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The temperature-compensated photoperiodic clock ‘programming’ development and pupal diapause in the flesh-fly, Sarcophaga argyrostoma
J. InsectPhyriol.,1971,Vol. 17,pp. 801to 812.Pergamn Press. Printed in Great Britain
THE TEMPERATURE-COMPENSATED PHOTOPERIODIC CLOCK ‘PROGRAMMING’ DEVELOPMENT AND PUPAL DIAPAUSE IN THE FLESH-FLY, SARCOPHAGA ARGYROSTOMA D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 16 October 1970) -t--The flesh-fly, Sarcophqga aqyrostoma, is a long day insect with a temperature-modified photoperiodic ‘clock controlling the onset of pupal diapause. The critical daylength is close to 13.5-14 hr light/24 at both 15 and 20°C, but diapause is practically eliminated at 25°C. The entire larval period constitutes the sensitive period, and long days have a greater photoperiodic effect than short days. Larvae of S. argyrostoma are able to ‘add up’ successive light : dark cycles and act accordingly. Moreover, the number of such cycles required is almost independent of temperature, and the induction of diapause depends on a ‘balance’ between a temperature-dependent process (the rate of larval development) and a temperature-compensated process (the summation of light : dark cycles). INTRODUCTION FRAENKELand Hsxao (l%Sa) showed that the flesh-&es, Sarcuphugu urgyrostonta and S. hllata, diapause as pupae, and demonstrated that the induction of this diapause in the former species was under photoperiodic control. During the development of the intra-puparial stagea, they ahowed that diapause occurred after head eversion, but before pupal-adult apolysis; resumption of development was marked by the appearance of the adult antennae. PupaI diapause seems to be uncommon in the Diptera, although FRAENKELand Hsrao (19680) quote other examples including the paraaitoid Pseudosarc&aga a@ttk (COPPEL et al., 1959; HOUSE, 1%7), the cabbage root maggot, Erticti biasszkue (HUGHES, 19&I), the spinach leaf miner, Pegomyia lrpmimi (ZAEHROV, l%l), the related P. be&e (MISSONNIER, 1963), the cherry fly, Rhagoletziccwi (LEGAY, 1962) and the carrot rust fly, PSZ’ZQ rosae (NATOPI’, 1966). It also occurs in the horn fly, Lyposia irritm (DEPNER, 1962). A considerable amount is known about the endocrinological events involved in pupation and adult development in two species of the Cyclorrhapha (SHAAYAand KARLSON, 1965; B~ITT and BIRT, 1970). In CalIiphora er-kx@~&, for example, SHMYA and KMUON (1965) showed that the titre of ecdysone in the haemolymph was at a peak at the time of puparium formation, fell to its lowest ebb about 2 days later, and rose again during adult differentiation. With this evidence 801
802
D. S.
k7NDBRS
from C. erythroccphalu, FRAENKELand HSL+O(1968b) suggested that diapause in Surc@ugu occurred at the point of lowest ecdysone titre, and proceeded to demonstrate that diapause could, in fact, be terminated by the injection of ecdysone into the thorax of dormant pupae. One of the ‘central’ problems in the photoperiodic control of insect diapause is the nature of the processes occurring between the reception of the photoperiod and the endocrine syndrome of diapause. Because these processes are often well separated in time and may span several intervening moults, they are thought to include the ‘programming’ of the neuro-endocrine system. Since the endocrine basis of diapause in Surcophtqa is reasonably well established it presents an opportunity to investigate these processes. This paper describes some of the propertiti of the photoperiodic clock controlling pupal diapause in S. aqyrostomu. It is shown that the ‘progmmming’ of development includes both the measurement of daylength and the summation of succmive light : dark cycles. Both of these pnxxases show a considerable degree of temperature-compensation, a feature considered important for accuracy in biological time measurement. MATERIALS AND METHODS
These experiments were done using a strain of Sarcophqa argymstoma(R.-D., 1830) ( = f&data, PandeU; Z&&u Thomson) which had been kept in Edinburgh since about 1961; before this its history is unknown. During this period it was kept at 25°C and 18 hr light/day, and no diapause was observed. The adult flies were supplied with water, sugar, and fresh meat daily. Pieces of meat containing newly deposited larvae were removed to biscuit tina for development, the meat being supplemented by an agar, dried milk, and yeast mixture. Mature larvae were allowed to form puparia in sawdust. Experimental conditiona of photoperiod and temperature were obtained in Gallenkamp cooled incubators fitted with Philips 8W striplights controlled by Londex time switches. ‘Batches’ of newly-deposited larvae were set up as deacribed above in white translucent polythene dishes and placed in the incubators. These ‘batches’ consisted of those larvae deposited on one piece of meat placed in an adult cage over a 24hr period. They were, therefore, the progeny of a number of different females, but from a very inbred population. Different experimental batches were set up on the same day, or on successive days, so that they were almost certainly the progeny of the same group of adults. The batches were of variable size, most commonly of between 100 and 700 larvae. At the end of each experiment the puparia were dissected after the first flies had emerged, or after a comparable time had elapsed. Developing individuals were recognized by their complete pigmentation or by emergence ; diapause pupae by the lack of pigmentation and of any sign of the development of the adult antennae (FRAENKELand HSIAO, 1%8a). Diapausing pupae remained in this condition for several montha.
PHOTOPERlODlC CLOCK AND PUPAL DIAPAUBE IN THE --FLY
803
RESULTS The Qiwk of photoperiod and temperature on the inductah of pupal diapawe in s. argyrwoma Batches of larvae were set up at 15°C and in a range of photoperiods, incMing the aperiodic regimes of continuous dark (DD) and continuous light (LL). A less comprehensive range of photoperiods was also eatabliahed at 20 and 25°C. The larvae and resuiting puparia were kept in these conditions until Mon. Fig. 1 shows that a typical long-day photoperiodic response curve was obtained at 15°C with a well-defined critical daylength at 13.5 to 14hr light/day. Photoperiods between LD 8 : 16 and LD 13 : 11 were ‘strong’ short daylengths and
Hours Ii@ per 24 FIG. 1. The eikt phg~ orgyror~:
of pbotopaiod on the induction of pupal dinpause in Siam, at 15°C; x----x, at 20°C;Ol+ 0, at
25°C. produced over 95 per cent diapausc in the pupae. Photoperiods over LD 15 : 9, on the other hand, were ‘strong’ long day&ths, and no diapause was recorded at LD 18:6orinLL. TheincidenceofdiapauseatLD6:18andLD4:20was less than that at the stronger short daylengths, and continuous darlums produced an ‘intermediate’ response, as described for many other species (Leas, 1968). At 20°C the critical daylength appeared to be in the same position as at lS”C, although the proportion of pupae entering diapause in the strong short daylcngth (LD8: 16toLD 13: 1l)wasnotsohighasatthelowertemperature. AsatlYC, LD 13 : 11 provided the strongest short-day signal. Very little diapause was recorded at 25”C, although, once again, LD 13 : 11 produced the most. These results demonstrate that S. argytartorrur has a temperature-modified photoperiodic response typical of a long-day species with a facuhative winter diapause (DE WILDE, 1962). This diapause is presumably equivalent to that
804
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SAUNDERS
observed by FRAENKELand HSIAO(1%8a) at 18”C, LD 8 : 16. Since the critical daylength is about the same at 15 and 20°C the results suggest that the mechanism controlling the induction of diapause is temperature-compensated, at least over this range of temperature. The elimination of diapause at high temperature is a wellknown phenomenon (LEES, 1948). In an experiment to test the reactivation of diapause in S. urgyrostomu a batch of larvae was set up at lS°C and at LD 8 : 16. The peak of puparium-formation occurred 22 days later, and 7 flies emerged after a further 36 to 37 days, indicating that the normal duration of development at this temperature was about 58 days. The remaining 5 11 pupae were firmly in diapause. On the eighty-second day of the experiment, one-third of the batch was dissected and confirmed to be in diapause, one-third was transferred to long daylength (LD 18 : 6) and the remainder left at LD 8 : 16. By the 194th day about 50 per cent of both the remaining groups of pupae had emerged as adult flies. This demonstrates that reactivation of the diapause pupae in S. argyrostoma is not under photoperiodic control, and that development was resumed and eventually completed after a period of over 5 months in diapause, regardless of the photoperiodic regime. A similar ‘spontaneous’ reaction of diapause pupae was described by FRAENKELand HSAIO (1968a). The ‘sensitive’ period In some insects the photosensitive stage is well separated in time from the stage at which diapause occurs, in others the two periods overlap or coincide. In the latter group the state of diapause is often under imminent control by the photoperiod and is terminated by the onset of long days (LEES, 1968). That this is not so in S. ufpyostomu suggests a temporal separation of the two stages. The sensitive period in S. mgymtoma was investigated by transferring larvae from short to long days, and vice versa at different periods of their development. Since the feeding stages of the larvae are within the meat or agar mixture and the three larval instars are diicult to recognize with certainty, the stages of development selected were: (a) the first 7 days, (b) the remainder of the feeding phase up to the time the larvae leave the meat, (c) the interval between leaving the meat and puparium-formation, and (d) the puparium itself. Transfers between stages (b) and (c) and between (c) and (d) were accomplished by moving the larvae or puparia; that between stage (a) and (b) by moving the whole larval culture. Table 1 shows the results of transferring larvae between short daylength (LD 12 : 12) and long daylength (LD 18 : 6), at a temperature of 15°C. When the entire period of larval development was spent at short daylength but-the puparia at long daylength (group 1) practically all the pupae entered diapause. Conversely, when larval development was completed at long daylength before transfer to short days as puparia (group 2), diapause was absent. This shows that the larvae are sensitive to daylength but the intra-puparial stages are not. When larvae were exposed to short days at any phase of larval development, except for the first 7 days (group 3) a proportion of the pupae entered diapause. Similarly, exposure to long days at any stage reduced the incidence of diapause. This demonstrates that the
PIiOTOPERIODIC CLOCK AND F’UPAL DIAPAIJSE IN THE FLESH-FLY
805
entire period of larval development constitutes the ‘sensitive period’. The degree of reversal obtained was always strongest when the hrvae experienced a transfer late in development. For instance, the incidence of diapause increased from 0 to 18 to 34 per cent when larvae experienced short days at stages (a), (b), and (c), respectively (groups 3,4, and 5) ; and the incidence fell from 70 to 31 to 22 per cent when larvae experienced long days at stages (a), (b), and (c) (groups 6, 7, and 8). TABLEl-Tn~
-CT
TO LONG DAY-LENGTH
OF TIN SFERFUNGs. bUhZtU FROM SHORT DAY-LENGTH (LD 12 : 12) (LD 18 : 6) ANDVICE VERSA AT DIFFERENT STAGES OF DEVELOPMENT, ALL AT 15°C
Larvae leave medium 4 Group 1 2 3 4 5
6 7 8
Puparium formation J
No. of puparia
Diapause pupae (%)
(a)
(b)
(c)
(d)
s
S L L
S L L
L S L
254 173 179
99.5 0 0
L
199
18.3
L
99
33.7
S
93
69.9
S
133
31.5
S
213
21.8
L s 7 cycles
\
12-17 cycles S L 7-8 cycles 3-11 cycles L s 7-16 cycles 13-14 cycles S L S 7 cycles l&l6 cycks S S L 7 cycles 9-6 cycles 8-l 4 cycles S S L \ 8-17 cycles 11-13 cycles L 7 cycles L
Stage (a) the first seven cycles; (b) until larvae leave the medium; (c) until puparium formation ; (d) intro-puparial stages. S-short daylength (LD 12 : 12) ; L-long daylength (LD 18 : 6).
This result may indiate that larvae are most susceptible later in their development or merely that the effect of earlier exposure to short or long daylength is reversed by later experience. The results of this experiment also suggest that long days are more effective than short days. For instance, even four to six cycles at LD 18 : 6 can reduce diapause to 31 per cent (group 7) and seven cycles experienced early in development can reduce it to 70 per cent (group 6), whereas similar periods of LD 12 : 12 have a much weaker diapause-inducing effect.
D. s.
806
SAUNDERS
Tke summation of light : dark cycles
Since the larvae of any one batch of S. argyrostoma form puparia over a period of several days, and the larval stages are sensitive to photoperiod but the puparial stages are not, it follows that those larvae which form puparia first experience fewer light : dark cycles than those which form puparia later. Furthermore, Fig. 2 shows that the incidence of pupal diapause rises with an increasing number of loor
FIG. 2. The ei?kct of the number of light : dark cycles experienced aa larvae on the incidence of pupal diapauat in S. argyrostoma: 04, at 2O”C, from left to right: LD 12 : 12, 10 : 14, 13 : 11, 8 : 16 and 14 : 10; l - - -4, at lS”C, from left to right: LD 10 : 14, 14 : 10 and 13 : 9.
short-day cycles experienced by the larvae. This was especially well-marked at 20°C where the proportion of diapausing pupae rose from about zero after twelve to thirteen cycles to about 100 per cent after seventeen to nineteen such cycles. At 15°C there was a similar increase, even at the rather long-day regimen of LD 15 : 9. Since these observations suggest a mechanism involving the summation of successive light : dark cycles, the following experiment was performed. Batches of larvae were set up at 16, 18, 20, 22, 24, and 26”C, all at LD 10 : 14. As the larvae formed puparia they were collected and separated from the rest of the group. Fig. 3 shows the ‘pattern’ of puparium-formation at the various temperatures, with the shaded portion representing the diapause pupae in each batch. The number of days to puparium-formation is clearly temperature-dependent, being about 9 days at 26°C and about 22 to 23 days at 16°C. At 26°C none of the pupae entered diapauae whereas at 18 and 16°C practically all did so. At the intermediate temperatures (24, 22, and 20°C) both developing and diapausing pupae were produced. The curvea for puparium-formation at these intermediate temperatures
PHOTOPERIODIC
CLOCK hND PUPAL
DIhPhUSE IN TXE FLESH-FLY
807
(Fig. 3) are in two cases clearly bimodal, with the developing individuals in the first peak and the diapausing individuals in the second. This suggests that those larvae which are destined for continuous development have a shorter developmental period than those which are destined to enter diapause, and this might account for the increase of diapause with age. However, when the incidence of
Days to puporium_(ormation FIG. 3. The effect of temperature on the induction of pupal diapause in S. mgyro~tomo at LD 10 : 14. The solid lines show the number of larvae forming puparia, the shaded portion of each curve represents the number of diapause pupae. Inset: The efkt of temperature on the proportion of diapause pupae at LD 10 : 14.
pupal diapause in the individual groups was plotted against the number of light : dark cycies experienced (Fii. 4) it became clear that there was a ‘family’ of curves ail with the same general and upward trend, which is compelling evidence that the larvae of S. urgyostonra are able to ‘add up’ the number of hght : dark cycles received and act accordingly. At 24,22, and 20°C the first larvae to form puparia (after nine to eleven cycles) showed continuous development as pupae, whereas those which experienced a greater number of short-day cycles became dormant. The larvae reared at 18 and 16°C experienced seventeen or more short-day cycles and practically all of them became diapause pupae. Fig. 5 shows that the rate of larval development is temperature-dependent, with Qr,, of about 2.70; this is normal for a physiological process of this kind. On the other hand, the temperature codGent for the number of light : dark cycles needed to raise the proportion of diapause pupae to 50 per cent is much nearer and therefore shows a high degree of temperature-compensation. unity (Pm= 144)
808
D.
s. SAUNDBRS
The mechanism controlling diapause induction, therefore, depends on a ‘balance’ between a temperature-dependent process (the rate of larval development) and a
FIG. 4. The effect of temperuture on the summation of light : dark cycles (LD 10 : 14): a-26, b-24, c-22, d-20, e-18, f-16%.
FIG. 5. The effect of tempemture on the rate of larval development ( x -X) and the number of light : dark CY&S (LD 10 : 14) required to raiw the proportion of diapau6e pupae to 50 per cent (0 -0).
partially temperature-compensated process (the summation of light : dark cycles). At 26°C larval development is so rapid that the larvae experience too few short-day cycles to enter diapause, but at 18” and 16OC it is so protracted that more than a
PHOTOPERIODIC CLOCK AND PUPAL DIAPAUSE IN THE FLESH-PLY
809
sufkicnt
number of cycles are experienced and practically all of the insects cease development as pupae. Fig. 4 suggests that the ‘critical number’ of short-day cycles is about thirteen to fourteen, and that about seventeen to nineteen are required to complete the ‘switch’ to diapause. DISCUSSION
In the majority of long-day insects, long daylength and high temperature operate together to eliminate diapause, and short daylength and low temperature act together to increase diapause. This effect of temperature appears to be twofold. Firstly, it may have a direct efkt on the incidence of diapause even at the ‘strong’ short daylengths and, secondly, it may affect the position of the critical daylength. These efkcts, however, may not be entirely independent, since lowering the proportion of insects entering diapause at points close to the critical daylength will obviously result in a shift of the critical daylength to shorter values, and vice versa. In some species, such as the cabbage white butterfly, picris brarricae, temperature up to about 28°C has little efkct on the critical daylength, but diapause does not occur, even at short daylength, above 30°C (mING and JORRRRR~S, 1%2; DANILEYSKII,1965). This species, therefore, is regarded as having a photoperiodic clock which is temperature-compensated over practically the entire range of ecologically important temperatures. The critical daylength in the aphid, Megouru vi&e, appears to be temperature-compensated between 10 and 20°C since it shows a shift to shorter values of only about 15 min for every 5” rise in temperature (Ltzs, 1963). At 23°C and above, however, short-day induction of oviparae fails completely. Similar evidence for temperature-compensation is evident for OJttinia a&&z&, in which the critical daylength at 19” is only about 30 min longer than at 29°C (BECK and HANE, 1960). In other species the critical daylength deceases steadily as the tempera&e rises. In Acnmycta rum&s, for example, it moves to shorter values by about l-5 hr for every 5” increment (&RYsiUN, 1955). Still other species, such as Pqpmytk hytwid and &itnkka brassicac, have a relatively unstable critical photoperiod which shortens by 3 and 4.5 hr, respectively, with a 7°C rise in temperature (ZmIROv, 1961). The results of the present investigation indicate that the photoperiodic response in S. Q~WWSWWW is of the intermediate type. The critical daylength appears to be in the same position at both 15 and ZO’C, but at 25°C diapause induction practically fails so that a critical value is no longer apparent. The question of temperature-compensation is evidently of importance in photoperiodism (PIYYR~~RIOHand MINJS, 1964) since it afkcts the accuracy of the photoperiodic clock; it is therefore worthy of closer examination. The present results have shown that the larvae of S. arwo&rr&z appear to ‘add up’ the number of short-day cycles and only enter diapause (as pupae) after thkteen to fourteen such cycles have been experienced. Furthermore, the number of cycles required is only slightly afkcted by temperature. Since larval development like the majority of physiological pmcesses, is clearly temperature-dependent, the proportion of
810
D. s.
SAUNDERS
pupae entering diapause is obviously the result of an interaction between the rate of process (the summation development (Qrs = 2.70) and a temperature-compensated of light : dark cycles) which has a Q1s much closer to unity (144). A very similar situation occurs in the parasitic wasp, Nastmriz titrr$ti (SAUNDERS, 1966, 1%9). In this insect the number of short-day cycles experienced by the parent female wasp and necessary for the ‘switch’ to the production of diapause progeny was practically the same at all temperatures between 15 and 30°C (Qm= 144). The rate of oviposition, however, was clearly temperature-dependent so that at high temperature most of the eggs were deposited before diapause determination was complete and the incidence of diapause in the resulting larvae was low. Conversely, at low temperature, the majority of the eggs were deposited after a sufficient number of short-day cycles had been experienced, so that the larvae showed a high incidence of diapause. Such a mechanism involving a ‘balance’ between a temperature-dependent process controlling the length of the sensitive period and a temperaturecompensated process controlling the number of light : dark cycles required (as in Fig. 5) could explain the temperature-modification of the photoperiodic response in the other species reviewed above. For instance, if the number of short-day cycles required is small they could casiiy be accommodated within the sensitive period, even up to quite high temperatures so that the insect would be relatively insensitive to temperature. On the other hand, if a larger number of cycles are required, the insect would show a steady decrease in the proportion of diapausing individuals at short daylength, and probably also in the critical daylength, as the temperature rose. In all cases, the elimination of diapause (or short-day effects) at high temperature could be caused by the duration of the sensitive period becoming leas than the number of short-day cycles nv to operate the ‘metabolic switch’. The discussion so far has presented the classical interpretation that diapause is ‘actively’ induced by short daylength. This is based on the view that continuous development must be regarded as the ‘primitive’ state, and that diapause is a later acquisition which provides a means for the synchronixation of growth and development with the favourable seasons. This argument has been followed in this paper in order to facilitate comparisons with earlier work. However, diapause is most frequently the result of an inactivation of the neuro-endocrine system, and it may be more profitable to regard continuous development as the actively induced state (LEES, 1968). Results from the present experiments show that long days have a stronger photoperiodic e&t than short days and therefore support this hypothesis. In addition, results from ‘night interruption’ experiments with a growing number of species show that the coincidence of light with a very short part of the night (the photoperiodically-inducible phase 4%)results in development, whereas if light falls outside +$ the insects become or remain dormant (PIITBNDRICH, 1966; SAUNDERS, 1969). In many species (but not all) the photoperiodic clock appears to be based on a circadian rhythm of sensitivity to light which is phase-set by the light : dark cycle (PITTENDRICH and MINIS, 1964; SAUNDEM,1969, 1970).
PHOTOPEFtIODlC CLOCK AND PUPAL DIAPAUSE IN THE FLESH-FLY
811
In an insect such as S. urgyr&omu, therefore, the ‘programming’ of the neuroendocrine system probably involves two p”cesses, both of which are associated with the photoperiodic clock, and temperature-compensated. These are: (1) the measurement of a long-day by tbe interaction of light with +<, and (2) the summation of a sufficient number of such interactions in successive light : dark cycles. The first of these pmcesaes would be temperature-compensated over quite a wide range of temperature because +r occurs at a particubtr phase of an oscillation which is phase-set by the light : dark cycle. The second process would also show a high degree of temperature-compensation because, although the metabolic pmcesses associated with the interaction between light and +r may be temperature-dependent, these interactions only occur as short and discrete events once every 24 hr. Since the second process is em&aged as the ‘active’ process, it implies that each interaction results in the production of a substance in ‘circadian increments’ to a level necessary to ensure continuous development. Converaely, at short daylength no such interactions occur, the apparent summation of short-day cycles in S. mgyro&MU sumting that the material essential for development runs out after thirteen to fourteen cycles, again in a circadian faahion. This hypothesis is obvioualy overGrnplified since diapause is ciearly a genetically determined state of suppreaaed development (BRcK, 1968): many of the characteristics of diapauae and photoperiodiam can be inherited and diapauae occurs at species-specific points in development, auggesGng aim&i&s to the built-in ‘inatar couILting rne&a&m’ d&unaed by WI~GLBW~RTH (1948). However, the
mu&s do suggest that the syntheks of a subatmcc in ‘circadianincrements’ should be looked for. Since the site of photomeption and probably the clock itself is located in the brain (LEES, 1964; WILLIAMS and ADKXSSON,1961; WILL-, 1%9), and the neum+ecre tory cells of the brain are known to undergo circadian oscillations in size, secretory activity and RNA synthesis (CYMROROWSIUand DUTKOWSKI,1%9), these cells would appear to be the most obvious candidates. Ac&~~~~&&emenr+-T&eauthor would like to thank M& HELENGBNLW for technical akstance.
The work was supported by a grant from the Sciaxt
Research Council.
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Science, Wush. M&601-
603. SHAAYA E. and KARLSON P. (1965) Der Ecdysontiter w&rend der Insektenentwickhmg-II.
Die postembryonale Entwicklung der Schmeissfliege Calliphora tvythcephuka Meig. r. Insect Physiol. 1, 65-49. Wtco~~s~owrn V. B. (1948) The functions of the corpus allatum in Rhodnivs prolixus (Hemiptera). _y. exp. BioZ. 25, 1-14. DE WILDE J. (1%2) Photoperiodiam in insects and mites. A. Rew. Ent. 7,1-26. WILLUMS C. M. (1969) Photoperiodism and the endocrine aspects of insect diapause. Symp. Sot. exp. Bid. 23,285-300. WILLIAN C. M. and ~lussoN P. L. (1964) Physiology of insect diapau~c-XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silkworm, Antheram pe~nyi. Biol. Bull., Woo& Hole 127, 511-525. ZABIROV S. M. (1961) Factors governing the seasonal development cycles of the spinach leaf miner (Pegomyia hyosciami Pans.) and the cabbage my(pot (Hybmyia brusticue Bouche) (Diptera, Anthomyiidae). Ent. Rev. U.S.S.R. 40, 148-151.
THE TEMPERATURE-COMPENSATED PHOTOPERIODIC CLOCK ‘PROGRAMMING’ DEVELOPMENT AND PUPAL DIAPAUSE IN THE FLESH-FLY, SARCOPHAGA ARGYROSTOMA D. S. SAUNDERS Department of Zoology, University of Edinburgh (Received 16 October 1970) -t--The flesh-fly, Sarcophqga aqyrostoma, is a long day insect with a temperature-modified photoperiodic ‘clock controlling the onset of pupal diapause. The critical daylength is close to 13.5-14 hr light/24 at both 15 and 20°C, but diapause is practically eliminated at 25°C. The entire larval period constitutes the sensitive period, and long days have a greater photoperiodic effect than short days. Larvae of S. argyrostoma are able to ‘add up’ successive light : dark cycles and act accordingly. Moreover, the number of such cycles required is almost independent of temperature, and the induction of diapause depends on a ‘balance’ between a temperature-dependent process (the rate of larval development) and a temperature-compensated process (the summation of light : dark cycles). INTRODUCTION FRAENKELand Hsxao (l%Sa) showed that the flesh-&es, Sarcuphugu urgyrostonta and S. hllata, diapause as pupae, and demonstrated that the induction of this diapause in the former species was under photoperiodic control. During the development of the intra-puparial stagea, they ahowed that diapause occurred after head eversion, but before pupal-adult apolysis; resumption of development was marked by the appearance of the adult antennae. PupaI diapause seems to be uncommon in the Diptera, although FRAENKELand Hsrao (19680) quote other examples including the paraaitoid Pseudosarc&aga a@ttk (COPPEL et al., 1959; HOUSE, 1%7), the cabbage root maggot, Erticti biasszkue (HUGHES, 19&I), the spinach leaf miner, Pegomyia lrpmimi (ZAEHROV, l%l), the related P. be&e (MISSONNIER, 1963), the cherry fly, Rhagoletziccwi (LEGAY, 1962) and the carrot rust fly, PSZ’ZQ rosae (NATOPI’, 1966). It also occurs in the horn fly, Lyposia irritm (DEPNER, 1962). A considerable amount is known about the endocrinological events involved in pupation and adult development in two species of the Cyclorrhapha (SHAAYAand KARLSON, 1965; B~ITT and BIRT, 1970). In CalIiphora er-kx@~&, for example, SHMYA and KMUON (1965) showed that the titre of ecdysone in the haemolymph was at a peak at the time of puparium formation, fell to its lowest ebb about 2 days later, and rose again during adult differentiation. With this evidence 801
802
D. S.
k7NDBRS
from C. erythroccphalu, FRAENKELand HSL+O(1968b) suggested that diapause in Surc@ugu occurred at the point of lowest ecdysone titre, and proceeded to demonstrate that diapause could, in fact, be terminated by the injection of ecdysone into the thorax of dormant pupae. One of the ‘central’ problems in the photoperiodic control of insect diapause is the nature of the processes occurring between the reception of the photoperiod and the endocrine syndrome of diapause. Because these processes are often well separated in time and may span several intervening moults, they are thought to include the ‘programming’ of the neuro-endocrine system. Since the endocrine basis of diapause in Surcophtqa is reasonably well established it presents an opportunity to investigate these processes. This paper describes some of the propertiti of the photoperiodic clock controlling pupal diapause in S. aqyrostomu. It is shown that the ‘progmmming’ of development includes both the measurement of daylength and the summation of succmive light : dark cycles. Both of these pnxxases show a considerable degree of temperature-compensation, a feature considered important for accuracy in biological time measurement. MATERIALS AND METHODS
These experiments were done using a strain of Sarcophqa argymstoma(R.-D., 1830) ( = f&data, PandeU; Z&&u Thomson) which had been kept in Edinburgh since about 1961; before this its history is unknown. During this period it was kept at 25°C and 18 hr light/day, and no diapause was observed. The adult flies were supplied with water, sugar, and fresh meat daily. Pieces of meat containing newly deposited larvae were removed to biscuit tina for development, the meat being supplemented by an agar, dried milk, and yeast mixture. Mature larvae were allowed to form puparia in sawdust. Experimental conditiona of photoperiod and temperature were obtained in Gallenkamp cooled incubators fitted with Philips 8W striplights controlled by Londex time switches. ‘Batches’ of newly-deposited larvae were set up as deacribed above in white translucent polythene dishes and placed in the incubators. These ‘batches’ consisted of those larvae deposited on one piece of meat placed in an adult cage over a 24hr period. They were, therefore, the progeny of a number of different females, but from a very inbred population. Different experimental batches were set up on the same day, or on successive days, so that they were almost certainly the progeny of the same group of adults. The batches were of variable size, most commonly of between 100 and 700 larvae. At the end of each experiment the puparia were dissected after the first flies had emerged, or after a comparable time had elapsed. Developing individuals were recognized by their complete pigmentation or by emergence ; diapause pupae by the lack of pigmentation and of any sign of the development of the adult antennae (FRAENKELand HSIAO, 1%8a). Diapausing pupae remained in this condition for several montha.
PHOTOPERlODlC CLOCK AND PUPAL DIAPAUBE IN THE --FLY
803
RESULTS The Qiwk of photoperiod and temperature on the inductah of pupal diapawe in s. argyrwoma Batches of larvae were set up at 15°C and in a range of photoperiods, incMing the aperiodic regimes of continuous dark (DD) and continuous light (LL). A less comprehensive range of photoperiods was also eatabliahed at 20 and 25°C. The larvae and resuiting puparia were kept in these conditions until Mon. Fig. 1 shows that a typical long-day photoperiodic response curve was obtained at 15°C with a well-defined critical daylength at 13.5 to 14hr light/day. Photoperiods between LD 8 : 16 and LD 13 : 11 were ‘strong’ short daylengths and
Hours Ii@ per 24 FIG. 1. The eikt phg~ orgyror~:
of pbotopaiod on the induction of pupal dinpause in Siam, at 15°C; x----x, at 20°C;Ol+ 0, at
25°C. produced over 95 per cent diapausc in the pupae. Photoperiods over LD 15 : 9, on the other hand, were ‘strong’ long day&ths, and no diapause was recorded at LD 18:6orinLL. TheincidenceofdiapauseatLD6:18andLD4:20was less than that at the stronger short daylengths, and continuous darlums produced an ‘intermediate’ response, as described for many other species (Leas, 1968). At 20°C the critical daylength appeared to be in the same position as at lS”C, although the proportion of pupae entering diapause in the strong short daylcngth (LD8: 16toLD 13: 1l)wasnotsohighasatthelowertemperature. AsatlYC, LD 13 : 11 provided the strongest short-day signal. Very little diapause was recorded at 25”C, although, once again, LD 13 : 11 produced the most. These results demonstrate that S. argytartorrur has a temperature-modified photoperiodic response typical of a long-day species with a facuhative winter diapause (DE WILDE, 1962). This diapause is presumably equivalent to that
804
D. s.
SAUNDERS
observed by FRAENKELand HSIAO(1%8a) at 18”C, LD 8 : 16. Since the critical daylength is about the same at 15 and 20°C the results suggest that the mechanism controlling the induction of diapause is temperature-compensated, at least over this range of temperature. The elimination of diapause at high temperature is a wellknown phenomenon (LEES, 1948). In an experiment to test the reactivation of diapause in S. urgyrostomu a batch of larvae was set up at lS°C and at LD 8 : 16. The peak of puparium-formation occurred 22 days later, and 7 flies emerged after a further 36 to 37 days, indicating that the normal duration of development at this temperature was about 58 days. The remaining 5 11 pupae were firmly in diapause. On the eighty-second day of the experiment, one-third of the batch was dissected and confirmed to be in diapause, one-third was transferred to long daylength (LD 18 : 6) and the remainder left at LD 8 : 16. By the 194th day about 50 per cent of both the remaining groups of pupae had emerged as adult flies. This demonstrates that reactivation of the diapause pupae in S. argyrostoma is not under photoperiodic control, and that development was resumed and eventually completed after a period of over 5 months in diapause, regardless of the photoperiodic regime. A similar ‘spontaneous’ reaction of diapause pupae was described by FRAENKELand HSAIO (1968a). The ‘sensitive’ period In some insects the photosensitive stage is well separated in time from the stage at which diapause occurs, in others the two periods overlap or coincide. In the latter group the state of diapause is often under imminent control by the photoperiod and is terminated by the onset of long days (LEES, 1968). That this is not so in S. ufpyostomu suggests a temporal separation of the two stages. The sensitive period in S. mgymtoma was investigated by transferring larvae from short to long days, and vice versa at different periods of their development. Since the feeding stages of the larvae are within the meat or agar mixture and the three larval instars are diicult to recognize with certainty, the stages of development selected were: (a) the first 7 days, (b) the remainder of the feeding phase up to the time the larvae leave the meat, (c) the interval between leaving the meat and puparium-formation, and (d) the puparium itself. Transfers between stages (b) and (c) and between (c) and (d) were accomplished by moving the larvae or puparia; that between stage (a) and (b) by moving the whole larval culture. Table 1 shows the results of transferring larvae between short daylength (LD 12 : 12) and long daylength (LD 18 : 6), at a temperature of 15°C. When the entire period of larval development was spent at short daylength but-the puparia at long daylength (group 1) practically all the pupae entered diapause. Conversely, when larval development was completed at long daylength before transfer to short days as puparia (group 2), diapause was absent. This shows that the larvae are sensitive to daylength but the intra-puparial stages are not. When larvae were exposed to short days at any phase of larval development, except for the first 7 days (group 3) a proportion of the pupae entered diapause. Similarly, exposure to long days at any stage reduced the incidence of diapause. This demonstrates that the
PIiOTOPERIODIC CLOCK AND F’UPAL DIAPAIJSE IN THE FLESH-FLY
805
entire period of larval development constitutes the ‘sensitive period’. The degree of reversal obtained was always strongest when the hrvae experienced a transfer late in development. For instance, the incidence of diapause increased from 0 to 18 to 34 per cent when larvae experienced short days at stages (a), (b), and (c), respectively (groups 3,4, and 5) ; and the incidence fell from 70 to 31 to 22 per cent when larvae experienced long days at stages (a), (b), and (c) (groups 6, 7, and 8). TABLEl-Tn~
-CT
TO LONG DAY-LENGTH
OF TIN SFERFUNGs. bUhZtU FROM SHORT DAY-LENGTH (LD 12 : 12) (LD 18 : 6) ANDVICE VERSA AT DIFFERENT STAGES OF DEVELOPMENT, ALL AT 15°C
Larvae leave medium 4 Group 1 2 3 4 5
6 7 8
Puparium formation J
No. of puparia
Diapause pupae (%)
(a)
(b)
(c)
(d)
s
S L L
S L L
L S L
254 173 179
99.5 0 0
L
199
18.3
L
99
33.7
S
93
69.9
S
133
31.5
S
213
21.8
L s 7 cycles
\
12-17 cycles S L 7-8 cycles 3-11 cycles L s 7-16 cycles 13-14 cycles S L S 7 cycles l&l6 cycks S S L 7 cycles 9-6 cycles 8-l 4 cycles S S L \ 8-17 cycles 11-13 cycles L 7 cycles L
Stage (a) the first seven cycles; (b) until larvae leave the medium; (c) until puparium formation ; (d) intro-puparial stages. S-short daylength (LD 12 : 12) ; L-long daylength (LD 18 : 6).
This result may indiate that larvae are most susceptible later in their development or merely that the effect of earlier exposure to short or long daylength is reversed by later experience. The results of this experiment also suggest that long days are more effective than short days. For instance, even four to six cycles at LD 18 : 6 can reduce diapause to 31 per cent (group 7) and seven cycles experienced early in development can reduce it to 70 per cent (group 6), whereas similar periods of LD 12 : 12 have a much weaker diapause-inducing effect.
D. s.
806
SAUNDERS
Tke summation of light : dark cycles
Since the larvae of any one batch of S. argyrostoma form puparia over a period of several days, and the larval stages are sensitive to photoperiod but the puparial stages are not, it follows that those larvae which form puparia first experience fewer light : dark cycles than those which form puparia later. Furthermore, Fig. 2 shows that the incidence of pupal diapause rises with an increasing number of loor
FIG. 2. The ei?kct of the number of light : dark cycles experienced aa larvae on the incidence of pupal diapauat in S. argyrostoma: 04, at 2O”C, from left to right: LD 12 : 12, 10 : 14, 13 : 11, 8 : 16 and 14 : 10; l - - -4, at lS”C, from left to right: LD 10 : 14, 14 : 10 and 13 : 9.
short-day cycles experienced by the larvae. This was especially well-marked at 20°C where the proportion of diapausing pupae rose from about zero after twelve to thirteen cycles to about 100 per cent after seventeen to nineteen such cycles. At 15°C there was a similar increase, even at the rather long-day regimen of LD 15 : 9. Since these observations suggest a mechanism involving the summation of successive light : dark cycles, the following experiment was performed. Batches of larvae were set up at 16, 18, 20, 22, 24, and 26”C, all at LD 10 : 14. As the larvae formed puparia they were collected and separated from the rest of the group. Fig. 3 shows the ‘pattern’ of puparium-formation at the various temperatures, with the shaded portion representing the diapause pupae in each batch. The number of days to puparium-formation is clearly temperature-dependent, being about 9 days at 26°C and about 22 to 23 days at 16°C. At 26°C none of the pupae entered diapauae whereas at 18 and 16°C practically all did so. At the intermediate temperatures (24, 22, and 20°C) both developing and diapausing pupae were produced. The curvea for puparium-formation at these intermediate temperatures
PHOTOPERIODIC
CLOCK hND PUPAL
DIhPhUSE IN TXE FLESH-FLY
807
(Fig. 3) are in two cases clearly bimodal, with the developing individuals in the first peak and the diapausing individuals in the second. This suggests that those larvae which are destined for continuous development have a shorter developmental period than those which are destined to enter diapause, and this might account for the increase of diapause with age. However, when the incidence of
Days to puporium_(ormation FIG. 3. The effect of temperature on the induction of pupal diapause in S. mgyro~tomo at LD 10 : 14. The solid lines show the number of larvae forming puparia, the shaded portion of each curve represents the number of diapause pupae. Inset: The efkt of temperature on the proportion of diapause pupae at LD 10 : 14.
pupal diapause in the individual groups was plotted against the number of light : dark cycies experienced (Fii. 4) it became clear that there was a ‘family’ of curves ail with the same general and upward trend, which is compelling evidence that the larvae of S. urgyostonra are able to ‘add up’ the number of hght : dark cycles received and act accordingly. At 24,22, and 20°C the first larvae to form puparia (after nine to eleven cycles) showed continuous development as pupae, whereas those which experienced a greater number of short-day cycles became dormant. The larvae reared at 18 and 16°C experienced seventeen or more short-day cycles and practically all of them became diapause pupae. Fig. 5 shows that the rate of larval development is temperature-dependent, with Qr,, of about 2.70; this is normal for a physiological process of this kind. On the other hand, the temperature codGent for the number of light : dark cycles needed to raise the proportion of diapause pupae to 50 per cent is much nearer and therefore shows a high degree of temperature-compensation. unity (Pm= 144)
808
D.
s. SAUNDBRS
The mechanism controlling diapause induction, therefore, depends on a ‘balance’ between a temperature-dependent process (the rate of larval development) and a
FIG. 4. The effect of temperuture on the summation of light : dark cycles (LD 10 : 14): a-26, b-24, c-22, d-20, e-18, f-16%.
FIG. 5. The effect of tempemture on the rate of larval development ( x -X) and the number of light : dark CY&S (LD 10 : 14) required to raiw the proportion of diapau6e pupae to 50 per cent (0 -0).
partially temperature-compensated process (the summation of light : dark cycles). At 26°C larval development is so rapid that the larvae experience too few short-day cycles to enter diapause, but at 18” and 16OC it is so protracted that more than a
PHOTOPERIODIC CLOCK AND PUPAL DIAPAUSE IN THE FLESH-PLY
809
sufkicnt
number of cycles are experienced and practically all of the insects cease development as pupae. Fig. 4 suggests that the ‘critical number’ of short-day cycles is about thirteen to fourteen, and that about seventeen to nineteen are required to complete the ‘switch’ to diapause. DISCUSSION
In the majority of long-day insects, long daylength and high temperature operate together to eliminate diapause, and short daylength and low temperature act together to increase diapause. This effect of temperature appears to be twofold. Firstly, it may have a direct efkt on the incidence of diapause even at the ‘strong’ short daylengths and, secondly, it may affect the position of the critical daylength. These efkcts, however, may not be entirely independent, since lowering the proportion of insects entering diapause at points close to the critical daylength will obviously result in a shift of the critical daylength to shorter values, and vice versa. In some species, such as the cabbage white butterfly, picris brarricae, temperature up to about 28°C has little efkct on the critical daylength, but diapause does not occur, even at short daylength, above 30°C (mING and JORRRRR~S, 1%2; DANILEYSKII,1965). This species, therefore, is regarded as having a photoperiodic clock which is temperature-compensated over practically the entire range of ecologically important temperatures. The critical daylength in the aphid, Megouru vi&e, appears to be temperature-compensated between 10 and 20°C since it shows a shift to shorter values of only about 15 min for every 5” rise in temperature (Ltzs, 1963). At 23°C and above, however, short-day induction of oviparae fails completely. Similar evidence for temperature-compensation is evident for OJttinia a&&z&, in which the critical daylength at 19” is only about 30 min longer than at 29°C (BECK and HANE, 1960). In other species the critical daylength deceases steadily as the tempera&e rises. In Acnmycta rum&s, for example, it moves to shorter values by about l-5 hr for every 5” increment (&RYsiUN, 1955). Still other species, such as Pqpmytk hytwid and &itnkka brassicac, have a relatively unstable critical photoperiod which shortens by 3 and 4.5 hr, respectively, with a 7°C rise in temperature (ZmIROv, 1961). The results of the present investigation indicate that the photoperiodic response in S. Q~WWSWWW is of the intermediate type. The critical daylength appears to be in the same position at both 15 and ZO’C, but at 25°C diapause induction practically fails so that a critical value is no longer apparent. The question of temperature-compensation is evidently of importance in photoperiodism (PIYYR~~RIOHand MINJS, 1964) since it afkcts the accuracy of the photoperiodic clock; it is therefore worthy of closer examination. The present results have shown that the larvae of S. arwo&rr&z appear to ‘add up’ the number of short-day cycles and only enter diapause (as pupae) after thkteen to fourteen such cycles have been experienced. Furthermore, the number of cycles required is only slightly afkcted by temperature. Since larval development like the majority of physiological pmcesses, is clearly temperature-dependent, the proportion of
810
D. s.
SAUNDERS
pupae entering diapause is obviously the result of an interaction between the rate of process (the summation development (Qrs = 2.70) and a temperature-compensated of light : dark cycles) which has a Q1s much closer to unity (144). A very similar situation occurs in the parasitic wasp, Nastmriz titrr$ti (SAUNDERS, 1966, 1%9). In this insect the number of short-day cycles experienced by the parent female wasp and necessary for the ‘switch’ to the production of diapause progeny was practically the same at all temperatures between 15 and 30°C (Qm= 144). The rate of oviposition, however, was clearly temperature-dependent so that at high temperature most of the eggs were deposited before diapause determination was complete and the incidence of diapause in the resulting larvae was low. Conversely, at low temperature, the majority of the eggs were deposited after a sufficient number of short-day cycles had been experienced, so that the larvae showed a high incidence of diapause. Such a mechanism involving a ‘balance’ between a temperature-dependent process controlling the length of the sensitive period and a temperaturecompensated process controlling the number of light : dark cycles required (as in Fig. 5) could explain the temperature-modification of the photoperiodic response in the other species reviewed above. For instance, if the number of short-day cycles required is small they could casiiy be accommodated within the sensitive period, even up to quite high temperatures so that the insect would be relatively insensitive to temperature. On the other hand, if a larger number of cycles are required, the insect would show a steady decrease in the proportion of diapausing individuals at short daylength, and probably also in the critical daylength, as the temperature rose. In all cases, the elimination of diapause (or short-day effects) at high temperature could be caused by the duration of the sensitive period becoming leas than the number of short-day cycles nv to operate the ‘metabolic switch’. The discussion so far has presented the classical interpretation that diapause is ‘actively’ induced by short daylength. This is based on the view that continuous development must be regarded as the ‘primitive’ state, and that diapause is a later acquisition which provides a means for the synchronixation of growth and development with the favourable seasons. This argument has been followed in this paper in order to facilitate comparisons with earlier work. However, diapause is most frequently the result of an inactivation of the neuro-endocrine system, and it may be more profitable to regard continuous development as the actively induced state (LEES, 1968). Results from the present experiments show that long days have a stronger photoperiodic e&t than short days and therefore support this hypothesis. In addition, results from ‘night interruption’ experiments with a growing number of species show that the coincidence of light with a very short part of the night (the photoperiodically-inducible phase 4%)results in development, whereas if light falls outside +$ the insects become or remain dormant (PIITBNDRICH, 1966; SAUNDERS, 1969). In many species (but not all) the photoperiodic clock appears to be based on a circadian rhythm of sensitivity to light which is phase-set by the light : dark cycle (PITTENDRICH and MINIS, 1964; SAUNDEM,1969, 1970).
PHOTOPEFtIODlC CLOCK AND PUPAL DIAPAUSE IN THE FLESH-FLY
811
In an insect such as S. urgyr&omu, therefore, the ‘programming’ of the neuroendocrine system probably involves two p”cesses, both of which are associated with the photoperiodic clock, and temperature-compensated. These are: (1) the measurement of a long-day by tbe interaction of light with +<, and (2) the summation of a sufficient number of such interactions in successive light : dark cycles. The first of these pmcesaes would be temperature-compensated over quite a wide range of temperature because +r occurs at a particubtr phase of an oscillation which is phase-set by the light : dark cycle. The second process would also show a high degree of temperature-compensation because, although the metabolic pmcesses associated with the interaction between light and +r may be temperature-dependent, these interactions only occur as short and discrete events once every 24 hr. Since the second process is em&aged as the ‘active’ process, it implies that each interaction results in the production of a substance in ‘circadian increments’ to a level necessary to ensure continuous development. Converaely, at short daylength no such interactions occur, the apparent summation of short-day cycles in S. mgyro&MU sumting that the material essential for development runs out after thirteen to fourteen cycles, again in a circadian faahion. This hypothesis is obvioualy overGrnplified since diapause is ciearly a genetically determined state of suppreaaed development (BRcK, 1968): many of the characteristics of diapauae and photoperiodiam can be inherited and diapauae occurs at species-specific points in development, auggesGng aim&i&s to the built-in ‘inatar couILting rne&a&m’ d&unaed by WI~GLBW~RTH (1948). However, the
mu&s do suggest that the syntheks of a subatmcc in ‘circadianincrements’ should be looked for. Since the site of photomeption and probably the clock itself is located in the brain (LEES, 1964; WILLIAMS and ADKXSSON,1961; WILL-, 1%9), and the neum+ecre tory cells of the brain are known to undergo circadian oscillations in size, secretory activity and RNA synthesis (CYMROROWSIUand DUTKOWSKI,1%9), these cells would appear to be the most obvious candidates. Ac&~~~~&&emenr+-T&eauthor would like to thank M& HELENGBNLW for technical akstance.
The work was supported by a grant from the Sciaxt
Research Council.
REFERENCES BARRITTL. C. and BIRT L. M. (1970) Prothoracic gIand hormone in the sheep blowfly, Lu&a cuptino. y. Itwcz lfhysiol. 16, 671677. BBCIC S. D. (1968) Issect Ahatopaiodimr, p. 135. Acdemic Prcsm,New York. BECKS. D. and HANX W. (1960) Diapause in the Europeen corn borer, purrnut nubilalis (Hifbn.). g. Inaz Phyrid. 4,304-318. B~MNINc E. and J~BRRENSG. (1962) Venuche uber den wg bei der photopcriodiachen Diapsuae-Induktion von pirris brasicae. 2. Natuqorxh. 17b, 57-61. CO~PXLH. C., HOUSE H. L., and MAW M. G. (1959) Studies on dipterous puasi#r of the spruce budworm Ctirossara jk$m (C&m.) (Lepidoptera: Tort&id@-VII. &ia q#K (Fail.) (D&era: !?arcophagida). C4n. 9: Zuol.37,817-830. C~NIBOROI~P~KI B. and DWHCO~SKIA. (1969) Circadian cftrager in RNA 8ynd#rir in the netmmcmoq da of the brain and a&oesophageal gangIion of the house cricket. g. Imsect PhysioI. 15,1187-1197. Dmunre~11 A. S. (1965) Photopdodirm ud Saorolrcil Lhdojmwnt of hects. Oliver & Boyd, Edinburgh.
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