Vitamin A is essential for two processes involved in the photoperiodic reaction in Pieris brassicae

J. Insect Physiol.

Vol. 38.No. 8,pp.569-514, 1992 Printed in Great Britain. All rights reserved

0022-1910/92 $5.00+ 0.00

Copyright 0 1992Pergamon Press Ltd

VITAMIN A IS ESSENTIAL FOR TWO PROCESSES INVOLVED IN THE PHOTOPERIODIC REACTION IN PIERIS BRASSICAE J. CLARET and N. VOLKOFF I.N.R.A. Laboratoire de Biologie des Invert&b&, 37, Bd du Cap, 06606 Antibes. France (Received 3 December

1991; revised 3 February 1992)

Abstract-In Pieris brassicae fed on a semi-synthetic diet without cabbage powder, point B of photosensibility to a l-h light pulse at the end of the scotophase in a 13 h light-l 1 h dark cycle disappears when light intensity is 300 lx, but remains when light is 50 lx. Despite the loss of B, larvae keep the ability to distinguish short- from long-days, and the 24 h photoperiod response curve is similar to the one observed with controls fed on cabbage. The supply of vitamin A (0.10 mg/g) allows a complete restoration of photosensitivity at B. The amount of vitamin A needed is 12 times higher than the amount (0.008 mg/g) necessary to distinguish between short- and long-day by larvae fed on this same artificial diet but carotenoid deficient. Hence, vitamin A interferes with two processes of the photoperiodic reaction. The first one is supposed to be photoreception, where the vitamin A requirement for pigment formation is low. The second one, requiring a higher quantity of vitamin A, is still unknown but is related to the nocturnal photosensitive point B. Implications of the loss of B on clock functioning are discussed. Another consequence of feeding P. brass&e on a semi-synthetic diet without cabbage is the reversal of the larval response to 12 h light60 h dark with a 300 lx light intensity used as a control in resonance experiments (Btinsow protocol). Larvae reared on artificial diet consider the photoperiod 12 h light-60 h dark as a short-day (100% diapause) while larvae fed on cabbage consider it as a long-day (3% diapause). However, with a 50 lx light, insects reared on artificial diet react in the same way as insects reared on cabbage. In the first case, a supply of vitamin A, even at a high concentration, does not restore the control response. Semi-synthetic diet, because of its deficiency, disturbs the photoperiodic responsiveness of P. brassicae and its use will allow us to study biochemical processes involved in photoperiodic time measurement by the biological clock which, until now, has always remained a “black box”. Key

Word Index:

Photoperiodism;

diapause; clock; hourglass; oscillator; vitamin A; Pieris

brassicae

INTRODUCTION

In photoperiod dependent biological events such as diapause, the supply of vitamin A or other carotenoids by food intake has been shown to be essential for several insect species and mites (Veerman and Helle, 1978; Van Zon et al., 1981; Veerman et al., 1983; Shimizu and Kato, 1984; Veerman et al., 1985; Claret, 1989; Overmeer et al., 1989; Takeda, unpublished). Supposedly, such a need for vitamin A is related to the formation of the brain pigment which is the photoperiodic receptor in insects and which is also, for at least one mite species (Van Houten et al., 1987; Van Houten and Veerman, 1990), the ther-

moperiodic receptor. Response to the photoperiodic signal is composed of several functional steps: reception of the information from the environment, then time measurement by the biological clock, summation of the daily signals by the counter and finally, neuro-endocrine and endocrine regulation. Resonance experiments (Nanda-Hamner protocol) with Pieris brassicae have indicated that biological clocks measuring the photoperiodic time for pupal diapause induction have a circadian component (Claret et al., 1981). Furthermore, night break experiments in a 24 h cycle with the same species revealed the existence of two light-sensitive points (long-day effect) at the beginning (point A) and at the

J.

570

CLARET and N. VOLKOFF

end (point B) of the scotophase (Dumortier and Brunnarius, 1981) as they also exist for many other species, but not all (cf. Saunders, 1982). The theoretical model which best explains these experiments is the external coincidence model developed by Pittendrigh (1966). This model consists of a single oscillator, phase-set by the “off’‘-signals, with a particular light-sensitive phase point (a,) being illuminated or not illuminated, to give long- or short-day effects respectively according to the length of the photoperiod. The second point B represents @, and a light-pulse occurring in the early scotophase (A) is “read” as a new dusk and thereby reduces the duration of effective scotophase and Qi occurred during the following photophase. Little is known about biochemical events underlying the complex clock-counter mechanism. Recently, Dumortier and Brunnarius (1987, 1989) found that larvae of P. brassicae fed on the David and Gardiner (1966) semi-synthetic diet without powdered cabbage presented different photoperiodic reactions compared with larvae reared on cabbage. Artificial diet leads to the elimination of one (B) of the two photosensitive points to a light-pulse in a long night and modifies the response of the insects submitted to a resonance experiment (Btinsow protocol). These authors conclude that artificial diet modifies the clock mechanism from a normal circadian one to an interval-timer or hourglass mechanism, but without affecting the measurement of photoperiodic time in 24 h. These results could be due either to the absence of a chemical in the food which is necessary for the normal course of the clock, or to the presence of a chemical that modifies this normal course. In this work, we have chosen to verify the first hypothesis. We have tried to find a compound which will restore the functioning of the clock so that it is identical to the one observed in insects fed on cabbage by supplementing the diet with different chemicals.

chemicals were conducted. In some experiments, 20&500 mg vitamin A palmitate was added (water dispersible, Sigma R 3750, containing antioxydants, 250,000 USP/g), corresponding respectively to 0.04-O. 10 mg of pure vitamin A/g of diet. A control for antioxydant activity was made using 80 mg vitamin A palmitate dissolved in oil (Sigma R 3500, without antioxydant, 1,600,OOOUSP/g), corresponding to 0.10 mg of pure vitamin A/g of diet. Experiments were conducted at 20 k 0.5 or 24 f 0.5% Light was produced by a fluorescent tube (8 W) with an approximate intensity of 300 lx at insect level. When needed, an intensity of 50 lx could be obtained by wrapping aluminium foil around part of the tube. Experimental conditions were maintained from the day of hatching till pupation. RESULTS

Normal response curve

The insect response to photoperiod (Fig. 1) in a die1 cycle is identical for both cabbage and artificial diets, and curves are well superposed. This indicates that the clock responsible for the photoperiodic time measurement differentiates between a short- and long-day cycle at the same values (between 13 and 14 h). Night interruption experiments

For P. brassicae fed on cabbage and exposed to a 13 h light-l 1 h dark cycle, a night-interruption with l-h supplementary light-pulses scanning the dark period reveals two points of photosensitivity (long-day effect). One is at the beginning (14-17 h) and the other at the end (21-23 h) of the night (Fig. 2). However, when reared on artificial diet, 100.

MATERIALS AND METHODS

Control insects are reared on cabbage. The others are fed on a semi-synthetic diet without powdered cabbage but containing sinigrin and linseed oil (David and Gardiner, 1966). The diet was modified by adding 70 mg phosphatidylethanolamin (Sigma P4264) and 0.500 g K,PO,,. 3H,O for 360 g of diet to improve insect growth (Agui et al., 1975; Junikkala, 1980). Our working hypothesis was that in larvae reared in artificial diet and submitted to a late night interruption by a 300 Ix light-pulse, photoreactivity was lost because a chemical needed for the response was lacking in the diet. In order to test this hypothesis, diet-supplementation experiments with several

60-

0

6

16

24

(h)

Fig. 1. Photoperiodic response curves of P. brassicae. Larvae were reared on cabbage (-) or artificial diet (---). Temperature: 20°C

Photoperiodic

reaction

in P. brussicae

571

Table 2. Action of temperature (24°C) on results of night interruption experiment with I h light pulse (22-23 h) in 13 h light-11 h dark Diet Control (without interruption) Cabbage Artificial diet 1 h light pulse Cabbage Artificial diet

Diapause (%)

n

22 78

115 205

0 4

62 100

Light intensity: 300 lx.

1 0

&

16

6

24

1

Fig. 2. Night interruption experiments in P. brassicue in 13 h light-l 1 h dark with 1 h light pulses scanning the scotophase. The graph shows A and B points of long-day effect when larvae were reared on cabbage (-) and only A when

fed on artificial diet (---). C: controls without interruption. Light intensity: 300 lx. Temperature: 20°C

larvae present only the first point of light sensitivity. These results are identical to those found by Dumortier and Brunnarius (1987). An experiment of dark interruption by a light pulse at 22-23 h in 13 h light-l 1 h dark was conducted at two light intensities: 300 and 50 lx. Larvae were reared on artificial diet in conditions of constant and alternating light intensities (Table 1). In constant conditions, a 300 lx light interruption does not induce a diapause inhibition. On the other hand, insects are very sensitive to a 50 lx light. When the two different intensities are combined, sensitivity is completely lost with a main photophase of 300 lx and a night interruption with a 50 lx light-pulse. When light intensity is reversed, the long-day effect is partial (3 1% diapause). Apparently, the loss of photosensitivity at the end of the scotophase is more dependent on the light intensity during the main photophase than on the intensity of the light-pulse used to interrupt the night. Photoperiodic reactions such as diapause only appear within certain temperature limits. The percentage of diapause in P. brassicae decreases when

the temperature increases (Danilevski, 1965). In order to see if temperature modifies the response of insects reared on artificial diet to a 1 h light break placed in the end of the night, an experiment was conducted at 24°C (Table 2). At this temperature, for insects reared on cabbage, diapause is induced by 13 h light-l 1 h dark in only a few individuals, but in a higher percentage for insects fed with artificial diet. Interruption by a 300 lx light is effective at 24°C but not at 20°C. Thus, the influence of artificial diet on the light-sensitivity at B is thermo-dependent. Therefore, two factors are involved in the loss of photosensitivity with a late interruption: high light intensity and moderate temperature. Vitamin A supplementation

Several chemicals were tested in our dietsupplementation experiments, but only vitamin A had an effect on insect response. The addition to the diet of 500 mg of vitamin A palmitate (Sigma, R 3750) in a dose of diet (equivalent to 0.10 mg of pure vitamin A/g) led to reappearance of larvae photoreactivity (Table 3). In order to verify that antioxydants present in the tested chemical have no effect on the result above, a similar experiment was conducted using an antioxydant-free compound, vitamin A palmitate solved in oil (Sigma R 3500). The result we get was similar, with re-establishment of a normal response (9% diapause, n = 89) when a quantity Table 3. The effects of various concentrations

Table 1. Effect of light intensity on results of night interruption experiment with 1 h light pulse (22-23 h) in 13 h light-11 h dark in larvae reared on artifical diet at 20°C Light intensity (lx) Photophase

Pulse

300

0

50

0

300 50 300 50

300 50 50 300

Diapause (%) . ,

n

100 97 100 0 100 31

131 37 86 53 64 173

of vitamin A in the artificial diet on the photoreactivity to a 1 h light break (22-23 h) in 13 h light-l 1 h dark Vitamin A palmitate (in mg/g of diet) 0.55 0.83 1.11 1.38

Diapause (%)

n

93 93 9 4

43 125 65 114

Vitamin A palmitate: Sigma (R 3500) 250.000 U.S.P./g, equivalent to 75 mg of pure vitamin A/g. Light intensity: 300 lx. Temperature: 20°C.

J. CLARETand N. VOLKOFF

512

equivalent to 0.10 mg of pure vitamin A/g of diet was added. Hence, artificial diet is deficient in vitamin A (or in its precursor carotenoids) and this chemical is necessary to get a response identical to the one observed when larvae are reared on cabbage. One experiment was chosen from the resonance experiments carried out by Dumortier and Brunnarius (1989) with a 12 h light-60 h dark cycle where the scotophase is scanned with a 2-h light break. We limited our own experiment to the unique portion where the response curves for the two diets differ, that is when the interruption by light occurs in the middle of the night (40-42 h after dawn). Two light intensities were tested: 50 and 300 lx. In our results (Table 4) the first observation was the influence of light intensity on the controls (without interruption). When light intensity was 50 lx in 12 h light-60 h dark, only 1 or 2% of the insects fed on either diet entered diapause. With a 300 lx light, 100% of the insects reared on artificial diet entered diapause, whereas only 3% of the animals reared on cabbage did. Twelve h light-60 h dark is considered as a long-day cycle by larvae reared on cabbage but as a short-day cycle by animals fed with an artificial diet. The second observation was the non-effectiveness of the light-pulse (4@42 h) since the insects, for both diets and for both light intensities, show a percentage of diapause more or less identical to their respective controls. Finally the response of insects reared on artificial diet supplemented with vitamin A is not re-established at 300 lx even though vitamin A restores photosensitivity in B at this intensity. In spite of vitamin A supplementation, semi-synthetic diet still lacks the necessary factor to restore a normal response of the insect’s biological clock when cycles are longer than 24 h. DISCUSSION The experiments show that the photoperiodic response mechanism of P. brassicae larvae reared on

the semi-synthetic diet of David and Gardiner (1966) without powdered cabbage differs from that of larvae reared on cabbage. For the latter, resonance experiments (Nanda-Hamner protocol) revealed the existence of a circadian component in the clock at the time of diapause induction (Claret et al., 1981). The clock of larvae fed on artificial diet remains able to distinguish between short- and long-days but its functioning presents some modified characteristics. In the die1 cycles, point B of nocturnal photosensitivity, found in several other species (see Saunders, 1982), is lacking. The theoretical model of Pittendrigh (1966), called “external coincidence model”, is the one that best fits the experimental results obtained with P. brassicae (Claret et al., 1981; Dumortier and Brunnarius, 1981; Claret, 1982). In this model, B is considered to represent the photoinductible point Qi of a circadian oscillator phased for the light-dark transition. This point is essential for the time measurement since the photoperiod will be considered as a short- or a long-day cycle depending on whether @, falls in a dark or in a light phase. The absence of B caused by food deficiency does not interfere with the ability to distinguish a short-day from a long-day cycle. Therefore, in experimental conditions, there are two possibilities. Either the clock works normally and, in this case, B does not have the importance given by the model and could be considered as accessory, or the clock, under the influence of food composition, modifies its functioning system and performs a time measurement according to another mode (hourglass?). The co-existence in the same individual of a circadian type of clock and an hourglass type of clock has been shown for two species: P. brassicae (Claret, 1985) and Ostrinia nubilafis (Skopik and Takeda, 1986). The first acts in the larvae when diapause is induced, the second when diapause is terminated. The switching of the clock mechanism could occur at metamorphosis. Another alternative could be the existence of two anatomically

Table 4. Effectsof diet on photoreactivity to a 2 h light break (4&42 h) in 12 h light-60 h dark Diapause (%) Artificial diet + vitamin A palmitate (mg/g) Cabbage Controls in LD12:60 50 lx 1 (90) 300 lx 3 (71) With light break 50 lx 300 lx

2 (41) 9 (68)

Artificial diet

1.38

2.77

2 (91) 100 (58)

16 (81) 82 (108)

100 (38)

0 (34) 96 (68)

0 (49) 95 (73)

The number of experimental insects is indicated in parentheses. Light intensity: 300 or 501x. Temperature: 20°C.

-

Photoperiodic

reaction in P. brassicae

different clocks, a larval and a pupal clock, each working in a different way. The first hypothesis is the most probable given the results presented on the disappearance of B. Results on diet supplementation suggest that the larval clock is able to work like an hourglass, which is a latent potentiality that would normally only be revealed after metamorphosis. However, our hypothesis is only conjectural. Temperature is also a factor that has been shown to modify responses of insects to resonance experiments (Saunders, 1982). Nanda-Hamner experiments in some insects resulted, at higher temperatures, in resonance peaks at circadian intervals but, at lower temperatures, in a response close to an hourglass mechanism. Carotenoids, or vitamin A, are supposed to be necessary for brain photoreceptor pigment formation. For P. brussicae, the diet of David and Gardiner without powdered cabbage, and specially treated in order to eliminate carotenoids, has to be supplemented with 0.008 mg of pure vitamin A/g to restore the larval response to the diapause inducing photoperiod (Claret, 1989). The use of non-treated diet, containing 0.001-0.02 mg of carotenoids for 1 g of diet (Rothschild ef al., 1977a, b), leads to the disappearance of B. Restoration of B is only obtained if 0.10 mg of vitamin A/g of diet are added to the non-treated diet described above. Therefore, there are two levels of need for vitamin A (separated by a factor 12). The first permits the distinction between short- and long-day cycle and, the second, the response at point B. If the first concentration (0.008 mg/g) is essential for the formation of the photoreceptor pigment, we do not know how the second (0.10 mg/g) acts on the clock or the counter mechanism. Loss of B is related to light intensity and to duration of exposure, since a photophase of 13 h at 300 lx has more importance on suppression of the response in B than a 1 h light break with the same intensity. However, does the 300 lx light act on the diet or on the P. brassicae larvae? We can suppose there is a destructive (or inactivator) effect of light on the carotenoids of the diet, and this effect would be reduced when light is 50 lx. But length of exposure at 300 lx is the same (13 + I) in the case of an effective interruption at the beginning of the night (A) and in the case of an ineffective light-pulse at the end of the scotophase (B). Therefore, if light does not interfere with the diet, the lack of response of the larvae to a late interruption by a 300 lx light might be related to a circadian rhythmicity of a biological process interfering with diapause induction. This process would need a rather large quantity of vitamin A in order to permit the response of insects at the end of the night. If the need for vitamin A is only related to the

573

pigment formation, the hypothesis of a unique brain receptor for the main photophase and the two peaks A and B is difficult to accept. It is more probable that there are two photoreceptors, one for the main photophase and the light at A and another one for the light at B, each with different needs in vitamin A. However, there is still the possibility that vitamin A not only plays a role in photoreception but also in the clock or counter mechanism, for which a higher quantity would be necessary. The action of temperature on the effectiveness of a light interruption at point B seems curious if we consider the destructive effect of light on carotenoids. So the question of how a temperature increase could act against light action still remains. With 72 h-cycles at 50 lx, results are the same on artificial and natural diet. At 300 lx, the nature of the diet does not change the response of insects submitted to a night interruption at 40-42 h but modifies the response of the controls. Vitamin A supplementation, even at the concentration of 0.20mg/g, does not change the results. Although related to light intensity, the influence of diet composition does not seem to come from a lack of carotenoids. Carotenoid levels in fresh cabbage present variety and seasonal variations, and data indicate levels ranging from 0.034 to 0.37 mg/g (Feltwel and Valadon, 1974; Rothschild et al., 1977b). However, vitamin A supplementation at levels close to the maximum measured on cabbage does not restore the response of controls. In their resonance experiments Dumortier and Brunnarius (1989) noticed an important difference between insects reared on cabbage and insects reared on artificial diet in their response to a light interruption in the middle of an extended night (60 h). Response curves were superposed for the two diets except on the central portion of the night. They concluded that the clock mechanism switched from a circadian type to an hourglass one. The results found in our work on night interruption by a light-pulse (300 lx) at 40-42 h are very close to their results (3% diapause on cabbage, 100% on artificial diet). The difference between the two studies is the lack of controls (12 h light-60 h dark without interruption) in their publication, which makes their results impossible to interpret. If controls had given the same percentages of diapause as in our work, then experiments on the central part of the night would not indicate a difference. This is because insects submitted to a light break react in both food conditions like their respective controls. Other authors (Veerman et al., 1988) noticed that the clock of P. brassicae reared on the David and Gardiner diet with powdered cabbage and 0.3 g/l vitamin A palmitate (0.022 mg pure vitamin A/g) functions more like an hourglass

514

than

J. CLARETand N. VOLKOFF an oscillator.

resonance

Their

experiments

and on experiments

observation

is based

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of temporary

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tests are negative Therefore

protocol)

transfer

dark. The latter allow them to conclude an interval-timer

on

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that there is However,

at 19°C and positive possible

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that food combut the food has still to be

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Van Houten Y. M., Overmeer W. P. J. and Veerman A. (1987) Thermoperiodically induced diapause in a mite in constant darkness is vitamin A dependent. Experientia 43, 933-93s.

Van Zon A. Q.. Overmeer W. P. J. and Veerman A. (1981) Carotenoids function in photoperiodic reaction in a predacious mite. Science 213, 1131-l 133. Veerman A. and Helle W. (1978) Evidence for the functional involvement of carotenoids in the photoperiodic reaction of spider mites. Nature 275, 234. Veerman A., Overmeer W. P. J., Van Zon A. Q., De Boer J. M., De Waard E. R. and Huisman H. 0. (1983) Vitamin A is essential for photoperiodic induction in an eyeless mite. Nature 302, 248-249. Veerman A., Slagt M. E., Alderlieste M. F. J. and Veenendaal R. L. (1985) Photoperiodic induction of diapause in an insect is vitamin A dependent. Exaerientia 41,-l 194-l 195. Veerman A., Beekman M. and Veenendaal R. L. (1988) Photoperiodic induction of diapause in the large white butterfly Pieris brassicae: evidence for hourglass time measurement. J. Insect Physioi. 33, 1063-1069.