SPONTANEOUSLY INDUCED RHYTHM OF TIDAL PERIODICITY IN LABORATORY-REARED CARCINUS

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1 J. Exp. Biol. (1967), With 2 text-figures Printed in Great Britain SPONTANEOUSLY INDUCED RHYTHM OF TIDAL PERIODICITY IN LABORATORY-REARED CARCINUS BY BARBARA G. WILLIAMS AND E. NAYLOR Department of Zoology, University College of Swansea {Received 7 April 1967) INTRODUCTION There is growing opinion that circadian rhythms have an inherent natural period approximating to that of environmental variables which, in field conditions, maintain exact period and appropriate phase (Pittendrigh, 1958, i960; Cloudsley-Thompson, 1961; Harker, 1964). Evidence supporting this view comes from the results of several workers (Harker, 1953; Pittendrigh, 1954; Aschoff & Meyer-Lohmann, 1954; Hoffman, 1955; Folk, 1955), who have initiated circadian rhythmicity in laboratory-reared organisms which had never experienced environmental rhythms of 24 hr. periodicity. No such evidence is available on the initiation of tidal rhythmicity, and present experiments were planned to investigate the rhythmicity of laboratory-reared shore crabs, Carcinus maenas (L.). Crabs were reared in the normal light/dark regime of the laboratory and away from the influence of tides. Their pattern of locomotor activity was recorded both before and after they were subjected to an environmental perturbation known to re-initiate tidal rhythmicity in crabs removed from the shore (Naylor, 1963). MATERIALS AND METHODS Specimens were reared from the egg, through four zoeal stages and one megalopa stage to the first crab stage, using as food the dinoflagellate Prorocentrum micans Ehrenberg and newly hatched nauplii of Artemia salina L. (Williams, 1967). During the larval stages the animals were kept at 12 C. and in an approximately normal light/dark regime. On completing metamorphosis the young crabs were transferred to circulating sea water in a laboratory aquarium and fed every few days on pieces of Mytilus. When they reached a suitable size of about 8 mm. carapace width their pattern of locomotor activity was recorded continuously over a period of several days. To obtain the recording a crab was enclosed in a Perspex tube through which sea water circulated slowly, and across which dim light from a source above impinged on a photo-electric cell beneath. The electrical apparatus recording movements of the crab between the light source and the photo-electric cell was based on that described by Southward & Crisp (1965). It was maintained in a constant temperature room at 15 C. and in which dim red light from a 40 W. bulb was a continuous source of background illumination. After the initial record of activity each crab was then chilled to a temperature of 4 0 C. for 15 hr. and its hourly locomotor activity was recorded again for a few days. Data obtained in these experiments have been analysed for rhythmicity using the method of periodogram analysis (see Enright, 1965). In this, the sequence of hourly

2 230 BARBARA G. WILLIAMS AND E. NAYLOR activity values (x v x t, #3,..., * n ) wa3 scanned for possible rhythms of whole-hour frequencies (/) from 8-30 hr. inclusive, covering a range from less than tidal to greater than circadian periodicity. Each scan of the data correspondingly gave 8-30 mean hourly activity values (X lt X s,..., X/) calculated thus: X 2f+1 x n-f+l X 2f+2 x n-/+2 Averages X 1 X e... X f This gives a form-estimate of possible rhythmicity at each of the twenty-two frequencies tested. Lack of rhythmicity in a scan is indicated when mean hourly values in a form-estimate tend to approximate to the value of over-all mean hourly activity. Rhythmicity is indicated when the means are distributed about a sine curve and therefore show extensive deviation from the over-all mean. Standard deviations of the hourly means were then calculated for each of the form estimates, applying the statistic and so obtaining a value of s for each of the 22 values of/, which may then be plotted on the periodogram. In a periodogram low values for deviation indicate lack of a rhythm of that frequency in the original data, while high deviation of the hourly means at a particular frequency may confirm the hypothesis of rhythmicity. Enright (1965) plots root mean square deviation as 'amplitude' in his periodograms, but in present results coefficient of variability (c.v.) is used. This expresses standard deviation as a percentage of over-all mean hourly activity and permits easier comparison of the results of different experiments. RESULTS Activity patterns were similar in four replicate experiments, each involving a reared crab freshly removed from the light/dark regime of the aquarium, and the average hourly activity of these four crabs over a period of 102 hr. is given in Fig. 1 a. Circadian rhythmicity is apparent in these results, with outbursts of locomotor activity coinciding with what would have been the hours of darkness. The periodogram derived from the results (Fig. 2 a) confirms that conclusion, since the coefficients of variability of the mean values for hourly activity are highest in the frequency range hr. Average hourly activity of the same crabs during a period of 79 hr. after chining is illustrated in Fig. 1 b. One crab unfortunately moulted and died during the chilling treatment, so these results are based on three replicate experiments. In all the survivors there was an outburst of activity immediately after chilling, which was followed by successive outbursts which coincide fairly closely with arrows marked off at 12-4 hr. (tidal) intervals after the time of return to the warm room. The occurrence of the rhythm of approximate tidal periodicity after chilling is confirmed in the periodogram (Fig. 2b), which exhibits peak values for coefficient of variability in the

3 Rhythm of tidal periodicity in laboratory-reared Carcinus 231 frequency range of hr- The peak at hr. in Fig. ib is a multiple of the h*- rhythm and appears as a result of the procedure of periodogram analysis. DISCUSSION In crabs collected from the shore and kept in the laboratory, chilling resets or re-initiates the rhythm of tidal periodicity (Naylor, 1963). Similarly, crabs from a non-tidal dock can be induced by chilling to show tidal rhythmicity when previously circadian rhythmicity was apparent (Naylor, 1963). Induced rhythms of this kind are, of course, not phased with external tides but they indicate that the ability to ( ) Time (hr.) Fig. 1. Average hourly activity of crabs reared in a 24 hr. light/dark regime: (a) on transfer to continuous dim light (4 crabs); (6) after subsequent chilling to 4 0 C. for 15 hr. (3 crabs). (M-midnight; arrows at 12-4 hr intervals after chilling.) show tidal rhythmicity is deep-seated in British Carcinus. Present results in which laboratory-reared crabs are also induced to show a rhythm of approximately tidal periodicity, without having experienced environmental changes of such a frequency, confirm that tidal rhythmicity is very deep seated and suggest that it may be inherited. In nature, tidal variables such as hydrostatic pressure, temperature change and periodical immersion no doubt add to the precision of the period, but these factors act primarily as synchronizers which maintain the correct phase (WiUiams, 1966).

4 232 BARBARA G. WILLIAMS AND E. NAYLOR Several authors have shown that animals reared in constant laboratory conditions can rapidly develop overt persistent rhythms with a natural period of approximately 24 hr. (Harker, 1953; Pittendrigh, 1954; Aschoff & Meyer-Lohmann, 1954; Hoffman, 1955; Folk, 1955). Harker (1953) initiated a persistent circadian swimming rhythm in laboratory-reared larvae of the mayfly Ecdyonurus torrentis after exposing them to only one 24 hr. cycle of light and dark, while Pittendrigh (i960) showed that a light flash of ^jjw sec- was sufficient to initiate a 24 hr. eclosion rhythm in aperiodic Drosophila. Present work differs somewhat from those results in that the crabs, though is? 50 - ~ 40 - ^ 30 H 20 H I «10-I d Frequency (hr.) o 20- c te 10 - I I I I I I I I I I I l^ I I I T I 1 I I I Frequency (hr.) Fig. a. Periodogram analyses of data in Fig. 1: (a) crabs before chilling; (b) crabs after chilling (see text for procedure). reared in the absence of tides, were exposed to 24 hr. cycles of light and dark during development. It is, however, unlikely that crabs in the present experiments learned the tidal rhythm as an approximate submultiple of the 24 hr. light/dark cycle in which they were reared. Carcinus from the relatively tideless Mediterranean are subjected to predominantly 24 hr. cycles in their environment but they do not exhibit a hr. rhythm when chilled (Naylor, 1961, 1963). In addition, it is unlikely that the crabs learned the new periodicity as a function of the 15 hr. chilling period, which was deliberately chosen not to be a simple multiple or submultiple of 12-4 hr. Moreover, it has previously been shown that similar approximately tidal rhythmicity is induced in crabs chilled for various lengths of time from 6-24 hr. (Naylor, 1963).

5 Rhythm of tided periodicity in laboratory-reared Carcinus 233 It is possible to take the view that the appearance of hr. rhythmicity after chilling for 6 hr. or more is entirely an artifact, of no significance in nature. However, the same phenomenon has recently been reported in shore fishes (Gibson, 1967) and it would be surprising to find such induced rhythms, so close to tidal frequency, which are not of adaptive significance. Moreover, temperatures of 4 0 C. are not far below environmental temperatures for Carcinus in winter, when their rhythmicity is observed to disappear. In addition, as indicated above, chilling does not induce approximately tidal rhythmicity in Carcinus from the non-tidal Mediterranean (Naylor, 1961). On present evidence it seems, therefore, that approximate tidal rhythmicity is an inherited and adaptive feature of the behavioural physiology of Carcinus in tidal waters. Synchronization by normal environmental variables or by sudden cold shock results in the expression of this property as overt tidal rhythmicity. These results are in general agreement with the ' multiple clock hypothesis' for the control of rhythmicity which postulates the presence of numerous clocks of approximately tidal and circadian (twice-tidal) periodicity in Carcinus (Naylor, i960). The rhythm of laboratory-reared crabs before chilling could be the expression of circadian clocks which are phased by the environmental light/dark cycle, while the rhythmicity after chilling is the expression of approximately tidal clocks which are synchronized for the first time by the chilling procedure. SUMMARY 1. Laboratory-reared shore crabs show circadian rhythmicity which is transformed to approximate tidal rhythmicity after a period of chilling to 4 0 C. 2. Periodogram analysis of the results characterizes the periodicity of the two types of rhythm, which suggest that the ability to show a rhythm of tidal periodicity is an inherited feature of shore-crab physiology. We are grateful to S.R.C. who provided financial support to one of us (B.G.W.) and to Mr G. Thomas who constructed the aktograph. REFERENCES ASCHOFF, J. & MEYER-LOHMANN, J (1954). Angeborene 24-Stunden Periodik beim Kttchen. Pflugers Arch. get. Phynol. 360, CLOUDSLEY-THOMPSON, J. L. (1961). Rhythmic Activity in Animal Physiology and Behaviour, 236 pp. New York and London: Academic Press. ENRIGHT, J. T. (1965). The search for rhythmicity in biological time-series. J. theoret. biol. 8, FOLK, G. E. (1955). Modification by light and feeding of the 34-hour rhythm of activity in rodents. Proc. V. Conference. Soc. pro. Studio Rhythmi Biologict, Stockholm. Ed. A. Sollberger. GIBSON, R. N. (1967). Experiments on the tidal rhythm of Blennaa pholu. J. mar. biol. Ass. U.K. 47, HARKER, J. E. (1953). The diurnal rhythm of activity of mayfly nymphs J. Exp. Biol. 30, HARKER, J. E. (1964). The Physiology of Diurnal Rhythms, 114 pp. Cambridge University Press. HOFFMAN, K. (1955). AktivitStsregistrierungen bei frisch geschlupften Eidechsen. Z. vergl. Physiol NAYLOR, E. (i960). Locomotory rhythms in Carcinus maenas (L.) from non-tidal conditions. J. Exp. Biol. 37, NAYLOR, E. (1961). Spontaneous locomotor rhythm in Mediterranean Carcinus. Pubbl. Stax. Zool. Napoli 33, NAYLOR, E. (1963). Temperature relationships of the locomotor rhythm of Carcinus. J. Exp. Biol. 40, Exp. B10L 47, 2

6 234 BARBARA G. WILLIAMS AND E. NAYLOR PITTENDRIGH, C. S. (1954). On temperature-independence in the clock-system controlling emergence time in Drosophila. Proc. natn. Acad. Sri. U.S.A. 40, PITTENDRIGH, C. S. (1958). Perspectives in the study of biological clocks. In Perspective! in Marine Biology, pp Ed. A. A. Buzzati-Traverse University California Press. PITTENDRIGH, C. S. (i960). Circadian rhythms and circadian organization of living systems. Cold Spring Harb. Symp. quant. Biol. 25, SOUTHWARD, A. J. & CRISP, D. J. (1965). Activity rhythms of barnacles in relation to respiration and feeding. J. mar. biol. An. UJC. 45, WILLIAMS, B. G. (1966). Studies on the development and control of rhythmic activity of Carrimu maenas (L.). Ph.D. Thesis: University College of Swansea, Wales. WILLIAMS, B. G. (1967). Laboratory rearing of the larval stages of Carrimu maenas (L.). J. Nat. Hist. (In press).