection and analysis protocol to process the gas chromatography PUFA chromatograms.  These techniques are being reduced to a routine which may be able to analyse the PUFA pattern of individual larval fish.  This approach promises to allow the assesment of dietary effects as well as the variance of those effects within the  cohort of aquacultured tautog.

Biochemical Methods.
The Kunkel lab is familiar with the techniques of lipid extraction, amino acid analysis and gravimetric determination of % lipid in biological samples (Kunkel and Pan, 1976).  The first few months of our project time were spent developing the lipid extraction and analysis techniques.  We are now capable of preparing lipid extracts of the samples to be provided by the Milford Lab at the single fish level. The polyunsaturated profiles are being analyzed under the direction of Associate Professor Eric Decker of the U. Mass. Department of Food Sciences.   Analysis of the protein, RNA and DNA content of the resultant eggs and larvae will be carried out by established protocols in the Kunkel laboratory.  We have obtained an 8 ml sample of tautog eggs with which to purify calmodulin and Lv and make an anti-tautog-Lv antiserum. Further, our procedures in this respect have been recently published as we applied them to Winter Flounder (Hartling et al., 1997, 1999)

Growth Measurements.
Morphometrics has experienced a revolution in the past decade.  The old statistical techniques are being abandoned in favor of a new approach that allows one to deal with the size and overall shape of an organism.  New tools for examining shape have been developed and are being appled to the tautog.

  1. Two workers trained in lipid extraction protocol and sample preparation for gas chromatography of PUFAs.
  2. External standard established and standards identified for PUFAs
  3. Standards for fish PUFAs have been analysed and used to estimate PUFA patterns of sample fish tissues.
  4. Three small batches of tautog embryos have been obtained from the Milford lab and two of these have been used so far to establish the morphometric approach to growth and development indices.
  5. Tautog eggs have been obtained for purification of Lv and calmodulin and one undergraduate has received training in CaM analysis.

The Kunkel laboratory has a published history and capability of applying both biochemical and morphometric approaches to the study of development, entrez author search on kunkel-jg .  We are particularly interested in applying our experience with measuring biochemical and morphometric properties of organisms during development.  We intend to use this opportunity to demonstrate the usefulness of using a combination of biochemical and morphometric assays to establish the effect of dietary factors on tautog development.

                                                          YR-2     2-YR
                                                         99-00    98-00
Personnel: 1 graduate student ($13.08/hr)              $13,604  $27,207
           Geo Fringe benefits ($1.525/hr)               1,586    3,172
           1 undergraduate student trainee                 500    1,000

Services:  PUFA analysis                                 1,000    1,300
           Amino Acid Analysis                             800      900
           Antibody Production                               0    1,000

Equip: Trinocular dissecting scope.                          0    4,000
         CCD Camera                                        500      500
         Dedicated computer                              2,000    2,000
         Frame grabber Board                             1,000    1,000

Supplies:  Chemicals, immunologicals, glassware          3,000    6,000

                                       Total:          $23,980  $48,069
An effective project to determine the effects of diet on tautog aquaculture will require at least two years of study.  The first year, underway, is being used to analyze the proposed tautog adult and larval diet items and to develop the micro assays which will be needed to analyze the effects of diet on the approximate 10 larvae per stage that will be available from the rearing facility at Milford.   All assays will be able to be applied to all stages with the exception of the PUFA analysis which requires several micrograms of lipid to do an analysis.  We are exploring how small a sample will do.  The exact stages upon which PUFA analysis will be performed will depend upon preliminary samples of various aged fish which will be provided by the second six months of the first year.
Year 1 1st 6 months a.  Graduate student training phase in PUFA analysis.  (Done)
b.  Purification of tautog Lv. (in progress)
c.  Characterization of tautog CaM reactivity. (in progress) 
d.  Analysis of diet PUFA components. (in progress)
2nd 6 months a.  Antiserum production. 
b.  Development of  morphometric analysis.  (in progress)
c.  Preliminary PUFA analyses on various aged fish.
last 3 months a.  Characterization of  anti-tautog-Lv and developing dot blot assay.
Year 2 1st six months a.  Collect and assay tautog growth samples for all feasible assays.
2nd six months a.  Prepare results for publication.

Hartling RC, JJ Pereira and JG Kunkel. 1997. Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus).  J. Exp. Zool. 278: 156-166.
Hartling RC, and JG Kunkel. 1999. Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.   J. Exp. Zool. (in press).
Kunkel JG, and ML Pan.  1976.   Selectivity of yolk protein uptake: Comparison of vitellogenins of two insects. J. Insect Physiol. 22: 809-818.
Perry, D. and L. Ramseyer. 1997. 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. Project Proposals to NOAA/CMER.