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The thermal resistance and thermal resistance acclimation of ciliary activity in the Mytilus gills
Camp. Biochem. Physiol., 1975. Vol. 51A. pp. 957 to 961. Pergamon Press. Printed in Great Britain
THE THERMAL RESISTANCE AND THERMAL RESISTANCE ACCLIMATION OF CILIARY ACTIVITY IN THE MYTILUS GILLS K. E. 0. S~NWS* Zoophysiological Laboratory, Department of Zoology, Christian Albrecht University, D-2300 Kiel, Germany, and Zoophysiological Laboratory, Department of Zoology, University of Turlcu, SF-2OSOO Turlcu 50, Finland (Received 4 April 1974) Ah&act-l. The thermal resistance and thermal resistance acclimation of ciliary activity in the gills of Mytibs edulis was studied. 2. The thermal resistance acclimation appeared in 4 days when intact animals were acclimated to __ 5, 15 and 25°C. 3. There was no thermal resistance acclimation in isolated surviving gills kept for 1 day at 4 or 24°C. 4. ACh and S-HT had no effect on the thermal resistance of ciliarv activity in isolated aills. 5. It is suggested that the thermal resistanceof ciliary activity in the Mytili gills is con&led rather
by neural mechanism than on the tissue level.
INTRODUCTION THEREhave been many investigations of the ciliary activity and its physiological regulation in Mytilus gills (Biilbring et al., 1953; Aiello, 1960, 1962; Gosselin, 1961; Gosselin et al., 1962; Aiello & Guideri, 1964; Paparo & Aiello, 1970). However, studies on the thermal resistance and thermal resistance acclimation of ciliary activity comparable with the thermal resistance investigations of the fresh water mussel, Anodontu cygneu celiensis (Lagerspetz & Dubitscher, 1966; Lagerspetz et al., 1970; Senius & Lagerspetz, 1974), have not been performed. In the present study, the thermal resistance and thermal resistance acclimation of ciliary activity in the Mytilus gill plates is investigated. Further, the effect of acetylcholine (ACh) and 5-hydroxytryptamine (5HT) on the thermal resistance of ciliary activity in the isolated gills is reported. MATERlALS AND METHODS The mussels (Mytilus edufis) were collected from the bay of Kiel (“Kieler F&de”, Baltic Sea) at the Falckenstein beach. The mean salinity of sea water was 18 parts per mill. Individuals with a shell length of 4-7 cm were stored in sea water at 5, 15 and 25°C in polyethylene containers for 3 weeks in order to determine the thermal resistance acclimation of ciliary activity in the gills of intact animals. Aeration and filtration of sea water were *Present address: Institute of Biomedical Sciences, University of Tampere, SF-33520 Tamperc 52, Finland.
provided but no food was given. The original salinity of sea water in the containers was adjusted every day. The method used to measure the thermal resistance of ciliary activity was essentially that described by Vernberg et al. (1963). The median gills were excised in the sea water, cut into small strips and transferred to 5Oml of sea water in containers kept in an accurate constanttemperature bath. In the experiments for determination of thermal resistance of ciliary activity in the isolated gills acclimated to different temperatures, the excised gills were previously kept for 1 day at 24”C, respectively at 4°C in Petri dishes in sea water before cutting into strips and transferring to a constant-temperature bath. In these experiments, the median gill from one side of the animal was kept at one temperature, the other median gill at the other. The sides were changed in successive experiments when the gills were acclimated either to 4 or 24°C. The temperature of the bath varied in different experiments from 35 to 39°C. At intervals of 10 min a piece of gill was removed from the bath container for the measurement of ciliary activity by the particle transport method (Lagerspctz & Dubitscher, 1966). The time in minutes from the onset of the experiment at the constant temperature to the middle of the interval, during which the movement of the frontal cilia became unobservable, was used as the measure of thermal resistance. In some experiments, with the isolated gills acclimated previously to different temperatures, different concentrations of ACh chloride or 5-HT creatinine sulphate were added into the containers kept in the constanttemperature bath. A statistical comparison of the results is calculated using the t-test and, in some cases, the analysis of variance. The vertical bars in the figures indicate standard errors. 957
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Thermal resistance of the gills of intact animais kept at different temperatures
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It has been shown previously that an acclimation period of 4 days is sufficient to produce thermal resistance acclimation of the ciliary activity in the gills of intact Anudonta mussels kept at 5 or 21°C (Lagerspetz & Dubitscher, 1966). In this study, intact MytiZus mussels collected from the 13°C sea water were acclimated to 5, 15 and 25°C for 1-16 days. After the acclimation period the thermal resistance of ciliary activity was measured at the incubation temperatures of 35 and 36°C.
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Fig. 2. The effect of temperature acclimation of intact animals on the thermal resistance of ciliary activity at 36°C. Abscissa, the acclimation period in days; ordinate, the resistance time in minutes; a, the thermal resistance of ciliary activity just after the collection of animals from the 13°C sea water; @, animals acclimated to 5°C; x, animals acclimated to 15°C (control); o, animals acclimated to 25°C. The number of experiments at each point was six.
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Fig. 1. The effect of temperature acclimation of intact animals on the thermal resistance of ciliary activity at 35°C. Abscissa, the acclimation period in days; ordinate, the resistance time in minutes; q, the thermal resistance of ciliary activity just after the collection of animals from the 13°C sea water; 0. animals acclimated to 5°C; x , animals acciimated to 15°C (control); o, animals acclimated to 25°C. The number of experiments at each point was five to six.
At 36°C jncubation tem~ratu~ (Fig. 2), no thermal resistance alteration appeared in the gills of animals kept for 2-16 days at 5°C when compared with controis kept at 15°C. On the first day only the thermal resistance was significantly (P
At 35°C incubation temperature (Fig. l), the thermal resistance acclimation of animals kept at 25°C appeared in 2 days (P
In order to confirm that the alteration of thermal resistance of ciliary activity in the gills of intact animals kept at different temperatures is an actual acclimation effect, and not produced for instance by starvation, the mussels kept for 9 days at 25°C or for 10 days at 5°C were reaccIimated to 15°C for several days. When measured at 35°C the difference of thermal resistance became, during a reacclimation period of 3 days, statistically non-si~i~cant between the group transferred from 25 to 15°C and that transferred from 5 to 15°C. In 8 days the acclimation effect between these groups almost completely disappeared (Fig. 3).
Thermal resistance in the Mytilus gill cilia 250
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Similar results were obtained at the incubation temperature of 36°C (Fig. 4). After the reacclimation of 8 days at 15°C the significant difference of thermal resistance between the groups kept previously at 5 or 25°C also disappeared. Thermal resistance of isolated gills kept at different temperatures
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Fig. 3. The reversion of thermal resistance of ciliary activity at 35°C after the 15°C reacclimation of intact animals acclimated previously for 10 days to 5°C or 9 days to 25°C. Abscissa, the reacclimation period in days; ordinate, the resistance time in minutes; l, animals acclimated previously to 5°C; x , control animals kept continuously at 15°C; 0, animals acclimated previously to 25°C. The number of experiments at each point was six.
As reported earlier (Lagerspetz & Dubitscher, 1966), the thermal resistance acclimation of ciliary activity appears in the gills of the fresh water mussel Anodonta when isolated gills are kept for 1 day at different temperatures. In order to study this kind of acclimation in the Mytilus gills, the thermal resistance of ciliary activity was measured at 3539°C using isolated gills kept for 1 day at 4 or 24°C. The gills were cut from mussels stored for 5-12 days at 5°C. The thermal resistance of the gills of intact animals collected from the sea (water temperature 13°C) just before the determination was also measured. The results are shown in Fig. 5. The thermal resistance times of cold- and warmacclimated isolated gills did not differ significantly
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Fig. 4. The reversion of thermal resistance of ciliary activity at 36°C after the 15°C reacclimation of intact animals acclimated previously for 10 days to 5°C or 9 days to 25°C. Abscissa, the reacclimation period in days; ordinate, the resistance time in minutes; l, animals acclimated previously to 5°C; x, control animals kept continuously at 15’C; o, animals acclimated previously to 25°C. The number of experiments at each point was six.
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Fig. 5. The effect of incubation temperature on the thermal resistance of ciliary activity. Abscissa, the incubation temperature in “C; ordinate, the resistance tune in minutes; x , gills excised from animals collected from the 13°C sea water just before the determination; l, isolated gills acclimated for 1 day at 4°C; 0, gills acclimated to 24”C, respectively. The number of experiments at each point was five to nine except at 35°C (0) and (0) where it was three.
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one from the other at any of the incubation temperatures tested. However, at 35°C the resistance time of the cold-acclimated as well as of the warmacclimated isolated gills was almost twice as long (PC 0.001) as that of the gills of intact animals. Also at 36°C the heat resistance of the warm-acclimated isolated gills was significantly higher (PC 0.02) than that of the gills of intact animals. At higher incubation temperatures there was no significant difference between the two groups of isolated gills and the gills of intact animals.
The efect
of ACh and S-HT on thermal resistance
It has been shown that the cholinergic system has an important role in the thermal resistance acclimation of ciliary activity in the Anodonta gills (Lagerspetz et al., 1970; Senius & Lagerspetz, 1974). A small effect of 5-HT on the thermal resistance of Anodonta gills has also been reported (Lagerspetz et al., 1970). In addition, S-HT is a cilioexcitatory substance in the MytiZus gills (Aiello, 1960, 1962; Gosselin, 1961; Gosselin et al., 1962). Hence, the effect of ACh and 5-HT added into the incubation medium on the thermal resistance of ciliary activity in the isolated Mytilas gills was measured. In these experiments, the gills of animals kept for 4-21 days at 5°C were used. The concentrations lo+, 10e5, 10e4 and 1O-3 g/ml of ACh chloride added into the 36 or 37°C incubation medium had no significant effect on the thermal resistance of ciliary activity. Likewise, 5-HT added into the incubation at 36 or 37°C did not alter the thermal resistance when concentrations of 10e8, 1O-5, lo-* and 1O-3 g/ml of 5-HT creatinine sulphate were used. DISCUSSION
The present results show that the acclimation of intact mussels to different temperatures causes in principle a similar acclimation effect in the thermal resistance of ciliary activity in the gills of Mytilus as in those of the fresh water mussel Anodonta studied earlier by Lagerspetz & Dubitscher (1966). The complete reversal of thermal resistance to the control level in the warm-acclimated group (25°C) as well as in the cold-acclimated group (5”C), when these mussels were put back to the original control temperature (15”C), shows that the effect is not due to starvation or irreversible tissue injury. In these experiments, the thermal resistance is measured at 35 and 36°C. The latter temperature has been used earlier in the thermal resistance determinations of ciliary activity of Mytilus gills (Friedrich, 1967; Theede & Lassig, 1967). However, present results show that the thermal resistance acclimation is not more at 36°C as is evident at 35°C. It is possible that the regulation mechanism of ciliary activity or
some other key factor is not functioning more adequately at 36°C or at higher temperatures. The thermal resistance times of ciliary activity obtained in this study at 36°C after the acclimation of intact animals to 5 or 15°C are compatible with the earlier observations of heat resistance of terminal cilia at 36°C in the gills of Mytilus mussels collected from the bay of Kiel and kept for 3-21 days at 10°C before the determination (Theede & Lassig, 1967, Fig. 2). The heat resistance of Mytilus, which has been reported to be dependent on the salinity of sea water (Theede & Lassig, 1967), is much smaller than that of the fresh water mussel Anodonta. In Myti/us, movement of the frontal cilia of isolated gills kept for 1 day at different temperatures as well as of the gills cut just before the determination from intact animals collected from 13°C sea water, stopped at the moment the gill strips were transferred to the 39°C incubation temperature. In the experiments of Theede (1972), the activity of terminal gill cilia in the Mytilus mussels collected from the North Sea (mean salinity30 parts per mill) became unobservable at 40°C when the incubation temperature was raised 0.2”C/min. In Anodonta, activity of the frontal cilia stopped immediately at 4344°C in the isolated gills acclimated for 1 day to 4 or 24°C (Senius & Lagerspetz, 1974). In the case of intact Anodonta mussels acclimated for several weeks either to 5 or 21°C the activity of the frontal cilia of excised gills disappeared at 4749°C when the incubation temperature was raised approximately O.S”C/min (Lagerspetz & Dubitscher, 1966). Contrary to earlier results obtained through studies of isolated Anodonta gills (Lagerspetz & Dubitscher, 1966; Lagerspetz et a/., 1970; Senius & Lagerspetz, 1974), there was no thermal resistance acclimation of ciliary activity in isolated Mytilus gills acclimated to different temperatures. However, when the isolated gills were kept for 1 day at 4 or 24°C the thermal resistance of ciliary activity was significantly increased in both groups at the 35°C incubation temperature when compared to the resistance time of the gills excised from mussels collected from 13°C sea water. As seen in the Results, the concentrations of ACh and 5-HT added into the incubation at 36 or 37°C had no effect on the thermal resistance of ciliary activity in the isolated gills. On the other hand, it has been shown in many experiments that ACh (Bulbring et a/., 1953) as well as .5-HT (Aiello, 1960, 1962; Gosselin, 1961; Gosselin et a/., 1962) has definite effects on the velocity of ciliary movement in the Mytilus gills. Enzymes involved in the cholinergic or serotonergic system have also been found in the Myfi/us gill tissue (Bulbring et al., 1953 ; Milton, 1959; Milton & Gosselin, 1960; Blaschko & Milton, 1960; Gosselin et al., 1962). Gosselin et al. (1962) have suggested that the ciliary activity is controlled by 5-HT functioning as a local
Thermal resistance. in the Mytilus gill cilia
hormone. However, it was shown later that the branchial nerve also takes part in that regulation (Aiello & Guideri, 1964; Paparo & Aiello, 1970). Also, the thermal resistance of ciliary activity in Mytilus seems to be controlled rather by neural mechanism than on the tissue level. Two facts mentioned before point out the possible neural control: (1) the thermal acclimation of ciliary activity could be induced by temperature change in whole animals but not in isolated gills, and (2) the storage of excised gills for 1 day caused a significant increase in thermal resistance of ciliary activity as compared to the gills excised from the mussels just before the thermal resistance determination. Acknowledgements-I wish to express my gratitude to Professors Herbert Precht. Ph.D.. and Harms-Dieter Jankowsky, Ph.D., of the Department of Zoology, Christian Albrecht University (Kiel), for providing pleasant and stimulating working conditions at their laboratory. Mr. Hans-Werner Huppert, candrernat., Mr. Falk Krebs, cand.rer.nat., Mr. Ekkehart Wodtke, Dr.rer.nat., as well as all other colleagues in Kiel are acknowledged for many fruitful discussions and practical aid. My grateful thanks are also due to my teacher, Professor Kari Y. H. Lagerspetz, Ph.D., of the Department of Zoology, University of Turku, for critically reading the manuscript. I am also grateful to Mrs. Sinikka Hillgren for drawing the figures. This study was supported financially by a grant from the “Deutscher Akademischer Austauschdienst”, BonnBad Godesberg, Germany. REFERENCES AIELLOE. L. (1960) Factors affecting ciliary activity on the gill of the mussel Mytilus edulis. Physiol. Zoiil. 33, 120-135. AIELL~ E. L. (1962) Identification of the cilioexcitatory substance present in the gill of the mussel Mytilus edulis. J. cell, camp. Physiol. 68, 17-21. AIELLO E. L. & GUIDES G. (1964) Nervous control of ciliary activity. Science, Wash., 146,1692-1693. BLA~CHKOH. & MILTON A. S. (1960) Oxidation of 5hydroxytryptamine and related compounds by Mytilus gill plates. Br. J. Pharmac. 15,4246.
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BULBRING E., BURN J. H. & SHELLEYH. J. (1953) Acetylcholine and ciliary movement in the gill plates of Mytilus edulis. Proc. R. Sot. B 141,445-466. FRIEDRICH L. (1967) Experimentelle Untersuchungen zum ProbIem zelluhtrer nichtgenetischer Resistenzlnderungen bei der Miesmuschel Mytilus edulis L. Kieler Meeresforsch. 23, 105-126. G~~.~.ELINR. E. (1961) The cilioexcitatory activity of serotonin. J. cell. camp. Physiol. 58, 17-25. GOSSELINR. E., Mooar K. E. & MILTON A. S. (1962) Physiological control of molluscan gill cilia by 5hydroxytryptamine. J. gen. Physioi. 46, 277-296. LAGERSPETZ K. Y. H. & DUB~WXER I. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. LAGERSPETZ K. Y. H., IMPIVAARAH. & SENXJSK. (1970) Acetylcholine in the thermal resistance acclimation of the ciliary activity in the gills of Anodonta. Comp. gen. Pharmac. 1, 236-240. _ MILTONA. S. (1959) Choline acetvlase in the ail1 elates of Mytilus edulis. Proc. R. Sac. R 150,240-2&L _ MILTONA. S. & GOWLIN R. E. (1960) Metabolism and cilia-accelerator action of 5-hydroxytryptophan (5HTP) in gill plates of MytiIus and Modioius. Fedn Proc. Fedn Am. Sots exp. Biol. 19, 126. PAPAROA. & AIELLOE. (1970) Cilio-inhibitory effects of branchial nerve stimulation in the mussel, Mytilus edulis. Comp. gen. Pharmac. 1.241-250. SENDS K. E. 0. & LAGERSPETZ K. Y. H. (1974) The role of cholinergic receptors in the thermal resistance. and therma resistance acclimation of the ciliary activity in the Anodonta gills. Comp. gen. Pharmac. 5,169-179. THEEDEH. (1972) Zur zellul&ren Hitzeresistenz mariner Evertebraten. Oecologia, Berl. 11, 17-31, THEEDEH. & LA~%GJ. (1967) Comparative studies on cellular resistance of bivalves from marine and brackish waters. Helgokinder wiss. Meeresunters. 16, 119-129. VERNBERGF. J., SCHLIEPERC. & SCHNEIDER D. E. (1963) The influence of temperature. and salinity on ciliary activity of excised gill tissue of molluscs from North Carolina. Comp. Biochem. Physiol. 8, 271-285. Key Word Index-Temperature acclimation; thermal resistance; ciliary activity; gill tissue; acetylcholine; 5-hydroxytryptamine; Mytilus edulis.
THE THERMAL RESISTANCE AND THERMAL RESISTANCE ACCLIMATION OF CILIARY ACTIVITY IN THE MYTILUS GILLS K. E. 0. S~NWS* Zoophysiological Laboratory, Department of Zoology, Christian Albrecht University, D-2300 Kiel, Germany, and Zoophysiological Laboratory, Department of Zoology, University of Turlcu, SF-2OSOO Turlcu 50, Finland (Received 4 April 1974) Ah&act-l. The thermal resistance and thermal resistance acclimation of ciliary activity in the gills of Mytibs edulis was studied. 2. The thermal resistance acclimation appeared in 4 days when intact animals were acclimated to __ 5, 15 and 25°C. 3. There was no thermal resistance acclimation in isolated surviving gills kept for 1 day at 4 or 24°C. 4. ACh and S-HT had no effect on the thermal resistance of ciliarv activity in isolated aills. 5. It is suggested that the thermal resistanceof ciliary activity in the Mytili gills is con&led rather
by neural mechanism than on the tissue level.
INTRODUCTION THEREhave been many investigations of the ciliary activity and its physiological regulation in Mytilus gills (Biilbring et al., 1953; Aiello, 1960, 1962; Gosselin, 1961; Gosselin et al., 1962; Aiello & Guideri, 1964; Paparo & Aiello, 1970). However, studies on the thermal resistance and thermal resistance acclimation of ciliary activity comparable with the thermal resistance investigations of the fresh water mussel, Anodontu cygneu celiensis (Lagerspetz & Dubitscher, 1966; Lagerspetz et al., 1970; Senius & Lagerspetz, 1974), have not been performed. In the present study, the thermal resistance and thermal resistance acclimation of ciliary activity in the Mytilus gill plates is investigated. Further, the effect of acetylcholine (ACh) and 5-hydroxytryptamine (5HT) on the thermal resistance of ciliary activity in the isolated gills is reported. MATERlALS AND METHODS The mussels (Mytilus edufis) were collected from the bay of Kiel (“Kieler F&de”, Baltic Sea) at the Falckenstein beach. The mean salinity of sea water was 18 parts per mill. Individuals with a shell length of 4-7 cm were stored in sea water at 5, 15 and 25°C in polyethylene containers for 3 weeks in order to determine the thermal resistance acclimation of ciliary activity in the gills of intact animals. Aeration and filtration of sea water were *Present address: Institute of Biomedical Sciences, University of Tampere, SF-33520 Tamperc 52, Finland.
provided but no food was given. The original salinity of sea water in the containers was adjusted every day. The method used to measure the thermal resistance of ciliary activity was essentially that described by Vernberg et al. (1963). The median gills were excised in the sea water, cut into small strips and transferred to 5Oml of sea water in containers kept in an accurate constanttemperature bath. In the experiments for determination of thermal resistance of ciliary activity in the isolated gills acclimated to different temperatures, the excised gills were previously kept for 1 day at 24”C, respectively at 4°C in Petri dishes in sea water before cutting into strips and transferring to a constant-temperature bath. In these experiments, the median gill from one side of the animal was kept at one temperature, the other median gill at the other. The sides were changed in successive experiments when the gills were acclimated either to 4 or 24°C. The temperature of the bath varied in different experiments from 35 to 39°C. At intervals of 10 min a piece of gill was removed from the bath container for the measurement of ciliary activity by the particle transport method (Lagerspctz & Dubitscher, 1966). The time in minutes from the onset of the experiment at the constant temperature to the middle of the interval, during which the movement of the frontal cilia became unobservable, was used as the measure of thermal resistance. In some experiments, with the isolated gills acclimated previously to different temperatures, different concentrations of ACh chloride or 5-HT creatinine sulphate were added into the containers kept in the constanttemperature bath. A statistical comparison of the results is calculated using the t-test and, in some cases, the analysis of variance. The vertical bars in the figures indicate standard errors. 957
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SENIUS
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Thermal resistance of the gills of intact animais kept at different temperatures
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It has been shown previously that an acclimation period of 4 days is sufficient to produce thermal resistance acclimation of the ciliary activity in the gills of intact Anudonta mussels kept at 5 or 21°C (Lagerspetz & Dubitscher, 1966). In this study, intact MytiZus mussels collected from the 13°C sea water were acclimated to 5, 15 and 25°C for 1-16 days. After the acclimation period the thermal resistance of ciliary activity was measured at the incubation temperatures of 35 and 36°C.
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Fig. 2. The effect of temperature acclimation of intact animals on the thermal resistance of ciliary activity at 36°C. Abscissa, the acclimation period in days; ordinate, the resistance time in minutes; a, the thermal resistance of ciliary activity just after the collection of animals from the 13°C sea water; @, animals acclimated to 5°C; x, animals acclimated to 15°C (control); o, animals acclimated to 25°C. The number of experiments at each point was six.
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Fig. 1. The effect of temperature acclimation of intact animals on the thermal resistance of ciliary activity at 35°C. Abscissa, the acclimation period in days; ordinate, the resistance time in minutes; q, the thermal resistance of ciliary activity just after the collection of animals from the 13°C sea water; 0. animals acclimated to 5°C; x , animals acciimated to 15°C (control); o, animals acclimated to 25°C. The number of experiments at each point was five to six.
At 36°C jncubation tem~ratu~ (Fig. 2), no thermal resistance alteration appeared in the gills of animals kept for 2-16 days at 5°C when compared with controis kept at 15°C. On the first day only the thermal resistance was significantly (P
At 35°C incubation temperature (Fig. l), the thermal resistance acclimation of animals kept at 25°C appeared in 2 days (P
In order to confirm that the alteration of thermal resistance of ciliary activity in the gills of intact animals kept at different temperatures is an actual acclimation effect, and not produced for instance by starvation, the mussels kept for 9 days at 25°C or for 10 days at 5°C were reaccIimated to 15°C for several days. When measured at 35°C the difference of thermal resistance became, during a reacclimation period of 3 days, statistically non-si~i~cant between the group transferred from 25 to 15°C and that transferred from 5 to 15°C. In 8 days the acclimation effect between these groups almost completely disappeared (Fig. 3).
Thermal resistance in the Mytilus gill cilia 250
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Similar results were obtained at the incubation temperature of 36°C (Fig. 4). After the reacclimation of 8 days at 15°C the significant difference of thermal resistance between the groups kept previously at 5 or 25°C also disappeared. Thermal resistance of isolated gills kept at different temperatures
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Fig. 3. The reversion of thermal resistance of ciliary activity at 35°C after the 15°C reacclimation of intact animals acclimated previously for 10 days to 5°C or 9 days to 25°C. Abscissa, the reacclimation period in days; ordinate, the resistance time in minutes; l, animals acclimated previously to 5°C; x , control animals kept continuously at 15°C; 0, animals acclimated previously to 25°C. The number of experiments at each point was six.
As reported earlier (Lagerspetz & Dubitscher, 1966), the thermal resistance acclimation of ciliary activity appears in the gills of the fresh water mussel Anodonta when isolated gills are kept for 1 day at different temperatures. In order to study this kind of acclimation in the Mytilus gills, the thermal resistance of ciliary activity was measured at 3539°C using isolated gills kept for 1 day at 4 or 24°C. The gills were cut from mussels stored for 5-12 days at 5°C. The thermal resistance of the gills of intact animals collected from the sea (water temperature 13°C) just before the determination was also measured. The results are shown in Fig. 5. The thermal resistance times of cold- and warmacclimated isolated gills did not differ significantly
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Fig. 4. The reversion of thermal resistance of ciliary activity at 36°C after the 15°C reacclimation of intact animals acclimated previously for 10 days to 5°C or 9 days to 25°C. Abscissa, the reacclimation period in days; ordinate, the resistance time in minutes; l, animals acclimated previously to 5°C; x, control animals kept continuously at 15’C; o, animals acclimated previously to 25°C. The number of experiments at each point was six.
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Fig. 5. The effect of incubation temperature on the thermal resistance of ciliary activity. Abscissa, the incubation temperature in “C; ordinate, the resistance tune in minutes; x , gills excised from animals collected from the 13°C sea water just before the determination; l, isolated gills acclimated for 1 day at 4°C; 0, gills acclimated to 24”C, respectively. The number of experiments at each point was five to nine except at 35°C (0) and (0) where it was three.
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one from the other at any of the incubation temperatures tested. However, at 35°C the resistance time of the cold-acclimated as well as of the warmacclimated isolated gills was almost twice as long (PC 0.001) as that of the gills of intact animals. Also at 36°C the heat resistance of the warm-acclimated isolated gills was significantly higher (PC 0.02) than that of the gills of intact animals. At higher incubation temperatures there was no significant difference between the two groups of isolated gills and the gills of intact animals.
The efect
of ACh and S-HT on thermal resistance
It has been shown that the cholinergic system has an important role in the thermal resistance acclimation of ciliary activity in the Anodonta gills (Lagerspetz et al., 1970; Senius & Lagerspetz, 1974). A small effect of 5-HT on the thermal resistance of Anodonta gills has also been reported (Lagerspetz et al., 1970). In addition, S-HT is a cilioexcitatory substance in the MytiZus gills (Aiello, 1960, 1962; Gosselin, 1961; Gosselin et al., 1962). Hence, the effect of ACh and 5-HT added into the incubation medium on the thermal resistance of ciliary activity in the isolated Mytilas gills was measured. In these experiments, the gills of animals kept for 4-21 days at 5°C were used. The concentrations lo+, 10e5, 10e4 and 1O-3 g/ml of ACh chloride added into the 36 or 37°C incubation medium had no significant effect on the thermal resistance of ciliary activity. Likewise, 5-HT added into the incubation at 36 or 37°C did not alter the thermal resistance when concentrations of 10e8, 1O-5, lo-* and 1O-3 g/ml of 5-HT creatinine sulphate were used. DISCUSSION
The present results show that the acclimation of intact mussels to different temperatures causes in principle a similar acclimation effect in the thermal resistance of ciliary activity in the gills of Mytilus as in those of the fresh water mussel Anodonta studied earlier by Lagerspetz & Dubitscher (1966). The complete reversal of thermal resistance to the control level in the warm-acclimated group (25°C) as well as in the cold-acclimated group (5”C), when these mussels were put back to the original control temperature (15”C), shows that the effect is not due to starvation or irreversible tissue injury. In these experiments, the thermal resistance is measured at 35 and 36°C. The latter temperature has been used earlier in the thermal resistance determinations of ciliary activity of Mytilus gills (Friedrich, 1967; Theede & Lassig, 1967). However, present results show that the thermal resistance acclimation is not more at 36°C as is evident at 35°C. It is possible that the regulation mechanism of ciliary activity or
some other key factor is not functioning more adequately at 36°C or at higher temperatures. The thermal resistance times of ciliary activity obtained in this study at 36°C after the acclimation of intact animals to 5 or 15°C are compatible with the earlier observations of heat resistance of terminal cilia at 36°C in the gills of Mytilus mussels collected from the bay of Kiel and kept for 3-21 days at 10°C before the determination (Theede & Lassig, 1967, Fig. 2). The heat resistance of Mytilus, which has been reported to be dependent on the salinity of sea water (Theede & Lassig, 1967), is much smaller than that of the fresh water mussel Anodonta. In Myti/us, movement of the frontal cilia of isolated gills kept for 1 day at different temperatures as well as of the gills cut just before the determination from intact animals collected from 13°C sea water, stopped at the moment the gill strips were transferred to the 39°C incubation temperature. In the experiments of Theede (1972), the activity of terminal gill cilia in the Mytilus mussels collected from the North Sea (mean salinity30 parts per mill) became unobservable at 40°C when the incubation temperature was raised 0.2”C/min. In Anodonta, activity of the frontal cilia stopped immediately at 4344°C in the isolated gills acclimated for 1 day to 4 or 24°C (Senius & Lagerspetz, 1974). In the case of intact Anodonta mussels acclimated for several weeks either to 5 or 21°C the activity of the frontal cilia of excised gills disappeared at 4749°C when the incubation temperature was raised approximately O.S”C/min (Lagerspetz & Dubitscher, 1966). Contrary to earlier results obtained through studies of isolated Anodonta gills (Lagerspetz & Dubitscher, 1966; Lagerspetz et a/., 1970; Senius & Lagerspetz, 1974), there was no thermal resistance acclimation of ciliary activity in isolated Mytilus gills acclimated to different temperatures. However, when the isolated gills were kept for 1 day at 4 or 24°C the thermal resistance of ciliary activity was significantly increased in both groups at the 35°C incubation temperature when compared to the resistance time of the gills excised from mussels collected from 13°C sea water. As seen in the Results, the concentrations of ACh and 5-HT added into the incubation at 36 or 37°C had no effect on the thermal resistance of ciliary activity in the isolated gills. On the other hand, it has been shown in many experiments that ACh (Bulbring et a/., 1953) as well as .5-HT (Aiello, 1960, 1962; Gosselin, 1961; Gosselin et a/., 1962) has definite effects on the velocity of ciliary movement in the Mytilus gills. Enzymes involved in the cholinergic or serotonergic system have also been found in the Myfi/us gill tissue (Bulbring et al., 1953 ; Milton, 1959; Milton & Gosselin, 1960; Blaschko & Milton, 1960; Gosselin et al., 1962). Gosselin et al. (1962) have suggested that the ciliary activity is controlled by 5-HT functioning as a local
Thermal resistance. in the Mytilus gill cilia
hormone. However, it was shown later that the branchial nerve also takes part in that regulation (Aiello & Guideri, 1964; Paparo & Aiello, 1970). Also, the thermal resistance of ciliary activity in Mytilus seems to be controlled rather by neural mechanism than on the tissue level. Two facts mentioned before point out the possible neural control: (1) the thermal acclimation of ciliary activity could be induced by temperature change in whole animals but not in isolated gills, and (2) the storage of excised gills for 1 day caused a significant increase in thermal resistance of ciliary activity as compared to the gills excised from the mussels just before the thermal resistance determination. Acknowledgements-I wish to express my gratitude to Professors Herbert Precht. Ph.D.. and Harms-Dieter Jankowsky, Ph.D., of the Department of Zoology, Christian Albrecht University (Kiel), for providing pleasant and stimulating working conditions at their laboratory. Mr. Hans-Werner Huppert, candrernat., Mr. Falk Krebs, cand.rer.nat., Mr. Ekkehart Wodtke, Dr.rer.nat., as well as all other colleagues in Kiel are acknowledged for many fruitful discussions and practical aid. My grateful thanks are also due to my teacher, Professor Kari Y. H. Lagerspetz, Ph.D., of the Department of Zoology, University of Turku, for critically reading the manuscript. I am also grateful to Mrs. Sinikka Hillgren for drawing the figures. This study was supported financially by a grant from the “Deutscher Akademischer Austauschdienst”, BonnBad Godesberg, Germany. REFERENCES AIELLOE. L. (1960) Factors affecting ciliary activity on the gill of the mussel Mytilus edulis. Physiol. Zoiil. 33, 120-135. AIELL~ E. L. (1962) Identification of the cilioexcitatory substance present in the gill of the mussel Mytilus edulis. J. cell, camp. Physiol. 68, 17-21. AIELLO E. L. & GUIDES G. (1964) Nervous control of ciliary activity. Science, Wash., 146,1692-1693. BLA~CHKOH. & MILTON A. S. (1960) Oxidation of 5hydroxytryptamine and related compounds by Mytilus gill plates. Br. J. Pharmac. 15,4246.
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BULBRING E., BURN J. H. & SHELLEYH. J. (1953) Acetylcholine and ciliary movement in the gill plates of Mytilus edulis. Proc. R. Sot. B 141,445-466. FRIEDRICH L. (1967) Experimentelle Untersuchungen zum ProbIem zelluhtrer nichtgenetischer Resistenzlnderungen bei der Miesmuschel Mytilus edulis L. Kieler Meeresforsch. 23, 105-126. G~~.~.ELINR. E. (1961) The cilioexcitatory activity of serotonin. J. cell. camp. Physiol. 58, 17-25. GOSSELINR. E., Mooar K. E. & MILTON A. S. (1962) Physiological control of molluscan gill cilia by 5hydroxytryptamine. J. gen. Physioi. 46, 277-296. LAGERSPETZ K. Y. H. & DUB~WXER I. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. LAGERSPETZ K. Y. H., IMPIVAARAH. & SENXJSK. (1970) Acetylcholine in the thermal resistance acclimation of the ciliary activity in the gills of Anodonta. Comp. gen. Pharmac. 1, 236-240. _ MILTONA. S. (1959) Choline acetvlase in the ail1 elates of Mytilus edulis. Proc. R. Sac. R 150,240-2&L _ MILTONA. S. & GOWLIN R. E. (1960) Metabolism and cilia-accelerator action of 5-hydroxytryptophan (5HTP) in gill plates of MytiIus and Modioius. Fedn Proc. Fedn Am. Sots exp. Biol. 19, 126. PAPAROA. & AIELLOE. (1970) Cilio-inhibitory effects of branchial nerve stimulation in the mussel, Mytilus edulis. Comp. gen. Pharmac. 1.241-250. SENDS K. E. 0. & LAGERSPETZ K. Y. H. (1974) The role of cholinergic receptors in the thermal resistance. and therma resistance acclimation of the ciliary activity in the Anodonta gills. Comp. gen. Pharmac. 5,169-179. THEEDEH. (1972) Zur zellul&ren Hitzeresistenz mariner Evertebraten. Oecologia, Berl. 11, 17-31, THEEDEH. & LA~%GJ. (1967) Comparative studies on cellular resistance of bivalves from marine and brackish waters. Helgokinder wiss. Meeresunters. 16, 119-129. VERNBERGF. J., SCHLIEPERC. & SCHNEIDER D. E. (1963) The influence of temperature. and salinity on ciliary activity of excised gill tissue of molluscs from North Carolina. Comp. Biochem. Physiol. 8, 271-285. Key Word Index-Temperature acclimation; thermal resistance; ciliary activity; gill tissue; acetylcholine; 5-hydroxytryptamine; Mytilus edulis.