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The effect of salinity, temperature, season and intertidal height on calcium uptake by Mytilus edulis… Robinson, Donald C. E. 1982

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THE EFFECT OF SALINITY, TEMPERATURE, SEASON AND INTERTIDAL HEIGHT ON CALCIUM UPTAKE BY MYTILUS EPULIS (LINNAEUS) by DONALD C.E. ROBINSON Bachelor of Science (University of B r i t i s h Columbia, 1977) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1982 (c) Donald C.E. Robinson, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of •pTrT'' (^Z  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date p*l ^ J A J SP-DE-6 (3/81) i i ABSTRACT This study has shown that season, s a l i n i t y , temperature and i n t e r t i d a l height a l l aff e c t the rate up calcium uptake by mussels. For summer-adapted mussels, calcium uptake was found to be temperature dependent over the range of acute temperatures measured (1°-23°C). When subjected to a range of s a l i n i t i e s over a three week period, summer-adapted mussels showed calcium-uptake rates which were s a l i n i t y dependent from 25%-75% SW, and which did not show any increase in uptake rate in s a l i n i t i e s greater than 75% SW. For winter-adapted mussels, calcium uptake was temperature independent over a temperature range from 5°-l7°C. At higher and lower temperatures, uptake was reduced. When subjected to a range of s a l i n i t i e s over a three-week period, winter-adapted mussels were also unable to compensate for the lower concentration of calcium in the seawater, and did not show any increase in the uptake rate in s a l i n i t i e s greater than 75% SW. It was found that high and low i n t e r t i d a l mussels had dif f e r e n t calcium uptake rates, and that transplantation could a l t e r the uptake rate of transplanted mussels to the uptake rate of untransplanted controls. In the i n t e r t i d a l zone a gradient of s h e l l size was found, which could be associated with the change in uptake range over the i n t e r t i d a l range. Differences in immersion time between the two. s i t e s could not explain a l l of the differences in uptake rate, but high i n t e r t i d a l mussels were found to have less t o t a l dry weight of soft parts than low mussels, and correcting for this difference accounted for the the remainder of the difference in calcium-uptake rate between the two s i t e s . The soft parts of the mussel were found to become saturated with " 5Ca afte r four hours, while the sh e l l accumulated calcium for the duration of the experiment. The mantle and g i l l tissue held the same amount of calcium when corrected for differences in weight, while the viscera held a greater pool of calcium. Accounting for real increases in the amount of calcium accumulated by the s h e l l showed that the uptake rates reported in t h i s study are about 59% of the absolute uptake rates. iv TABLE OF CONTENTS ABSTRACT i i LIST OF FIGURES . v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 MATERIAL AND METHODS 10 Col l e c t i n g s i t e 10 Col l e c t i o n of animals 10 F i e l d measurements 11 General laboratory methods 12 1. S a l i n i t y experiments 14 2. Acute temperature response experiments 15 3. I n t e r t i d a l transplant experiment 16 4. Size gradient study 16 5. Time course uptake study 18 Data analysis 19 RESULTS 22 S a l i n i t y experiments 22 Summer experiment (August 1979) 22 Winter experiment (February 1980) 33 Acute temperature response experiments 46 Summer experiment (August 1980) 46 Winter experiment (November 1979) 49 In t e r t i d a l transplant experiment 56 Size gradient study 66 V Time course uptake study 82 DISCUSSION 101 S a l i n i t y experiments 101 Acute temperature response experiments 105 I n t e r t i d a l transplant experiment 110 Size gradient study 114 Time course uptake study 118 SUMMARY 123 LITERATURE CITED 126 v i LIST OF FIGURES Figure 1. Summer adapted regressions, Day 0 and 7 . .. 23 Figure 2. Summer adapted regressions, Day 14 25 Figure 3. Summer adapted regressions, Day 21 27 Figure 4. Summer adapted s a l i n i t y response 31 Figure 5. Winter adapted regressions, Day 0 and 7 34 Figure 6. Winter adapted regressions, Day 14 36 Figure 7. Winter adapted regressions, Day 21 38 Figure 8. Winter adapted s a l i n i t y response 42 Figure 9. Calcium uptake in summer and winter 44 Figure 10. Summer adapted temperature regressions 47 Figure 11. Summer adapted temperature response 50 Figure 12. Winter adapted temperature regressions 52 Figure 13. Winter adapted temperature response 54 Figure 14. 2.2 meter transplant regressions 57 Figure 15. 1.2 meter transplant regressions 59 Figure 16. 0.2 meter transplant regressions 61 Figure 17. I n t e r t i d a l transplantation response 64 Figure 18. I n t e r t i d a l size-frequency histograms 67 Figure 19. Dry weight s h e l l regressions 70 Figure 20. Dry weight mantle regressions 72 Figure 21. Dry weight g i l l regressions 74 Figure 22. Dry weight viscera regressions 76 Figure 23. Shell height-length regressions 78 Figure 24. Shell width-length regressions 80 v i i F i g u r e 25. S h e l l w e i g h t - d i s p l a c e m e n t r e g r e s s i o n s 83 F i g u r e 26. S h e l l d i s p l a c e m e n t - l e n g t h r e g r e s s i o n s 85 F i g u r e 27. S h e l l u p t a k e r e g r e s s i o n s 88 F i g u r e 28. M a n t l e u p t a k e r e g r e s s i o n s 90 F i g u r e 29. G i l l u p t a k e r e g r e s s i o n s 92 F i g u r e 30. V i s c e r a u p t a k e r e g r e s s i o n s 94 F i g u r e 31. S h e l l and t i s s u e u p t a k e o v e r 32 h o u r s 97 v i i i ACKNOWLEDGEMENTS I wish to acknowledge the support of Dr. P.A. Dehnel during the course of thi s research. In addition to supervising my studies, he has given considerable time and attention to the preparation of this manuscript. I would also l i k e to acknowledge the assistance of Drs. T.H. Carefoot and J.M. Gosline for their advice and c r i t i c i s m during my research, and the valuable help of Dr. David Z i t t i n and the staff of the Biology Data Centre during data analysis and the preparation of th i s manuscript. I also wish to thank Wendy Hird for her continual support through the course of thi s study. F i n a l l y , I acknowledge the generosity of the Royal Vancouver Yacht Club for allowing me to use their wharf for establishing my i n t e r t i d a l trays, and the Vancouver Public Aquarium for supplying seawater. This research was made possible by a 1980 Summer Graduate Fellowship from the Department of Zoology, and by an NSERC grant (67-7088) to Dr. P.A. Dehnel. 1 INTRODUCTION The study of ion transport and ionic and osmotic regulation in freshwater and marine molluscs i s well documented (Potts 1954; L i t t l e 1965; Pierce 1970; Pierce and Greenberg 1971; Greenaway 1971a, 1971b; Shumway 1977a; Shumway et a l . 1977). Ty p i c a l l y , marine bivalve molluscs maintain their blood in osmotic equilibrium with their surrounding media (Segal and Dehnel 1962), but this does not mean that each blood ion species is in equilibrium with i t s external counterpart. Signif i c a n t differences in the internal concentrations of potassium, calcium and carbonate ions are common among marine molluscs (Potts 1954), but other than these three ions, the blood composition is similar to that of seawater. In addition, marine molluscs have been shown to excrete i n t r a c e l l u l a r free amino acids, notably glycine and taurine, in response to osmotic stress (Pierce 1970; Hoyaux e_t a_l. 1976). This reaction is thought to reduce ion losses by reducing the i n t r a c e l l u l a r osmotic pressure which i s contributed by free amino acids, thus, reducing the t o t a l osmotic gradient. Comparisons of the blood ions of marine and freshwater molluscs by Potts (1954) (Mytilus edulis and Anodonta cygnea) and Chaisemartin et §_1. (1969) (Margar i t i f era and Lymnaea) have demonstrated that freshwater molluscs display remarkable differences in blood ion concentrations, -when compared to marine molluscs. Blood ions of freshwater molluscs are hyperionic to the corresponding external ions for sodium, potassium, calcium, magnesium, chloride, sulphate, carbon dioxide and phosphate. 2 Thus, although freshwater molluscs maintain blood ionic and osmotic concentrations lower than marine molluscs, the animals have a demonstrated a b i l i t y to maintain an ionic gradient between themselves and their environment. Of the blood ions, calcium i s of part i c u l a r importance with respect to the function of nerve and muscle c e l l s . A low concentration of i n t r a c e l l u l a r calcium i s essential to maintain a balance of sodium and potassium ions in squid neurons (Hodgkin and Keynes 1957). Recent studies point to the importance of calcium in the response of bursting pacemaker neurons in Aplysia (Barker and Gainer 1973; Johnston 1976). When i n t r a c e l l u l a r calcium concentrations are reduced experimentally, the c e l l membrane becomes less able to maintain ionic and e l e c t r i c gradients necessary for normal function. Calcium is also necessary to couple actin and myosin f i b r i l s during s t r i a t e d muscle contraction (Szent-Gyorgyi 1975). In addition to the other constituents of seawater and hemolymph, calcium plays a part in the process of osmoregulation (Pierce and Greenberg 1971; Shumway 1977a, 1977b; Shumway et a l . 1977). Pierce and Greenberg (1971) have shown that when the blood calcium levels of Mytilus are lowered below those found in the blood of mussels in f u l l strength seawater, the mantle tissue becomes less able to withstand osmotic stress. Kirschner (1963) believes that lowered blood calcium concentration causes the mantle of clams (no species given) to become leaky with respect to the transport of sodium and potassium, and interferes with i t s a b i l i t y to maintain an e l e c t r i c gradient. 3 Besides i t s importance in c e l l u l a r physiology, calcium i s mineralized by animals from every invertebrate phylum (Lowenstam 1981). Among the marine invertebrates there are three major phyla which depend upon calcium carbonate for their exoskeleton: the scleracterian coelenterates (Goreau 1959), the crustacean arthropods (Robertson 1937), and the molluscs (Wilbur and Jodrey 1952). Among the molluscs and coelenterates the p r e c i p i t a t i o n of calcium carbonate is a continuous process which occurs throughout the l i f e of the organism. Crustaceans secrete a new exoskeleton following periodic molting (Travis 1955; McWhinnie et a l . 1969). Prior to the molt, calcium i s dissolved from the integument and held in --the blood and hepatopancreas (Travis 1955). Freshwater c r a y f i s h hold calcium carbonate stores in ga s t r o l i t h s located in the cardiac stomach (McWhinnie 1962, Chaisemartin 1965). Following molt, these calcium stores are returned to the new integument. Two recent studies have investigated the kinetics of calcium transport across the molluscan mantle. Greenaway's study (1971b) of changes in the e l e c t r i c potential across the mantle tissue of the aquatic gastropod Lymnaea stagnalis indicate that calcium uptake is active when the external calcium concentration ranges between 0.06 and 0.3 mM. Above this concentration, calcium i s accumulated by d i f f u s i o n along an e l e c t r i c a l and chemical gradient. In a related study, Greenaway (1971a) has shown that when the external calcium ion concentration f a l l s below the threshold of uptake of the calcium transport system (0.06 mM), calcium i s dissolved from the inner surface of the 4 exi s t i n g s h e l l to make up for e f f l u x losses. E a r l i e r work (Kirschner e_t a_l. 1960; Kirschner 1963) also has shown the existence of a calcium dependent potential of 20-70 mV across the mantle of clams (no species given). Calcium may be absorbed from the external environment by a number of tissues, but in molluscs i t s p r i n c i p a l place of deposition i s the s h e l l (Wilbur and Jodrey 1952; Jodrey 1953). It has been known for some time that calcium dissolved in the aquatic medium is taken up by the mantle tissues (Schoffeniels 1951a, 1951b). The g i l l is also known to play a part in uptake in the marine bivalve Hyriopsis schlegeli i (Horiguchi 1958) and in the sea mussel Mytilus californianus (Rao and Goldberg 1954). Both g i l l and foot may function as temporary storage sites in Viviparus bengalensis (Sen Gupta 1977) and the freshwater bivalve Cr i star ia p i i c a t a (Numanoi 1939). The gastropod Lymnaea has been shown to derive about 20% of i t s calcium requirements from i t s food (van der Borght and van Puymbroeck 1966). Because of the predominance of calcium carbonate deposition in the s h e l l in molluscs, there i s considerable l i t e r a t u r e describing this process. Some studies have focussed on the s i t e s of calcium carbonate secretion and upon the relationship between the existing s h e l l , protein matrix, and newly secreted s h e l l . (Kapur and Gibson 1968; Timmermans 1969; Hirata 1953; Bubel 1973; Sminia et a l . 1977). Molluscan blood is normally saturated or supersaturated with calcium carbonate (Potts 1954), and i t is thought that c r y s t a l l i n e p r e c i p i t a t i o n is induced by the conformation of the protein matrix of the e x i s t i n g s h e l l 5 (Kapur and Gibson 1968; Timmermans 1969). Changes in the pH of the blood are also known either to favour p r e c i p i t a t i o n (under alkaline conditions) or di s s o l u t i o n of the s h e l l (under acidic conditions) (Akberali et a l . 1977). Anaerobic metabolism is known to occur during the t i d a l emersion of Venus mercenaria (Dugal 1939). During these periods of anaerobic metabolism, calcium carbonate is dissolved from the s h e l l in order to buffer pH changes resulting from anaerobic succinate production and ammonia excretion (Dugal 1939; Akberali et a l . 1977). Since most of the calcium absorbed from the environment is l a i d down as new s h e l l , increases in s h e l l dimension have been used commonly as a parameter for the measurement of molluscan growth. Studies measuring growth by calcium uptake or s h e l l deposition have been made by many workers (Orton 1928; Galtsoff 1934; Newcombe 1935; Fox and Coe 1943; Loosanoff and Nomejko 1949; Wagge 1952; Wilbur and Jodrey 1952; Horiguchi et a l . 1954; Horiguchi 1958; Bonham 1965; Seed 1968; Zischke et a l . 1970). Some have noted the seasonal effects of temperature on sh e l l growth (Coulthard 1929; Galtsoff 1934; Loosanoff and Nomejko 1949; Orton 1928). Wilbur and Jodrey (1952) have studied the s i t e s of deposit ion.of calcium carbonate on the s h e l l , and found that newly secreted calcium- is concentrated at the posterior and ventral margins of the s h e l l . Two studies have assessed calcium budgets of the bivalve Hyriopsis schlegeli i (Horiguchi et a_l. 1954; Horiguchi 1958), and Helix aspersa (Wagge 1952). Bonham (1965) has used 9 0 S r deposition from the radioactive f a l l o u t of nuclear weapons to measure the rate of 6 growth of the bivalve Tridacna qigas. A study by Marbach and Wilbur (1973) has examined the effects of a changing l i g h t regime on the dail y deposition of calcium carbonate by the limpet Patella rota. Dehnel (1956) has measured the growth of mussels and found s i g n i f i c a n t differences in the rates of growth between southern and northern populations, and between and high and low i n t e r t i d a l populations of Mytilus ca l i f o r n i a n u s . His study demonstrated that high i n t e r t i d a l mussels have lower growth rates than low i n t e r t i d a l mussels, and that mussels from northern and southern locations grow at similar rates in spite of temperature differences between the two l o c a l i t i e s . Newcombe (1935) and Seed (1968) also found that inte r t i d a l " height and the density of the community affe c t the rate of s h e l l growth. In spite of the number of studies on growth and calcium metabolism, I have been unable to locate any study which has systematically examined the effect of altered environmental conditions upon calcium uptake in molluscs. There are, however, systematic studies of other parameters as they affect various metabolic functions; for example, the effect of temperature on oxygen consumption (Widdows 1973) and osmoregulation in Mytilus edulis (McLachlan and Erasmus 1974). The purpose of thi s study was to examine the effect of s a l i n i t y , temperature, i n t e r t i d a l height and season upon the uptake rate of calcium by the bay mussel Mytilus edulis (Linnaeus), the genus being common to the i n t e r t i d a l region of coasts throughout the world (Soot-Ryen 1955). Since the Vancouver Harbour study area is subject to seasonal variations 7 in s a l i n i t y , this study has examined the e f f e c t of long-term decreased and increased s a l i n i t i e s upon summer- and winter-adapted mussels. This was done to determine whether the calcium-uptake c a p a b i l i t i e s of mussels were altered with respect to s a l i n i t y in the summer and winter environment. The temperature of subsurface water (1.0 meter) at the f i e l d s i t e in Vancouver harbour was found to vary between 2° and 20°C between January and August, respectively. It seems l i k e l y that both acute and seasonal changes in temperature may result in changes in calcium uptake, a finding which Loosanoff and Nomejko (1949) reported for Ostrea edulis, and which Coulthard (1929) reported for Mytilus edulis. The present study determined the effect of acute temperature-changes upon the uptake rate of summer- and winter-adapted mussels. Because of the wide i n t e r t i d a l d i s t r i b u t i o n of Mytilus, this study further examined the calcium-uptake rate of transplanted and untransplanted mussels from the high and low i n t e r t i d a l s i t e s . Ecological factors have been implicated as the cause of i n t e r t i d a l size gradients (Vermeij 1972; Paine 1976; Bertness 1977). It seems l i k e l y that differences in the length of immersion would result in an i n t e r t i d a l size gradient, but i t is not known whether the reduction of immersion time af f e c t s the growth of the s h e l l and the soft parts equally. It is possible that at increased i n t e r t i d a l heights the proportions of the soft parts may d i f f e r in order to maximize calcium r e t r i e v a l from the environment. Differences between high and low i n t e r t i d a l populations are examined in a study of the size d i s t r i b u t i o n of Mytilus edulis 8 in the i n t e r t i d a l zone. A comparison of the soft part weights and s h e l l weights of high and low i n t e r t i d a l mussels was also made. The f i n a l experiment of th i s study examined the transport of calcium into the soft parts and s h e l l over a 32 hour period. In a l l of these experiments the calcium-uptake rate i s calculated as the mean hourly uptake rate over a 24 hour period. The potential effects of diurnal or t i d a l rhythms, such as those reported in the limpet Patella rota (Marbach and Wilbur 1973), are removed by t h i s method. Calcium uptake is measured by the use of a radioactive isotope, " 5Ca, which is used to label the calcium present in seawater. The use of an isotope in the measurement of calcium uptake requires the estimation of two unknown parameters. The f i r s t of these values i s the amount of unlabelled calcium which resides in the soft parts, and which does not actually contribute to s h e l l growth. The second unknown value i s the length of time required for the r a t i o of labelled/unlabelled calcium in the seawater (the s p e c i f i c a c t i v i t y ) to equi l i b r a t e in the calcium pools in the soft tissues. U n t i l there is an equilibrium between the external and internal pools, the actual rate of calcium uptake cannot be determined. These two parameters are estimated in a 32 hour time-course study of the passage of calcium into the mantle, g i l l , viscera and s h e l l . A review of studies of calcium-uptake rate and s h e l l growth has shown few which accounted for the size of the experimental animals. It is not known a p r i o r i whether the calcium-uptake rate is dependent or independent of weight, or whether the 9 uptake rate s h i f t s between weight dependence and independence. Unless uptake can be expressed on a per gram basis, the foundation for comparison between studies i s l i m i t e d . As Zeuthen (1947) pointed out, the lack of consideration of size can often be ascribed to the small size range of the animals used (Wilbur and Jodrey 1952), but some authors seem to be undecided as to the possible effect of size on uptake rate (Loosanoff and Nomejko 1949). This study, therefore, has used a range of animal sizes, allowing the cal c u l a t i o n of the regression l i n e of the rate of calcium uptake as a function of t o t a l dry weight. The results of th i s study contribute to the understanding of the patterns of growth of marine molluscs as they are influenced by d i f f e r e n t physical factors. 10 MATERIAL AND METHODS Col l e c t i n g s i t e The mussel Mytilus edulis (Linnaeus) was co l l e c t e d from the north jetty of the Royal Vancouver Yacht Club, situated east of Spanish Bank in Vancouver harbour. The t i d a l datum of the s i t e was found by marking the water l i n e of a p i l i n g at successive dates and comparing the time and height with the Canadian Hydrographic Survey prediction for Vancouver tides (Anonymous 1979, 1980). Measurements of i n t e r t i d a l position are accurate to 10 cm r e l a t i v e to datum. Collect ion of animals A column of six rectangular p l a s t i c trays was assembled and hung from the jet t y . The trays were 30 cm long, 23 cm wide, and 12 cm deep. A 5 cm hole in the floor of each tray allowed water to drain out when i t was emersed. The trays were suspended in a v e r t i c a l l i n e with 0.5 m distance between trays. The lowest tray was 0.2 m above the zero datum point. The corners of each tray were fastened by knots in a 1/4 inch polypropylene rope which passed through the corners of a l l six of the trays. This assembly was suspended at the top by a 1/4 inch polypropylene rope t i e d to the four l i n e s at the upper tray, and which passed through a pulley to a cleat attached to one of the jetty's p i l i n g s . The trays were held taut at the bottom by a similar l i n e which passed down through a submerged pulley, then back up 11 to fasten to another c l e a t . The submerged pulley was attached to a heavy concrete block. This movable assembly allowed the trays to be hauled out of the water to retrieve samples. Mussels were obtained by transplantation from the p i l i n g s into the trays, and covered the floor of each tray, approximately 300-400 mussels to a tray. Care was taken to insure that the mussels which were transplanted from the p i l i n g s into trays remained at the same equivalent i n t e r t i d a l height. Spat f a l l on the trays was also included in experiments. Subtidal animals were taken from about 50 cm below the waterline of mooring flo a t s which were d i r e c t l y adjacent to the trays. F i e l d measurements Temperature measurements were made by means of a Tempscribe recorder, and are accurate to ±1°C. S a l i n i t i e s were determined by means of a Buchler-Cotlove chloridometer. Deep-water seawater samples, obtained from the Vancouver Public Aquarium, indicated that the s a l i n i t y of harbour water at 60 m remained constant at about 480 mM C l ' / l i t e r . This s a l i n i t y , which is equivalent to 31.8 parts/thousand, was established as 100% seawater. The millimolar concentrations of the major ions present in 100% seawater (SW), calculated from Barnes (1954), are C l " , 480 mM; Na +, 412 mM; Mg + 2, 47 mM; SO,,"2, 25 mM; K+, 9 mM; C a + 2 , 9mM; and C0 3" 2, 2 mM. The 9 mM concentration of calcium in 100% SW corresponds to 0.395 grams c a l c i u m / l i t e r seawater. 1 2 General laboratory methods Seawater was supplied by the Vancouver Public Aquarium. Glass d i s t i l l e d water was used to d i l u t e t h i s seawater to the experimental s a l i n i t i e s below 100% SW. Higher s a l i n i t i e s were made by adding the appropriate inorganic s a l t s to 100% seawater (Barnes 1954). Calcium uptake was measured by the transfer of * 5Ca from seawater into the tissues and s h e l l of the animal. Normal dosage was 50 microcuries. The isotope was supplied by Amersham Corp. as c a r r i e r - f r e e aqueous " 5 C a C l 2 at -pH 5-7. The ra t i o of labelled/unlabelled calcium ( s p e c i f i c a c t i v i t y ) in 100% SW was 0.0087. Animals brought from the f i e l d were maintained in an environmental chamber with a lightrdark regime of 12 hr:12 hr. Control s a l i n i t i e s for the summer and winter seasons were established from measurements of the s a l i n i t y of the environmental seawater 1 meter below the mooring f l o a t s . These measurements were made weekly for one month before the mussels were brought to the laboratory. The calculated mean s a l i n i t i e s were 47% and 90% SW for the summer and winter seasons respectively. On the basis of these values, the summer and winter control s a l i n i t i e s were established as 50% and 100% SW. The winter and summer control temperatures were established as the mean of a two week continuous measurement (determined every three hours) made 1 meter below the mooring f l o a t s . On the basis of these values, the summer control temperatures were established as 15°C for 1979 and 17°C for 1980, while the winter 13 control temperature was 5°C for both years. In the environmental chamber, mussels were held in aerated p l a s t i c aquaria i d e n t i c a l to the trays used in the f i e l d . For experiments which required long-term holding, the water of each aquarium was replaced twice weekly by 5 l i t e r s of new water. With the exception of two experiments described l a t e r , mussels were not fed. Before an exper iment,,.- -each animal was scraped clean and placed in a test dish with 200 ml. of seawater, one mussel to a dish. The sample size for each test was 15 animals, but was made smaller when high mortality occurred within an experimental group. The temperature of the test dishes was maintained by a c i r c u l a t i n g water bath accurate to ±1°C, and a period of 24 hours was allowed to recover from cleaning and handling. Following the recovery period, the water in each test dish was supplied with 50 microcuries of fl5CaCl2. The water was s t i r r e d , then the mussel was l e f t undisturbed and allowed to take up the isotope for 24 hours. After the 24 hour period, a l l the mussels were rinsed in clean seawater and frozen. Later, the animals were thawed, the s h e l l and soft parts dissected and dried at 100°C for 24 hours. Following t h i s they were weighed to an accuracy of ±0.1 mg, and the presence of radioactive calcium measured by means of a Nuclear-Chicago planchette proportional counter. Details of the counting procedure are given in the Treatment Of Data And S t a t i s t i c a l Analysis section found at the end of the Material and Methods. Contaminated seawater l e f t after each experiment was dil u t e d to prescribed concentrations and disposed of by drain. 1 4 J_. S a l i n i t y experiments The purpose of thi s experiment was to determine the calcium-uptake rate of mussels exposed to long-term changes in s a l i n i t y . Subtidal mussels were removed from the mooring f l o a t s during the summer (August 1979) and winter (February 1980) seasons, and maintained as described above. The duration of the experiment was three weeks, and the calcium-uptake rates of groups of mussels were measured at weekly intervals using the methodology described in the General Laboratory Methods section. The experimental s a l i n i t i e s used were 25%, 50% (summer control), 75%, 100% (winter control) and 125% SW. The control temperature was 15°C for the summer and 5°C for the winter. Before placing any of the mussels in experimental s a l i n i t i e s , a group from the f i e l d was placed under control conditions and the i n i t i a l calcium-uptake rate was measured. This is presented in the Results section as the Day 0 group. During the summer t r i a l , i t became apparent that after two days the animals held in the 125% seawater were not opening. This s a l i n i t y was discarded from the summer t r i a l . After the experiment was performed, i t came to my attention that the Week 0 control s a l i n i t y (50%) for the August 1979 experiment had been carried out in c o r r e c t l y . I had erroneously tested the Day 0 group in 100% seawater, instead of 50% seawater. As a result, beginning in December 1980, I began to adapt a group of winter-adapted mussels to summer conditions. This was done by bringing subtidal animals to the laboratory. Their f i e l d temperature and s a l i n i t y were 7°C and 90% at the 15 time they were removed. They were transferred to an environment of 15°C and 50% for 12 days, and maintained on a culture of the diatom Skeletonema costatum at a concentration of 30,000/ml. High mortality after 14 days in these conditions prevented allowing t h i s group a longer period of adaptation. In the Results section, the calcium-uptake rate for the summer-adapted Week 0 control group represents this group of mussels. 2. Acute temperature response experiments This experiment was designed to determine the acute response of the calcium-uptake rates of summer- and winter-adapted mussels to changes in temperature. Subtidal mussels were removed from the mooring f l o a t s during the summer (August 1980) and winter (November 1979) seasons. Mussels were cleaned as described above, then transferred to environmental chambers. The experimental temperatures were 1°, 5° (winter co n t r o l ) , 12°, 17° (summer contr o l ) , and 23°C. While in the environmental chambers, the mussels were held in darkness. The s a l i n i t i e s were 100% seawater for the winter and 50% seawater for the summer. Again, i t came to my attention in December 1980 that the summer t r i a l had been improperly made. I had erroneously used 100% seawater as the summer c o n t r o l - s a l i n i t y , instead of 50% seawater. As a result, winter mussels adapted to summer conditions, taken from the Seasonal S a l i n i t y Experiment described above, were used to rerun the summer experiment. 16 3_. I n t e r t i d a l transplant experiment The purpose of thi s experiment was to determine the calcium-uptake rates of mussels at di f f e r e n t i n t e r t i d a l heights, and to see i f the uptake rate was modified by reciprocal transplantation to higher and lower i n t e r t i d a l positions. In October 1979, mussels were removed from the trays in the f i e l d located at i n t e r t i d a l heights of 0.2, 1.2 and 2.2 m. Transplants of about 50 mussels were made from each tray into the other two trays: mussels from 0.2 m were transplanted to 1.2 m and 2.2 m; mussels from 1.2 m were transplanted to 0.2 m and 2.2 m; and mussels from 2.2 m were transplanted to 0.2 m and 1.2 m. After 34 days, 15 individuals from each of these nine groups were transported to the laboratory. Three groups were from the untransplanted controls, and six groups were from the transplanted experimental groups. They were cleaned and allowed 24 hours to recover from handling. After the recovery period, the calcium-uptake rate of each of the sample groups was measured according to the methods described in the General Laboratory Methods section. 4. Size gradient study This study was made to determine whether the s i z e - d i s t r i b u t i o n of Mytilus varied with i n t e r t i d a l height. Tissue weights of mussels taken from the trays at equivalent i n t e r t i d a l heights of 0.2 and 2.2 m were measured to determine whether the proportionate weights of the s h e l l , mantle, g i l l and 17 viscera remained constant r e l a t i v e to the t o t a l dry weight of soft parts between high and low i n t e r t i d a l mussels. The dry weight of the s h e l l and soft parts were determined after 24 hours of drying at 100°C. The volume of the two valves were compared by measuring the length, width and height of individual s h e l l s , and then measuring their displacement volume. In this context, s h e l l displacement volume refers to the volume of the sh e l l material of the two valves, and not to the inner s h e l l volume. Shell displacement volume was measured by placing the s h e l l valves in a graduated cylinder, and then measuring the change in volume of butyl alcohol dispensed from a calibr a t e d burette which was required to f i l l the graduated cylinder. Shells that were too large to f i t in the graduated cylinder were broken into pieces. Butyl alcohol was used because of the small miniscus i t produced in the graduated cylinder. The volume measurements are accurate to ±0.02 ml. In January 1979 a study of the v e r t i c a l d i s t r i b u t i o n of Mytilus was made. A 6 cm diameter cable suspended from the jetty and adjacent to the trays was chosen as the c o l l e c t i n g _ s i t e , since i t was inaccessible to the mussels' p r i n c i p a l predators Pisaster ochraceous and Thais lamellosa. Beginning at the top of the d i s t r i b u t i o n of Mytilus and working down the cable in increments of between 20 and 40 cm v e r t i c a l distance, a l l the mussels were removed and returned to the laboratory for measurement. Size measurements accurate to ±0.1 mm were recorded by d i a l c a l i p e r s as the greatest distance along the anterior-posterior axis. These values were plotted as histograms 18 at 2 mm increments. Since the mussels were taken from d i f f e r i n g lengths of cable, the frequency scale of each histogram was adjusted so that each histogram represented a surface area of 100 square centimeters. 5_. Time course uptake study The purpose of this study was to determine the amount of l a b e l l e d calcium taken up by the s h e l l , mantle, g i l l and viscera as a function of time. This permitted the determination of the length of time necessary for the s p e c i f i c a c t i v i t y (ratio of labelled/unlabelled calcium) to equilibrate between the external seawater and the mantle, g i l l and viscera. It also allowed the c a l c u l a t i o n of a correction factor between the uptake rates given in this study, and the absolute uptake rates. In February 1981, 50 subtidal mussels were removed from the mooring f l o a t s and transported to the laboratory. They were cleaned and allowed a recovery period as described in the General Laboratory Methods section, and held at winter control conditions (5°C; 100% SW). After the recovery period, a l l the mussels received a standard dose of isotope. Beginning one hour l a t e r , 10 mussels were removed and frozen. After that, at 2, 4, 8, 16 and 32 hours, 10 mussels at each time in t e r v a l were removed, rinsed in clean seawater and frozen. Later they were thawed, and the s h e l l , mantle, g i l l and viscera dissected. The s h e l l and tissues were dried and weighed, and the calcium isotope present in the s h e l l and each of the tissues measured. 19 Data analysis The measurement of r a d i o a c t i v i t y , expressed as disintegrations/minute by the proportional counter, was recalculated as microcuries of " 5Ca. This was done by measuring the d i s i n t e g r a t i o n rate of standards of known a c t i v i t y , and then determining the equation which described the relationship between the real rate of disintegration (determined by the h a l f - l i f e of the isotope), and the less e f f i c i e n t rate which the proportional counter measured. This calculation did not correct for self-absorption by the samples. An attempt was made to spread out the soft parts before drying, rendering them as thin as possible after drying. It was then assumed that the drying of the tissues made them thin enough to reduce the effects of self-absorption. Preliminary experiments showed that after 24 hours, 75-95% of the r a d i o a c t i v i t y was found in the s h e l l . Since th i s newly deposited calcium would be present on the inner surface of the s h e l l , the surface which was counted, the effect of self-absorption in the s h e l l would be n e g l i g i b l e . Radioactivity measurements underwent further adjustment to compensate for differences in the calcium content of seawater of di f f e r e n t s a l i n i t i e s , differences in the duration of the experiments, differences in isotope dosage, and isotope decay. After these corrections, the f i n a l result was expressed as pg calcium/gram t o t a l dry weight/hour. Total dry weight refers to the combined dry weight of the shel l s and dry weight of soft parts. For each experiment, the regression: log 1 0 ( r<g calcium uptake/gram t o t a l dry weight/hour) was plotted as a function of 20 log,o(grams t o t a l dry weight). By t h i s method, the slope of the regression l i n e corresponds to a rate constant for each experimental group, and the Y-intercept corresponds to the logarithm of the calcium uptake rate of a 1.0 gram t o t a l dry weight mussel. The use of 1.0 gram t o t a l dry weight mussels when making comparisons between experiments is ar b i t r a r y , and based upon the si m p l i c i t y of deriving the uptake rate from the Y-intercept of the regression. In those experiments which showed low slopes, the use of thi s weight made l i t t l e difference to the uptake rate. However, in those experiments which showed large negative slopes ( t y p i c a l l y during the summer), small mussels, because of their higher r e l a t i v e surface area, showed higher calcium-uptake rates than large mussels. This p e c u l i a r i t y must be borne in mind when comparing seasonal differences in uptake rate. The n u l l hypothesis for each s t a t i s t i c a l comparison of experiments was that there was no difference between the slopes or intercepts of the regression l i n e s . Analyses of covariance between experiments were made by a PDP 11/45 computer, and were considered to be s i g n i f i c a n t l y d i f f e r e n t for Alpha less than 0.05. Therefore, in the context of comparisons between experiments, the use of the term s i g n i f i c a n t difference indicates that the analysis of covariance resulted in an F-test value which rejected the n u l l hypothesis when the probability of common variance between sets of compared data was less than 5%. In those instances where the analysis of covariance revealed that the slopes of the lines were not s i g n i f i c a n t l y d i f f e r e n t from one another, comparisons of the Y-intercept were made. 21 These comparisons were also considered s i g n i f i c a n t for Alpha less than 0.05. 22 RESULTS S a l i n i t y experiments Summer experiment (August 1979) The purpose of this experiment was to observe the response of summer-adapted mussels to changes in s a l i n i t y at a control temperature of 15°C. Measurement of the calcium-uptake rate of mussels held at 50% SW (control s a l i n i t y ) was made i n i t i a l l y (Day 0). Thereafter, the calcium-uptake rate was measured in 25%, 50%, 75% and 100% SW at 7, 14 and 21 days. At each weekly interval and at each experimental s a l i n i t y the regression l i n e of calcium uptake as a function of t o t a l dry weight was calculated. The regression l i n e s calculated for the summer experiment are found in Figures 1-3. Figure 1 shows the regression l i n e and data points for the control s a l i n i t y at Day 0 and Day 7. Figures 2 and 3 show the regression lines for each of the experimental s a l i n i t i e s at Day 14 and 21. In Figure 1, the data points for each s a l i n i t y i s shown, while Figures 2 and 3 show only the data points for the control s a l i n i t y . When the results of the control group (50% SW) shown in Figures 1-3 are considered, there i s no s i g n i f i c a n t change in the intercept value of the regression l i n e of the control group between Figures 1 through 3. That i s , there is no s i g n i f i c a n t change in the calcium-uptake rate of a mussel of 1.0 gram t o t a l dry weight during the three-week experiment. However, the control group does show a s i g n i f i c a n t change in slope between 23 Figure 1 . Day 7 summer-adapted regression lines of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight at 25%, 50%, 75% and 100% SW. Individual measurements of the s a l i n i t i e s are marked as follows: 25% SW, ( o ) ; 50% SW-, (•); 75% SW, (n); 100% SW, (•). The equations of the lines are: 25% SW; log Y = -1.129 log X + 0.717 (n=l0) 50% SW; log Y = -0.916 log X + 1.124 (n=15) 75% SW; log Y = -1.126 log X + 1.524 (n=l5) 100% SW; log Y = -1.063 log X + 1.622 (n=15) The summer-adapted regression l i n e for the Day 0 control s a l i n i t y (15°C, 50% SW) i s shown for comparison. Individual measurements are indicated by (A). The equation of this l i n e i s : 50% SW; log Y = -0.150 log X + 0.982 n=!0 300 200 100 E CL 13 < 50 30 20 10 3 2 DAY 0 "50% o. o o o \ M i l l c r - o l 0.1 0.2 0-3 0.5 TOTAL DRY WEIGHT (gm.) 25 Figure 2. Day 14 summer-adapted regression l i n e s of calcium uptake/gram dry weight/hour as a function of t o t a l dry weight at 25%,' 50%, 75% and 100% SW. Individual measurements of the control s a l i n i t y (50% SW) are marked by (•). The summer-adapted regression l i n e for the Day 0 control s a l i n i t y i s included for comparison. The equations of the lines are: 25% SW; log Y 50% SW; log Y 75% SW; log Y 100% SW; log Y 0.928 log X + 0.622 1.101 log X + 0.901 0.812 log X + 0.966 1.063 log X + 1.506 n=9 n=15 n=1 5 n=1 5 TOTAL DRY WEIGHT (gm.) 27 Figure 3. Day 21 summer-adapted regression lines of calcium uptake/gram dry weight/hour as a function of t o t a l dry weight at 50%, 75% and 100% SW. Individual measurements of the control s a l i n i t y (50%) are marked by (•). The summer adapted regression l i n e for the Day 0 control s a l i n i t y i s included for comparison. The equations of the l i n e s are: 50% SW; log Y = 75% SW; log Y = 100% SW; log Y = -0.524 log X + 0.912 -0.579 log X + 1.032 -0.349 log X + 1.578 n = 5 n=1 1 n=1 5 TOTAL DRY WEIGHT (gm.) 29 Day 0 and Day 7. The slope of the l i n e increases from -0.15 to -0.92, as shown in Figure 1. This difference i s discussed l a t e r . At Day 7 no s i g n i f i c a n t differences occur among the slopes of the regression l i n e s of the experimental s a l i n i t i e s shown in Figure 1. The mean slope of the experimental s a l i n i t i e s i s -1.06. However, there are s i g n i f i c a n t differences between the intercepts of some of the regression l i n e s . The intercepts of the 25%, 50% and 75% SW groups in Figure 1 are a l l d i f f e r e n t from one another, while the intercept of the 100% SW group i s not d i f f e r e n t from the intercept of the 75% SW group. Since the intercepts of the regression l i n e s show differences while the slopes show no such differences, i t i s apparent that the mechanism of uptake is similar in a l l cases (hence the same slope), and that the actual rate of calcium uptake (the Y-intercept value) is dependent upon the external s a l i n i t y . Thus, there is a dire c t relationship between the external s a l i n i t y and the uptake rate, as the differences between the 25%, 50% and 75% SW groups show. Since there i s no difference between the 75% and 100% SW groups, i t may be that the uptake mechanism becomes saturated with respect to calcium in s a l i n i t i e s greater than 75% SW. At Day 14 (Fig. 2), mussels in 25%, 50% and 100% SW show no s i g n i f i c a n t change in either the slope or the intercept of the regression l i n e , when compared with experiments made at the same s a l i n i t y the previous week (Fig. 1). The mean value for the slopes of the regression l i n e s in Figure 2 is -0.87. Mussels in 75% SW show no s i g n i f i c a n t change in slope either, but do show a 30 s i g n i f i c a n t decrease in the intercept, indicating a decrease in the calcium-uptake rate. The decrease in calcium-uptake rate in the 75% SW group could be attributed to continued osmotic stress, which is discussed l a t e r . At Day 21, the remaining experimental groups shown by the regression l i n e s of Figure 3 show no s i g n i f i c a n t change in either slope or intercept, when compared with the experimental groups of the same s a l i n i t y shown in Figure 2. That i s , the 50% and 75% SW groups are not d i f f e r e n t from one another, while the intercept of the 100% SW group is greater than the control group. In addition to the results just described, a l l the mussels in 25% seawater died after Day 14. This was not surprising, since they had shown the highest mortality rate during the f i r s t two weeks. In fact, there seemed to be a c o r r e l a t i o n between the s a l i n i t y and mortality rate. Although I did not record mortality data, I noticed a continuous high mortality among the 25% SW group, and v i r t u a l l y no mortality among the 100% SW mussels. The intercepts of the regression lines of each of the experimental s a l i n i t i e s shown in Figures 1-3 have been used to plot Figure 4, which shows the calcium-uptake rate of a summer-adapted 1.0 gram t o t a l dry weight mussel at each experimental s a l i n i t y as a function of time. Figure 4 demonstrates that during the summer the calcium-uptake rate is correlated with s a l i n i t y and that this c o r r e l a t i o n is evident after 7 days. Figure 4 also shows that summer-adapted mussels are not able to raise t h e i r calcium-uptake rates to compensate 31 Figure 4. The calcium-uptake rate of summer-adapted mussels of 1.0 gram t o t a l dry weight expressed as a function of time. The control temperature and s a l i n i t y are 15°C and 50% SW, experimental s a l i n i t i e s are 25%, 75% and 100% SW. V e r t i c a l bars on the figure indicate ±1 S.E. about the mean of each point. 5z_ 33 for lower s a l i n i t i e s . F i n a l l y , the figure shows that mussels in 75% SW are not able to maintain a calcium-uptake rate intermediate between the 50% and 100% SW groups, since at 14 days the uptake rate decreases to the rate of the control group (50% SW). Winter experiment (February 1980) The purpose of t h i s experiment was to observe the response of winter-adapted mussels to changes in s a l i n i t y at a control temperature of 5°C. Measurement of the calcium-uptake rate of mussels held at the control s a l i n i t y (100% SW) was made i n i t i a l l y (Day 0). Thereafter, the calcium-uptake rate of mussels held at 25%, 50%, 75%, 100% and 125% SW was measured at 7, 14 and 21 days. At each weekly interval and at each experimental s a l i n i t y the regression l i n e for calcium uptake as a function of t o t a l dry weight was calculated. The regression lines calculated for the winter experiment are shown in Figures 5-7. Figure 5 shows the regression l i n e and data points for the control s a l i n i t y at Day 0 and at Day 7. Figures 6 and 7 show the regression l i n e s for the Day 0 control s a l i n i t y and for each of the experimental s a l i n i t i e s at Day 14 and 21. In Figures 6 and 7 only the data points for the control group (100% SW) are shown. The mussels of the control group (100% SW) show no s i g n i f i c a n t change in either slope or calcium-uptake rate during the three weeks of the experiment. At Day 7 the regression l i n e slopes of the five experimental groups shown in Figure 5 have a mean slope of -0.14, which is not di f f e r e n t from the value of 34 Figure 5. Day 7 winter-adapted regression l i n e s of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight at 25%, 50%, 75%, 100%, and 125% SW. Individual measurements of the control s a l i n i t y (100% SW) are marked by (•). The equations of the l i n e s are: 25% SW; log Y = -0.304 log X + 0.980 n=14 50% SW; log Y = -0.188 log X + 1.353 n=l5 75% SW; log Y = 0.058 log X + 1.696 n=l5 100% SW; log Y = -0.114 log X + 1.654 n=l5 125% SW; log Y = -0.165 log X + 1.584 n=15 the winter-adapted regression l i n e for the Day 0 control s a l i n i t y (5°C, 100% SW) is shown for comparison. Individual measurements are marked by (A). The equation of t h i s l i n e i s : 100% SW; log Y = -0.026 log X + 1 .897 n=15 TOTAL DRY WEIGHT (gm.) 36 Figure 6. Day 14 winter-adapted regression l i n e s of calcium uptake/gram dry weight/hour as a function of t o t a l dry weight at 25%, 50%, 75%, 100% and 125% SW. Individual measurements of the control s a l i n i t y (100%) are marked by (•). The winter adapted regression l i n e for the Day 0 control s a l i n i t y i s included for comparison. The equations of the li n e s are: 25% SW 50% SW 75% SW 100% SW 125% SW log Y log Y log Y log Y log Y 0.632 log X + 0.841 0.252 log X + 1.191 0.318 log X + 1.397 0.132 log X + 1.733 0.214 log X + 1.532 n=1 5 n=1 5 n= 1 4 n= 1 4 n= 1 5 3=h 0.1 0.2 0.3 0.5 I 2 3 5 TOTAL DRY WEIGHT (gm.) 38 Figure 7. Day 21 winter-adapted regression l i n e s of calcium uptake/gram dry weight/hour as a function of t o t a l dry weight at 25%, 50%, 75%, 100% and 125% SW. Individual measurements of the control s a l i n i t y (100% SW) are marked by (•). The winter adapted regression l i n e for the Day 0 control s a l i n i t y i s included for comparison. The equations of the lines are: 25% SW; log Y = = -o. 1 48 log X + 1 .095 n= 1 3 50% SW; log Y = = 0. 01 1 log X + 1 .525 n = 1 5 75% SW; log Y = = 0. 249 log X + 1 .745 n = 1 5 100% SW; log Y = = -o. 1 58 log X + 1 .757 n = 1 5 125% SW; log Y = = -0. 01 1 log X + 1 .796 n= 1 5 TOTAL DRY WEIGHT (gm.) 40 -0.03 shown by the control s a l i n i t y at Day 0. The intercepts of the 75%, 100% and 125% SW groups show no difference from one another or from the intercept of the Day 0 regression, but there are s i g n i f i c a n t differences between the intercepts of the 25%, 50% and the 75% SW groups. This result i s similar to that found at Day 7 in summer-adapted mussels. In both cases there is a dir e c t correlation between s a l i n i t y and uptake rate, and in both cases the rate of calcium uptake reaches a plateau, above which an increase in the external s a l i n i t y has no s i g n i f i c a n t effect upon the uptake rate. At 14 days (Fig. 6), the intercepts of the regression l i n e s of mussels held at 75%, 100% and 125% SW show no s i g n i f i c a n t difference from one another or from the intercept values of mussels held at the same experimental s a l i n i t i e s at Day 7 (Fig. 5). Mussels in 25% and 50% SW also show no change in calcium-uptake rate when compared to mussels held at the same s a l i n i t y at Day 7. The slope of the l i n e of the 25% SW group is -0.63, which i s s i g n i f i c a n t l y d i f f e r e n t from a l l the other regression lines shown in Figure 6, which have a mean slope of -0.16. A comparison of the regression lines at Day 21 (Fig. 7) and Day 14 (Fig. 6) shows that there are no s i g n i f i c a n t changes in any of the intercepts of the regression l i n e s . There i s , however, a s i g n i f i c a n t change in the regression slope of the 25% SW group, which changes from -0.63 in Figure 6 to -0.15 in Figure 7. The intercepts of the regression lines of each of the 41 experimental s a l i n i t i e s shown in Figures 5-7 have been used to plot Figure 8. This figure shows the calcium uptake of a winter-adapted 1.0 gram t o t a l dry weight mussel at each experimental s a l i n i t y as a function of time. The decrease in the uptake rate of the control group between Day 0 and Day 7 is not s t a t i s t i c a l l y s i g n i f i c a n t , and the increase in uptake rate shown by the 50%, 75% and 125% SW groups between the second and t h i r d week is not s i g n i f i c a n t . A comparison of the calcium-uptake rate response of summer-and winter-adapted mussels to changes in s a l i n i t y shows that after one week mussels from both seasons show a direct c o r r e l a t i o n between s a l i n i t y and the calcium-uptake rate. Mussels from the summer and winter seasons show a plateau in the rate of calcium uptake in seawater above 75% s a l i n i t y . Neither summer- nor winter-adapted mussels are able to raise their calcium-uptake rate in order to compensate for a reduction in s a l i n i t y . Under winter conditions in s a l i n i t i e s greater than 75% SW, calcium uptake is not limited by external concentration, but rather by the a b i l i t y of Mytilus to take up calcium. This i s shown in Figure 9, which compares calcium uptake rate as a function of external s a l i n i t y for the summer and winter seasons. The uptake rates shown in th i s figure are taken from the Day 7 data points of Figures 4 and 8. A plateau in the uptake rates is v i s i b l e in both seasons, but a constant plateau i s not apparent among the summer-adapted mussels, as Figure 4 indicates. 42 Figure 8. The calcium-uptake rate of winter-adapted mussels of 1.0 gram t o t a l dry weight expressed as a function of time. The control temperature and s a l i n i t y are 5°C and 100% SW, experimental s a l i n i t i e s are 25%, 50%, 75% and 125% SW. V e r t i c a l bars on the figure indicate ±1 S.E. about the mean of each point. < 3 2 WINTER 1 4 2 1 DAYS 44 Figure 9. Calcium uptake by summer-adapted (15°C, 50% SW) and winter-adapted (5°C, 100% SW) mussels of 1.0 gram t o t a l dry weight as a function of s a l i n i t y . V e r t i c a l bars indicate ±1 S.E. about the mean of each point. SALINITY (%) 46 Acute temperature response experiments Summer experiment (August 1980) The purpose of this experiment was to determine the calcium-uptake rate of summer-adapted mussels in response to acute changes in temperature. The calcium-uptake rate of mussels in 50% SW was measured at the 1980 summer control temperature, 17°C, and at 1°, 5°, 12°, and 23°C. The regression l i n e of calcium uptake as a function of t o t a l dry weight was calculated at each of these temperatures and plotted in Figure 10. In this figure only the data points for the summer control temperature (17°C) are shown. A l l five of the groups plotted in Figure 10 have regression slopes which show no s i g n i f i c a n t difference from one another. Their mean value i s -1.02, which is not s i g n i f i c a n t l y d i f f e r e n t from the slope of -0.92 shown by summer-adapted mussels after one week of holding (Fig. 1). The experimental group at 17°C were taken from winter-adapted mussels which were subsequently adapted to summer conditions in the laboratory. This group shows a slope which is s i g n i f i c a n t l y d i f f e r e n t from those of the Day 0 control group shown in Figure 1, which . had a similar history. Possible reasons for th i s difference are given in the Discussion. In addition, there are no s i g n i f i c a n t differences between the intercepts of the adjacent temperature groups from 5° to 23°C. There is a s i g n i f i c a n t difference between the intercepts of the 12° and 23°C experiments, and between the 1°C group and a l l the other temperatures. 47 Figure 10. Summer-adapted regression l i n e s of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight, acutely tested at 1°, 5°, 12°, 17° and 23°C in 50% SW. Individual measurements at the summer control temperature (17°C) are marked by (•). The equations of the l i n e s are: 1 °C; log Y = = - o .714 log X + 0. 079 n = 1 3 5°C; log Y = = - o .822 log X + 0. 519 n = 1 4 1 2°C; log Y = = -1 .439 log X + 0. 580 n = 1 5 1 7°C; log Y = • -1 .272 log X + 0. 881 n = 1 5 23°C; log Y = = -0 .870 log X + 0. 935 n = 13 V>8 TOTAL DRY WEIGHT (gm.) 49 The intercepts of the regression l i n e s of each of the experimental temperatures shown in Figure 10 have been used to plot Figure 11. This figure shows the calcium-uptake rate of summer-adapted 1.0 gram t o t a l dry weight mussels at each of the experimental temperatures. These results suggest that the uptake rate i s temperature dependent between 1° and 23°C. Winter experiment (November 1979) The purpose of this experiment was to determine the calcium-uptake rate of winter-adapted mussels in response to acute changes in temperature. The calcium-uptake rate of mussels in 100% SW was measured at the winter control temperature, 5°C, and at 1°, 12°, 17° and 23°C. The regression l i n e of calcium uptake as a function of t o t a l dry weight was calculated at each of these temperatures and plotted in Figure 12. In th i s figure only the data points for the control temperature (5°C) are shown. The regression l i n e s from Figure 12 cart be divided into two groups on the basis of the slope of the regression l i n e s . One group, comprising the 1° and 23°C experimental groups, has a mean slope of -0.54. The other group, composed of the 5°, 12° and 17°C groups, has a mean slope of +0.08. This indicates that calcium uptake by winter-adapted mussels i s independent of weight between 5° and 17°C, but weight dependent above and below those l i m i t s . This i s shown in Figure 13, which plots the calcium-uptake rate of winter-adapted 1.0 gram t o t a l dry weight mussels at each of the experimental temperatures. Figure 13 50 Figure 11. The calcium-uptake rate of summer-adapted mussels of 1.0 gram t o t a l dry weight expressed as a function of acute temperature. The s a l i n i t y i s 50% SW. Experimental temperatures are 1°, 5°, 12°, 17° and 23°C. V e r t i c a l bars on the figure indicate ±1 S.E. about the mean of each point. T E M P E R A T U R E ( ° C ) 52 Figure 12. Winter-adapted regression l i n e s of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight, acutely tested at 1°, 5°, 12°, 17° and 23°C in 100% SW. Individual measurements of the winter control temperature (5°C) are marked by (•). The equations of the lines are: 1°C; log Y = -0.601 log X + 1.004 n=14 5°C; log Y = -0.114 log X + 1.654 n=l5 12°C; log Y = -0.054 log X + 1.595 n=14 17°C; log Y = 0.166 log X + 1.498 n=15 23°C; log Y = -0.477 log X + 0.852 n=15 TOTAL DRY WEIGHT (gm.) 54 Figure 13. The calcium-uptake rate of winter-adapted mussels of 1.0 gram t o t a l dry weight expressed as a function of acute temperature. The s a l i n i t y i s 100% SW. Experimental temperatures are 1°, 5°, 12°, 17° and 23°C. V e r t i c a l bars on the figure indicate ±1 S.E. about the mean of each point. 5 5 " TEMPERATURE (°C) 56 shows a plateau in calcium-uptake rates between 5° and 17°C, and a reduction in uptake at temperatures about these points. The differences in uptake rate among the experimental groups at 5°, 12° and 17°C are not s t a t i s t i c a l l y s i g n i f i c a n t , and the calcium uptake rate within t h i s range i s , therefore, independent of temperature. I n t e r t i d a l transplant experiment The purpose of thi s experiment was twofold: to determine whether i n t e r t i d a l height affected the rate of calcium uptake in mussels; and to see i f the rate of calcium uptake could be altered by changing the i n t e r t i d a l height of mussels, by comparing the uptake rates of transplanted mussels with those of untransplanted control mussels. Mussels from the trays located at 2.2, 1.2 and 0.2 m above datum were transplanted r e c i p r o c a l l y from 2.2 m to 0.2 and 1.2 m; from 1.2 m to 2.2 and 0.2 m; and from 0.2 m to 2.2 and 1.2 m. One month after transplantation, the calcium-uptake rates of the transplanted and the untransplanted mussels were measured in the laboratory, employing 1979 summer control conditions (15°C, 50% SW). The regression l i n e s of calcium uptake as a function of t o t a l dry weight were calculated for the experimental and control groups located at 2.2, 1.2 and 0.2 m, and plotted in Figures 14, 15 and 16 respectively. In each of the figures, only the data points of the untransplanted control groups are shown. A comparison of the slopes of the regression lines in Figure 14 shows that there are no s i g n i f i c a n t differences 57 Figure 14. Reciprocal transplant regression lines of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight. Individual measurements of the untransplanted controls (2.2 m equivalent i n t e r t i d a l height) are marked by (•).,..The equations of the li n e s are: 2.2 m; log Y 1.2 m; log Y 0.2 m; log Y -0.171 log X + 1.388 -0.693 log X + 0.997 -0.290 log X + 1.254 n=1 0 n=1 1 n=1 0 5% 0 . 0 1 ure 15. Reciprocal transplant regression lines of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight. Individual measurements of the untransplanted controls (1.2 m equivalent i n t e r t i d a l height) are marked by (•). The equations of the li n e s are: 2.2 m; log Y = -0.142 log X + 1.562 n=l0 1.2 m; log Y = -0.210 log X + 1.468 n=l2 2.2 m; log Y = 0.006 log X + 1.552 n=l0 6o TOTAL DRY WEIGHT (gm.) Figure 16. Reciprocal transplant regression lines of calcium uptake/gram t o t a l dry weight/hour as a function of t o t a l dry weight. Individual measurements of the untransplanted controls (0.2 m equivalent i n t e r t i d a l height) are marked by (•). The equations of the li n e s are: 2.2 m; log Y = 1.2 m; log Y = 0.2 m; log Y = -0.042 log X + 1.573 0.060 log X + 1.708 0.356 log X + 1.777 n=1 2 n=1 2 n= 1 1 TOTAL DRY WEIGHT (gm.) 63 between any of the experimental groups. The mean slope of the three groups i s -0.38. There are no s i g n i f i c a n t differences between any of the intercepts in this figure. Figure 15 shows the regression l i n e s of the experimental groups at 1.2 m. As in Figure 14, there are no s i g n i f i c a n t differences between the slopes of the regression l i n e s of any of the experimental groups. The mean value of the slope i s -0.12. In addition, there are no s i g n i f i c a n t differences between any of the intercepts of any of the experimental groups. The regression lines of the experimental groups at 0.2 m are shown in Figure 16. As in Figures 14 and 15, there are no s i g n i f i c a n t differences between either the slopes or intercepts of the regression lines of the experimental groups. The mean value of the slope is +0.12. A comparison of the regression lines from Figures 14, 15 and 16 shows no s i g n i f i c a n t difference between the slopes of the regression l i n e s . There is a s i g n i f i c a n t difference between the uptake rates of mussels at the upper and lower extremes, although t h i s difference is obscured by the large scatter found in some of the experiments. This difference is i l l u s t r a t e d in Figure 17, which shows the calcium-uptake rate of 1.0 gram t o t a l dry weight mussels of transplanted and untransplant-ed groups from each of the i n t e r t i d a l trays. In addition, there is a trend in the slopes of the regression l i n e s ; -0.38 at 2.2 m to +0.12 at 0.2 m. These values are the mean slopes of the controls and transplants at the upper and lower trays. 64 Figure 17. The calcium-uptake rate of untransplanted and r e c i p r o c a l l y transplanted i n t e r t i d a l mussels of 1.0 gram t o t a l dry weight expressed as a function of i n t e r t i d a l height. The l i n e between i n t e r t i d a l heights connects untransplanted controls. O r i g i n a l i n t e r t i d a l heights of transplants are marked above the individual measurements. V e r t i c a l bars on the figure indicate ±1 S.E. about the mean of each point. 4 ^ 0 0.2 1.2 2*2 3 INTERTIDAL HEIGHT (m ) 66 Size gradient study The purpose of thi s study was threefold: to see whether an i n t e r t i d a l size gradient existed among mussels; to see whether there were differences in the dry weight of the mantle, g i l l or viscera of high and low i n t e r t i d a l mussels; and to see whether there were differences in the size, shape or displacement of the shell s of high and low i n t e r t i d a l mussels. Differences between high (2.2 m) and low (0.2 m) mussels with respect to any of these physical c h a r a c t e r i s t i c s could y i e l d information useful to the interpretation of calcium-uptake differences between the two heights. The size measurements obtained from the mussels removed from the cable were plotted as histograms in Figure 18. The ordinate of Figure 18 indicates the i n t e r t i d a l height from which the mussels used in each histogram were taken, and the abscissa indicates the sh e l l length size intervals of the mussels in each histogram. An examination of the histograms shows that the modal sh e l l length (indicated by the shaded in t e r v a l of each histogram in Figure 18) decreases steadily with increasing i n t e r t i d a l height. Since the s i t e was inaccessible to i t s major predators (as described in the Materials and Methods), th i s suggests that physical factors are able to produce a size gradient. This is consistent with the results of the reciprocal transplant experiment, which showed that high i n t e r t i d a l mussels had lower rates of calcium uptake, and, presumably, lower rates of s h e l l growth, when compared to low i n t e r t i d a l mussels. Mussels from the 0.2 and 2.2 m trays were compared for 67 Figure 18. The size-frequency d i s t r i b u t i o n of mussels as a function of i n t e r t i d a l height. The intersection of each histogram with the ordinate marks the i n t e r t i d a l height from which the s h e l l length data for that histogram were co l l e c t e d . The abscissa marks the size increments for the histograms. The density of mussels in each histogram i s given by the legend bar marked 0 to 30 at the 2.2 m . histogram. The modal size i n t e r v a l i s indicated by shading. SHELL LENGTH (mm.) 69 differences in the relationship of the t o t a l dry dry weight of a l l soft parts to the dry weight of the s h e l l , mantle, g i l l and viscera. The dry weights of the s h e l l , mantle, g i l l and viscera are plotted as a function of the t o t a l dry weight of soft parts in Figures 19-22. Figure 19 demonstrates that there is a s i g n i f i c a n t difference in the weight of the she l l s of high and low mussels. High mussels have dry s h e l l weights 710% of the t o t a l dry weight of soft parts, while low she l l s have weights 230% of the t o t a l dry weight of soft parts; a difference of approximately three times. There is no s i g n i f i c a n t difference between the two Y-intercepts in Figure 19. Figures 20, 21 and 22 indicate that there are no s i g n i f i c a n t differences between the dry weight of mantle, g i l l or v i s c e r a l tissue as a function or the t o t a l dry weight of soft parts between high (2.2 m) and low (0.2 m) mussels. Expressed as a percentage of the t o t a l dry weight of soft parts, these are 39% for mantle, 8% for g i l l and 53% for viscera. The difference in s h e l l weights between high and low populations may be attributed to differences in the t o t a l dry weight of the soft parts, or to differences in the amount of calcium carbonate in she l l s of similar shape, or to a combination of these two factors. Figures 23 and 24 show the relat i o n s h i p between s h e l l height and length, and s h e l l width and length, respectively. There are no s i g n i f i c a n t differences between the high and low groups in either figure. This demonstrates that the she l l s are of similar shape. Figure 25 shows that there is no s i g n i f i c a n t difference in the density of 70 Figure 19. The regression l i n e s of dry weight of s h e l l as a function of t o t a l dry weight soft parts of mussels from 2.2 m ( o ) and 0.2 m (•). The equations of the li n e s are: 2.2 m; Y = 7.126 X - 0.063 n=44 0.2 m; Y = 2.285 X + 0.266 n=45 DRY WEIGHT SOFT PARTS (gm.) gure 20. The regression l i n e s of dry weight of mantle as a function of t o t a l dry weight of soft parts of mussels from 2.2 m (o) and 0.2 m (•). The equations of the l i n e s are: 2.2 m; Y = 0.389 X - 0.043 n=44 0.2 m; Y = 0.407 X - 0.073 n=45 0 0.05 0.10 0.15 0.2 DRY WEIGHT SOFT PARTS (gm.) 74 Figure 21. The regression lines of dry weight of g i l l as a function of t o t a l dry weight of soft parts of mussels from 2.2 m (o) and 0.2 m (•). The equations of the li n e s are: 2.2 m; Y = 0.077 X + 0.003 n=44 0.2 m; Y = 0.074 X + 0.005 n=45 DRY WEIGHT SOFT PARTS (gm.) 76 Figure 22. The regression l i n e s of dry weight of viscera as a function of t o t a l dry weight of soft parts of mussels from 2.2 m (o) and 0.2 m (•). The equations of the lines are: 2.2 m; Y = 0.530 X + 0.001 n=44 0.2 m; Y = 0.520 X + 0.002 n=45 0 0.05 0.10 0.15 0.2 DRY WEIGHT SOFT PARTS (gm.) 78 Figure 23. The regression l i n e s of s h e l l height as a function of s h e l l length in mussels from 2.2 m (o) and 0.2 m (•) The equations of the li n e s are: 2.2 m; Y = 0.518 X + 0.860 n=36 0.2 m; Y = 0.507 X + 0.716 n=36 SHELL LENGTH (mm.) ure 24. The regression lines of s h e l l width as a function of s h e l l length in mussels from 2.2 m ( o ) and 0.2 m (•) The equations of the lines are: 2.2 m; Y = 0.382 X + 0.175 n=36 0.2 m; Y = 0.352 X + 0.098 n=36 SHELL LENGTH (mm.) 82 the s h e l l valves from high and low s i t e s . The mean of the two slopes of the regression lines i s 0.40 ml./gm. The inverse of thi s value, 2.51 gm./ml., approximates the published value for the density of c a l c i t e , 2.7-2.9 grams/ml. (Weast, 1974). Figure 26 shows that there are no s i g n i f i c a n t differences between high and low mussels with respect to the volume of the s h e l l valves as a function of s h e l l length. In summary, Figures 23-26 indicate that the she l l s of high and low mussels show no s i g n i f i c a n t difference with respect to shape, density or valve displacement. Thus, the differences in s h e l l dry weight as a function of the t o t a l dry weight of soft parts (Fig. 19) i s due to a difference in the dry weights of the soft parts between high and low mussels. Time course uptake study The purpose of this study was to follow the movement of la b e l l e d calcium from the seawater to the s h e l l , mantle, g i l l and viscera over 32 hours. The experiment was performed under winter control conditions (5°C, 100% SW) . I n i t i a l l y , 60 mussels received a dose of " 5Ca. After inte r v a l s of 1, 2, 4, 8, 16 and 32 hours, ten mussels were removed from the seawater. The mussels were dissected into s h e l l , mantle, g i l l and viscera, and the r a d i o a c t i v i t y of the s h e l l and each of the tissues measured. From the s h e l l and from each tissue, the regression l i n e for calcium uptake as a function of t o t a l dry weight was calculated at each of the six time in t e r v a l s . These regression lines are plotted in Figures 27-30 for the s h e l l , mantle, g i l l and 83 Figure 25. The regression l i n e s of s h e l l valve displacement as a function of t o t a l dry weight s h e l l in mussels from 2.2 m (o) and 0.2 m (•). The equations of the lines are: 2.2 m; Y = 0.381 X + 0.015 n=36 0.2 m; Y = 0.415 X + 0.001 n=36 SHELL WEIGHT (gm.) 85 Figure 26. The regression lines of s h e l l valve displacement as a function of s h e l l length in mussels from 2.2 m (o) and 0.2 m (•). The equations of the li n e s are: 2.2 m; log Y = 2.136 log X - 3.685 n=36 0.2 m; log Y = 2.118 log X - 3.705 n=36 SHELL LENGTH (mm.) 87 viscera, respectively. In each of the figures, only the data points from the f i n a l i n t e r v a l (32 hours) are shown. Differences between intercepts in the Figures 27-30 are a r t i f a c t s produced when the regression lines are calculated as an hourly uptake rate. Therefore, these differences are of no real signifigance, and are not discussed further. Figure 27 shows the calculated regression l i n e s for calcium uptake by the s h e l l as a function of dry s h e l l weight. The slopes of the regression lines show no s i g n i f i c a n t differences, and have a mean value of -0.09. Figure 28 shows the regression l i n e s for calcium uptake by the mantle as function of the dry weight of mantle. The slopes of the regression l i n e s show no s i g n i f i c a n t differences, with a mean slope of -0.01. Figure 29 shows the regression lines for calcium uptake by the g i l l as a function of the dry weight of g i l l . The slopes of the regression show no s i g n i f i c a n t differences and have a mean value of +0.23. F i n a l l y , Figure 30 shows the regression l i n e s for calcium uptake by the viscera as a function of the dry weight of the viscera. Again, the slopes of the regression show no s i g n i f i c a n t differences, and have a mean value of -0.11. Using information from the Size Gradient Study, i t was possible to calculate the amount of isotope taken up by, the s h e l l and tissues of a subtidal mussel of 1.0 gram t o t a l dry weight. The s h e l l and tissue weights were calculated to be 0.695 grams s h e l l ; 0.104 grams mantle; 0.024 grams g i l l ; 0.177 grams viscera. Using the equations of the regression l i n e s in Figures 27-30 given in the legends of each figure, the uptake rate of 88 Figure 27. The regression l i n e s of s h e l l calcium uptake/gram dry weight shell/hour as a function of dry weight s h e l l at 1, 2, 4, 8, 16 and 32 hours. Individual measurements of the 32 hour experiment are marked by (•). The equations of the lines are: 1 hr 2 hr 4 hr 8 hr 16 hr 32 hr log Y log Y log Y log Y log Y log Y 0.160 log X + 2.822 0.054 log X + 2.382 0.120 log X + 2.494 0.376 log X + 1.895 0.475 log X + 1.636 0.388 log X + 1.864 n= 1 0 n=l0 n= 1 0 n = 9 n = 9 n = 9 O i |. ...I„„.i n , i, ii, n i n 11 i t 0.1 0.2 0.3 0.5 I 2 3 5 DRY WT. SHELL (gm.) ure 28. The regression l i n e s of mantle calcium uptake/gram dry weight mantle/hour as a function of dry weight mantle at 1, 2, 4, 8, 16 and 32 hours. Individual measurements of the 32 hour experiment are marked by (•). The equations of the l i n e s are: 1 hr log Y = - o .068 log X + 2 .421 n = 9 2 hr log Y = 0 .056 log X + 2 .544 n= 1 0 4 hr log Y = 0 .332 log X + 2 .587 n=9 8 hr log Y = -0 .052 log X + 1 .686 n = 9 1 6 hr log Y = - o .254 log X + 1 .414 n=9 32 hr log Y = -0 .097 log X + 1 .060 n=8 92 Figure 29. The regression l i n e s of g i l l calcium uptake/gram dry weight gill / h o u r as a function of dry weight g i l l at 1, 2, 4, 8 , 16 and 32 hours. Individual measurements of the 32 hour experiment are marked by (•). The equations of the l i n e s are: 1 hr log Y = 0. 347 log X + 3 . 463 n = 7 2 hr log Y = 0. 109 log X + 2 . 852 n = 7 4 hr log Y = 0. 474 log X + 3 . 206 n = 8 8 hr log Y = - o . 135 log X + 1 . 612 n = 5 1 6 hr • log Y = 0. 1 1 8 log X + 1 . 2 9 5 n = 9 32 hr log Y = 0. 488 log X + 2 . 042 n = 9 (w6) 119 1H9I3M AHQ 94 Figure 30. The regression l i n e s of v i s c e r a l calcium uptake/gram dry weight viscera/hour as a function of dry weight viscera at 1, 2, 4, 8, 16 and 32 hours. Individual measurements of the 32 hour experiment are marked by (•). The equations of the li n e s are: 1 hr 2 hr 4 hr 8 hr 16 hr 32 hr log Y log Y log Y log Y log Y log Y -0.074 log X + 2.525 -0.238 log X + 2.501 -0.064 log X + 2.490 0.026 log X + 1.726 -0.265 log X + 1.669 0.165 log X + 1.728 n = 9 n=1 0 n=9 n=8 n= 1 0 n = 9 DRY WEIGHT VISCERA (gm.) 96 each tissue was calculated. F i n a l l y , each hourly uptake rate was multiplied by the duration of the experiment and the s p e c i f i c a c t i v i t y of the isotope, 0.0087, to give the t o t a l amount of isotope present in the s h e l l and each of the tissues for each time i n t e r v a l of the experiment. This' information i s displayed in Figure 31, and shows the amount of " 5Ca present in the s h e l l , mantle, g i l l and viscera of a mussel of 1.0 gram t o t a l dry weight, measured at intervals over a period of 32 hours. In th i s figure, the l i n e s connecting the o r i g i n of the graph with the mantle, viscera and s h e l l have been omitted for c l a r i t y , leaving only the l i n e connecting the o r i g i n to the g i l l at 1 hour. It -was assumed that e q u i l i b r a t i o n of the s p e c i f i c a c t i v i t y of the soft tissues and the seawater occurred at the fourth hour, and that i t was possible to calculate the amount of calcium in the tissues, based upon the amount of " 5Ca present. This assumption is defended in the. Discussion. The calcium taken up by the mantle does not increase after 1 hour, and remains at a low l e v e l throughout the experiment. This indicates that the unlabelled calcium in the mantle tissue equilibrates with the a 5Ca in the seawater very quickly, and accumulates no net calcium over the course of the experiment. The calcium in the g i l l shows a similar response at 1 hour, and shows no s i g n i f i c a n t change throughout the experiment. The viscera show no s i g n i f i c a n t change after 4 hours, except for a decrease at 8 hours, followed by an increase at 16 hours to the amount of calcium taken up at 4 hours. The s h e l l removes calcium from the seawater during the course of the experiment, but shows 97 Figure 31. " 5Ca uptake by the s h e l l , mantle, g i l l and viscera of a 1.0 gram t o t a l dry weight winter-adapted mussel (5°C, 100% SW) as a function of time. The dry s h e l l and dry tissue weights are 0.695 grams s h e l l , 0.104 grams mantle, 0.024 grams g i l l , and 0.177 grams viscera. V e r t i c a l bars on the figure represent ±1 S.E. about the mean of each point, the marks along the top of the figure indicate t i d a l maxima and minima (measured in meters above datum) which occurred during the course of the experiment, and which may explain some of the variation between data points at 4 and 8 hours. NOTE: These data were log-transformed to make possible their presentation in one figure. Therefore, care must be taken in interpreting differences along the ordinate. 19> HOURS 99 wide fluctuations in the amount of calcium taken up, es p e c i a l l y at 1 hour and 4 hours. These fluctuations may be caused by t i d a l rhythms, and are dealt with in the Discussion. In summary, the u 5Ca in seawater reaches equilibrium with the calcium of the mantle and g i l l at 1 hour, and with the viscera at 4 hours. After t h i s e q u i l i b r a t i o n period, the s h e l l accumulates calcium from the seawater. In this experiment, a sizeable fract i o n of the radioactive calcium taken from the seawater is found in the soft tissues. If the time course of this experiment had been longer, the r e l a t i v e amount of calcium in the soft parts would have been lower compared to the amount of calcium taken into the s h e l l , and, therefore, most of the l a b e l l e d calcium taken up would represent s h e l l growth. In addition, much of the calcium taken up by the s h e l l occurred in the process of e q u i l i b r a t i o n , and does not represent true calcium uptake. In order to estimate the true uptake rate, the slope of the shell-uptake l i n e was estimated between the 4 and 32 hour in t e r v a l s . This l i n e yielded an average uptake rate of 32 i/g Ca/hr, compared to an estimate of 55 */g Ca/hr which i s calculated from the amount of calcium in the s h e l l and soft parts, determined at 24 hours in Figure 31. Therefore, the values of calcium-uptake rate presented in Figures 4, 8, 11, 13 and 17 are overestimated by a factor of 55/32, or 1.7. The presence of a correction factor, while reducing the absolute values of calcium-uptake rate, does not affect the differences between uptake rates which have been shown in these figures. It should also be noted that this correction factor could be 100 di f f e r e n t for summer-adapted mussels, since they show weight-dependent regression slopes, and may therefore be taking up calcium by a dif f e r e n t mechanism. 101 DISCUSSION S a l i n i t y experiments A comparison of the summer and winter experiments shows that the summer regression-lines have slopes which are t y p i c a l l y steeper than -0.5, while the winter regression-lines show slopes which are often near zero. This difference is probably not a calcium-specific response, and l i k e l y represents a change in the over a l l metabolism of summer- and winter-adapted mussels --summer-adapted mussels being less active metabolically than winter-adapted ones. When the results of the three-week experiments shown in Figures 4 and 8 are pooled to give an average uptake rate for the 100% SW experimental groups from each season, winter mussels of 1.0 gram t o t a l dry weight show maximum calcium-uptake rates about 1.5 times greater than summer mussels in the same s a l i n i t y : 59 ^g Ca/hr compared to 38 t>q Ca/hr. A comparison of the control s a l i n i t y uptake rates reveals an even greater contrast: 10 »q Ca/hr for the summer control group, and 59 »»g Ca/hr for the winter control group. In 25% SW, winter mussels show calcium-uptake rates twice those of summer mussels: 10 ^g Ca/hr compared to 5 ^g Ca/hr. Further, there is a consistent relationship between the seawater s a l i n i t y and the rate of calcium uptake during both seasons. The uptake rates of the control groups, as shown in Figures 4 and Figure 8, show no si g n i f i c a n t change between Days 0, 7, 14 and 21. A reduction in the s a l i n i t y causes a decrease in calcium-uptake rate when the s a l i n i t y i s below 75% SW, while in most cases an increase in the 1 02 s a l i n i t y above 75% SW causes no increase in the uptake rate. During both seasons, mussels were not able to increase their calcium uptake rate in response to extended periods in d i l u t e seawater. However, during the summer experiment shown in Figure 4, the experimental group in 75% SW showed a response between the f i r s t and second week of the experiment which could be interpreted as regulation of the uptake rate. In the case of the summer-adapted mussels (Fig. 4), the 75% seawater group showed an increase at Day 7 compared with the Day 0 control s a l i n i t y , but returned to the Day 0 control l e v e l at Day 14. This change cannot be attributed to starvation, since i t would have presumably affected the 100% seawater group s i m i l a r l y . Differences of slope would indicate differences in the metabolic states of the experimental groups, but, with the exception of the Day 0 control, there are no differences among any of the regression slopes in Figures 1-3. Mussels are known to be poor osmoregulators in d i l u t e seawater, and show higher mortality in low s a l i n i t i e s than in high ones (Potts 1954; McLachlan & Erasmus 1974; Hoyaux et a l . • 1976). It follows that summer-adapted mussels are subject to osmotic stress which is not l e t h a l , but for which they do not compensate their blood ion composition. Because of th i s prolonged stress, summer-adapted mussels may have a limited a b i l i t y to respond to increases in s a l i n i t y by increasing their calcium-uptake rate, and thus show only a temporary increase in uptake rate when maintained in 75% SW. This interpretation is speculative, but may be supported by the fact that summer regression-lines (Fig. 1-3) are usually 103 quite steep. Although the slopes of the regressions must be interpreted with caution, steep slopes probably indicate low metabolic a c t i v i t y . If low metabolic a c t i v i t y i s c h a r a c t e r i s t i c of the summer-adapted group, i t could predispose them to a reduced a b i l i t y to respond to the increases in s a l i n i t y desribed previously. If thi s i s true, i t further suggests that the Day 0 summer-adapted control group, which represented the summer-adapted winter mussels referred to in the Material and Methods, showed a low regression slope because they had not acclimated to summer conditions in the time allowed, and that a longer period of adaptation might have resulted in slopes similar to those shown by the Week 1 regression l i n e s (Fig. 1). When the calcium-uptake rates reported in these experiments are compared with published reports of calcium-uptake rates, i t is usually necessary to refer to studies which have measured s h e l l growth rate over monthly, or longer, in t e r v a l s . Fortunately, since most calcium is used for sh e l l growth, calcium uptake measurements and s h e l l growth rates are comparable. Although s h e l l growth and growth of the soft parts are not synonymous, s h e l l size i s commonly used as an indicator of growth. Galtsoff (1934) for example, found that s h e l l growth in Crassostrea v i r g i n i c a continued after low-temperature fasting had begun, and that the weight of the soft parts did not increase during periods of fasting at low temperatures. In his study, Galtsoff found that the s h e l l weight of C. V i r g i n i c a increased from 60 to 130 grams over 11 months. Assuming that the calcium-uptake rate was independent of weight (as my study has 1 04 found i t to be under most conditions), this indicates an average uptake rate of 39 «/g Ca/gram/hr. In their study of the sea mussel Mytilus californianus , Fox and Coe (1943) reported an increase in s h e l l weight from 15 grams to 105 grams over a period of 30 months. This corresponds to an average calcium-uptake rate of 30 j*g Ca/gram/hr. When one considers that these two values are annual averages and do not account for seasonal differences in s a l i n i t y or temperature, the reported values are in general agreement with the uptake rates found in this study, namely 59 */g Ca/hr during the winter season and 38 A»g Ca/hr during the summer season (based on a 1.0 gram t o t a l dry weight mussel in 100% SW). The agreement i s even closer i f the correction factor of 1.7 (referred to previously) i s applied to my data, giving winter and summer uptake rates of 30 and 22 >/g Ca/hr. In a study of s h e l l growth in Crassostrea (Ostrea) v i r g i n i c a , Wilbur and Jodrey (1952) found s h e l l growth to be 0.92 grams/month, determined at 22°C and 35 parts/thousand seawater. However, Wilbur and Jodrey reported only the length of the she l l s used in their study: 8-9 cm. In an attempt to estimate the weight of shel l s of this length, in order to compare Wilbur and Jodrey's data, I used two valves of C. gigas from the U.B.C. Invertebrate Museum c o l l e c t i o n , and calculated the weight of a valve of 9 cm as being 36 grams. Table 2 of Quayle (1969) indicates that oysters with s h e l l s of this weight would have a t o t a l dry weight of approximately 80 grams. Using these assumptions, the uptake rate of the oysters was 7 ug Ca/gram/hr. This is a very low uptake rate (probably due to the 105 dissection technique), and does not agree with Galtsoff's measurement of sh e l l growth based on 11 months growth: 39 Mg Ca/gram/hr. The large difference between these two values underscores the necessity to validate growth measurements based on isotope uptake against actual measurements of increase in sh e l l weight. In conclusion, a survey of s h e l l growth studies indicates that Myt i l u s c a l i fornianus and Crassostrea v i r g i n i c a show similar calcium-uptake rates compared to those reported here for Mytilus edulis. This lends credence to the results given here, since I was concerned with recording uptake rates in short term experiments, while the experiments of Fox and Coe and Galtsoff were concerned with long-term s h e l l growth. Acute temperature response experiments The temperature experiments summarized in Figures 11 and 13 display d i s t i n c t seasonal differences in the response to acute changes in temperature. During the summer, calcium uptake is temperature dependent from 1° to 23°C, although the uptake rates of adjacent experimental temperatures are not usually s i g n i f i c a n t l y d i f f e r e n t . During the winter season calcium uptake is temperature-independent between 5° and 17°C, as shown by Figure 13. The mean regression slope for these temperatures is +0.08 (Fig. 12). The mean slopes of the upper and lower temperatures, 1° and 23°C, are -0.54. Referring to the summer experiment, i t should be noted that s i g n i f i c a n t differences were found between the slopes of the two groups of summer-adapted 106 winter mussels: the Day 0 summer control group from the Seasonal S a l i n i t y Experiments and the summer Acute Temperature Response Experiment. These two groups had i d e n t i c a l previous h i s t o r i e s , and were not separated u n t i l the experiments were performed. The only difference between the experimental conditions was the absence of l i g h t in the case of the acute-temperature response experiment. It may be, then, that the l i g h t i n g conditions caused thi s difference. According to Galtsoff (1934), fasting occurred at temperatures below 4°C in Crassostrea v i rgin ica , but he indicated that s h e l l growth was not reduced at temperatures lower than t h i s . Loosanoff and Nomejko (1949) reported fasting in C. v i r g i n i c a at temperatures below 5°C, along with reduced growth in s h e l l weight. They also reported that s h e l l growth was greatest between 15° and 20°C. Richards (1935) has reported that s h e l l growth of Mytilus edulis was reduced at temperatures above 20° and below 5°C, l i m i t s which agree with the results of the experiment on winter-adapted mussels in thi s study. Seed (1968) reported that the s h e l l of Mytilus edulis in England showed l i t t l e linear increase during the period from October to A p r i l , and 90% of their annual s h e l l growth during the remaining i n t e r v a l . Quayle (1969) reported that both the s h e l l and soft parts of Crassostreas gigas ceased growing between November and A p r i l , when the water temperature was less than 10°C. In the present study, however, winter-adapted Mytilus edulis continued to take up calcium independently of temperature as low as 5°C. If t h i s abrupt reduction in uptake rate is a general feature of 107 marine bivalves at low temperatures, i t helps to explain the reduction of s h e l l growth at low temperatures reported by Richards (1935), Loosanoff and Nomejko (1949) and Seed (1968), and may explain some of the apparent contradictions between studies which report winter growth and those which report no winter growth. It does not explain the basis for the change in uptake rate, but the fasting at low temperatures as reported by Galtsoff (1934) may be responsible for the cessation of a number of metabolic a c t i v i t i e s , including calcium uptake. The reduction of uptake rate among winter-adapted mussels at high temperatures .was noted by Coe and Fox (1942) in Mytilus californianus (season not given) at temperatures above 20°C. This temperature may represent an upper physiological l i m i t which Mytilus  c a l i fornianus is capable of t o l e r a t i n g . On a seasonal basis, the difference between the regression slopes of summer- and winter-adapted mussels may be attributed to seasonal differences in s a l i n i t y and food resources between the two seasons. Quayle (1969) reported that winter shell-growth rates in Crassostrea gigas were low in the absence of high phytoplankton concentrations, while soft-part growth, s h e l l growth and the accumulation of glycogen stores (=fattening) were correlated with seasonal increases in phytoplankton concentrations. Quayle also reported a high increase in s h e l l weight in August in the absence of high phytoplankton concentrations, but I suspect that t h i s single observation was not representative of the long-term a v a i l a b i l i t y of phytoplankton. 108 The correlation between phytoplankton concentration, glycogen reserves, and s h e l l growth i s obscured by temperature and s a l i n i t y effects which cannot be separated in f i e l d studies. This study has shown that starvation has no effect upon the calcium uptake rates of summer- and winter-adapted mussels measured over a three week period in the laboratory. This conclusion is based upon the fact that the uptake rates of summer- and winter-control mussels did not change s i g n i f i c a n t l y during the s a l i n i t y experiments. However, I think that the reason for seasonal differences in the metabolic states of the mussels l i e s in the long-term osmotic stress to which summer-adapted mussels are subjected. If t h i s i s true, the annual pattern of winter and summer temperature and s a l i n i t y conditions l i m i t s the calcium uptake of mussels in the following way. During winter, mussels experience l i t t l e low s a l i n i t y stress, and except at temperatures below 5°C, are able to take up calcium faster than at any other time of year. However,the phytoplankton which they require for their metabolism are t y p i c a l l y in very low concentration during the winter (Fox & Coe 1943). In addition, winter temperatures are l i k e l y to f a l l below 5°C occasionally. During the summer the opposite condition e x i s t s . Mussels are under osmotic stress, and, therefore, unable to take up much calcium. This occurs at a time when phytoplankton concentrations are high. Since calcium uptake is only one of the metabolic a c t i v i t i e s of mussels, i t is reasonable to expect that during the summer they make use of high phytoplankton concentrations for the growth of soft parts, 109 for gonadal development, and for glycogen stores. This has been shown to take place in Ostrea edulis (Orton 1928), in Mytilus  californianus (Fox & Coe 1943), and in Crassostrea v i r g i n i c a (Galtsoff 1934; Loosanoff and Nomejko 1949). Thus, although summer-adapted Mytilus are not well-adapted to take up calcium, they may be expected to feed and increase the weight of their soft parts and accumulate energy reserves. Winter-adapted Mytilus may show reduced growth of soft parts, but given temperatures greater than 5°C are able to continue s h e l l growth •in the absence of high phytoplankton concentrations by drawing upon their glycogen reserves. Orton (1928) and Coe and Fox (1942) suggest that an antagonistic mechanism exists which i n h i b i t s calcium deposition during periods when glycogen is being synthesized (Orton 1928; Coe and Fox 1942), but this suggestion i s untested and may simply be a r e f l e c t i o n of changes in s h e l l growth rate as a function of seasonal changes in s a l i n i t y . It may be suggested that gonadal development could interfere with calcium uptake and s h e l l deposition by competing for glycogen stores. At the c o l l e c t i n g s i t e at Spanish Bank, I noticed spawning mussels from about March u n t i l September, and developing gonadal tissue invading the mantle tissue at a l l times of the year. Since gonadal development occurs throughout most of the year, i t i s not possible to answer th i s question based on my data. However, Loosanoff and Nomejko (1949) have found that spawning did not affect the rate of s h e l l growth in Crassostrea v i r g i n i c a . Thus, the relationship between gonadal 110 development and calcium uptake remains unresolved. In conclusion, the pattern of calcium uptake shown by summer-adapted mussels i s much d i f f e r e n t from that of winter-adapted ones. These differences are evidenced by the differences in the slopes of the regression l i n e s , and by differences in the uptake rates themselves (Figs. 4 and 8). I believe that these differences are caused by the persistence of summer control conditions, namely low s a l i n i t y and high temperature, which reduce the a b i l i t y of summer-adapted Mytilus to take up calcium independently of weight at any temperature. This is also reflected in the effect of acute changes in temperature upon calcium uptake by summer-adapted mussels. I n t e r t i d a l transplant experiment The results of the reciprocal transplant experiment are shown in Figure 17. These data show that mussels which were transplanted to new i n t e r t i d a l heights had calcium-uptake rates similar to those of untransplanted mussels at the same i n t e r t i d a l height. The variances are large among mussels transplanted from 0.2 m compared with mussels from 2.2 m, and t h i s suggests that there i s less variation in the uptake rates of high i n t e r t i d a l mussels. Figure 17 also suggests that these differences in variation p e r s i s t for at least one month, since mussels originating at 0.2 m generally had uptake rates with large standard errors, while those originating at 2.2 m had uptake rates with small standard errors which persisted at 1.2 and 0.2 m. There is a change in the mean value of the regression 111 slopes, from -0.38 at 2.2 m to +0.12 at 0.2 m,'and although these differences are not s i g n i f i c a n t , they demonstrate a trend in the regression slopes which I believe i s r e a l , and which suggests that mussels high in the i n t e r t i d a l zone are less metabolically active than mussels low in the i n t e r t i d a l zone. This interpretation is supported by the work of Segal et. a l . (1953), and Rao (1954) who found that the water-pumping rates of high i n t e r t i d a l Mytilus californianus were more weight-dependent than their low i n t e r t i d a l counterparts. Although the reciprocal transplant experiment of the present study demonstrates that both the uptake rate and the regression slopes of mussels within the i n t e r t i d a l zone can be altered by transplantation to d i f f e r e n t heights, i t does not demonstrate how this occurs. Rao (1954) has shown that pumping rates of Myt i l u s eduli s and Myt i l u s c a l i fornianus are synchronous with t i d a l rhythms, and can be altered by transplantation. His study also showed that t i d a l pumping rhythms were present in subtidal mussels which were not subject to t i d a l emersion. It i s , therefore, not d i f f i c u l t to imagine that the a c t i v i t y rhythms of i n t e r t i d a l mussels could be reset by the occurrence of new immersion/emersion patterns. Tidal emersion would c l e a r l y l i m i t the amount of time available for f i l t e r i n g , gas exchange, and other a c t i v i t i e s . At the the Spanish Bank c o l l e c t i n g s i t e , the average amount of time emersed each day at each of the i n t e r t i d a l positions are as follows: 0.2 m, 0 hours; 1.2 m, 2.6 hours; 2.2 m, 7.0 hours. These emersion times are taken from Quayle (1969), who based his calculations 1 12 upon annual Canadian Hydrographic Survey t i d a l records for Point Atkinson. Even when the uptake rate of mussels at 2.2 m are adjusted to account for only 17 hours immersion (by multiplying the uptake rate by 24/17), they s t i l l have uptake rates much lower than those at 0.2 m. Assuming, for the sake of comparison, that the duration and rates of pumping of mussels at the two heights were the same, the uptake rate at 2.2 m would only change from 18 ^g Ca/hr to 25 »q Ca/hr, s t i l l much lower than the uptake rate of 49 >/g Ca/hr shown by mussels at 0.2 m. Since the soft parts comprise the s i t e s of calcium uptake before deposition on the s h e l l edge and inner surface, i t seems reasonable to presume that the difference in calcium-uptake rates may be correlated with the differences in the weight of soft parts between the high and low mussels. The results of the Size Gradient Study show that mussels from low i n t e r t i d a l heights have greater t o t a l dry weight of soft parts than those from high i n t e r t i d a l heights. Using the regressions given in the legends of Figure 21, i t can be shown that mussels from the upper and lower heights having a t o t a l dry weight of 1.0 gram have a t o t a l dry weight of soft parts equalling 0.123 and 0.241 grams, respectively. Assuming, for the moment, that t h i s difference somehow aff e c t s the calcium-uptake rate, then the difference between calcium-uptake rates of high and low mussels is removed when these rates are calculated based on the t o t a l dry weight of soft parts, and both groups show i d e n t i c a l uptake rates: 203 »q Ca/gram t o t a l dry weight soft parts/hr i f this were true, then mussels transplanted from 0.2 m to 2.2 m might 113 have been expected to show higher uptake rates than the 0.2 m controls. Since th i s did not occur after one month, the mussels transplanted from 0.2 to 2.2 m may have reduced their weight of soft parts. Segal (1956a) has shown that the limpet Acmaea 1imatula can resorb gonadal tissue after one month of transplantion from low i n t e r t i d a l to high i n t e r t i d a l s i t e s . So i t is possible that the weight of the soft parts did actually decrease. Rao (1953a) found that high i n t e r t i d a l Mytilus californianus had less soft parts than low i n t e r t i d a l mussels. In th i s study, he also showed that high i n t e r t i d a l mussels had lower water pumping rates than low i n t e r t i d a l mussels from the same location, even when measured as a function of the wet weight of the soft parts. So in the case c i t e d by Rao, differences in weight could not be pointed to as the sole cause for the difference in water pumping rates at d i f f e r e n t i n t e r t i d a l heights. Although other factors may be involved, this leads me to suggest that differences in the weight of soft parts between high and low i n t e r t i d a l mussels are the p r i n c i p a l reason for differences in measurements of calcium-uptake rate between these two groups. However, the underlying causes for the difference in the weight of the soft parts of high and low mussels remains unresolved, since i t cannot be explained simply by differences in immersion time. 114 Size gradient study The present study is not alone in noting differences in growth rates between v e r t i c a l l y separated i n t e r t i d a l bivalves. Newcombe (1935) recorded the length of Mytilus edulis taken from 3 feet (1 m) above the mean of the lower low waters (MLLW) in England. The annual growth of these mussels was from 9.2 to 16.0 mm. Assuming, for the sake of comparison, that the s h e l l and soft parts relationships are the same as those described in this study, t h i s indicates a change in s h e l l weight of 0.121 grams and an average calcium uptake rate of 37 >/g Ca/gram/hr at about 1 m above datum, compared to 34 »q Ca/gram/hr measured at 1.2 m in t h i s study. This comparison shows that the two uptake rates are approximately equal, but since the t i d a l patterns at Newcombe's s i t e are unknown, and since there i s no assurance that mussels from d i f f e r e n t locations have the same sh e l l and soft parts morphology (Fox & Coe 1943), the agreement may be fortuitous. Seed (1968) has also recorded s h e l l length data of v e r t i c a l l y and geographically separated populations of Mytilus  edulis in England. In th i s study, Seed calculated the age of mussels on the basis of incremental growth rings. At one s i t e he recorded the length of 9 year old mussels from 'high shore' and 'low shore' s i t e s as 5.5 and 4.0 cm respectively,. At another s i t e he recorded lengths from high shore and low shore mussels as 4.0 and 2.0 cm. Employing the same assumptions as the previous example, the uptake rates from mussels at a l l of these si t e s are about 7 »q Ca/gram/hr, indicating that the 1 15 calcium-uptake rates are i d e n t i c a l among v e r t i c a l l y separated mussels from these s i t e s . This is a very low uptake rate when compared with those rates found in thi s study, and also when compared with those found for another population of Myt i l u s  edulis from England, 37 Ca/gram/hr (Newcombe 1935). However, the mussels used in Seed's study were a l l presumably very old, and neither the present study nor Newcombe's dealt with animals that old. These low values do raise doubts, though, about the age estimates which Seed made in his study. In a study of the growth rate of i n t e r t i d a l l y and l a t i t u d i n a l l y separated populations of Mytilus californianus , Dehnel (1956) calculated growth rates of southern C a l i f o r n i a mussels from 0.3 m and 1.0 m above MLLW. He recorded the absolute growth of mussels over 30 days from these i n t e r t i d a l heights, and found s h e l l length increases of 2.3 mm and 0.96 mm from animals from 0.3 and 1 m respectively. From these measurements, instantaneous growth rates . of 0.002 and 0.0008 were calculated. Using these length increase measurements and growth rates, the t o t a l length of the mussels can be calculated to be 38.3 and 40.0 mm for the mussels from 0.3 and 1 m. For the sake of comparison, I have used regression equations given by Coe and Fox (1942) which calculate the weight of the s h e l l and the dry weight of the soft parts of Mytilus c a l i fornianus, in order to calculate the calcium-uptake rates for these two i n t e r t i d a l l y separated populations. Using these regressions, Myt i l u s c a l i fornianus of 38.3 and 40.0 mm length have calculated t o t a l dry weights of 3.64 and 4.08 grams. Mussels of thi s size 1 16 show s h e l l weight increases of 0.562 grams and 0.245 grams at 0.3 and 1 m respectively, and these increases correspond to calcium-uptake rates of 79 »q Ca/gram/hr at 0.3 m, and 32 ?q Ca/gram/hr at 1 m, without correcting for differences in immersion time. Therefore, i t can be shown from Dehnel (1956) that Mytilus californianus which are v e r t i c a l l y separated in the i n t e r t i d a l zone also show differences in calcium uptake rate which are similar to those shown by the present study of Mytilus  edulis. Judging from the s h e l l growth studies referred to above, differences in the calcium-uptake rate at d i f f e r e n t i n t e r t i d a l heights may be common to bivalves. The results shown by Figure 18 indicate that the differences in calcium-uptake rates discussed above may contribute to the gradient of s h e l l lengths which occurs within the i n t e r t i d a l d i s t r i b u t i o n of Mytilus  edulis. The c o l l e c t i o n s i t e had been free from Pisaster or Thais predation for the l i f e of the mussels at the s i t e , and this lends support the interpretation that physical factors are the cause for the size gradient. A size gradient might also have been produced by d i f f e r e n t i a l s e t t l i n g of spat at lower i n t e r t i d a l heights, followed by higher s e t t l i n g during later spat f a l l s . It does seem unlikely, though, that subsequent spat f a l l would have been at sequentially higher i n t e r t i d a l heights. It seems most l i k e l y that the size gradient shown in Figure 18 is the result of longer emersion times at higher i n t e r t i d a l heights, and as a consequence, reduced feeding time. However, i f differences in feeding time were the only l i m i t a t i o n , i t would 1 1 7 be expected that mussels would simply grow more slowly. Although t h i s may be the case, Figure 19 demonstrates that they also show d i f f e r e n t i a l growth of the s h e l l with respect to the t o t a l dry weight of the soft parts. This i s similar to the findings of Segal (1956b), who reported that the soft parts of high i n t e r t i d a l Acmaea limatula weighed less than low i n t e r t i d a l limpets. However, in the case of A. 1imatula, high i n t e r t i d a l limpets were also found to have thicker s h e l l s . If s h e l l growth requires l i t t l e metabolic energy, then i t is conceivable that mussels may expend l i t t l e energy growing larger s h e l l s . Segal (1956b) and Segal and Dehnel (1962), have reported that the limpet A. limatula l i v i n g high in the i n t e r t i d a l retains more water in the mantle cavity than i t s counterparts low in the i n t e r t i d a l zone. They have suggested that during emersed periods and high temperatures, high i n t e r t i d a l limpets allow this water to evaporate, and escape overheating by evaporative cooling of the mantle water. Besides t h i s heat buffering e f f e c t , they suggest that the retention of a larger volume of mantle water would also buffer the osmotic stress caused by water loss from the soft parts during long periods of emersion. This hypothesis may explain how mussels l i v i n g high in the i n t e r t i d a l zone adapt to their location, since they have a r e l a t i v e l y larger mantle volume than low i n t e r t i d a l mussels. In another study of mussels, Rao (1953b) found that the weight of soft parts varied as a function of i n t e r t i d a l height for both Mytilus edulis and Mytilus ca l i f o r n i a n u s . In the case of Mytilus edulis, he found that low i n t e r t i d a l mussels had greater s h e l l weights for the 118 same wet weight of soft parts when compared with high i n t e r t i d a l mussels, in contradiction to the results shown in Figure 19 of t h i s study. In Rao's report, he compared Mytilus edulis underneath f l o a t i n g wharves with mussels growing on p i l i n g s at 0.5-0.6 m and found that for 10 grams wet weight of soft parts, the subtidal mussels had she l l s weighing 26 grams, while the i n t e r t i d a l mussels had shells weighing 17.5 grams. From these data, he concluded that the weight of the s h e l l was dependent upon the immersion time. These results are d i f f i c u l t to explain, since they c o n f l i c t with his e a r l i e r (1953a) report, with the results of t h i s study, and with the results of Segal (1956b) for A. limatula. Time course uptake study The results of the time-course study indicate that the soft parts become saturated with " 5Ca within the f i r s t four hours of immersion in lab e l l e d seawater, and with some variations, maintain a constant l e v e l of isotope after that period. In a similar study by Wilbur & Jodrey (1952), the mantle of Crassostrea v i r g i n i c a required only 30 minutes to reach isotope saturation. The weight of the soft part tissue appears to be correlated with the length of time required for saturation. For example, in a mussel of 1.0 gram t o t a l dry weight, the g i l l , with a dry weight of 0.024 grams, was saturated after one hour, the mantle, weighing 0.104 grams,.was saturated after two hours, and the viscera, weighing 0.177 grams, became saturated after four hours. There is also a co r r e l a t i o n between the dry weight 119 of the tissue and calcium-pool s i z e . The calcium pools of the mantle, g i l l and viscera in a mussel of 1.0 grams t o t a l dry weight were determined by the mean le v e l of calcium in each tissue after reaching saturation with " 5Ca, and found to contain 62, 19 and 249 micrograms respectively. When adjusted for differences in weight, however, i t becomes apparent that the mantle and g i l l behave s i m i l a r l y in the amount of calcium they carry in their tissues. The g i l l c a r r i e s about 770 »q Ca/gm, and the mantle about 600 >/g Ca/gm. By comparison, the viscera carry the highest concentration of calcium, 1400 ><g Ca/gm. Since calcium i s always associated with muscular a c t i v i t y (Szent-Gyorgyi 1975), th i s difference may be attributed to the presence of muscle tissue in the viscera, compared with i t s absence in the g i l l and the small amounts found in the mantle. When the soft parts are considered in t o t a l , the average concentration of calcium i s 0.12%, which is in agreement with a chemical determination made by Fox and Coe (1943), who reported a concentration of 0.15% calcium in the soft parts of Mytilus  ca l i f o r n i a n u s . This supports the conclusion that the soft parts are saturated with " 5Ca at 4 hours, and that the unlabelled calcium can be determined from the isotope a c t i v i t y . . In comparison to the soft parts, the s h e l l , after the four hour e q u i l i b r a t i o n , continues to accumulate calcium for the remainder of the experiment, and shows a real uptake rate of about 32 >»g Ca/hr. In addition to the results discussed above, there are some noteworthy variations in tissue calcium l e v e l s . The g i l l reaches 120 a plateau in calcium l e v e l after one hour, and thereafter shows variations which are obscured by the large variances found throughout the course of the experiment. The reason for the high v a r i a b i l i t y in the g i l l calcium l e v e l is unknown, but has been noted by Chaisemartin et a l . (1969) in a study of the freshwater bivalve Marqaritifera (no species given) in a study of the marine bivalves Pteria martensi i and Hyriopsis  s c h l e g e l i i , Horiguchi (1958) has reported that the g i l l i s active in calcium uptake and storage, but that the calcium found in the g i l l i s extremely l a b i l e , and may be turned over in as l i t t l e as 10 minutes. This is in agreement with a report by Rao and Goldberg (1954), who, in a autoradiographic study, showed that the g i l l of Mytilus californianus i s the f i r s t tissue to take up calcium. They also found that the mucus which coated the g i l l s adsorbed calcium rapidly, and they concluded that most calcium uptake took place by adsorption onto the mucous sheet, followed by ingestion into the gut. A similar study by Fretter (1953), using 9 0 S r , reached the same conclusion. The s h e l l shows a s i g n i f i c a n t variation in calcium at the second hour. However, since the soft parts did not reach equilibrium u n t i l the fourth hour, changes in the the s p e c i f i c a c t i v i t y of " 5Ca in the s h e l l may have produced this a r t i f a c t during the e q u i l i b r a t i o n of the s p e c i f i c a c t i v i t y in the whole animal. A more serious question is raised by the combined va r i a t i o n of the s h e l l and viscera at 8 hours. This variation indicates a drop in the s h e l l calcium from 825 to 500 >>g and a decrease in v i s c e r a l calcium from 240 to 70 M g ; a combined loss of almost 500 »q calcium which 121 represents a 54% decrease in the t o t a l amount of calcium present at the fourth hour. It i s possible that t h i s loss of calcium i s due to a change in water pumping rhythms or to closing of the valves for a period of time. This is consistent with Rao's report (1954) that pumping rates of subtidal Mytilus  c a l i fornianus are synchronous with their i n t e r t i d a l counterparts, that water pumping rates are highest at high tide, and lowest at low tide, and that the synchrony of pumping is maintained for long periods when Mytilus c a l i fornianus is held in the laboratory. Thus, during the course of t h i s experiment, the mussels would have maintained t i d a l rhythms synchronous with those in .the f i e l d . The Canadian Hydrographic Survey t i d a l predictions (Anonymous 1981) for the day of the experiment (Hour 0 = 0830 27 January 1981) have provided the t i d a l information shown in Figure 31. These t i d a l records indicate that the tide was high from Hour 2 u n t i l Hour 4, but at i t s lowest point around Hour 8. If the valves were closed for a period around the time of low tide, then i t is possible that the mussels were subject to anaerobic conditions. Under these conditions bivalves have been shown to dissolve calcium from the s h e l l in order to buffer pH changes in the blood (Akberali et a l . 1977). For the time periods after.Hour 8, t i d a l e f f e c t s would not be detected because of the long intervals between samplings. In conclusion, the soft parts of Mytilus edulis become saturated with labelled calcium within four hours of the introduction of the l a b e l . The s h e l l continues to accumulate calcium, and is at a l l times the largest pool of calcium. The 1 22 dynamics of calcium transport between tissue compartments are not well understood, but may be affected by variations in a c t i v i t y that are synchronous with the tides. i 1 23 SUMMARY 1. The calcium-uptake rate of Mytilus edulis (Linnaeus) was examined under di f f e r e n t seasonal, s a l i n i t y , temperature and i n t e r t i d a l height conditions. The relationship between the t o t a l dry weight of soft parts and the weight of s h e l l was studied at two i n t e r t i d a l heights. 2. There were s i g n i f i c a n t differences between summer- and winter-adapted mussels subjected to three weeks immersed in seawater varying in s a l i n i t y from 25% to 125% SW (100% SW = 480 mEq C l ' / l i t e r ) . The calcium-uptake rate was d i r e c t l y correlated with s a l i n i t y in both seasons, but showed no increase in uptake rate in s a l i n i t i e s above 75% SW. Summer-adapted mussels had lower uptake rates at a l l s a l i n i t i e s when compared to winter-adapted mussels. For example, the calcium-uptake rates of summer and winter-adapted mussels of 1.0 gram t o t a l dry weight were 38 ?q Ca/hr and 59 t,q Ca/hr in 100% SW. 3. There were differences between summer- and winter-adapted mussels subjected to acute changes in temperature. Summer-adapted mussels were more temperature dependent than winter-adapted mussels, which took up calcium independently of temperature between 5° and 17°C. 4. There were s i g n i f i c a n t differences in the calcium-uptake rates of i n t e r t i d a l mussels from 0.2 and 2.2 m above datum. 124 These differences were shown to be interchangeable by reciprocal transplantation. Mussels from the high s i t e (2.2 m) had lower calcium-uptake rates than mussels from the low s i t e (0.2 m); 18 */g Ca/hr versus 49 */g Ca/hr. Differences in the immersion times at the two s i t e s were not s u f f i c i e n t to account for a l l of th i s difference. If the uptake rates of mussels from the high and low i n t e r t i d a l s i t e s were based upon the t o t a l dry weight of soft parts, instead of the t o t a l dry weight, then i t was found that the difference in uptake rate between the two s i t e s disappeared. It was shown that there was an i n t e r t i d a l size-gradient in the shel l s of mussels. This gradient may be related to the reduction in the calcium-uptake rate among mussels higher in the i n t e r t i d a l zone. 5. Mussels from 0.2 and 2.2 m were found to have similar dry weights of mantle, g i l l and viscera compared to the t o t a l dry weight of soft parts, but mussels at 2.2 m had less t o t a l dry weight of soft parts in proportion to the t o t a l dry weight, compared to mussels at 0.2 m. 6. A time-course uptake study showed that the soft parts became saturated with * 5Ca within four hours, but that s i g n i f i c a n t fluctuations took place between sampling times. It was shown that these fluctutions might be explained by t i d a l opening or pumping rhythms of the mussels. After the e q u i l i b r a t i o n period, the soft parts did not accumulate any calcium, while the s h e l l showed a net accumulation of calcium 125 throughout the experiment. 7. A correction factor was established, based on the net uptake of the s h e l l a f t e r the e q u i l i b r a t i o n of the s h e l l and soft parts. It was found that absolute uptake rates were about 59% of the uptake rates measured here. 126 LITERATURE CITED Akberali, H.B., Marriott, K.R.M. and E.R. Trueman. 1977. Calcium u t i l i z a t i o n during anaerobiosis induced by osmotic shock in a bivalve mollusc. Nature, Lond. 266:852-853. Anonymous. Canadian tide and current tables. 1979, 1980, 1981. Printing and Publishing, Supply and Services. Ottawa, Canada. Barker, J.L. and H. Gainer. 1973. The role of calcium in seasonal modulation of pacemaker a c t i v i t y in a molluscan neurosecretory c e l l . Nature, Lond. 245:462-464. Barnes, H. 1954. Some tables for the ionic composition of seawater. J. exp. B i o l . 31:582-588 . Bertness, M.D. 1977. Behavioral and ecological aspects of shore le v e l size gradients in Thais lamellosa and Thais  emarginata. Ecology 58:86-97. 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A mantle-shell preparation for in v i t r o studies. B i o l . B u l l . (Woods Hole, Mass.) 104:394-397. Hodgkin, A.L. and R.D. Keynes. 1957. The movement of calcium in squid giant axon. J. Physiol. 138:253-281. Horiguchi, Y. 1958. Biochemical studies on Pteria (Pinctada) martensi i (Dunker) and Hyriopsis schlegeli i (V. martens). IV. Absorption and transference of 4 5Ca in Hyriopsis  s c h l e g e l i i (V. martens). B u l l . Jap. Soc. Scient. Fish. 23:710-715. Horiguchi, Y., Miyake, M., Yoshii, G., Okada, Y., Inoue, Y. and M. Miyamura. 1954. Biochemical studies with radioactive isotopes on Pteria (Pinctada) martensi i (Dunker) and Hyriopsis s c h l e g e l i i (V. martens). I. Ca metabolism by "^Ca tracer in Hyriopsis schlegeli i (V. martens). B u l l . Jap. Soc. Scient. F i s h . 20:101-106. Hoyaux, J., G i l l e s , R., and C. Jeuniaux. 1976. Osmoregulation in mollusks of the i n t e r t i d a l zone. Comp. Biochem. Physiol. 53A:361-365. Jodrey, L.H. 1953. Studies on s h e l l formation. I I I . Measurement of calcium deposition in s h e l l and calcium turnover in mantle tissue using the mantle-shell preparation and u 5Ca. B i o l . B u l l . (Woods Hole, Mass.) 104:398-407. Johnston, D. 1976. Voltage clamp reveals basis for calcium regulation of bursting pacemaker potentials in Aplysia neurons. Brain Res. 107:418-423. 129 Kapur, S.P. and M.A. Gibson. 1968. A histochemical study of the development of the mantle-edge and s h e l l in the fresh water gastropod, Helisoma duryi eudiscus ( P i l s b r y ) . Can. J. Zool. 46:481-491. Kirschner, L.B. 1963. Tra n s e p i t h e l i a l e l e c t r i c a l phenomena in the molluscan mantle. J. Gen. Physiol. 46:362A-363A. Kirschner, L.B., Sorensen, A.L. and M. Kriebel. 1960. Calcium and e l e c t r i c potential across the clam mantle. Science, N.Y. 131:735. L i t t l e , C. 1965. Osmotic and ionic regulation in the prosobranch gastropod mollusc, Viviparus viviparus Linn. J. exp. B i o l . 43:23-37. Loosanoff, V.L. and C.A. Nomejko. 1949. Growth of oysters, 0. v i r g i n i c a , during d i f f e r e n t months. B i o l . B u l l . (Woods Hole, Mass.) 97:82-94. Lowenstam, H.A. 1981. Minerals formed by organisms. Science, N.Y. 211:1126-1131. McLachlan, A. and T. Erasmus. 1974. Temperature tolerances and osmoregulation in some estuarine bivalves. Zool. Afr. 9:1-13. McWhinnie, M.A. 1962. G a s t r o l i t h growth and calcium s h i f t s in the freshwater c r a y f i s h Oreonectes v i r i l i s . Comp. Biochem. Physiol. 7:1-14. McWhinnie, M.A., Cahoon, M.O. and R. Johanneck. 1969. Hormonal eff e c t s on calcium metabolism in Crustacea. Am. Zool. 9:841-855. Marbach, A. and K.M. Wilbur. 1973. Influence of environmental factors on deposition of calcium carbonate in mollusks. Isr. J. Zool. 22:200. Newcombe, C.L. 1935. A study of the community relationships of the sea mussel, Mytilus edulis L. Ecology 16:234-243. 130 Numanoi, H. 1939. Di s t r i b u t i o n of calcium in the soft parts of the freshwater bivalve C r i s t a r i a p i i c a t a . Jap. J. Zool. 8:353-356. Orton, J.H. 1928. On the rhythmic periods of s h e l l growth of 0. edulis with a note on fattening. J. Mar. B i o l . Assoc. U. K. Jj5:365-427. Paine, R.T. 1976. Size limited predation: an observational and experimental approach with the Mytilus-Pisaster interaction. Ecology 57:858-873. Pierce, S.K. 1970. The water balance of Modiolus (Mollusca: B i v a l v i a : Mytilidae); osmotic concentrations in changing s a l i n i t i e s . Comp. Biochem. Physiol. 36:521-533. Pierce, S.K. and M.J. Greenberg. 1971. Ionic basis of c e l l u l a r volume regulation in molluscs. Am. Zool. 11:663• Potts, W.T.W. 1954. The inorganic composition of the blood of Mytilus edulis and Anodonta cygnea. J. exp. B i o l . 3_l:376-385. Quayle, D.B. 1969. P a c i f i c oyster culture in B r i t i s h Columbia. Fish . Res. Bd. Canada. B u l l . 169, 192 pp. Rao, K.P. 1953a. Rate of water propulsion in Mytilus californianus as a function of l a t i t u d e . B i o l . B u l l . (Woods Hole, Mass.) 104: 171-181. Rao, K.P. 1953b. Shell weight as a function of i n t e r t i d a l height in a l i t t o r a l population of pelecypods. Experientia 9: 465-466. Rao, K.P. 1954. Tidal rhythmicity of rate of water propulsion in Mytilus, and i t s modifiabi1ity by transplantation. B i o l . B u l l . (Woods Hole, Mass.) j_06: 353-359. Rao, K.P. and E.D. Goldberg. 1954. U t i l i z a t i o n of dissolved calcium by a pelecypod. J. C e l l . Comp. Physiol. 43: 283-292. 131 Richards, O.W. '1935. The growth of the mussel Mytilus edulis at Woods Hole Massachusetts. Anat. Rec. 64 (Supplement): 68. Robertson, J.D. 1937. Some features of the calcium metabolism of the shore crab (Careinus maenas Pennant). Proc. R. Soc. Lond. B. 124:162-182. Schoffeniels, E. 1951 a. 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Handbook of Chemistry and Physics. 55th e d i t i o n . CRC Press, Cleveland, Ohio. Page B-193. Widdows, J. 1973. Effect of temperature and food on the heart beat, v e n t i l a t i o n rate and oxygen uptake of Mytilus edu l i s . Mar. B i o l . 20:269-276. 1 Wilbur, K.M. and L.H. Jodrey. 1952. Studies on s h e l l formation I. measurement of the rate of s h e l l formation using u sCa B i o l . B u l l . (Woods Hole, Mass.) 103:269-276. Zeuthen, E. 1947. Body size and metabolic rate in the animal kingdom with special regard to the marine micro-fauna. C. R. trav. lab. Carlsberg, Ser. chim. 26:17-161. Zischke, J.A., Watabe, N. and K.M. Wilbur. 1970. Studies on s h e l l formation: measurement of growth in the gastropod Ampullarius glaucus. Malacologia 10:423-439. 

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