VOLUME REGULATION IN MOPALIA MUSCOSA (MOLLUSCA: POLYPLACOPHORA) A Thesis Presented to the Graduate Faculty of California State University, Hayward In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology By Mike Moran August, 1976 ABSTRACT Volume regulation ln Mopalia muscosa was investigated. The chiton does not behave as a perfect osmometer, and volume regulates in salinities as low as 60% SW; little volume regulation occurs in hyperosmotic sea water. Changes ln tissue free amlno acid concentrations are accounted for by tissue hydration. Thus, free amino acids are not involved in volume or tissue water + content regulation. Na and Cl are concentrated considerably in the foot tissue and may be involved ln volume and tissue water content regulation. ++ . Hemolymph Ca lS regulated ln dllute sea water and this may be related to ciliary action (which is dependent on Ca++) and urine production by the kidney. Temperatures as high as 19.4°C do not affect volume regulation, whereas low temperatures of about 7°C affect the response; this is demonstrated by a decreased water influx and reduced ability to volume regulate. Fall and Spring volume regulation experiments indicate the possibility of a seasonal response. Individuals take up significantly more water in the Fall ll than in the Spring though further work is needed to verify the possibility of a seasonal response in M. muscosa. lll TO: My Mother, Alvin, Winks and Beckie. v ACKNOWLEDGEMENTS I would like to sincerely thank Dr. John Martin for use of the atomic absorption spectrophotometer. Thanks is also glven to Steve Lasley for providing storage space in the chem lab, to Connie Driscoll for typing the rough draft of the thesis and to Lynn McMasters for drawing Figure l. A thanks is glven to Dr. Robin Burnette of Hopkins Marine Station for discussions on the biology of M. muscosa and for reading the manuscript. Thanks are given to Dr. Michael Foster for reading the manuscript. A very special thank you goes to my major advisor, Dr. Richard Tullis for guidance in my graduate program. His tremendous teaching abilities are hopefully reflected in this thesis. Vl TABLE OF CONTENTS Page LIST OF TABLES Vlll LIST OF FIGURES lX INTRODUCTION . l MATERIALS AND METHODS 6 RESULTS 16 DISCUSSION 65 SUMMARY 83 LITERATURE CITED 84 Vll LIST OF TABLES TABLE Page + + ++ ++ l. Na , K , Ca and Mg concentrations of normal sea water and sea water with instant ocean added 18 2. Comparison of observed maximal weight changes to that of a perfect osmometer 38 3. Tissue ion concentrations at different salinities . 44 4. Hemolymph ion concentrations at different salinities . 46 Vlll LIST OF FIGURES FIGURE Page 1. Description of experimental apparatus 8 2. Fall volume regulation . 20 3. Regression of plate wt. vs. body wt. 24 4. Regression of maximum percent increase in wet wt. of soft parts vs. soft part wet wt. 26 5. Spring volume regulation . 28 6. Volume regulation at different temperatures 31 7. Graphical plot of P (osmotic perme- ability) OS 34 8. Volume regulation of a perfect osmometer 36 9. Foot water content at different salinities 42 10. Tissue NPS at different salinities . 49 11. Hemolymph K+ at different salinities 53 12. Hemolymph Ca++ at different salinities 55 13. Difference between hemolymph and sea water ca++ and K+ at different salinities 57 14. Time Experiment, Cl concentration of the hemolymph 60 + 15. Time Experiment, Na concentration of the hemolymph 62 16. Time Experiment, K+ concentration of the hemolymph 64 lX INTRODUCTION Intertidal marine invertebrates are subject to salinity variations. Fresh water runoff, tidal fluctuations of sea water (SW) in estuaries, and diluted surface waters after heavy rains which splash intertidal zones will cause osmotic problems. There- fore, the ability of an intertidal organism to control its water content 1n the face of a salinity stress is extremely important. Intracellular isosmotic regulation, a term applied for the ~ontrol of the intracellular osmotic pressure 1n changing salinities, is an adaptive mech­ anlsm many marine invertebrates use to control tissue water content (Pierce, l97la). Intracellular isosmotic regulation is a process that involves removal of solute from the cell, resulting'in a decreased osmotic pressure (Florkin and Schoffeniels, 1969). The solute may be removed by extrusion or merely rendered osmotically inactive (Bedford, 1971; Freel, et al., 1973). In either case the osmotic pressure within the cell 1s adjusted to approximate that of the body fluids. In the anisosmotic regulating (i.e., the regula­ tion of body fluid osmotic pressure) decapod crustacea, the necessity for intracellular isosmotic regulation is l 2 not as great as ln the osmotic conformers (Pierce, l97la). The ability to regulate the body fluid com- position upon the introduction to an altered salinity allows for the maintenance of a small osmotic gradient between the body fluids and the cytoplasm, thus lessening the requirement for osmotic pressure adjustments within the cell. Anisosmotic regulation is lacking ln marlne molluscs (Webber and Dehnel, 1968; Boyle, 1969, Pierce, 1970; Little, 1972; Simonsen, 1975). Those marine molluscs which are not capable of avoiding a salinity stress, as is true for many lamellibranchs, will have to rely on physiological mechanisms for controlling tissue water content. For long term survival in a reduced salinity, intracellular isosmotic regulation becomes very important to marine osmoconformers. Without this adaptive mechanism a salinity reduction could cause cellular swelling which may lmpalr locomotion, nerve impulse propagation, feeding and reproductive capacity (Pierce, 1971; Oglesby, 1975). Duchateau et al. (1952) reported that marlne lamellibranchs had greater concentrations of tissue e amino acids (ninhydrin positive substance, NPS) than closely related fresh water forms. This suggested a role for amino acids in osmoregulation, and it is now 3 well documented that free amino acids are involved in regulating the cytoplasmic osmotic pressure in inverte- brates. (For review, see Florkin and Schoffeniels, 1969). The use of ninhydrin positive substance in intracellular isosmotic regulation has been demonstrated in almost all marine invertebrate phyla as well as in fish (Pierce and Greenberg, 1972; Ahokas and Duerr, 1975). The amino acids involved are primarily the nonessential ones (Claybrook, 1976). For example, in the Chinese crab Eriocheir sinensis, alanine, arginine, glutamic acid, glycine and the imino acid proline are involved along with trimethyl amine oxide, taurine and betaine (Florkin and Schoffeniels, 1969). In the lamellibranch Modiolus demissus granosissimus, taurine, glycine, alanine and proline are the main osmotic effectors (Pierce and Greenberg, 1972). Volume regulation, the process by which body volume or weight is returned towards the original weight after an osmotic uptake or loss of water, may be achieved by intracellular isosmotic regulation (Pierce, l97la). Though free amino acids are usually involved in volume regulation and the control of tissue water content ln marine invertebrates, this lS not always the case. Hoyaux- et a 1 . ' (1976) have shown that a good tissue free amino acid response in relation to a reduced salinity 4 does not insure greater tissue water content regulation than in an animal lacking such a response. For example, the gastropod Patella vulgatta decreases its tissue free amino acid concentration by 100 mM/kg wet weight and increases its tissue hydration 7.8% when in 50% SW. The polyplacophoran Acanthochitona discrepans alters its free amino acid pool by 2 mM/kg wet weight when subjected to 50% SW yet its tissue water content changes by only 7.5%. The barnacle Balanus improvisus contains tissue free amino acids plus proline at 211 m-molal in 1000 mOsmol SW (100% SW) (Fyhn and Costlow, 1975); this lS only 20% of the total tlssue osmolarity. The decrease ln NPS plus proline is 115 m-molal when the sea water decreases by 500 mOsmol and tissue water content is regulated away from the line for a perfect osmometer. Though tissue water content is regulated in Balanus and an active free amino acid response occurs, the magnitude of the response is limited due to the low concentration of free amino acids in the tissue. Other physiological mechanisms are most likely aiding in limiting tissue hydration, or as the authors suggest, some other form of solute is involved along with free amino acids (Fyhn and Costlow, 1975). Until 1969, osmoregulatory studies in regard to polyplacophora were for the most part non-existant 5 (Boyle, 1969). Papers of a biochemical nature existed, especially in reference to seasonal biochemical changes (Vasu and Giesa, 1966; Giesa and Hart, 1967). The reason for the lack of interest in the polyplacophora is certainly puzzling. These hardy molluscs survive well under laboratory conditions and are common inhabitants of the rocky intertidal zone throughout the world (Hyman, 1967). Mopalia muscosa, the subject of the present investig~tion, is an abundant inhabitant of the rock jetty at the mouth of Elkhorn Slough. These chitons will face osmotic stresses ln this habitat. Norris (1969) has reported salinities as low as 20%o (60% SW) in the surface water in the area where chitons were collected for this study. This investigation was undertaken to determine if M. muscosa faced with a salinity stress was: l) capable of volume regulation; 2) whether or not the chitons behaved as perfect osmometers; 3) if a volume regulating response was exhibited, were free amino acids involved; 4) hemolymph and tissue ion regulation; 5) temperature effects on the volume regulating response, if exhibited. MATERIALS AND METHODS M.. muscosa were collected at lower low water from flat rock surfaces on the south jetty of Elkhorn Slough. All chitons were taken within the approximate tidal range of -0.5 to +2.0 feet. Chitons for this study were restricted to this intertidal range to limit intra-specific differences due to habitat var- iation.