W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 1977 Characterization of Carbohydrates in Oyster (Crassostrea virginica) Hemolymph Robert Joel Lowy College of William and Mary - Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Biochemistry Commons Recommended Citation Lowy, Robert Joel, "Characterization of Carbohydrates in Oyster (Crassostrea virginica) Hemolymph" (1977). Dissertations, Theses, and Masters Projects. Paper 1539617477. https://dx.doi.org/doi:10.25773/v5-9v6d-k328 This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. CHARACTERIZATION OF CARBOHYDRATES IN OYSTER (CRASSOSTREA VIRGINICA) HEMOLYMPH A Thesis Presented to The faculty of the School of Marine Science The College of William and Mary in Virginia In Partial Fulfillment Of the Requirements for the Degree of Master of Arts by R. Joel Lowy 1977 APPROVAL SHEET This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Arts Roberge Joel Lowy Approved, August, 1977 DexterS.Haven, M.S. Frank 0. Perkins, Ph.D. y ± 7 ( j u J U & Bruce J. Nea<Lson, Ph.D Ernest Warriner, III, M.A. Richard L. Wetzel, Pn.D. 11 TABLE OF CONTENTS page ACKNOWLEDGMENTS. .............. V LIST OF TABLES.............. v i LIST OF FIGURES. ......................... vii ABSTRACT. ......... ....................... „ c............ viii INTRODUCTION . 2 METHODS AND MATERIALS................... 4 SOURCE OF OYSTERS .............. 4 COLLECTION OF HEMOLYMPH SAMPLES ........... 5 COLORIMETRIC ANALYSIS ..... 8 HEMOLYMPH EXTRACTION AND FRACTIONATION PROCEDURES... .12 THIN LAYER CHROMATOGRAPHY.......... 17 COLORIMETRIC RESULTS EVALUATION OF EXTRACTION TECHNIQUES FOR HEMOLYMPH CARBOHYDRATES................. 22 COMPARISON OF TOTAL APS, FCHO AND PCHO IN HEMOLYMPH FROM FIELD SAMPLES 2 5 SEASONAL VARIATION IN TOTAL APS, FCHO, PCHO AND A-B CHO IN FIELD AND LABORATORY OYSTERS.......... 31 SPECIFIC ENZYMATIC COLORIMETRIC METHODS FOR GLUCOSE AND TREHALOSE.............................. 36 SUMMARY OF CARBOHYDRATE COMPOSITION OF OYSTERS BASED ON COLORIMETRIC FACTS................... 47 TABLE OF CONTENTS (CONTINUED) Page THIN LAYER CHROMATOGRAPHY RESULTS........................ 49 EVALUATION OF SEVERAL EXTRACTION AND DESALTING TECHNIQUES ....... 49 IDENTIFICATION OF OYSTER HEMOLYMPH FREE CARBOHYDRATE.52 DISCUSSION. ........ ....... 72 ANALYTICAL TECHNIQUES ... 72 COMPOSITION OF OYSTER HEMOLYMPH........................ 74 GEOGRAPHIC AND SEASONAL VARIATION......... .79 SUMMARY. ............. 87 '^APPENDIX A - AN THRONE REACTION........................... .88 :>APPENDIX B - HEXOSAMINE REACTION..........................95 ‘ APPENDIX C - GLUCOSE OXIDASE DETERMINATION OF GLUCOSE.101 'APPENDIX D — FLUOROMETRIC DETERMINATION OF GLUCOSE....106 '.APPENDIX E - ENZYMATIC.. TREHALOSE DETERMINATION........ 112 APPENDIX F - TLC VISUALIZATION METHODS..................115 BIBLIOGRAPHY. ............................................. 120 VITA................... 125 iv ACKNOWLEDGEMENTS The author expresses his appreciation to the members of the committee for their helpful suggestions and critical reading of the manuscript. Particular thanks is due to Mr. Dexter Haven for his support and encouragement throughout the program. Special gratitude is extended to Mr. Haven, Dr. Perkins, Mr. Warinner and Dr. Zubfoff, Dr. Wetzel, and the members of their departments, as well as the Aquaculture Department, for use of laboratory equipment and facilities, and their help and courtesy. Dr. Paul Zubkoff and Nancy Windsor also provided many fruitful discussions and helpful suggestions. Additional thanks is extended to Teresa Wilburn for her typing skills and the VIMS Art Department, particularly June Hoagman. Finally, I extend my appreciation and gratitude to my parents and my Lord Jesus Christ, without whose loving support and encouragement this thesis would not have been possible. v LIST OF TABLES Table Page 1. Hemolymph Sampling Conditions.................. 6 2. Comparison of Carbohydrate Extraction Methods........ 23 3. Field Survey Data................... .................... 3 0 4. Summary of Colorimetric Data for Hemolymph Carbohydrates . ................... 35 5. Glucose Oxidase Determination of Hemolymph Glucose. ........................... 38 6. Hexokinase-NADPH Determination of Hemolymph Glucose......... 42 7. Enzymatic Assay of Trehalose 4 5 8. Thin Lciyer Chromatography Mobility Data...............68 i 9. Comparative Hemolymph Carbohydrate Data...............84 10. Anthrone Standard Curves........................ 91 11. Acid-Base Hydrolysis and Anthrone Determination of Representative Carbohydrates. ..... 92 12. Hexosamine Standard Curve........ 98 13. Glucose Oxidase Standard Curve........................103 14. Hexokinase-NADPH Glucose Determination - Standard Curve...... 109 15. Trehalose Assay Reaction Mixtures.................... 114 16. TLC Visualization Reagent Results.................... 118 vi LIST OF FIGURES Figure Page 1. Oyster hemolymph carbohydrate assays,........ 9 2. Fractionation and extraction procedures.............13 3 . Total APS versus wet weight. ......... 24 4. Total APS versus dry weight............ ,.29 5. Time variation in hemolymph and environmental factors............................. 34 6. Effect of salinity on glucose and trehalose mobility .......... 57 1\ Comparison of extraction - desalting methods for TLC................................ 59 8. Comparison and preliminary identification of carbohydrates in three hemolymph samples...... 61 9. TLC identification of hemolymph carbohydrates II...63 10. Identification of hemolymph carbohydrates II........65 11. Identification of carbohydrates by differential visualization methods ................... 67 12. TLC mobilities versus hexose unit number............ 71 13. Anthrone standard curve.................. 94 14. Hexosamine standard curve..................... 100 15. Glucose oxidase standard curve....... 105 16. HK-NADPH glucose determination standard curve..... 110 ABSTRACT Colorimetry and thin 3_ayer chromatography techniques have been developed and used to characterize oyster hemolymph carbohydrate (CHO). The■non-specific anthrone determination has been coupled with TCA-ETOH fractionation procedure to measure total anthrone positive substances (total APS) , TCA-ETOH soluble substances (FCHO) and TCA- ETOH insoluble substances (PCHO). The FCHO fraction was further treated by an acid-base technique to obtain highly chemically stable carbohydrates (A-B CHO). The total APS changed from 1100 yg/ml in the winter, falling to 234 yg/ml by April and returned to 1200 yg/ml by May. Most of this ..change was due to fluctuations in the PCHO. As a proportion fof the total APS, PCHO was least constant and FCHO most iccrhstant. The hemolymph CHO change seemed too early to ^correlate with gametogenesis, but did follow condition index. «Tt was hypothesised that, the change is nutritionally caused, -due ,to an increase in metabolic activity above assimilation. Field samples taken from VIMS (York River), the Rappahannock River and Eastern Shore showed little difference in any ■fraction regardless of geographic origin, animal size, or ?,salinity. Although there is a great deal of variation in •total APS between individual oysters, total APS or other 'ihemolymph fractions could be used as indicators of nutritional state in field and laboratory experiments. The glucose oxidase and a fluororoetric modification of hexokinase-NADPH enzymatic glucose determinations were tried. Oyster hemolymph was found to have approximately 40 yg/ml of glucose, which is below or near the detection limits of these methods. Am enzymatic determination of trehalose, using trehalase, was not completely successful, but did indicate that levels are quite low. Glucose was concluded to be the primary "blood sugar". A large proportion of the FCHO and A-B CHO remained unidentified. It is hypothesised a large amount of these fractions may be maltotriose or other oligosaccharides, but the physiological function is unknown. The TLC data showed the following carbohydrates to be present, in order of concentration: maltotriose, glucose, maltose/trehalose, and galactose. In concentration and composition oyster hemolymph is quite similar to that determined for other mollusca and the crustaceans. CHARACTERIZATION OF CARBOHYDRATES IN OYSTER (CRASSOSTREA VIRGINIA) HEMOLYMPH INTRODUCTION Despite considerable information about the carbohydrate metabolism of the mollusca, relatively little is known about the carbohydrates of the hemolymph and which ones are metabolically active as blood sugar (Goudsmit, 1972). Most values, with the exception of the gastropods, are relatively old and done with procedures which measure total reducing carbohydrates rather than glucose or even free monosaccharides specifically (Martin, 19 61; Goddard and Martin, 1966)-. Information on the lamellibranchs is particularly sparce. Therefore, an attempt to characterize the free carbohydrates and identify the blood sugar of the oyster is a useful undertaking. Glucose was considered the most likely major constitutent of the hemolymph and blood sugar, with trehalose a secondary possibility. Glucose is of widespread importance, including