Photoperiodic induction of pupal diapause in Sarcophaga argyrostoma: Temperature effects on circadian resonance

J. Insect Physiof., Vol. 28, No. 4, pp. 305-310, 1982 Printed in Great Britain.

(X322-1910/82/040305~6$03.00/0 0 1982 Pergamon Press Ltd

PHOTOPERIODIC INDUCTION OF PUPAL DIAPAUSE IN SARCOPHAGA ARGYROSTOMA: TEMPERATURE EFFECTS ON CIRCADIAN RESONANCE D. S. SAUNDERS Department of Zoology, West Mains Road, Edinburgh EH9 3JT. U.K. (Receioed 18 August

1981)

Abstract-Larval cultures of the flesh-fly Sarcophaga argyrostoma maintained in circadian ‘resonance’ experiments produced a high incidence of pupal diapause when the period of the light cycle was close to (r) 24.48 or 72 hr, but a low incidence of diapause at T 36, 60 or 84 hr. Cultures pre-programmed for diapause by exposing pregnant females to long nights indicated the induction of non-diapause development at T 36.60 and 84, whereas cultures pre-programmed for diapause-free development by exposing females to continuous light indicated the induction of diapause at T 24, 48 and 72. Raising the temperature reduced the heights of the diapause peaks whereas lowering the temperature raised them. With progeny from long-night-reared flies the lowest temperature tested (18°C) produced a result indistinguishable from an ‘hour-glass’ response, warning that ‘negative’ resonance experiments may merely indicate non-permissive conditions for demonstrating the involvement of circadian rhythmi-

city in insect photoperiodism. The results of the ‘resonance’ experiments and the effects of temperature are interpreted in terms of a multioscillator ‘external coincidence-photoperiodic counter’ model for the clock. Key Word Index: Photoperiodism, pupal diapause, circadian clock, resonance, Sarcophaga argyros-

INTRODUCHON THE USE of daylength or nightlength for the control of seasonally appropriate developmental or reproductive strategies (photoperiodism) is widespread in animals and plants, particularly those in temperate, terrestrial environments. In a very wide phylogenetic range (plants, arthropods, vertebrates) such photoperiodic induction is known to be a function of the circadian system (FOLLETTand FOLLETT,1981) although, in a few organisms, notably the aphid Megoura viciae (LEES,1973) and the lizard Anolis carolinensis (UNDERWOOD. 1981), night or daylength appears to be measured by a nonoscillatory ‘hour-glass’. The most useful experiments to discriminate between circadian and hour-glass clocks are those in which different groups of animals are exposed to short light periods (say 8 or 12 hr) coupled with variable amounts of darkness to give a series of ‘environmental’ light cycles (of period T) from, say, 18-72 hr or more. These experiments are generally known as ‘resonance’ or Nanda-Hamner protocols (HAMNER, 1960; RTTENDRIGH,1981a; SAUNDERS,l981a). If, as in M. oiciae, short-night responses are obtained until the variable period of darkness exceeds a critical value, the photoperiodic clock is considered to be an hour glass (LEES,1973). On the other hand, if short-day or long-night responses (e.g. diapause, avian testis regression, or flowering in short-day plants) are obtained at cycle lengths close to 24, 48 and 72 hr, but an opposite response (e.g. non-diapause development, testis growth etc.) at T 12, 36 and 60 hr, the conclusion is that the mechanism of time measurement is a function of the circadian system, although quite how circadian rhythmicity is involved is not revealed. In the best

examples of photoperiodic resonance, numerous T-values in small incremental steps are used, and these record the circadian periodicity (7) of the photoperiodic oscillator. In Sarcophaga argyrostoma, for example, 7 is close to 24 hr (SAUNDERS,1973). in Pieris brassicae it is close to 21 hr (CLARET, in DUMORTIER and BRUNNARIUS,1981) and in the spider mite, Tetrunychus urticae, it is close to 20 hr (VEERMAN and VAZ NUNES, 1980). The distinction between an hour-glass and a circadian response is not always so clear, however. In the flesh fly Sarcophaga argyrostoma (SAUNDERS, 1973) and the fruit fly Drosophila aurariu (PITENDRIGH, 1981a), circadian resonance is obtained at higher temperature, but if the temperature is lowered (to lS-16”C), results similar to those in M. uiciae (LEES, 1973) are obtained. For this reason, the present paper examines the effects of temperature on the resonance response in S. argyrostoma in greater detail.

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MATERIALS

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Adults of S. argyrostoma were maintained at 25°C in either continuous light (LL) or 12 hr of light followed by 12 hr of darkness (LD 12:12). They were provided with a piece of fresh meat daily and water and sugar ad lib&urn. Since this species is ovoviviparous and the embryos within the maternal uterus are particularly sensitive to photoperiod (DENLINGER, 1971; SAUNDERS,1980), adults in LL deposit larvae on the meat which are ‘pre-programmed’ for uninterrupted or diapause-free development, whereas those in LD 12: 12 deposit larvae ‘pre-programmed’ for dia-

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pause in the pupal instar. Larvae produced by both of

these stocks were set up in light-proof cabinets in ‘resonance’ experiments consisting of 12-hr light pulses repeated in light/dark cycles from T 18 to 87 hr (i.e. LD 12:6-12:75) at a range of constant temperatures from 14 to 28°C (*OS’ or less) in 2°C steps. Lights were provided by Philips 4W fluorescent tubes, water-jacketted to minimise temperature fluctuations within the cabinets. Light cycles were controlled by Venner time-switches. Larvae were fed during the experiments on meat supplemented by an agar/dried milk/yeast medium; each culture contained about 20&500 insects. Fullyfed mature larvae were allowed to disperse into dry sawdust for puparium-formation. At puparium formation the larval ‘sensitive period’ was considered to have ended (SAUNDERS,1979) and the puparia were sifted out and stored in the dark at 1%19°C. Ten to 14 days after puparium formation the puparia were opened to ascertain whether they contained pigmented developing pharate adults, or unpigmented diapausing pupae (FRAENKELand HSIAO, 1968). For each experimental group the data were calculated as a percentage entering diapause.

to an overall decline in diapause incidence as T increases, although with the exception of the 18°C panel, this decline is slight. (6) As the temperature rises, the overall proportion of the pupae entering diapause falls. For cultures derived from LL females, 3 resonance peaks were still evident at 2o”C, but were extinguished at 22°C (data not shown). At the lowest temperature tested (14°C) resonance was still apparent. For the cultures derived from LD females circadian resonance with 3 clear peaks was seen at 28°C. At 18”C, however, a result indistinguishable from a “Megoura-like” hour-glass response was obtained.

DISCUSSION Before proceeding with a discussion of the present results it is necessary to recall current views of the

RESULTS The results of the resonance experiments are summarised in Fig. 1. Since they contain such an abundance of information, the results are item&d below: (1) The incidence of pupal diapause in the LL to DD controls (left-hand panels) was zero at all temperatures from 14 to 20°C. For the LD 12:12 to DD controls (right-hand panels), however, diapause incidence was high (99-95%) until 24”C, then fell to 41% at 26°C and to 18% at 28°C. These results parallel those described earlier (SAUNDERS,1980). (2) All resonance curves show a low incidence of diapause at T 18 and T 21 but then a sharp rise to a peak at T 24-27. This response illustrates the induction of diapause once the night length exceeds its critical value ( -9$ hr) (SAUNDERS,1973). (3) The intervals between successive diapause peaks, or between successive non-dipause troughs, are close to 24 hr in all cases. This suggests that the circadian period (T) for that part of the system involved in photoperiodism is, like that for the eclosion rhythm (SAUNDERS,1976a, 1979), close to 24 hr. (4) For those cultures derived from LL-bred females (left-hand panels) pupal diapause is induced at T-values of 24-30, 48-54 and 72-78 hr, but not, or less so, at T 36-42, 60-66 and 84. On the other hand, circadian resonance in the cultures derived from LD-bred females (right-hand panels) appears to result from an induction of non-diapause development at T 36, 60 and 84, whereas diapause incidence remains high at T 24, 48 and 72. (5) The data also suggest that for cultures derived from LD-bred females (right-hand panels), induction of non-diapause development is strongest at T 36, weaker at T 60, and weakest at T 84, giving rise to an overall increase in diapause as T increases from 36 to 84. For cultures derived from LL-bred females (lefthand panels), however, diapause induction appears to be strongest at T 24 and weakest at T 72, giving rise

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Fig. 1. The incidence of pupal diapause in cultures of the flesh fly Sarcophaga argyrostoma raised as larvae in ‘resonance’ experiments and at a series of constant temperatures (14-28°C). L&hand panels: larvae deposited by female flies kept in continuous light (LL), 25”C, and then moved to resonance protocols at the temperature indicated. Right-hand panels: larvae deposited by female flies kept in long nights (LD 12:12), 25°C. and then moved to resonance protocols at different temperatures. T-period, in hr, of the experimental light cycle which consisted of 12 hr of light and a variable amount of darkness (i.e. LD 12:x) repeated throughout the larval sensitive period. The incidence of pupal diapause in the control groups (continuous darkness following embryonic pretreatment) was zero in left-hand panels, but is indicated by horizontal dotted lines in the right-hand panels.

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induction of pupal diapause in Sarcophaga argyrostoma

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photoperiodic clock in S. argyrostoma. The clock lished observations). However, a multi-oscillator model adopted as a working hypothesis in this species external coincidence clock has still to be distinguished is a multi-oscillator version (SAUNDERS,1978; unpubunequivocally from other theoretical possibilities, lished observation) of ‘external coincidence’ (PIT-TEN- such as ‘internal coincidence’ (PITTENDRIGH,1972, DRIGH, 1966, 1972). In its original form Pittendrigh’s 1981b), but is consistent with most of the experimenmodel consists of a circadian oscillation or pacemaker tal data obtained for S. argyrostom and, therefore, phase set by the light/dark cycle in such a way that a will be followed in this paper. hypothetical photo-inducible phase (&) falls in the In S. argyrostomo and a number of other species light of a long-day (short-night) regime, but in the (see SAUNDERS,1981a for review) the clock is seen as a dark of a short-day (long-night) regime. The illuminadevice which discriminates qualitatively between long tion or non-illumination of 4i thus controls the switch and short nights by some aspect of the entrained cirfrom one seasonal pathway to the other. Since the cadian system. In addition there appears to be a quanoscillation is always reset to a characteristic phase titative clock-related process, called the ‘photoperio(called Circadian time, Ct. 12) at the end of a light die counter’ (SAUNDERS,1971. 1981a) which accumuperiod longer than about 10 hr (PITTENDRIGH,1966; lates (‘adds up’) successive long nights to a hypothetiSAUNDERS,1981b), the clock effectively measures night cal internal threshold controlling the release or retenlength. In such cicles, the photo-inducible phase tion of prothoracicotropic hormone (PTTH) in the centres about 9f hr (the ‘critical night length’) after pupal brain. Whilst the rate of development and hence the duration of the embryonic and larval dusk. ‘sensitive period’ is temperature dependent, the acThe circadian system of a complex multicellular organism, such as a fly, is undoubtedly composed of cumulation of long nights is, like the periodicity of the many oscillations (PITTENDRIGH,1974). Some of these circadian ‘clockwork’, temperature compensated. The are temperature-compensated, light-sensitive, circainteraction between these two components with differdian pacemakers. Others are ‘slave’ oscillations, probent temperature coefficients then gives rise to the wellably not temperature-compensated or light-sensitive, known temperature effect on diapause induction but receiving their temporal organisation via entrain(SAUNDERS.1981a). For example, at higher temperament to the pacemakers. In turn, the slaves control tures the sensitive period is too short to accommodate overt rhythmicity such as pupal eclosion (PII-IENthe required number of long nights so that diapause DRIGH, 1981a). Although nothing is known about incidence is low or zero, whereas at lower temperapacemaker-slave interactions in S. argyrostoma, it is tures, a sufficient number of long nights are seen probable that such an organisation exists in this spebefore puparium formation cuts short the protracted cies, and that c#+is part of a slave rather than a pacesensitive period, and diapause incidence is high. At intermediate temperatures the first larvae to pupate maker (SAUNDERS,1976b). A number of oscillations, develop to the adult instar without arrest, whilst those perhaps even a high number of oscillations (both which pupate later enter diapause (SAUNDERS,1971). pacemakers and slaves), are probably therefore involved in the photoperiodic clock. A multi-oscillaThe present results will be interpreted in terms of this dual ‘external coincidence-photoperiodic counter’ tor view of the clock not only brings it into line with current concepts of the circadian system (PITTEN- model. DRIGH, 1974). but also accounts for such features as Circadian resonance the drop in diapause incidence in very short and ecoFigures 2 and 3 concern the interpretation of circalogically unnatural photoperiods (SAUNDERS,unpub-

T, h

Fig. 2. Sarcophaga argyrostom: Computed phase relationship of the photo-inducible phase (&) to the light cycle in ‘resonance’ experiments. PI, P2 and P,--three consecutive 12 hr light pulses covering a range of cycle lengths from T 18 (LD 12:6) to T 84 (LD 12:72). A, B and C-the primary, secondary and tertiary ranges of entrainment of the Sarcophaga circadian pacemaker. Within each range of entrainment, +i falls in the light with the shorter values of T (20-22, 44-46, and 68-70) and then passes into dark.

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T, hours Fig. 3. Sarcophaga nrgyrostoma: An explanation for the rhythmic incidence of diapause in ‘resonance’ experiments. Open circles and heavy solid and broken linesdiapause incidence. Closed circles and thin line-arrhythmicity within populations of enclosing Sarcophaga adults (data from SAUNDERS, 1978) in similar light regimes. A, B and C--the primary, secondary and tertiary ranges of entrainment of the Sarcophaga pacemaker (taken from Fig. 2). a, b and c--the T-values at which 6i passes from light to

dark. Within each range of entrainment, where rhythmicity is high (low R-values), diapause incidence is first low when $+ coincides with the light; it then rises steeply to the peak as 4i passes into the dark. Diapause incidence then drops between the peaks as arrhythmicity multi-oscillator circadian system.

dian resonance in terms of the multi-oscillator ‘external coincidence’ model. Using the circadian rhythm of pupal eclosion in S. argyrostoma as an overt indicator rhythm (SAUNDERS, 1976a, 1978) it is possible to compute, from the phase response curve for 12-hr light pulses, the ranges of entrainment of the circadian pacemaker to light cycles from LD 12:6 (T 18) to LD 12:72 (T 84) (Fig. 2). Although the hypothetical photo-inducible phase (tii) is probably part of a driven slave, and the phase relationship (J/) between the pacemaker and slave will change with T (PITTENDRIGH,1981b), it is necessary to plot $ of #i as though the latter were part of the pacemaker since we know little or nothing about pacemaker-slave relationships in S. argyrostoma. Nevertheless, there are clearly 3 ranges of entrainment for the pacemaker to the light cycle: the primary range from T 20 to T 29, the secondary range from T 44 to T 53, and the tertiary range from T 68 to T 77. Between each of these ranges, entrainment of the pacemaker to the light is impossible and a steady-state value for the phase relationship of & cannot be computed. In each of the 3 ranges of entrainment & lies in the light at the shorter T-values (T 20-22, T 4446, and T 68-70) but then passes into the dark at T 23-29, T 47-53, and at T 71-77. Figure 3 compares diapause induction in a resonance experiment (Fig. 1; 16”C, larvae from LLfemales) with both the computed ranges of entrainment (from Fig. 2), and with a measure of population arrhythmicity (the R-values of WINFREE, 1970) observed among flies emerging from their puparia in similar light regimes (SAUNDERS,1978). As before, therefore, pupal eclosion is being used as an overt indication of the state of the photoperiodic clock. Three vertical lines (a, b and c) have been added to

rises (high R-values) within the

Fig. 3 to show the T-values at which di is computed to pass from the light into the dark. Within each of the 3 ranges of entrainment (A, B and C) diapause incidence is initially low, but then rises steeply as pi moves from light to dark: this is in accordance with ‘external coincidence’. It can also be seen that within the ranges of entrainment, pupal-eclosion behaviour is highly rhythmic (low R-values). Assuming that coherent eclosion peaks reflect a similar coherence among the constituent oscillators of the photoperiodic clock, it may be supposed that discrimination between short and long nights (illumination or nonillumination of 4i) is most effective when the multioscillator system is stably entrained (SAUNDERS, unpublished). Between each of the 3 ranges of entrainment, however, arrhythmicity or R-values rise sharply to maxima at T 36,60 and 84, and this rise is correlated with a fall in diapause incidence. This is consistent with the idea (SAUNDERS, unpublished) that incoherence within the multi-oscillator clock reduces the efficiency of night-length measurement. The illumination or nonillumination of a photo-inducible phase, coupled with the coherence or incoherence within a population of oscillators, can thus explain the rhythmic induction of diapause in resonance experiments. Induction of diapause or development?

Since diapause incidence in the control (DD) cultures derived from LD-females (Fig. 1, right-hand panels) is high, whereas diapause inciden& in control cultures from LL-bred females is zero (Fig. 1, lefthand panels), it is evident that certain light cycles (T 36-42, T 60-66, and T 84 + ) are development inductive, whereas others (T 24-30, T 48-54, and T 72-78) are diapause inductive. In terms of the external co-

Photoperiodic

induction of pupal diapause in Sarcophaga argyrostotna

incidence model, these observations imply that illumination and non-illumination of I#+are both to be regarded as ‘inductive’ events, although these data cannot distinguish a ‘positive’ induction in a physiological sense from a removal of inhibition. However, since pupal diapause in S. argyrostoma is an evolved character which enables the species to pass the winter, and is only induced in the autumn months after a certain number of long nights, it seems likely that long-night measurement and the induction of diapause is a ‘real’ physiological phenomenon involving the accumulation of a product which eventually prevents PTTH production (SAUNDERS,1981a). Therefore, the effect of light coinciding with $i, is possibly an inhibition or active reversal of this product accumulation, allowing PTTH secretion to proceed. Promotion of non-diapause development by a coincidence of light with rbi is a part of the model in its original form (PITTENDRIGH,1966), and the declining strength of this response between T 36 and T 84 (Fig. 1, right-hand panels) is presumably correlated with a declining number of such coincidences within the sensitive period of the insects. The diapause-promoting effects of non-illumination of fji (i.e. long nights), on the other hand, does not feature in the original version of the model, but is a component of the photoperiodic counter hypothesis (see above; SAUNDERS,1971, 1981a). The present data show that the amplitude of the 3 diapause peaks shows little decline as T increases (with the exception of the third peak at 18”C, left-hand panels), suggesting that the photoperiodic system of which (bi is part free-runs in the extended night. The effects of temperature

Constant temperature could effect diapause induction in a number of ways. For example, high temperature might directly promote the rate of synthesis and/or release of neurohormones. Alternatively temperature might operate indirectly via an interaction between the temperature-dependent sensitive period and the temperature-compensated accumulation of long nights, as in the counter hypothesis. It is also likely that temperature alters the phase relationships between circadian pacemakers and their driven slaves (PIT-IENDRIGH,1981a, b), and these altered phase relationships may be important at the level of the clock. Here, however, the effects of constant temperature will be interpreted in terms of the first two possibilities. The data in Fig. 1 show a systematic decline in the amplitude of the 3 diapause peaks (T 24-30; T 48-54; T 72-78) as the temperature rises. Whilst much of this might be attributed to direct temperature effects (see above), it is also likely that the larval sensitive period shortens with temperature, causing a drop in the number of long nights experienced before puparium formation and a consequent reduction in diapause. There are apparently two reasons for the drop in diapause between the diapause peaks. The first (at T 3342; T 57-66; T 81-84) may be associated with rising arrhythmicity within the clock; the second (at T 4245 and T 66-69) may be attributed to coincidences between & and light (Fig. 3). The resonance curves show that the non-diapause troughs progressively fill up as the temperature falls. For coincidences

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between di and light one might have predicted a greater induction (i.e. lower diapause) as the temperature fell because an increase in the larval sensitive period would allow an increased experience of short nights. However, whilst the accumulation of long nights is a temperature-compensated process (SAUNDERS, 1971,1981a), evidence suggest that the accumulation of short nights is not (CHESTER and SAUNDERS, unpublished observations). For this reason, unlike long-night accumulation, the interaction between short nights and the sensitive period would be essentially neutral, allowing non-diapause development to be over ridden by direct temperature effects as the temperature falls. Hence the non-diapause troughs gradually fill in, first at T 81-84, then at T 5749, and finally at T 3345, until a result indistinguishable from an ‘hour-glass’ response (18’C, right-hand panel) is obtained. The caveat inherent in this and earlier reports (SAUNDERS,1973; PITTENDRIGH,198Ia, b) is that the finding of an ‘hour-glass’ response in resonance experiments is no evidence that circadian oscillations are not involved in photoperiodism: it may merely mean that ‘non-permissive’ conditions have been used. In resonance experiments generally a large number of different T-cycles should be used. For example, if only cycles of T 24, 36, 48, 60 and 72 hr are investigated, peaks and troughs may be be ‘missed’, particularly if the period of the photoperiodic oscillator is not close to 24 hr. In addition it is essential to conduct experiments with poikilotherms at more than one temperature. Initial experiments with S. argyrostoma (SAUNDERS,1973) and with Drosophila auraria (PI~NDRIGH, 1981a, b) for example, indicated ‘hour-glass’ clocks, but circadian rhythmicity was revealed when the temperature was raised. In the aphid Megoura oiciae, however, raising the temperature did not evoke rhythmicity (LEES, personal communication); this species therefore remains the strongest case for ‘hour-glass’ timing. Acknowledgements-Thanks are due to KATHLEENROTHfor technical assistance, and to the Nuffield Foundation and the Science Research Council for financial support. The computer programme for calculating entrainment of the Sarcophaga pacemaker to the experimental light cycles was written by JOHN DEAG and further developed by STEPHENGOLDSON. WELL

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