INVESTIGATORS: John J. Stegeman, Senior Scientist, Michael J. Moore, Research Specialist, Bruce R. Woodin, Research Associate, Michael S. Morss, Guest Student. Biology Department Woods Hole Oceanographic Institution Woods Hole, MA 02543-1049 jstegeman@whoi.edu mmoore@whoi.edu GRANT PERIOD: 1 September 1993 to 31 August 1996
The specific project goals are:
Quantitation of immunohistochemistry--PCNA and BrdU labelling indices are estimated for each cell type present in each specimen. PCNA expression is either calculated as a ratio of the number of labelled nuclei per 1000 nuclei (labelling index) or estimated as a semi-quantitative index. For the labelling index one thousand nuclei are examined for each cell type in each specimen, except in cases where the total number of nuclei of a particular cell type was less in the available section. In those cases the index is normalized to a fraction of 1000. PCNA expression and BrdU incorporation can also be estimated on a score of 0 (absent) through 4 (very common).
Goal 2. Establish which organs or tissues in copepods and larval fish have measurable increases in cell proliferation during body growth that are predictive of growth of the whole organism.
These methods have shown high levels of proliferation in the pancreas, liver, intestine and integument of larval cod and winter flounder. The intestinal contents appear not to give any reactivity with the PC 10 antibody suggesting that prey items should not interfere with whole organism nuclear extracts as used in the slot blot assay. In copepodites we have seen proliferation in the caudal region of the cephalothorax and the appendages, and in adults gonadal maturation is the most active proliferative event. PCNA expression in C5 and adult females was higher than in males. The low level of PCNA in males as compared to the other two classes is remarkable and presumably reflects the greater growth investment in eggs than sperm.
Goal 3. Compare a) the cell proliferation growth index (CPGI) with other commonly used indices of growth such as size, weight, egg production and RNA/DNA ratios by collaborating as necessary, and b) the influence of temperature and food availability on copepod and larval fish growth using the CPGI.
This goal is still being actively pursued. In collaboration with Scott Gallager and Phil Alatalo here at WHOI Pseudodiaptomus coronatus copepodites were reared at two temperatures, and either fed or starved. Larval codfish were reared on three different diets. Proliferation indices were compared with physical measurements made at the time of sampling. PCNA expression was found to be significantly elevated on a diet of nauplii as compared to a mixed diet, or the initial condition. We have also completed data generation from a series of experiments conducted in parallel with Dr.'s Clarke, Huntley, Lopez and Crawford in Hawaii. PCNA expression does appear to correlate with feeding regime in a manner comparable to citrate synthase, whereas PCNA expression did not correlate with egg productivity. This presumably reflects a mismatch in the timing of PCNA expression during gonadal differentiation vs. the actual shedding of mature eggs.
Goal 4. Further optimize the CPGI methodology to establish a robust and rapid shipboard assay for growth in larval fish and copepods.
We have expended substantial effort in developing this assay with considerable success. For individual larval fish, and 1-5 copepods, depending on size, we have developed an in vitro assay for PCNA content on frozen samples. The method was first established using a western blot technique, and is now routinely run 40 samples at a time, using a slot blot technique, as described above. Samples are homogenized in a buffer, centrifuged to remove cell debris, and applied to a slot blot apparatus. Blots are then visualized using a chemiluminescence method, with digital quantitation. This method has been successfully applied both in larval cod and winter flounder, Pseudodiaptomus coronatus, and a number of copepod species and crab zoae from Hawaii.
We are also currently evaluating other cell cycle associated proteins for potential application in this project. In particular we will determine which proteins might have a shorter half life than PCNA and thus be sensitive to short term changes in environmental conditions.
Goal 5. Utilize the field method to compare growth in larval fish and copepods between areas of low and high productivity, comparing the effects of physical and biological parameters, such as temperature, stratification, mixing, and food availability on the CPGI and compare growth between groups of organisms at the micro-patch scale.
We participated in three US-GLOBEC cruises aboard the R/V Seward Johnson in the Spring of 1995 (SJ-9503, SJ-9505, SJ-9507). A total of 1457 individual samples of larval cod and haddock, and adult female Calanus finmarchicus were preserved in either liquid nitrogen or formalin for slot blot and immunohistochemical analysis respectively. These analyses are underway at present.
Goal 6. Examine cell proliferation in predators, such as Cyanea sp. to establish the changes in growth rate resulting from altered predation rates, as assessed by dietary studies.
This goal will be pursued as time allows in the coming year.
Goal 7. Utilize available histological samples to assess the impact of chemical contaminants and infectious disease on the growth and survival of larval cod and haddock.
This important question has not been addressed to date in the two target species, but we can report some important observations in another Georges Bank groundfish species, the winter flounder (Pleuronectes americanus). In other studies we have shown a strong correlation between exposure to persistent halogenated hydrocarbons, and the presence of hydropically vacuolated cells in the liver of this species. These cells have been shown to have increased proliferative activity. In a comparison of cell proliferation, chemical exposure, prevalence of vacuolation and water temperature at the time of collection, it appeared the strongest correlation was between cell proliferation and water temperature. Thus climate change as evidenced by changes in water temperature has the potential to be a significant factor in the physiology and pathology of this species at least, even in areas where chemical exposure is also of known significance.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85.
Ortego, L., Hawkins, W., Walker, W., Krol, R., Benson, W. In press. Detection of proliferating cell nuclear antigen (PCNA) in tissues of 3 small fish species. Biotechnic and Histochemistry
Ellwart, J., Dormer, P. 1985. Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdU/Hoechst quenching effect. Cytometry 6: 513-520.
Moore, M.J., Stegeman, J.J. 1992. Bromodeoxyuridine uptake in hydropic vacuolation and neoplasms in winter flounder liver. Marine Environmental Research 34: 13-18.
Moore, M.J., Leavitt, D.F., Shumate, A.M., Alatalo, P., Stegeman, J.J. 1994. A cell proliferation assay for small fish and aquatic invertebrates using bath exposure to bromodeoxyuridine. Aquat Toxicol 30: 183-188.