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Diurnal rhythms of heart rate in the snail Helix pomatia L.
0300-9629/9 I $3.00+ 0.00 0 1991Pergamon Press pIc
Vol. 99A, No. 3, pp. 41S-417, I991
Comp. Biochem. Physioi.
Printed in Great Britain
DIURNAL
RHYTHMS
OF HEART RATE IN THE SNAIL L.
HELM POMATIA W. W~~ENBEBG
Institute of Zoology, University of Kiel, O~shau~ns~. 40-60, D-2300 Kiel, Germany Telephone: (0431) 880-418I (Received 1 October 1990) Abstract-l. Under constant temperature and humidity conditions and a 12: 12 light-dark cycle heart rates of Helix porn&a show a diurnal rhythm with minimum values at 12.00 hr and maximum values at 24.00 hr. 2. Diurnal rhythm persists during estivation. 3. In hihematina snails neriodic fluctuations of heart rate occur which are not synchronized by the prevailing photop&od. -
~ODU~ION
Under natural conditions many life processes of pulmonate land snails are affected by nychthemeral fluctuations of environmental factors. In the slug Agriolimax reticulatus, e.g., the daily rhythm of locomotor activity follows the diurnal rhythm of the ambient temperature (T,) with the major portion of activity during the night (Dainton, 1954), while in Helix pomatia the nocturnal increase of activity is controlled by the relative humidity (r.h.) (Blaika, 1953), and the photoperiod (Tischler, 1973). Moreover, studies in Helix aspersa and Helix pomatia indicate that under natural conditions the endocrine dorsal bodies have a diurnal rhythm of activity with regard to hormone synthesis and release (Mom&h et al., 1988; Dorliichter, 1989). The existence of circadian rhythms is well documented for Apiysia californica (Block and Lickey, 1973; Lickey et al., 1976; Benson and Jacklet, 1977). There are, however, only a few studies on land snails in which periodic fluctuations of environmental factors were excluded. One of these studies indicates that in Helix pomatia, kept under constant environmental conditions for 20 days, a circadian rhythm of metabolic rate, but not of locomotor activity, occurs (Blaika, 1953). The locomotor behaviour of Limax maximus and L~~~~~s, on the other hand, shows a circadian rhythm which persists in constant darkness or light and was en~ainabIe to light-dark (LD) cycles (Sokolove et al., 1977; Beiswanger et al., 1981). The present study was performed to investigate the diurnal course of heart rate in Helix pomatia at constant T, and r.h. Of peculiar interest was the question whether diurnal rhythms exist during dormant states such as estivation and hibernation. MATERIALS AND METHODS Experiments were carried out in adult specimens of Helix pomatia collected in the field. Whole body mass (including shell) ranged between 15 and 27 g. Snails were maintained in the laboratory at T, = 2&22”C and a 12: 12 LD cycle and fed on lettuce. Egg-shell fra~ents were provided as a calcium source.
Heart rate was determined by re~s~ation of the electrocardiogram (ECG) which was picked up by means of fine stainless steel electrodes. For implantation of the electrodes the snails were immobilized by a light anaesthesia as described by Chung (1985). A small hole (diameter 3-4 mm) was drilled into the shell and the electrodes were attached to the epithelium over the heart region by a tissue adhesive. Dental cement was used to close the hole in the shell and to fix the electrodes. The leads connecting the electrodes with the preamplifier during the experiment were flexible and light so that the behaviour of the snail was not obviously disturbed. One week following the implantation of the electrodes the snails were placed into a climatic chamber. T. and r.h. in this chamber oscillated in a range of &O.S”C and + 5% respectively, with a periodic&y of 4Omin. Light in~nsity was appro~mately 1501~~. With the exception of experiments on estivating snails, animals were kept in indi~du~ Plexiglas cages the floor of which was covered with soil, leaves and moss pads. Estivation was induced by starvation and dehydration. Snails were placed into Plexiglas boxes which were perfused by air that was dried by silicate gel. Hibernation was evoked by exposure to cold (+5 f 05°C) and a 8:16 LD cycle. ECG was registered at 2 hr intervals for periods of 10 min. In the statistical treatment of the values the level of significance was set to 0.05 and calculated by Student’s r-test and by means of discriminatory analysis.
RESULTS
In all snails used in the present study heart rates could be determined by registration of the ECG (Fig. 1). Usually an undisturbed record of ECG was possible over periods of 8-12 weeks in active animals and up to 10 months in snails which had long phases of dormancy. In a first set of experiments 13 snails were exposed to T#= 18 ItO.S”C, r.h. = 70f 5%, and a 12:12 LD cycle. Figure 2 shows the average values of heart rates in the course of three days. Mean values of heart rates amounted to 35.5 f 5.6 min-’ during the scotophase (24:OO) and declined to an average value of 29 f 2.5 min-’ during the day (12:OO). Differences between highest and lowest values are significant (P < 0.05).
415 CllP %4/3--K
416
W.
I
0.5 mV
WUNNENBERG
5s.
12
Fig. 1. Electrocardiogram
of the snail Helix pornaria.
In eight snails estivation was induced by exposure to dehydrating conditions (T, = 25 +_0.5”C, r.h. < 10%). In these experiments no significant diurnal rhythm of heart rate was observed during the first three days (Fig. 3). In the following days, however, again a diurnal rhythm with significant differences between maximum and minimum values was reestablished. In comparison to the results shown in Fig. 2, in estivating snails maxima of heart rates seem to be shifted towards the beginning of the scotophase. Figure 3 further suggests that there is a gradual decrease of heart rates in the course of the 6 days of dormancy. Nine snails were exposed to T, = 5 + 0.5”C, r.h. = 80 f 5%, and a 8:16 LD cycle. Six of these animals entered hibernation, i.e. they secreted one or more calcareous epiphragms across the shell aperture. Figure 4 shows, as an example, portions of records obtained in four snails 34 months after the beginning of dormancy. In the dormant animals again a distinct rhythm of heart rates occurred, which, however, is not synchronized with photoperiod. As in estivating animals, the average minimum heart rates during hibernation were significant lower as compared with active snails exposed to the same environmental temperature (Table 1). DISCUSSION
The present study shows that registration of heart rates is a suitable method for investigations of diurnal or circadian rhythms. Compared with other biological functions, such as locomotor activity, the registration of heart rates has the advantage that it can also be used for studies in dormant snails-even in animals that are dug into the soil. Furthermore, recording of the ECG by means of chronically implanted electrodes allows the determination of heart rates in undisturbed animals. This might explain the
2L
12
TIME
2‘
12
21
2‘
12
2L
12
2‘
TIME
2L
12
OF DAY
12
lhl
2‘
12
24
12
Fig. 3. Average heart rates of eight dormant snails exposed to T, =25 +O.S”C, r.h. i IO%, and a 12:12 LD cycle. Vertical bars indicate SD.
fact that heart rates recorded in this study are lower than corresponding data reported by other authors (Biering, 1929; Skramlik, 1941; Schwartzkopff, 1956). Diurnal rhythms of heart rates were synchronized by photoperiod in active as well as in estivating snails that were completely withdrawn into their shells. The mechanisms mediating photoentrainment of these rhythms in Helix pomatiu have not been defined. Experiments in blinded garden slugs (Limax maximus, Limax Jlavw) indicate that the eyes are not necessary for either maintenance of rhythmicity or for LD entrainment (Beiswanger et al., 1981). Indications for the existence of an extraocular photoreception were also found in Helix pomatia (Schultz, 1938). In hibernating snails no distinct correlation between heart rates and LD cycles was observed. It should be noted, however, that these animals were more or less dug into the substrate. Thus, no reliable statements on light perception of these snails can be made. Possibly, the results obtained in this group of experiments can be interpreted as free-running rhythms. In dormant snails heart rates are significant lower than heart rates of non-dormant animals exposed to the same T, (Table 1). This result corroborates the well known finding that dormancy leads to a decline of many physiological functions such as metabolic
12
OF DAY [hl
Fig. 2. Average heart rates of 13 snails exposed to T. = 18 f O.S”C, r.h. = 70 f 5%, and a 12: 12 LD cycle. Vertical bars indicate SD.
TIME OF DAY Ihl
Fig. 4. Heart rates of four snails during hibernation. (T, = 5 &-O.S”C, r.h. = 70 + 5%, 8: 16 LD cycle).
Diurnal rhythm of heart rate in Helix pomatia Table 1. Minimum observed heart rates (mean + SD) of active and dormant snails (N = number of animals) T, = 5 * 0.w
Active Dormant
9.2 k 1.2min-’
T. = 25 f 0.5”C
44.5k 6.1 min-’
(N=9)
(N = 12)
5.03 + 0.68 min-’
28.3 k 4.7 min-’
(N=61
IN=R\
rate (Herreid, 1977), evaporative water loss (Bamhart and McMahon, 1987), enzymatic activity (Bielawski and Kesa, 1986), and the reproductive system (Gomot and Gomot, 1989). REFERENCES Bamhart M. C. and McMahon B. R. (1987) Discontinuous carbon dioxide release and metabolic depression in dormant land snails. J. EXD. Biol. 128. 123-138. Beiswanger C. M., Sokolove P. G. and Prior D. J. (1981) Extraocular photoentrainment of the circadian locomotor rhythm of the garden slug Limax. J. Exp. Zool. 216, 13-23. Benson J. A. and Jacklet
J. W. (1977) Circadian rhythm of output from neurones in the eye of Aplysia IV. A model of the clock:differential sensitivity to light and low temperature pulses. J. Exp. Biol. 70, 195-211. Bielawski J. and Kesa H. (1986) Seasonal variation of phosphatase activity in the hepatopancreas of the snail Helix pomatia L. Camp. Biochem. Physiol. 83A, 105-107. Biering P. (1929) Untersuchungen iiber das Kreislaufsystem bei den Weichtieren II. Z. uergl. Physiol. 10, 465-496. Blaika P. (1953) Der tagliche Rhythmus des Gesamtmetabolismus in der Weinbergschnecke (Helix pomatia L). Acta Sot. Zool. Bohemosl. 17, 251-265. Block G. D. and Lickey M. E. (1973) Extraocular photoreceptors and oscillators can control the circadian rhythm
417
of behavioral activity in Aplysia. J. Camp. Physiol. 84, 367-374.
Chung D. (1985) An anesthetic for internal operations on the land snail Helix aspersa Miiller. Veliger 27, 331-335.
Dainton B. H. (1954) The activity of slugs 1. The induction of activity by changing temperatures. J. Exp. Biol. 31, 165-187. Dorliichter
M. (1989) Diurnal changes of the endocrine mediodorsal body cells in Helix pomatia L (Gastropoda, Pulmonata). Zool. Jb. Phvsiol. 93, 37-51. Gomot P. and Gomot L: (1989). Etude experimentale in oiuo du role du cerveau dans faction de la temperature sur la spermatogenese de 1’Escargot Helix aspersa en hibernation. Acad. Sci. Paris 308, Sirie III, 135-140. Herreid C. F. (1977) Metabolism of land snails (Otala lactea) during dormancy, arousal and activity. Camp. Biochem. Physiol. 56A, 211-215.
Lickey M. E., Block G. D., Hudson D. J. and Smith J. T. (1976) Circadian oscillators and photoreceptors in the gastropod Aplysia. Photochem. Photobiol. 5, 253-273. Mounzih K.. Griffond B. and Roubos E. W. (1988) Investigations of the diurnal activity of the endocrine dorsal bodies in Helix aspersa. Int. J. Inv. Reprod. Dev. 14, 229-244.
Schultz F. (1938) Bau und Funktion der Sinneszellen in der Korperoberflache von Helix pomatia. Z. Morph. okol. Tiere 33, 555-58 1. Schwartzkopff J. (1956) Herzfrequenz und KiirpergrdBe bei Mollusken. Verh. Dtsch. Zool. Ges. 1956,463469. Skramlik E. v. (1941) Uber den Kreislauf bei den Weichtieren. Erg. Biol. 18, 88-286. Sokolove P. G., Beiswanger C. M., Prior D. J. and Gelperin A. (1977) A circadian rhythm in the locomotor behavior of Limax maximus. J. Exp. Biol. 66, 47-64. Tischler W. (1973) Zur Biologie und dkologie der Weinbergschnecke (Helix pomatia). Faun. -iikol. Mitt. 4, 283-298.
Vol. 99A, No. 3, pp. 41S-417, I991
Comp. Biochem. Physioi.
Printed in Great Britain
DIURNAL
RHYTHMS
OF HEART RATE IN THE SNAIL L.
HELM POMATIA W. W~~ENBEBG
Institute of Zoology, University of Kiel, O~shau~ns~. 40-60, D-2300 Kiel, Germany Telephone: (0431) 880-418I (Received 1 October 1990) Abstract-l. Under constant temperature and humidity conditions and a 12: 12 light-dark cycle heart rates of Helix porn&a show a diurnal rhythm with minimum values at 12.00 hr and maximum values at 24.00 hr. 2. Diurnal rhythm persists during estivation. 3. In hihematina snails neriodic fluctuations of heart rate occur which are not synchronized by the prevailing photop&od. -
~ODU~ION
Under natural conditions many life processes of pulmonate land snails are affected by nychthemeral fluctuations of environmental factors. In the slug Agriolimax reticulatus, e.g., the daily rhythm of locomotor activity follows the diurnal rhythm of the ambient temperature (T,) with the major portion of activity during the night (Dainton, 1954), while in Helix pomatia the nocturnal increase of activity is controlled by the relative humidity (r.h.) (Blaika, 1953), and the photoperiod (Tischler, 1973). Moreover, studies in Helix aspersa and Helix pomatia indicate that under natural conditions the endocrine dorsal bodies have a diurnal rhythm of activity with regard to hormone synthesis and release (Mom&h et al., 1988; Dorliichter, 1989). The existence of circadian rhythms is well documented for Apiysia californica (Block and Lickey, 1973; Lickey et al., 1976; Benson and Jacklet, 1977). There are, however, only a few studies on land snails in which periodic fluctuations of environmental factors were excluded. One of these studies indicates that in Helix pomatia, kept under constant environmental conditions for 20 days, a circadian rhythm of metabolic rate, but not of locomotor activity, occurs (Blaika, 1953). The locomotor behaviour of Limax maximus and L~~~~~s, on the other hand, shows a circadian rhythm which persists in constant darkness or light and was en~ainabIe to light-dark (LD) cycles (Sokolove et al., 1977; Beiswanger et al., 1981). The present study was performed to investigate the diurnal course of heart rate in Helix pomatia at constant T, and r.h. Of peculiar interest was the question whether diurnal rhythms exist during dormant states such as estivation and hibernation. MATERIALS AND METHODS Experiments were carried out in adult specimens of Helix pomatia collected in the field. Whole body mass (including shell) ranged between 15 and 27 g. Snails were maintained in the laboratory at T, = 2&22”C and a 12: 12 LD cycle and fed on lettuce. Egg-shell fra~ents were provided as a calcium source.
Heart rate was determined by re~s~ation of the electrocardiogram (ECG) which was picked up by means of fine stainless steel electrodes. For implantation of the electrodes the snails were immobilized by a light anaesthesia as described by Chung (1985). A small hole (diameter 3-4 mm) was drilled into the shell and the electrodes were attached to the epithelium over the heart region by a tissue adhesive. Dental cement was used to close the hole in the shell and to fix the electrodes. The leads connecting the electrodes with the preamplifier during the experiment were flexible and light so that the behaviour of the snail was not obviously disturbed. One week following the implantation of the electrodes the snails were placed into a climatic chamber. T. and r.h. in this chamber oscillated in a range of &O.S”C and + 5% respectively, with a periodic&y of 4Omin. Light in~nsity was appro~mately 1501~~. With the exception of experiments on estivating snails, animals were kept in indi~du~ Plexiglas cages the floor of which was covered with soil, leaves and moss pads. Estivation was induced by starvation and dehydration. Snails were placed into Plexiglas boxes which were perfused by air that was dried by silicate gel. Hibernation was evoked by exposure to cold (+5 f 05°C) and a 8:16 LD cycle. ECG was registered at 2 hr intervals for periods of 10 min. In the statistical treatment of the values the level of significance was set to 0.05 and calculated by Student’s r-test and by means of discriminatory analysis.
RESULTS
In all snails used in the present study heart rates could be determined by registration of the ECG (Fig. 1). Usually an undisturbed record of ECG was possible over periods of 8-12 weeks in active animals and up to 10 months in snails which had long phases of dormancy. In a first set of experiments 13 snails were exposed to T#= 18 ItO.S”C, r.h. = 70f 5%, and a 12:12 LD cycle. Figure 2 shows the average values of heart rates in the course of three days. Mean values of heart rates amounted to 35.5 f 5.6 min-’ during the scotophase (24:OO) and declined to an average value of 29 f 2.5 min-’ during the day (12:OO). Differences between highest and lowest values are significant (P < 0.05).
415 CllP %4/3--K
416
W.
I
0.5 mV
WUNNENBERG
5s.
12
Fig. 1. Electrocardiogram
of the snail Helix pornaria.
In eight snails estivation was induced by exposure to dehydrating conditions (T, = 25 +_0.5”C, r.h. < 10%). In these experiments no significant diurnal rhythm of heart rate was observed during the first three days (Fig. 3). In the following days, however, again a diurnal rhythm with significant differences between maximum and minimum values was reestablished. In comparison to the results shown in Fig. 2, in estivating snails maxima of heart rates seem to be shifted towards the beginning of the scotophase. Figure 3 further suggests that there is a gradual decrease of heart rates in the course of the 6 days of dormancy. Nine snails were exposed to T, = 5 + 0.5”C, r.h. = 80 f 5%, and a 8:16 LD cycle. Six of these animals entered hibernation, i.e. they secreted one or more calcareous epiphragms across the shell aperture. Figure 4 shows, as an example, portions of records obtained in four snails 34 months after the beginning of dormancy. In the dormant animals again a distinct rhythm of heart rates occurred, which, however, is not synchronized with photoperiod. As in estivating animals, the average minimum heart rates during hibernation were significant lower as compared with active snails exposed to the same environmental temperature (Table 1). DISCUSSION
The present study shows that registration of heart rates is a suitable method for investigations of diurnal or circadian rhythms. Compared with other biological functions, such as locomotor activity, the registration of heart rates has the advantage that it can also be used for studies in dormant snails-even in animals that are dug into the soil. Furthermore, recording of the ECG by means of chronically implanted electrodes allows the determination of heart rates in undisturbed animals. This might explain the
2L
12
TIME
2‘
12
21
2‘
12
2L
12
2‘
TIME
2L
12
OF DAY
12
lhl
2‘
12
24
12
Fig. 3. Average heart rates of eight dormant snails exposed to T, =25 +O.S”C, r.h. i IO%, and a 12:12 LD cycle. Vertical bars indicate SD.
fact that heart rates recorded in this study are lower than corresponding data reported by other authors (Biering, 1929; Skramlik, 1941; Schwartzkopff, 1956). Diurnal rhythms of heart rates were synchronized by photoperiod in active as well as in estivating snails that were completely withdrawn into their shells. The mechanisms mediating photoentrainment of these rhythms in Helix pomatiu have not been defined. Experiments in blinded garden slugs (Limax maximus, Limax Jlavw) indicate that the eyes are not necessary for either maintenance of rhythmicity or for LD entrainment (Beiswanger et al., 1981). Indications for the existence of an extraocular photoreception were also found in Helix pomatia (Schultz, 1938). In hibernating snails no distinct correlation between heart rates and LD cycles was observed. It should be noted, however, that these animals were more or less dug into the substrate. Thus, no reliable statements on light perception of these snails can be made. Possibly, the results obtained in this group of experiments can be interpreted as free-running rhythms. In dormant snails heart rates are significant lower than heart rates of non-dormant animals exposed to the same T, (Table 1). This result corroborates the well known finding that dormancy leads to a decline of many physiological functions such as metabolic
12
OF DAY [hl
Fig. 2. Average heart rates of 13 snails exposed to T. = 18 f O.S”C, r.h. = 70 f 5%, and a 12: 12 LD cycle. Vertical bars indicate SD.
TIME OF DAY Ihl
Fig. 4. Heart rates of four snails during hibernation. (T, = 5 &-O.S”C, r.h. = 70 + 5%, 8: 16 LD cycle).
Diurnal rhythm of heart rate in Helix pomatia Table 1. Minimum observed heart rates (mean + SD) of active and dormant snails (N = number of animals) T, = 5 * 0.w
Active Dormant
9.2 k 1.2min-’
T. = 25 f 0.5”C
44.5k 6.1 min-’
(N=9)
(N = 12)
5.03 + 0.68 min-’
28.3 k 4.7 min-’
(N=61
IN=R\
rate (Herreid, 1977), evaporative water loss (Bamhart and McMahon, 1987), enzymatic activity (Bielawski and Kesa, 1986), and the reproductive system (Gomot and Gomot, 1989). REFERENCES Bamhart M. C. and McMahon B. R. (1987) Discontinuous carbon dioxide release and metabolic depression in dormant land snails. J. EXD. Biol. 128. 123-138. Beiswanger C. M., Sokolove P. G. and Prior D. J. (1981) Extraocular photoentrainment of the circadian locomotor rhythm of the garden slug Limax. J. Exp. Zool. 216, 13-23. Benson J. A. and Jacklet
J. W. (1977) Circadian rhythm of output from neurones in the eye of Aplysia IV. A model of the clock:differential sensitivity to light and low temperature pulses. J. Exp. Biol. 70, 195-211. Bielawski J. and Kesa H. (1986) Seasonal variation of phosphatase activity in the hepatopancreas of the snail Helix pomatia L. Camp. Biochem. Physiol. 83A, 105-107. Biering P. (1929) Untersuchungen iiber das Kreislaufsystem bei den Weichtieren II. Z. uergl. Physiol. 10, 465-496. Blaika P. (1953) Der tagliche Rhythmus des Gesamtmetabolismus in der Weinbergschnecke (Helix pomatia L). Acta Sot. Zool. Bohemosl. 17, 251-265. Block G. D. and Lickey M. E. (1973) Extraocular photoreceptors and oscillators can control the circadian rhythm
417
of behavioral activity in Aplysia. J. Camp. Physiol. 84, 367-374.
Chung D. (1985) An anesthetic for internal operations on the land snail Helix aspersa Miiller. Veliger 27, 331-335.
Dainton B. H. (1954) The activity of slugs 1. The induction of activity by changing temperatures. J. Exp. Biol. 31, 165-187. Dorliichter
M. (1989) Diurnal changes of the endocrine mediodorsal body cells in Helix pomatia L (Gastropoda, Pulmonata). Zool. Jb. Phvsiol. 93, 37-51. Gomot P. and Gomot L: (1989). Etude experimentale in oiuo du role du cerveau dans faction de la temperature sur la spermatogenese de 1’Escargot Helix aspersa en hibernation. Acad. Sci. Paris 308, Sirie III, 135-140. Herreid C. F. (1977) Metabolism of land snails (Otala lactea) during dormancy, arousal and activity. Camp. Biochem. Physiol. 56A, 211-215.
Lickey M. E., Block G. D., Hudson D. J. and Smith J. T. (1976) Circadian oscillators and photoreceptors in the gastropod Aplysia. Photochem. Photobiol. 5, 253-273. Mounzih K.. Griffond B. and Roubos E. W. (1988) Investigations of the diurnal activity of the endocrine dorsal bodies in Helix aspersa. Int. J. Inv. Reprod. Dev. 14, 229-244.
Schultz F. (1938) Bau und Funktion der Sinneszellen in der Korperoberflache von Helix pomatia. Z. Morph. okol. Tiere 33, 555-58 1. Schwartzkopff J. (1956) Herzfrequenz und KiirpergrdBe bei Mollusken. Verh. Dtsch. Zool. Ges. 1956,463469. Skramlik E. v. (1941) Uber den Kreislauf bei den Weichtieren. Erg. Biol. 18, 88-286. Sokolove P. G., Beiswanger C. M., Prior D. J. and Gelperin A. (1977) A circadian rhythm in the locomotor behavior of Limax maximus. J. Exp. Biol. 66, 47-64. Tischler W. (1973) Zur Biologie und dkologie der Weinbergschnecke (Helix pomatia). Faun. -iikol. Mitt. 4, 283-298.