THE CIRCADIAN RHYTHM OF LOCOMOTOR ACTIVITY IN THE LIZARD HOPLODACTYLUS PACIF1CUS, AND ITS POSSIBLE TAXONOMIC USE

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1 TANE 22, 1976 THE CIRCADIAN RHYTHM OF LOCOMOTOR ACTIVITY IN THE LIZARD HOPLODACTYLUS PACIF1CUS, AND ITS POSSIBLE TAXONOMIC USE by J.A. Benson Department of Biological Sciences, State University of New York at Albany, Albany, NY 12222, U.S.A. SUMMARY The New Zealand gecko, Hoplodactylus pacificus has been shown to exhibit a circadian rhythm of locomotor activity which is controlled by an endogenous "biological clock". Two populations of H. pacificus, the small, coastal and the large, bush-dwelling types, can be distinguished on the basis of the properties of their locomotor rhythms. The coastal group does not show a free-running rhythm in the constant experimental conditions described here, and entrains to environmental light-dark cycles of abnormally short period, though not to light-dark cycles of period greater than 24 hours. The bush-dwelling population shows a clear free-running rhythm of period greater than 24 hours, which obeys Aschoff s Rule. Temperature cycles are neither sufficient nor necessary for the entrainment of the locomotor rhythm. It is suggested that the analysis of the circadian rhythms of some populations of geckos could provide a useful supplementary diagnostic feature for use in the separation of species of New Zealand geckos. INTRODUCTION The daily cycles of activity exhibited by almost all animals are controlled by endogenous circadian "clocks" which are entrained by cyclic environmental stimuli, in order that the animal may carry out its various activities at the appropriate time of day or night. Characteristically, in the absence of external time-cues, the circadian clock "free-runs" so that activity follows a cycle of period approximately, but rarely exactly, 24 hours. The control of locomotor rhythms has been investigated in a number of lizard species, first in diurnal lizards by Barden (1942) who showed that the locomotor rhythm of Cnimidophorus sexlineatus persisted in constant conditions and could be entrained by light-dark cycles. Later, Hoffman (1955, 1957a, 1957b, 1959, 1960) demonstrated that the activity rhythms of Lacerta sicula and Lacerta agilis were endogenous and temperature compensated. Evans (1966) investigated rhythmic locomotor activity in the diunal aguanid, Ufa stansburiana, and, for the first time in a nocturnal lizard, in the gekkonid Coleonyx variegatus. The former may be entrained by temperature cycles independently of conflicting light-dark cycles, while the latter shows an interaction between the effects of environmental light and temperature cycles. Taylor & Tschirgi (1960) found that the circadian rhythm of activity in Sceloporus magister entrained to temperature cycles. Gelderloos (1975, 1976) has examined the circadian rhythms and light preferences of the desert iguana, Dipsosaurus dorsalis. 119

2 Most recently, work on the clock systems of lizards has shown that light sensitive clocks can be entrained to light-dark cycles in blinded Anolis carolinensis via extraretinal photoreceptors. These photoreceptors are probably located in the brain (Underwwod & Menaker 1970; Underwood 1973, 1975). Quay (1970), studying a field population of the lizard Sceloporus occidentalis, found that the 5-HT concentration in the brain appeared to be related to time of day and temperature, and Doshi, Huggins & Fitzgerald (1975) have detected a circadian rhythm in the brain serotonin levels of Anolis carolinensis. Hoplodactylus pacificus, the Pacific gecko, occurs throughout New Zealand. All populations are included in a single species, but many of the more distinct colour varieties are known to breed true and possibly have separate geographical ranges (Sharrell 1966). General observations of caged specimens have confirmed the behavioural data systematized by McCann (1955). H. pacificus is nocturnal, but occasionally moves out from cover during the day to bask in small patches of sunlight. Benson (1970), in unpublished experiments, investigated the effects of temperature cycles and light cycles on H. pacificus, and showed that the coastal and bush varieties of this lizard have circadian rhythms of locomotor activity that differ significantly. This article includes the results of those experiments, together with a discussion of the possibility of using such behavioural differences in the revision of the taxonomy of New Zealand geckos. Mclvor (1973) confirmed that there is a daily cycle of activity in H. pacificus and that it is under endogenous control. He showed that absolute amount of activity decreases with temperature and that there is a rhythm in the rate of breathing movements. METHODS AND MATERIALS The specimens of H. pacificus used in these investigations were as follows: Type A: Slate green in colour: size range 10cm to 12.5cm in length; captured at Port Jackson, Coromandel Peninsula, amongst litter in the upper maritime zone of an open coast. Type B: Brown, with distinct patterning; size range 16cm to 17.5cm in length; captured near Wanganui amongst forest litter in dense inland bush. Locomotor activity was recorded by means of pivoted aktographs writing directly on 1 rev. per day kymographs. Water but not food was provided throughout each experiment. Artificial light-dark cycles were produced by tungsten bulbs heat-filtered through 15cm of water. Constant temperature was provided by a temperaturecontrolled room held at 19.0 ± 0.5 C. Temperature cycle experiments were carried out in an incubator, with the cold phase held at 13.0 ± 0.5 C and the warm phase at room ambient. Temperature cycles were recorded continuously on a Cassella Thermohydrograph. Pen records of activity have been traced and are presented with consecutive days one beneath the other. RESULTS Activity in natural light and temperature cycles Locomotor activity was recorded in a roof-top herbarium open to natural 120

3 daily light and temperature cycles. Fig. 1 shows that activity began shortly after sunset and persisted for most of the night, decreasing towards dawn, when it ceased, except for a few brief forays during daylight hours. In longer records, the level of activity decreased with duration of confinement in the aktograph tray. Activity in natural light cycles at constant temperature Records of activity in these conditions are indistinguishable from those of activity recorded in natural temperature cycles together with natural light-dark cycles. Temperature cycles were therefore not essential entraining stimuli. Activity in constant light intensity and constant temperature In conditions of constant light and temperature, qualitative differences between the activity of Type A and Type B geckos became apparent. Fig. 2 illustrates the activity of a Type A specimen in constant light (2.0 ± 1.0 lux). A free-running rhythm cannot be clearly distinguished, except perhaps on Days 2A. All other records of Type A lizards in constant conditions show completely random activity, with both high and low total activity levels (i.e. there is no masking due to high overall activity). Fig. 3 shows a record typical of Type B activity in constant conditions (8.0 ± 1.0 lux). A distinct free-running rhythm occurred. Experiments carried out in 2.0 ± 1.0 lux light intensity show similar behaviour but with a decrease in period from approximately 27 hours, as in Fig. 3, to 25 hours in 2.0 ± 1.0 lux. This is in accordance with Aschoff's Rule which states that in nocturnal organisms the free-running period increases with increase in the light intensity (Aschoff 1960). These results suggest that endogenous control of activity may not be as strong in Type A H. pacificus as in Type B. Entrainment to artificial light-dark cycles It is a characteristic of circadian clocks that they will not entrain to light-dark cycles of periods more than a few hours greater or less than their free-running periods. Experiments in constant conditions failed to show convincingly that there was any endogenous control of locomotor activity in Type A lizards. For this reason, tests for limits of entrainment were applied mainly to Type A lizards. If endogenous clock control was involved, distinct limits close to 24 hours for the range of entrainable periods would be expected. A series of experiments was carried out with light-dark cycles ranging from LD 12:12 to LD 6:6. No lower limit to entrainment was found. Figs. 4, 5 and 6 show entrainment, or at least conformity of behaviour, to LD 11:11, LD 9:9 and LD 6:6 respectively for Type A H. pacificus. Activity is confined to the dark phase of the light-dark cycles down to a remarkably short period. A Type B lizard is shown by Fig. 7 to entrain, after a single transient, to a LD 10:10 cycle. Type B lizards were not tested in light dark cycles of period shorter than 20 hours. However, it is hypothesized that they would show a lower limit of entrainment closer to 24 hours, both because of the greater amount of control by the circadian clock, and because their free-running period is greater than 24 hours. Using the same light intensities, Type A animals were tested for an upper 121

4 limit of entrainment. Unexpectedly, it proved impossible to entrain Type A lizards to light-dark cycles of LD 13:13 or longer periods. Fig. 8 shows an example of the effects of LD 13:13 on the activity of a Type A lizard. A clear rhythm can be observed, but its period is slightly less than 26 hours. There is an initial burst of activity some hours before onset of darkness although a higher level of activity appears during the dark phase of the light-dark cycle. This is a clear case of relative co-ordination (Wever 1972), and was also seen to occur in LD 14:14 and LD 15:15 (e.g. Fig. 9). In some records, the rhythm is masked quite heavily by random activity, while in others the entrainment is almost complete, with the onset of activity stable at a positive phase angle to the entraining light-dark cycle (i.e. activity beginning before onset of darkness by the same interval each day), as shown in Fig. 9. Activity in constant light and 24 hour temperature cycles An attempt was made to entrain the activity rhythm of H. pacificus to a temperature cycle of period 24 hours and range 19 to 13 C. Fig. 10 is a record of a Type A lizard exposed to the temperature cycle as illustrated. On Days 4 to 7 there is more activity during the cool phase than during the warm phase of the temperature cycle. With Type B animals the active phase began later in the cool phase and extended for a few hours into the warm phase. This difference in phase angle suggests that in the former case the lizard was reacting directly to the ambient temperature, while in the case of the Type B animal there may have been true entrainment. However, Mclvor (1973) found that in the population that he used, when they^ were^kept in constant darkness, the activity decreased from 14 until at 9.5 to 7 C activity was extremely low. The experiments reported here, for 2.0 ± 1.0 lux, indicate that activity is greater at 13 C in comparison with 19 C, as would be expected in a nocturnal animal. It is therefore most likely that 13 to 14 C is the optimum temperature for activity by H. pacificus. DISCUSSION Hoplodactylus pacificus exhibits a circadian rhythm of locomotor activity which can be entrained to light-dark cycles of natural period. The rhythm is manifested clearly in constant conditions of light and temperature by the large, bush-dwelling type of this species, but not by the smaller coastal type in the conditions applied during these experiments. The coastal Type A lizards could not be completely entrained to light-dark cycles of period greater than 24 hours, even though light-dark cycles of period less than 24 hours to as low as 12 hours could produce ostensible entrainment with the same light intensities. This indicates that there is some endogenous control of activity in Type A lizards, but it is probably not as strong as that in bush-dwelling Type B lizards. This could be due to ecological adaptation to different natural light-dark cycle intensities, the bush-dwelling variety have a less marked external time cue than geckos living on relatively open coastal zones. It is tentatively suggested that the properties of activity rhythms could be used as diagnostic features in the determination of the limits of some species. Rhythmic activity in lizards can be easily and cheaply monitored, and can be 122

5 24 HRS Fig. 1. (Type B) Activity in natural light and temperature cycles. SS is sunset (1748) and SR is sunrise (0650). 24 HRS Fig. 2. (Type Activity in constant light intensity (2.0 ± 1.0 lux) and constant temperature (19 C). 123

6 24 HRS ft a temperatu^a9 C). ACtiVily C O n S t a n t light intensity ( 8 -» ± 1.0 lux) and constant 0 ON OFF 22 HRS Fig. 4. (Type A) Activity in LD 11:11 at constant temperature (19 C). 124

7 0 ON OFF 18 HRS Fig. 5. (Type A) Activity in LD 9:9 at constant temperature (19 C). O F F ON O F F ON 24 HRS Fig. 6. (Type A) Activity in LD 6:6 at constant temperature (19 C). 125

8 OFF ON 24 HRS VIM WW Fig. 7. (Type B) Activity in LD 10:10 at constant temperature (19 C). O F F ON 24 HRS Mk^M^ - Fig. 8. (Type A) Activity in LD 13:13 at constant temperature (19 C). A recording failure occurred on Day

9 numerically quantified for statistical analysis (e.g. period; phase relationship to entraining light-dark cycles of various periods; activity to rest ratio; period changes in response to changes in light intensity). For example, the free-running period of Type B H. pacificus ranges from 25 hours to 27 hours, depending on the light intensity. If the free-running period of the underlying clock in Type A lizards were the same, entrainment to light-dark cycles of period greater than 24 hours would be expected. The fact that entrainment occurred only to light-dark cycles of short period suggests a short intrinsic free-running period for Type A H 127

10 pacificus, a period which is probably significantly different from that of Type B animals. Qualitative differences, such as entrainability or otherwise to temperature cycles of various ranges, could be equally useful. In species of gulls, for example, distinctive behaviour patterns are accepted by taxonimists as essential diagnostic features. Rhythmic behaviour patterns might provide equally precise information about populations of New Zealand geckos where other features have proved inadequate for a satisfactory classification of species. ACKNOWLEDGEMENTS The author thanks Dr R.D. Lewis and Assoc. Prof. J. Robb for critical comments on an earlier draft of this report. This work was carried out under the supervision of Dr Lewis at the Zoology Dept., University of Auckland, Auckland 1, New Zealand. REFERENCES Aschoff, J. 1960: Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol. 25: Barden, A. 1942: Activity of the lizard, Cnemidophorus sexlineatus. Ecology 23: Benson, J.A. 1970: "Locomotor activity in the lizard Hoplodactylus pacificus". Zoology IIIB Project, University of Auckland. Doshi, E.C., Huggjns, S.E., & Fitzgerald, J.M. 1975: Circadian rhythms in brain serotonin concentrations of the lizard Anolis carolinensis. Comp. Biochem. Physiol. 5IC: Evans, K.J. 1966: Responses of the locomotor activity rhythms of lizards to simultaneous light and temperature cycles. Comp. Biochem, Physiol. 19: Gelderloos, O.G. 1975: Circadian periodicities and light preference in the desert iguana, Dipsosaurus dorsalis. Chronobiologica, in press. Gelderloos, O.G. 1976: Circadian activity patterns in the desert iguana. Dipsosaurus dorsalis. Physiolog. Zool. 49: Hoffman, K. 1955: Aktivitatsregistrierungen bei frisch geschluften Eidechsen. Z. vergl. Physiol. 37: Hoffman, K. 1957a: Angeborene Tagcspcriodik bei Eidechsen. Naturwiss. 44: Hoffman, K. 1957b: Uber den Einfluss der Temperatur auf die Tagesperiodik bei Poikilofhermen. Naturwiss. 44: 358. Hoffman, K. 1959: Die Aktivitatsperiodik von im 18- und 36-Studen-tag erbruteten Eidechsen. Z. vergl. Physiol. 42: Hoffman, K. 1960: Versuche zur Analyse der Tagesperiodik-1. Der Einfluss der Lichtintensitat. Z. vergl. Physiol. 43: McCann, C. 1955: The lizards of New Zealand. Dominion Museum Bulletin, No. 17. Wellington, New Zealand. Mclvor, I. 1973: Activity and daily breathing patterns of the gecko, Hoplodactvlus pacificus Gray. Mauri Ora 1: Quay, W.B. 1970: Experimental studies on brain 5-hydroxytryptamine and monoamine oxidase in a field population of the lizard Sceloporus occidentalis. Physiol. Zool. 43: Sharrell, R. 1966: The tuatara, lizards and frogs of New Zealand. Collins, London. Taylor, J.L. & Tschirgi, R.D. 1960: Behavioral effects of random temperature cycles in lizards. Fed. Proc. 19: 54. Underwood, H. 1973: Retinal and extraretinal photoreceptors mediate entrainment of the circadian locomotor rhythm in lizards. J. comp. Physiol. 83: Underwood, H. 1975: Extraretinal light receptors can mediate photoperiodic photoreception in the male lizard Anolis carolinensis. J. comp. Physiol. 99: Underwood, H. & Menaker, M. 1970: Extraretinal light perception: Entrainment of the biological clock controlling lizard locomotor activity. Set, 170: Wever, R. 1972: Virtual synchronisation towards the limits of the range of entrainment. J. theor. Biol. 36: