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Diel oxygen uptake rhythms in diapausing pupae of Pieris brassicae and Papilio machaon
J. Insect Phvsiol.. Vol. 25, pp. 647 to 652. Pergamon Press Ltd. 1979 Printed in Great Britain.
DIEL OXYGEN UPTAKE RHYTHMS IN DIAPAUSING PUPAE OF PZERZS BRASSZCAE AND Z’APZLZOMACHAON A. J. G. CROZIER Department of Zoology. University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN. Scotland (Received 22 September 1978; revised 19 April 1979) Abstract-Die1
rhythms of oxygen uptake are described for P. brassicae and P. machaon. The rhythms are bimodal in both species at lO”C, with a main midday peak, a smaller peak in the afternoon or early evening and low nocturnal uptake rates under natural and artificial (LD 9:15) light regimes. In P. brussicae, the rhythm of oxygen uptake is linked with a die1 rhythm of the incidence of short-term oxygen uptake cycles. Summated batch curves for both species contain significant elements of individual variation. In P. machaon, the timing of daily peaks in oxygen uptake is related directly to the level of metabolism. Kev Word Index: Diaoause. Pieris brassicae. Paoilio machaon, die1 rhythm. supradian cycles. oxygen . uptake, individual variafion
one-hourly intervals and converted ~1hr-l g-l live weight at STP.
INTRODUCTION metabolism remains relatively diapause (WIGGLESWORTH, 1950; BUCK, 1962; BUCK and KEISTER, 1974), some diapausing species display systematic oxygen uptake cycles between 1 hr and 14 days (BECK, 1964; DENLINGERet al., 1972; HAYESet al., 1972; CROZIER, 1979) Die1 oxygen uptake rhythms occur in many non-diapausing insects (BECK, 1964; RICHARDSand HALBERG, 1964; STLJSSI, 1972; ULAN~~KI and MCCIFFETT,1972; BELCHERand BRETT,1973, CHIBAet al., 1973; NASCIMENTO,1973; BANKS et al., 1975), but amongst diapausing species are known only for Laspeyresia (HAYES et al., 1972) and Ostrinia (BECK, 1964). Die1 rhythms of oxygen uptake in diapausing species are notable because they are not obviously related to feeding or activity cycles (BRADY, 1974). This paper describes similar die1 oxygen uptake rhythms for Pieris brassicae and P. machaon, and gives details of the influence of individual variation on the average curves for groups of pupae. The possibility of linkage between the die1 rhythm and other forms of systematic variation is also considered.
to units
of
ALTHOUGH general
constant
during
MATERIALS
AND METHODS
Diapausing pupae of Pieris brassicae L. and Papilio machaon L. were obtained from a commercial breeder, and pupae of P. brassicae were also reared from ova in an artificial light regime (LD 9: 15) at 20+ 3°C. All pupae were kept under the same conditions (light: 9: 15 LD or natural) as in the experiments, and allowed to equilibrate at the experimental conditions for at least 1 week before any measurements took place. The term batch refers to a group of pupae reared at the same time and used for one set of experiments; it therefore denotes their temporal rather than genetic homogeneity. Oxygen uptake was measured using a Scholander constant pressure microrespirometer (SCH~LANDER, 1942) with 20% potassium hydroxide as the carbon dioxide absorbent. Readings were taken at 30 min or 641
RESULTS The diel rhythm in P. brassicae
Similar die1 rhythms of oxygen uptake were recorded for individual pupae and batches of pupae at 5, 10, 15 and 20°C. Examples of average curves for individual pupae at 10°C and for batches of pupae at 5, 10 and 20°C are shown in Fig. la and b, but the data are incomplete since nocturnal hourly uptake rates are represented by a mean uptake rate taken from values for total uptake between working hours. In general, pupae displayed a major midday peak, a smaller peak in the afternoon or early evening and low nocturnal rates. Peaks in oxygen uptake were generally ‘sharper’ for individual pupae, and were up to 3 times the mean nocturnal uptake rates. A clear bimodal rhythm of oxygen uptake with delayed ‘dawn’ and ‘dusk’ peaks can be seen in the average batch curve of Fig. lc. Nocturnal values were obtained by varying the recording period over 24 hr and the average curve therefore includes data-missing intervals. Complete die1 average curves for the individual pupae from the same batch (D) were obtained by the same method, but were smoothed with a 3-point moving average (Fig. 2). This was because these particular pupae had low uptake rates, associated with a fall in metabolism preceding postdiapause development (CROZIER,1979). This suggests an increased experimental error. The ‘smoothed’ curves in Fig. 2 are highly significant (PC O.OOl), when the distribution of phases of length d is tested (KENDALLand STUART, 1966; CROZIER.1979).
The curves in Fig. 2 suggest that the die1 variation in pupae of the same age under the same experimental conditions can vary considerably. Further evidence for this is supplied by the data for more pupae from the same batch in Fig. 3. In contrast to the smooth, bimodal rhythm shown by the whole batch in Fig. lc,
A. J. G.
648
CROZIER
(a)
(b)
2or
3
9
12
45
h
18
21
TIME
0
3
6
TIME
24
(
( Hours
la
12
15
,A8
21
9
Hours
9
5Oc
18
21
24
GMT )
15
24
3
GMT )
Fig.
1. Die1 average curves of oxygen uptake in P. brassicae. Solid bars on the x-axis represent the scotophase, and the letter in each example refers to the number of the batch (as defined in the Materials and Methods section). N, mean overnight values. Differences between means are significant (P
individual pupae have curves with between 3 and 5 peaks of variable size and timing. No significant correlation (P> 0.05) was found between the number or timing of uptake peaks and the general bvel of metabotism. In every case, however, maximal uptake rates in the unsmoothed data occurred between 900 and 1900 hr GMT in two groups (900-12,00 1500-1900 hr; PcO.05) corresponding to the two peaks in Fig. ic. Single day records’ of oxygen uptake for pupae undergoing post~iapau~ development (when mean QO, X50 $0, hr- l g-r) matched the shape of the average curves in Fig. 1. However, although maximal daily uptake rates during stable diapause generally occur around midday, the nature of die1 variation in single day records is disguised by the presence of shortterm (supradian) cycles of oxygen demand (CROZIER, 1979). One would not expect the supradian cycles to
show linkage with the die1 uptake rhythm, because their frequency is metabolism-dependent. (CROZIER, 1979). However, the recorded incidence of maxima and minima during individual supradian cycles, recorded over the course of several months to avoid short-term serial correlation, showed peaks during the 24-hr cycle at intervals of about 3 hr at 10°C. Examples for a single pupa and a batch of pupae are shown in Fig. 4. Disturbance of the pupae may give rise to the observed die1 rhythm, but there was no si~ifi~ant trend (P> 0.90) in the initial readings of experiments started at various times during the 24-hr period. However, experiments started at a particular time of the day showed significant trends (P
Oxygen uptake in Pieris brassicaeand Papiliomachaon
649
‘b.
3 ?
0
6
12
18
24
0
6
12
18
24
,
1
I
ti
I
TIME
( Hours
I
!
GMT1
Fig. 2. Two examples of complete die1average curves for individual pupae of P. brassicueat WC. -IJ-,
mean hourly uptake rates (raw data), -O-
The variation in the average curves of Figs. 2 and 3 may be affected by the rhythm in the incidence of supradian cycles (Fig. 4). However, the recorded intervals between peaks in Figs. 2 and 3 are significantly different (P
data smoothed with a 3-point moving average.
frequency/24 hr = 1.99 + 0.88 (mean QO,); PcO.025) Unlike P. brassicae, the incidence of supradian cycles is not linked with the die1 uptake rhythm and shows no significant deviation (P > 0.10) from the expected even distribution arising from a random drift in cycles over 24 hr. Longer-term systematic variation in oxygen uptake was detected by smoothing single day records for individual pupae with a 3-point moving average (Fig. 6). The timing of daily peaks varied between 1000 and 1800 hr GMT. There was no obvious trend in the timing of successive daily peaks (e.g. a steady drift), but the timing of a daily peak was related directly to its size (Fig. 6). The data of Fig. 6 therefore, suggests that the apparently stable midday peak in average curves contains an element that is metallic-de~ndent.
Similar die1 average curves of oxygen uptake were found for the pupae of P. muchaon. Incomplete curves for a single pupa and a batch of four pupae are shown in Fig. 5. Like P. brassicae, the average curves include a main midday peak with a possible peak in the early DISCUSSION evening and low nocturnal rates. Short-term cycles of oxygen uptake lasting 1-6 hr, Diet average curves for both species (Figs. 1 and 5) similar to those found for P. brussicae (CROZIER, display delayed ‘dawn’ and ‘dusk’ peaks similar to 1979), were recorded and their frequency shown to be those present in some insect circadian rhythms related directly to metabolism at 10°C (cycle (BRADY, 1974), but contain significant elements of
650
A. J. G. CROZER
TIME ( Hours
GMT )
Fig 3. Smoothed average curves at 10°C for 8 pupae of P. brassicae from the same batch as the pupae in Fig. 2.
individual variation (Figs. 2,3 and 6). If these rhythms are in fact circadian, then the observed variation may reflect phase relatjonship changes between physiological responses driven by an oscillator, and not an inherent instability in the circadian system. Alternatively, individual responses may be similar in nature to recent ‘spontaneous feedback oscillation’ models of circadian systems (HASTINGSand TYSON, 1975; SEL’KOV,1975; WINFREE,1975a). Although their batch curves are similar (Figs. 1 and S), there are significant differences between P.brassicae and P. machaon. In P. brassicae, maximal daily oxygen uptake occurs around midday, and suggests that the associated die1 rhythm of supradian cycles (Fig. 4) may be due to the increased probability of bursts at this time. Maximal daily uptake rates in P. machaon, however, vary in their timing and are not linked with a die1 rhythm of supradian cycles. The temporal dependence of daily maxima on metabolism (Fig. 6) suggests some form of involvement with longer-term variation. Infradian oxygen uptake cycles of 3-5 days, similar to those observed for P. brussicue
(CROZIER,in press), have been recorded in P. machaon (unpublished data). The functional significance of these die1 rhythms is obscure, particularly as the diurnal maxima will theoretically increase metabolism and water loss under natural temperature conditions. There are at least four possibilities; the rhythms may reflect the general circadian organization that is apparently necessary for homoeostasis in eukaryotes (SAUNDERS, 1977); their significance may relate to a limited number of events in the animal’s lifetime such as eclosion (WJNFREE, 1975b); or they may influence factors affecting survival such as water loss across the spiracles. Alternatively, they may be involved in the control of diapause development, possibly through some kind of ‘coincidence model’ mechanism (F’ITTENDRIGH,1966, 1972; PITTENDRJGHand MINIS, 1964; SAUNDERS, 1978). In addition, interaction between a circadian oscillator and metabolismdependent infradian cycles (CROZIER, 1979) could operate a long-term ‘counter’ system that could be affected by temperature.
651
Oxygen uptake in Pieris brussicae and Pupilio machaon
60
3
6
9 TIME
12 ( Hours
15
18
21
24
GMT)
Fig. 4. Die1 rhythm of supradian cycles in P. brassicae at 10°C. Each histogram bar records the proportion (%) of hourly oxygen uptake readings within a given hour-class, which were maxima (upright bars) or minima (inverted bars) during the course of individual supradian oxygen uptake cycles. Upper record, single pupa (P
A
‘p 9
1:
15
18
)
12
15
18
6 15
l
y: 33 0 -161x .
. .
10
.
. .
.N 5
\
9
12 TIME
15
I Hours
18
.
. .
l
.
. .
12
'5
TIME (Hours
18
GMT)
GMT1
Fig. 5. Die1 average curves of oxygen uptake for a single pupa (A) and a batch of P. machaon pupae (B, n = 4) at 1OC. Differences between means are significant (P
Fig. 6. A. Some examples of smoothed single day records of oxygen uptake for individual pupae of P. machaon at 10°C. B. The relationship betwe& the amplitude of maximal hourly readings in single day records and their time of occurrence. The linear plot is significant (PcO.05).
A. J. G. CROZIER
652
Acknowledgements-I should like to thank Dr. D. F. HOULIHANfor all his help and advice, and also the Science Research Council for financial assistance. REFERENCES BANKSW. M., BRUCEA. S. and PEARTH. T. (1975) The effects of temperature, sex and circadian rhythm on the oxygen uptake of two swcies of cockroaches. Coma. Biichem. >h_v.viol52A, 223-228. BECK S. D. (1964) Time measurement in insect photoperiodism. Am. Nat. 98, 329-346. BELCHER K. S. and BRETTW. J. (1973) Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster. J. Insect Physiol. 19, 271-286.
BRADYJ. (1974) The physiology of insect circadian rhythms. Adv. Insect Physiol. 10, l-l 15. BUCKJ. (1962) Some physical aspects of insect respiration. A. Rev. Ent. I, 27-56. BUCK J.
and KEISTERM. (1974) Respiration: some exogenous and endogenous effects on the rate of respiration. In The Physiology of Insecta (Ed. by ROCKSTEINM.) Vol. 6 Academic Press, London. CHIBA,Y., CUTKOMPL. K. and HALBERGF. (1973) Circadian oxygen consumption rhythm of the flour beetle, Tribolium confusum. J. Insect Physiol. 19, 2163-2172.
A. J. G. (1979) Supradian and infradian cycles of oxygen uptake in diapausing pupae of Pieris brassicae. J.
CROZIER
Insect Physiol. 25, 575-582.
DENLINGERD. L.. WILLISJ. H. and FRAENKELG. (1972) Rates and cycles of oxygen consumption during pupal diapause in Sarcophaga fleshflies. J. Insect Physiol. 18, 871-882.
HASTINGS J. W. and TYSONS. (1975) Oscillations in a negative feedback cellular control system. Biophys. J. 15, 179a. HAYESD. K., HORTONJ., SCHECTER M. S. and HALBERGF. (1972) Rhythm of oxygen uptake in diapausing larvae of the codling moth at several temperatures. Ann. ent. SOC. Am. 65, 93-96.
KENDALLM. G. and STUARTA. (1966) The Advanced Theory of Statistics. Vol. 3, Design & Analysis & Time Series. Griffin, London. NASCIMENTO I. A. (1973) Variacoes no consumo de oxigenio una praga de coqueiro, Homalinotus coriaceus Gyll.-Biol. Zool. Biol. Mar. (Sao Paula). 30. 161-183. PITTENDRIGHC. S: (1966) T%e circadian oscillation in Drosophila pseudoobscura pupae: a model for the photoperiodic clock. Z. Pjlanzenphysiol. 54, 275-307. PITTENDRIGHC. S. (1972) Circadian surfaces and the diversity of possiblk roles of circadian organization in photoperiodic induction. Proc. natn. Acad. Sci. U.S.A. 69, 2734-2737. PITTENDRIGH C.S. and MINISD. H. (1964) The entrainment of circadian oscillations by light and their role as photoperiodic clocks. Am. Nat. 98, 261-294. RICHARDSA. G. and HALBERGF. (1964) Oxygen uptake rhythms in a cockroach gauged by variance spectra. Experientia 20, 40-42.
SAUNDERSD. S. (1977) An Introduction to Biological Rhythms. Blackie, Glasgow. SAUNDERS D. S. (1978) Internal and external coincidence and the apparent diversity of photoperiodic clocks in insects. J. camp. Physiol. 127A, 197-207. SCHOLANDER P. F. (1942) Volumetric respirometers. Rev. scient. Instrum. 13, 32-33.
SEL’KOVE. (1975) Stabilization of energy change, generation of oscillations and multiple steady states in energy metabolism as a result of purely stoichiometric regulation. Eur. J. Biochem. 59, 151-157.
STLJSSIT. (1972) L’heterothermie
de I’abielle. Archs. Sci.
physiol. 26, 131-139.
ULANOSKIJ. T. and MCDIFFETTW. F. (1972) Diurnal respiration in mayfly nymphs. Physiol. Zoo/. 45, 97-105. WIGGLESWORTH V. B. (1950) The Princioles of Insect ’ Physiology. 4th Edition, Meihuen, Londoh. WINFREEA. T. (1975a) Unclocklike behaviour of biological clocks. Nature, Lond. 253, 315-319. WINFREEA. T. (1975b) Unclocklike behaviour of a biological clock. Biophys. J. 15, 119d.
DIEL OXYGEN UPTAKE RHYTHMS IN DIAPAUSING PUPAE OF PZERZS BRASSZCAE AND Z’APZLZOMACHAON A. J. G. CROZIER Department of Zoology. University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN. Scotland (Received 22 September 1978; revised 19 April 1979) Abstract-Die1
rhythms of oxygen uptake are described for P. brassicae and P. machaon. The rhythms are bimodal in both species at lO”C, with a main midday peak, a smaller peak in the afternoon or early evening and low nocturnal uptake rates under natural and artificial (LD 9:15) light regimes. In P. brussicae, the rhythm of oxygen uptake is linked with a die1 rhythm of the incidence of short-term oxygen uptake cycles. Summated batch curves for both species contain significant elements of individual variation. In P. machaon, the timing of daily peaks in oxygen uptake is related directly to the level of metabolism. Kev Word Index: Diaoause. Pieris brassicae. Paoilio machaon, die1 rhythm. supradian cycles. oxygen . uptake, individual variafion
one-hourly intervals and converted ~1hr-l g-l live weight at STP.
INTRODUCTION metabolism remains relatively diapause (WIGGLESWORTH, 1950; BUCK, 1962; BUCK and KEISTER, 1974), some diapausing species display systematic oxygen uptake cycles between 1 hr and 14 days (BECK, 1964; DENLINGERet al., 1972; HAYESet al., 1972; CROZIER, 1979) Die1 oxygen uptake rhythms occur in many non-diapausing insects (BECK, 1964; RICHARDSand HALBERG, 1964; STLJSSI, 1972; ULAN~~KI and MCCIFFETT,1972; BELCHERand BRETT,1973, CHIBAet al., 1973; NASCIMENTO,1973; BANKS et al., 1975), but amongst diapausing species are known only for Laspeyresia (HAYES et al., 1972) and Ostrinia (BECK, 1964). Die1 rhythms of oxygen uptake in diapausing species are notable because they are not obviously related to feeding or activity cycles (BRADY, 1974). This paper describes similar die1 oxygen uptake rhythms for Pieris brassicae and P. machaon, and gives details of the influence of individual variation on the average curves for groups of pupae. The possibility of linkage between the die1 rhythm and other forms of systematic variation is also considered.
to units
of
ALTHOUGH general
constant
during
MATERIALS
AND METHODS
Diapausing pupae of Pieris brassicae L. and Papilio machaon L. were obtained from a commercial breeder, and pupae of P. brassicae were also reared from ova in an artificial light regime (LD 9: 15) at 20+ 3°C. All pupae were kept under the same conditions (light: 9: 15 LD or natural) as in the experiments, and allowed to equilibrate at the experimental conditions for at least 1 week before any measurements took place. The term batch refers to a group of pupae reared at the same time and used for one set of experiments; it therefore denotes their temporal rather than genetic homogeneity. Oxygen uptake was measured using a Scholander constant pressure microrespirometer (SCH~LANDER, 1942) with 20% potassium hydroxide as the carbon dioxide absorbent. Readings were taken at 30 min or 641
RESULTS The diel rhythm in P. brassicae
Similar die1 rhythms of oxygen uptake were recorded for individual pupae and batches of pupae at 5, 10, 15 and 20°C. Examples of average curves for individual pupae at 10°C and for batches of pupae at 5, 10 and 20°C are shown in Fig. la and b, but the data are incomplete since nocturnal hourly uptake rates are represented by a mean uptake rate taken from values for total uptake between working hours. In general, pupae displayed a major midday peak, a smaller peak in the afternoon or early evening and low nocturnal rates. Peaks in oxygen uptake were generally ‘sharper’ for individual pupae, and were up to 3 times the mean nocturnal uptake rates. A clear bimodal rhythm of oxygen uptake with delayed ‘dawn’ and ‘dusk’ peaks can be seen in the average batch curve of Fig. lc. Nocturnal values were obtained by varying the recording period over 24 hr and the average curve therefore includes data-missing intervals. Complete die1 average curves for the individual pupae from the same batch (D) were obtained by the same method, but were smoothed with a 3-point moving average (Fig. 2). This was because these particular pupae had low uptake rates, associated with a fall in metabolism preceding postdiapause development (CROZIER,1979). This suggests an increased experimental error. The ‘smoothed’ curves in Fig. 2 are highly significant (PC O.OOl), when the distribution of phases of length d is tested (KENDALLand STUART, 1966; CROZIER.1979).
The curves in Fig. 2 suggest that the die1 variation in pupae of the same age under the same experimental conditions can vary considerably. Further evidence for this is supplied by the data for more pupae from the same batch in Fig. 3. In contrast to the smooth, bimodal rhythm shown by the whole batch in Fig. lc,
A. J. G.
648
CROZIER
(a)
(b)
2or
3
9
12
45
h
18
21
TIME
0
3
6
TIME
24
(
( Hours
la
12
15
,A8
21
9
Hours
9
5Oc
18
21
24
GMT )
15
24
3
GMT )
Fig.
1. Die1 average curves of oxygen uptake in P. brassicae. Solid bars on the x-axis represent the scotophase, and the letter in each example refers to the number of the batch (as defined in the Materials and Methods section). N, mean overnight values. Differences between means are significant (P
individual pupae have curves with between 3 and 5 peaks of variable size and timing. No significant correlation (P> 0.05) was found between the number or timing of uptake peaks and the general bvel of metabotism. In every case, however, maximal uptake rates in the unsmoothed data occurred between 900 and 1900 hr GMT in two groups (900-12,00 1500-1900 hr; PcO.05) corresponding to the two peaks in Fig. ic. Single day records’ of oxygen uptake for pupae undergoing post~iapau~ development (when mean QO, X50 $0, hr- l g-r) matched the shape of the average curves in Fig. 1. However, although maximal daily uptake rates during stable diapause generally occur around midday, the nature of die1 variation in single day records is disguised by the presence of shortterm (supradian) cycles of oxygen demand (CROZIER, 1979). One would not expect the supradian cycles to
show linkage with the die1 uptake rhythm, because their frequency is metabolism-dependent. (CROZIER, 1979). However, the recorded incidence of maxima and minima during individual supradian cycles, recorded over the course of several months to avoid short-term serial correlation, showed peaks during the 24-hr cycle at intervals of about 3 hr at 10°C. Examples for a single pupa and a batch of pupae are shown in Fig. 4. Disturbance of the pupae may give rise to the observed die1 rhythm, but there was no si~ifi~ant trend (P> 0.90) in the initial readings of experiments started at various times during the 24-hr period. However, experiments started at a particular time of the day showed significant trends (P
Oxygen uptake in Pieris brassicaeand Papiliomachaon
649
‘b.
3 ?
0
6
12
18
24
0
6
12
18
24
,
1
I
ti
I
TIME
( Hours
I
!
GMT1
Fig. 2. Two examples of complete die1average curves for individual pupae of P. brassicueat WC. -IJ-,
mean hourly uptake rates (raw data), -O-
The variation in the average curves of Figs. 2 and 3 may be affected by the rhythm in the incidence of supradian cycles (Fig. 4). However, the recorded intervals between peaks in Figs. 2 and 3 are significantly different (P
data smoothed with a 3-point moving average.
frequency/24 hr = 1.99 + 0.88 (mean QO,); PcO.025) Unlike P. brassicae, the incidence of supradian cycles is not linked with the die1 uptake rhythm and shows no significant deviation (P > 0.10) from the expected even distribution arising from a random drift in cycles over 24 hr. Longer-term systematic variation in oxygen uptake was detected by smoothing single day records for individual pupae with a 3-point moving average (Fig. 6). The timing of daily peaks varied between 1000 and 1800 hr GMT. There was no obvious trend in the timing of successive daily peaks (e.g. a steady drift), but the timing of a daily peak was related directly to its size (Fig. 6). The data of Fig. 6 therefore, suggests that the apparently stable midday peak in average curves contains an element that is metallic-de~ndent.
Similar die1 average curves of oxygen uptake were found for the pupae of P. muchaon. Incomplete curves for a single pupa and a batch of four pupae are shown in Fig. 5. Like P. brassicae, the average curves include a main midday peak with a possible peak in the early DISCUSSION evening and low nocturnal rates. Short-term cycles of oxygen uptake lasting 1-6 hr, Diet average curves for both species (Figs. 1 and 5) similar to those found for P. brussicae (CROZIER, display delayed ‘dawn’ and ‘dusk’ peaks similar to 1979), were recorded and their frequency shown to be those present in some insect circadian rhythms related directly to metabolism at 10°C (cycle (BRADY, 1974), but contain significant elements of
650
A. J. G. CROZER
TIME ( Hours
GMT )
Fig 3. Smoothed average curves at 10°C for 8 pupae of P. brassicae from the same batch as the pupae in Fig. 2.
individual variation (Figs. 2,3 and 6). If these rhythms are in fact circadian, then the observed variation may reflect phase relatjonship changes between physiological responses driven by an oscillator, and not an inherent instability in the circadian system. Alternatively, individual responses may be similar in nature to recent ‘spontaneous feedback oscillation’ models of circadian systems (HASTINGSand TYSON, 1975; SEL’KOV,1975; WINFREE,1975a). Although their batch curves are similar (Figs. 1 and S), there are significant differences between P.brassicae and P. machaon. In P. brassicae, maximal daily oxygen uptake occurs around midday, and suggests that the associated die1 rhythm of supradian cycles (Fig. 4) may be due to the increased probability of bursts at this time. Maximal daily uptake rates in P. machaon, however, vary in their timing and are not linked with a die1 rhythm of supradian cycles. The temporal dependence of daily maxima on metabolism (Fig. 6) suggests some form of involvement with longer-term variation. Infradian oxygen uptake cycles of 3-5 days, similar to those observed for P. brussicue
(CROZIER,in press), have been recorded in P. machaon (unpublished data). The functional significance of these die1 rhythms is obscure, particularly as the diurnal maxima will theoretically increase metabolism and water loss under natural temperature conditions. There are at least four possibilities; the rhythms may reflect the general circadian organization that is apparently necessary for homoeostasis in eukaryotes (SAUNDERS, 1977); their significance may relate to a limited number of events in the animal’s lifetime such as eclosion (WJNFREE, 1975b); or they may influence factors affecting survival such as water loss across the spiracles. Alternatively, they may be involved in the control of diapause development, possibly through some kind of ‘coincidence model’ mechanism (F’ITTENDRIGH,1966, 1972; PITTENDRJGHand MINIS, 1964; SAUNDERS, 1978). In addition, interaction between a circadian oscillator and metabolismdependent infradian cycles (CROZIER, 1979) could operate a long-term ‘counter’ system that could be affected by temperature.
651
Oxygen uptake in Pieris brussicae and Pupilio machaon
60
3
6
9 TIME
12 ( Hours
15
18
21
24
GMT)
Fig. 4. Die1 rhythm of supradian cycles in P. brassicae at 10°C. Each histogram bar records the proportion (%) of hourly oxygen uptake readings within a given hour-class, which were maxima (upright bars) or minima (inverted bars) during the course of individual supradian oxygen uptake cycles. Upper record, single pupa (P
A
‘p 9
1:
15
18
)
12
15
18
6 15
l
y: 33 0 -161x .
. .
10
.
. .
.N 5
\
9
12 TIME
15
I Hours
18
.
. .
l
.
. .
12
'5
TIME (Hours
18
GMT)
GMT1
Fig. 5. Die1 average curves of oxygen uptake for a single pupa (A) and a batch of P. machaon pupae (B, n = 4) at 1OC. Differences between means are significant (P
Fig. 6. A. Some examples of smoothed single day records of oxygen uptake for individual pupae of P. machaon at 10°C. B. The relationship betwe& the amplitude of maximal hourly readings in single day records and their time of occurrence. The linear plot is significant (PcO.05).
A. J. G. CROZIER
652
Acknowledgements-I should like to thank Dr. D. F. HOULIHANfor all his help and advice, and also the Science Research Council for financial assistance. REFERENCES BANKSW. M., BRUCEA. S. and PEARTH. T. (1975) The effects of temperature, sex and circadian rhythm on the oxygen uptake of two swcies of cockroaches. Coma. Biichem. >h_v.viol52A, 223-228. BECK S. D. (1964) Time measurement in insect photoperiodism. Am. Nat. 98, 329-346. BELCHER K. S. and BRETTW. J. (1973) Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster. J. Insect Physiol. 19, 271-286.
BRADYJ. (1974) The physiology of insect circadian rhythms. Adv. Insect Physiol. 10, l-l 15. BUCKJ. (1962) Some physical aspects of insect respiration. A. Rev. Ent. I, 27-56. BUCK J.
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