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 second quarter, Jan. through Mar. 1999.

Progress Outline:

1. Personnel: Student training
2. Lipid Sample Assay: Developing the skills for lipid analysis
3. Lipid Data Analysis: Multivariate analysis of variance.
4. Cooperation with NMF Collaborators: Dean Perry and Laurel Ramseyer.

1. Personnel: Student training has proceeded with Joe Zydlewski working closely with Ray Moniz, a junior undergraduate in Biochemistry and Molecular Biology, on the tautog lipid analysis project.  Ray has learned to do the chloroform: methanol extraction procedure and cooperates with Joe Zydlewski in running the samples through the gas chromatography system. Ray has also worked on transcribing the data from the dot matrix output to the spreadsheet format that we use to begin analysis.

 
2. Lipid Sample Assay: Our objective is to be able to extract and analyze lipid from small groups of embryos and single juvenile fish.  We had established the chloroform : methanol lipid extraction protocol in the first quarter of this grant cycle and now have proceeded to assay the lipids of our available samples in the Food Science gas chromatography facility of Eric Decker.  The three standard lipid mixtures used as a calibration standard for our fatty acid (FA) assay include the oil of menhaden.  These FA standards allow us to identify the vast majority of FAs in our unknown mixtures, Table I.  On average, 85% of  the FA peak areas of our 30 unknown tautog project samples have been identified.   This represents between 16 to 34 peaks identified for each sample.  The three alga cultures contained 21, 22, and 23 FAs respectively but 31 FAs collectively.  The adult tautog food items (two species of crabs and mussels) encompassed 39 different FAs collectively.  The tautog samples including eggs, larvae and juveniles had 39 different FAs collectively.  These sets of FAs in foods and larvae do not perfectly coincide thus it is clear that analysis will involve not a one to one supply of FAs by a food item to a predator but a factorial effect of  a food item composition on the predator.  We will be able to supply the data for such a factorial analysis.
    
3. Lipid Data Analysis: We have started the process of establishing an analytical approach to understanding the PUFA patterns we are observing using mathematical procedures for simplifying complex data structures.  The FA patterns of 40 FAs spanned by the food and tautog samples may be able to be simplified using principal components analysis.  Toward that end we have applied such an analysis to FA samples from 39 shad gill lipid extracts (Fig. 1) obtained using the above extraction protocols.  The gill samples have comprised average wet weights of  47 mg, which is about the lower limit from which we have successfully recovered FAs.  Smaller samples resulted in smaller efficiencies of lipid extraction and lower numbers of identified peaks.   The resultant extracts were sufficient for several runs on the gas chromatograph, which is able to provide estimates of quantitative error in our technique.  Gill extracts may be a more sensitive approach to understanding dietary effects on PUFAs since this is a more highly metabolic tissue than whole body extracts and might signal the effect of the diet sooner.  However, the gill provides less tissue than the whole juvenile and would only be able to be applied to groups of juveniles.  It might be possible to assay a larger highly metabolic tissue such as the liver also.  The shad gill extracts contained 39 FAs.  The three top principle components of FA mole percent variation in these samples comprised 70%, 12% and 5% respectively of the total FA mole percent variability.   The 6 treatments spanned by these 39 samples could be meaningfully interpreted by viewing the average responses of the three principle components, Fig. 2.  This is an encouraging development because it suggests that the complex response of PUFA content may be able to be understood from a relative compositional perspective.
    
4. Cooperation with NMF Collaborators: We are in contact with Dean Perry and Laurel Ramseyer. We have developed our capabilities in analysing larval and juvenile tautog and will need to organise the collection of samples from the Milford lab based on our capabilities and how they can be maximally applied to the growth protocols developed at Milford. The development of tautog procedes rapidly starting some time in mid June. We will be meeting with Dean and Laurel in order to determine the precise food and juvenile sample analyses they plan to need from the current seasons aqua culture.