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Change in photoperiodic sensitivity during larval development of Pieris brassicae
Pergamon
0022-1910(94)00072-7
J. Insect Physiol. Vol. 41, No. 1, pp. 77-83, 1995 Copyright 0 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved oozz-1910/95 S9.50 + 0.00
Change in Photoperiodic Sensitivity During Larval Development of Pieris brassicae HUBERT
R. SPIETH*
Received I1 March 1994; revised 14 June 1994
Sensitivity to light/dark cycles in Pieris brussicae starts with the second larval stage at a low level, reaches a maximum l-2 days after the moult to the last larval stage, and then decreases rapidly to zero just before pupation. This peak in photoperiodic sensitivity is an invariable character of the species. It is not affected by temperature or by the strength of the photoperiodic signal, “or does it vary between different populations. This age-dependent sensitivity is a scarcely noticed quantitative component in arthropod time measurement. This study discusses, whether the change in photoperiodic sensitivity is an adaptation to minimize the risk of taking the wrong developmental pathway in late summer. Pieris brassicae
Photoperiodic
sensitivity
Quantitative
INTRODUCTION
time measurement
Fomenko, 1983, 1991; Hardie, 1990; Zaslavski, 1992), in different Drosophila species (Kimura, 1990), and in the large white butterfly, Pieris brussicue (Spieth and Sauer, 1991). These results suggest that the photoperiodic response of an individual not only depends on the mere frequency of a light/dark regime, but also on its inductive strength. Therefore, the concept of the photoperiodic counter has changed over the last years. In the species mentioned above, it seems to be clear now that the counter does not count qualitative signals, but accumulates the quantitative information generated by the clock. It is supposed__that the information is stored as an unknown “diapatise inducing substance”. The daily synthesized amount of this substance depends on both duration of the scotophase (or photophase) and temperature. Diapause is induced when the titre of the accumulated substance, called “induction sum” (Vaz Nunes and Veerman, 1982), exceeds a certain threshold. Recent models of the photoperiodic clock include the quantitative measurement of photoperiodic events to a varying degree (Lewis and Saunders, 1987; Saunders and Lewis, 1987; Zaslavski, 1988; Vaz Nunes et al., 1991a, b). In the model presented by Lewis and Saunders (1987) it is pointed out that the production of the induction sum is not only affected by the number of photocycles and by temperature, but also by the agl of the individual. Until now, this factor was rarely considered. Previous examinations on the large white butterfly could not detect any significant change of photoperiodic sensitivity during larval development (David and Gardiner, 1962; Danilevsky, 1965). Biinning and Jiirrens (1960) mentioned that in P. brussicue the photoperiodic sensitivity was higher after the third
In diapause research, the term “sensitive period” describes a distinct phase in an organism’s life cycle, during which, in contrast to other phases, light/dark regimes affect its photoperiodic response. Until now, the reason for the restriction of this sensitivity to a specific developmental phase has never been examined. Without knowing the underlying physiological mechanisms, experiments dealing with the photoperiodic response in arthropods show that daily measurement of the photoperiodic clock is recorded and stored only during an individuals sensitive period. Saunders (1971, 1981) developed the generally accepted concept of the “photoperiodic counter”. According to this concept, the photoperiodic clock defines a certain light/dark regime as either long-night or short-night, and the photoperiodic counter adds up the number of such qualitative events. The mode of development then depends on whether the number of short-nights (or long-nights), called the “required day number”, exceeds a certain internal threshold. Recent papers on diapause behaviour in arthropods showed that the photoperiodic clock does not classify a certain photocycle as either long-night or short-night in all cases, but rather measures the absolute length of the photo- or scotophase. Evidence for this quantitative measurement of photocycles was found in the knotgrass moth, Acronycta rumicis (Zaslavski and Fomenko, 1980), in the vetch aphid, Megoura viciue (Zaslavski and
*Lehrstuhl fiir Evolutionsforschung, Fakultlt fiir Biologie, Universitlt Bielefeld, UniversitltsstraDe 25, 33615 Bielefeld, Germany. 77
78
HUBERT
moult, but did not provide the experimental evidence to support this statement. Claret (1972) later confirmed this in a detailed investigation and stated that the sensitive period started after the third mouft. However, his results also indicated that the photoperiodic sensitivity is not strictly bound to certain larval stages, but that there may be a gradual change in the effectivity of a diurnal light/dark regime. Veerman et al. (1988) emphasized the change in photoperiodic sensitivity, and claimed that the inductive value of a long-night cycle varies in a quantitative manner with the age of the larvae. This paper examines how the diapause inducing effect of diurnal long-night or short-night regimes varies during the larval development of P. brassicae. I also investigate, whether photoperiodic sensitivity changes with temperature or between individuals of different geographical origin.
MATERIALS
AND METHODS
For the experiments, caterpillars from four laboratory stocks of different geographical origin (Horsens in Denmark, 56”N/ 1O”O; Flensburg in Germany, 55”N/lO”O; Anglian Heights in England, .52”N/O”O; and Banyuls in France, 42.5”N/3”0) were used. Eggs and caterpillars were kept in plastic boxes (10 x 10 x 7 cm) with gaze lids under controlled light regimes at constant temperature. Each box contained about 20 individuals. The whole batch of larvae tested under a certain light/dark regime comprised descendants of at least 20 females. Once a day the larvae were fed fresh cauliflower leaves. Pupae were stored in the dark at room temperature until the non-diapause individuals had emerged. The photoperiodic sensitive stage in P. bra&cue is the caterpillar. Unless otherwise noted, the larvae were initially reared under a long-night regime. To induce a non-diapause response in a proportion of the individuals, the long-night background was interrupted by a few short-nights (19 h light-5 h dark). This “short-night sequence” was inserted into the long-night background at different larval stages. With this experimental set-up a change of photoperiodic sensitivity should be indicated as a change in diapause response. A change of photoperiodic sensitivity can be most precisely determined if the short-night sequence consists of one or only a few photocycles with high inductive strength. In P. brassicae, the inductive strength of a short-night is enhanced if the strength of the long-nights, which were used as a background regime, is weakened (Spieth and Sauer, 1991). Long-nights with long photophases have a weak diapause inductive strength. Therefore, in each experiment long-nights with photophases of maximum length were used. This particular photophase at which diapause is induced in all individuals of a population was determined by the photoperiodic response of that population. In this way, the effect of the short-night sequence could be optimized.
R. SPIETH
RESULTS
The sensitive period After eclosion, caterpillars of the Flensburg-population were reared in a L/D regime of 14 h 20 min/9 h 40 min at 20°C. Under this regime less than 1% of the animals (1 of 125) responded with non-diapause. This long-night background was interrupted by two (2SN), three (3SN), and four (4SN) short-nights of L/D 19 h/5 h. As can be seen in Fig. 1, the 2 SN experiment shows that a non-diapause response started with the fourth larval stage and ended just before the prepupa. It was restricted to the 4th and 5th larval stage (L4 and L5). Furthermore, it turned out that the response attained a maximum of 43% non-diapause 1 day after moulting to L5. This indicates a very short ontogenetic phase of maximum photoperiodic sensitivity, which gradually declines to both sides without reaching a phase of saturation at the chosen conditions. This applied to 43% of the individuals. For the remaining 57%, two short-nights are a too weak stimulus for a non-diapause (Spieth and Sauer, 1991). If the larvae were exposed to three short-nights (Fig. 1, 3SN), the position of highest sensitivity remained unchanged at 1-2 days after the moult to L5. The percentage of non-diapause at this phase was about twice as high (89%) as with two short-nights. After L5/2 the photoperiodic sensitivity decreased rapidly and ended, as was the case with two short-nights, just before the prepupal stage. But the course of sensitivity before the L5 stage was different because the sensitivity started earlier. Some sensitivity was even visible in L2 and was obvious with 17% non-diapause in L3. With three short-nights, 11% still responded with diapause at the phase of highest sensitivity. These individuals needed a stronger stimulus of at least four short-nights to develop without diapause. The result (Fig. 1, 4SN) indicates an obvious photoperiodic sensitivity in the L2 stage. A sensitivity in the Ll stage is not proved by this measurement, but can be expected in face of the course of the 4SN curve. An induction of diapause in the first larval stages may only be prevented with a stronger stimulus, that is a higher number of short-night cycles. But in the Ll stage more cycles would be required than this stage actually lasts. From these experiments we can deduce that P. brassicae is photoperiodic sensitive during its whole larval life. Photoperiodic
sensitivity
to long-night
cycles
To test the effect of long-nights an experiment under inverse conditions was carried out. Larvae were reared in a short-night regime interrupted by a long-night sequence. In order to strengthen the effect of long-nights, the photophase of the background (L/D 15 h 45 min/8 h 15 min) was selected to just prevent diapause in all individuals (120 of 122). The long-night sequence comprised 4 cycles of L/D 10 h/14 h. The results are shown in Fig. 2, where the response is directly compared with the 3SN curve of the inverse
PHOTOPERIODIC
0
4
2
t Ll/O
t wo
6
SENSITIVITY
6
t L3/0
lo
79
12:
14
16
t L5lO
t L4lO
16
t t PP P
Larval Development [Days] FIGURE 1. Photoperiodic sensitivity in P. brassicae to short-night cycles. The sensitivity is measured as change of the non-diapause response of individuals which were reared in a diapause inducing regime of L/D 14 h 20 min/9 h 40 min, interrupted by two (2 SN), three (3 SN), and four (4 SN) short-nights (L/D 19 h/5 h) during different stages of development. As in all subsequent figures, the measured values are depicted midway through the short-night sequence, e.g. by a 2 short-night sequence after 1 day. The abscissa indicates the duration of larval development and the moulting stages (Ll/&L5/0 = larval stages; PP = prepupa; P = pupa) are drawn. n = 118-232 for each point.
0
t LllO
4
2
t wo
6
6
t L3/0
10
t L4lO
12:
t L5/0
14
16
16
t t PP P
Larval Development [Days] FIGURE 2. Photoperiodic sensitivity in P. brussicae to non-diapause response of individuals which were reared interrupted by four long-nights (L/D 10 h/4 h) during short-night response (SN), also depicted in Fig.
long-night cycles (LN). The sensitivity is measured as change of the in a non-diapause inducing regime of L/D 15 h 45 min/l h 15 min, different stages of development. The curve is compared with the 1. For the abscissa see Fig. 1. n = 122-170 for each point.
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HUBERT
R. SPIETH
experiment depicted in Fig. 1. Though the courses are inverse, the configuration of both curves is to a large extent identical. The maximum photoperiodic sensitivity appeared at the L5/1 stage. This indicates that the change in photoperiodic sensitivity during the larval development is not affected by the quality of L/D regimes, which may be experienced by individuals in nature. Geographical
comparison
of photoperiodic
short-nights caused a response that is more or less identical with that of the other populations (Fig. 3). The eflect of temperature Some populations were examined for their photoperiodic sensitivity at 15°C a temperature at which the developmental time of the larvae was 33 days (Fig. 4) which is nearly twice as long as at 20°C. The scotophase of the long-night background was adapted for each population according to its respective photoperiodic response at 15°C as already mentioned (Flensburg: 14 h 30 min; Banyuls: 12 h 55 min; Horsens: 15 h 05 min). All these regimes induce more than 99% diapause. The short-night sequence comprised 6 cycles of L/D 19 h/5 h. The results are depicted in Fig. 4. The change of photoperiodic sensitivity showed the same pattern in all populations, confirming the results at 20°C (Fig. 3). The maximum sensitivity was identical in all three cases and was at the same position on the developmental axis as at 20°C. From these results we can state that temperature, within a range often experienced by individuals in nature, does not influence the course of photoperiodic sensitivity. A difference in the intensity of a non-diapause response among the populations was obvious (Fig. 4). The Flensburg-population showed the strongest response (80% non-diapause). The other two responded about as half as strongly. The reasons for this behaviour were not examined. It may be that the Horsenspopulation needs a stronger short-night stimulus, as already elaborated above. In the Banyuls-population the
sensitivity
The conclusions deduced from the previously described experiments are valid for the Flensburg-population. But no general statement can be made for the species as a whole. Therefore, individuals of different geographical origin were tested. The photophase of the long-night background for the three populations (Anglian Heights: 14 h; Flensburg: 14 h 20 min; Horsens: 14 h 55 min) was determined by their photoperiodic response, as described above. The background regime was interrupted by two short-nights of L/D 19 h/5 h. Figure 3 shows that there were no differences in the peak sensitivity between the populations from England and Germany. Also the configuration of the curves and their position on the horizontal axis are identical. Non-diapause development invariably starts with the fourth and ends with the last larval stage. The population from Denmark behaved differently in that only a few individuals responded to two shortnights with non-diapause. But when the inducing signal was strengthened by an increase in the number of cycles, an appreciable response occurred. A doubled number of
m
0
4
2
Flensburg
B
England
6
6
t
t
WO
L3lO
10
t
L4lO
Larval Development
m
12:
Horsens
14
t L5lO
16
16
t
t
PP P
[Days]
FIGURE 3. Comparison of the photoperiodic sensitivity in populations of P. brassicae from different geographical origins. The non-diapause response induced by two (Anglian Heights = England; Flensburg) and four (Horsens) short-nights (L/D 19 h/5 h) was recorded in individuals which were reared in a long-night regime with population specific photophases (see text). For the abscissa see Fig. 1. n = 83-241 for each point.
PHOTOPERIODIC
0
4
8
12
81
SENSITIVITY
16
20
i
24
28
32
I
+ LiYO
4 L3lO
+ Lao
Larval Development FIGURE 4. Photoperiodic sensitivity origin are compared (H = Horsens; F 19 h/5 h) was recorded in individuals text).
long-night background may adapted to the photoperiodic
4 L5lO
t PP
I
4 P
[Days]
in P. brass&e at a lowered temperature of 15°C. Populations of different geographical = Flensburg; B = Banyuls). The non-diapause response induced by six short-nights (L/D which were reared in a long-night regime specifically adapted for each population (see For the abscissa see Fig. 1. n = 93-175 for each point.
not have been optimally response at 15°C.
DISCUSSION
Individuals of the large white butterfly, P. brassicae, reared under a long-night regime responded to a shortnight sequence, which was shifted through the different with changing proportions of nonlarval stages, diapause. The response started at 0% in newly emerged larvae, increased gradually reaching a maximum l-2 days after the moult to the 5th larval stage, and then decreased again to 0% just before pupation. These results show for the first time that short-nights in the second stage lead to non-diapause in a proportion of the tested individuals. The course of the non-diapause response can be recorded as a gradual change of photoperiodic sensitivity, with a maximum l-2 days after the moult to the L5 stage. The phase of highest photoperiodic sensitivity corresponds to a great extent to Claret’s (1972) conclusions. However, the results did not confirm his statement that no inducing effect of photoperiods could be measured before the third moult of a larva. Rather, the experiments support the findings of an earlier paper (Spieth and Sauer, 1991) that photoperiodic events experienced by larvae of P. brassicae during their first three stages effect the mode of development decisively. If a non-diapause response was induced at the beginning of larval development, then it was not cancelled by long-nights, even if experienced in the phase of highest photoperiodic sensitivity. In their paper on the mechanism of the photoperiodic
clock Veerman et al. (1988) suggested for the first time that in P. brassicae an unchanged long-night cycle does not have the same inductive strength during larval development even in the phase of high photoperiodic sensitivity. The described results confirm this presumption. There is no saturation, but a definite peak of maximum sensitivity (Figs 1 and 3). The inductive strength of a photoperiodic event changed gradually during all larval stages. To prevent a diapause response in the first larval stages or at the end of larval development the caterpillars require a stronger stimulus than in the phase of high photoperiodic sensitivity, e.g. a higher number of short-night cycles. The temporal pattern of the change of photoperiodic sensitivity in P. brassicae is very constant. The response in each geographical population tested at 20 or 15°C was very similar. A separate sensitivity and different maxima for the alternative regimes of short-night and long-night, as mentioned by Claret (1972, 1983), could not be found. According to the present results, the temporal pattern of photoperiodic sensitivity is not variable, but a character of the species. The changing photoperiodic sensitivity is very important for the design of models to describe photoperiodic responses. In recently developed models it is postulated that diapause is induced by the accumulation of an unknown substance. This diapause-inducing substance is synthesized during the scotophase of a photoperiodic cycle and subsequently passed to an internal storage. The daily synthesized amount of the substance determines the inductive strength of a 24 h light/dark cycle. More substance is produced with in-
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HUBERT
creasing duration of the scotophase (Vaz Nunes et al., 199 1b; Spieth and Sauer, 199 1) and decreasing temperature (Lewis and Saunders, 1987). Both factors are thought to alter properties of the photoperiodic clock, so that its output changes quantitatively. The required day number, often seen as a separate response inducing factor, is only a dependent variable of this output. Besides temperature and duration of the scotophase, an age-dependent change of photoperiodic sensitivity, as shown in this paper, is a further process which influences the photoperiodic response in a quantitative way. According to the discussed clock model, the gradual change of sensitivity is equivalent to a change of the inductive strength of a L/D cycle. Until now we did not know which level of the measuring system, the receptor, the clock, or a process subsequent to the clock, is affected by age. Zaslavski (1988, 1992) suggests that if the clock is exposed to a constant photoperiodic stimulus, it generates a constant signal independent of the larval stage. If this is true, then the photosensitivity of an individual (in the sense of receptivity to light or dark) does not really change with age. Some evidence also comes from a comparison of the temperature experiments (Fig. 4). As the peak sensitivity at 15°C as well as 20°C is achieved in the first days after the moult to L5, irrespective of the large difference in duration of larval development at both temperatures, the change of sensitivity does not appear to be triggered by a clock mechanism, but seems rather to be a stage specific character. If not the clock but one of the processes subsequent to the clock, such as those involved in transition and storing of the generated clock signals, are modified or temporarily inhibited with increasing age, then the photoperiodic sensitivity has nothing to do with a sensitivity to light or dark. It merely describes the age-dependent processing of an unchanged light/dark generated photoperiodic clock signal. An interesting question is, why does photoperiodic sensitivity in P. brassicae change at all? Are there physiological constraints, or is it a matter of adaptation to certain life cycles? In arthropod species examined to date the course of photoperiodic sensitivity is so heterogeneous (reviews in: Beck, 1980; Saunders, 1982; Danks, 1987; Veerman, 1992) that physiological contraints cannot be an explanation. On the other hand, the heterogenity also does not allow a general statement about the adaptive value. Therefore, it is necessary to formulate a separate hypothesis for each case (Danks, 1987). In the following this was tried for the adaptive value in P. brassicae. The large white butterfly belongs to a group of butterflies passing through a facultative diapause which is triggered by daylength. The diapause is very strong; it lasts in the field about 7 months, and is terminated by winter temperatures (Feltwell, 1982; Spieth, 1985). In central Europe, caterpillars hatching in August have a developmental time of about 3540 days under normal climatic conditions. During this time they have two choices: either they overwinter as diapausing pupae or they use the remaining food supply in the field
R. SPIETH
and reproduce again before winter. The latter provides the change to propagate its genes in the spring generation as multiple copies, but on the other hand it includes the risk that unfavourable climatic conditions could slow down the development of the progeny and they could either starve or be killed by frost. The decision to hibernate or not, is determined by daylength but modified to a large extent by the highly unpredictable weather conditions. Low temperature prolongs the development decisively so that caterpillars may experience much shorter daylengths at the peak sensitivity than at high temperature. If the phase of highest photoperiodic sensitivity occurs long before the pupal stage, subsequent unfavourable changes of external factors would have no or only minor effect on the mode of development. Against this background, the peak sensitivity during a phase just before the diapause stage makes sense. With a long lasting larval development a relatively late decision can diminish the risk to take the wrong path of development corresponding to season and climate.
REFERENCES Beck S. D. (1980) Insect Photoperiodism, 2nd edn. Academic Press, New York. Biinning E. and Joerrens G. (1960) Tagesperiodische antagonistische Schwankungen der Blauviolettund Gelbrot-Empfindlichkeit als Grundlage der photoperiodischen Diapause-Induktion bei Pieris brassicae. 2. Naturf. 15b, 205-213. Claret J. (1972) Period de sensibilitl des chenilles de Pieris brassicae i la photoptriode controlant la diapause. C. R. hebd. Sane. Acad. Sci., Paris 274, 1055-1058. Claret J. (1983) Signification differentielle des photoperiodes pour l’horloge biologique de Pieris brassicae (Lepidoptera). J. interdiscipl. Cycle Res. 14, 63-73. Danilevsky A. S. (1965) Photoperiodism and Seasonal Development of Insects. Oliver & Boyd, London. Danks H. V. (1987) Insect Dormancy: An Ecological Perspective. Biological Survey of Canada, Ottawa. David W. A. L. and Gardiner B. 0. C. (1962) Observations on the larvae and pupae of Pieris brassicae (L.) in a laboratory culture. Bull. ent. Res. 53, 417436. Feltwell J. (1982) Large White Butterfly: The Biology, Biochemistry, and Physiology of Pieris brassicae (Linnaeus). Dr W. Junk Publishers, The Hague. Hardie J. (1990) The photoperiodic counter, quantitative day-length effects and scotophase timing in the vetch aphid Megoura viciae. J. Insect Physiol. 36, 939-949. Kimura M. T. (1990) Quantitative response to photoperiod during reproductive diapause in the Drosophila auraria species-complex. J. Insect Physiol. 36, 147-152. Lewis R. D. and Saunders D. S. (1987) A damped circadian oscillator model of an insect photoperiodic clock. I. Description of the model based on a feedback control system. J. theor. Biol. 128, 47-59. Saunders D. S. (1971) The temperature-compensated photoperiodic clock ‘programming’ development and pupal diapause in the fleshfly, Sarcophaga argyrostoma. J. Insect Physiol. 17, 801-812. Saunders D. S. (1981) Insect photoperiodism-the clock and the counter: a review. Physiol. Ent. 6, 99-l 16. Saunders D. S. (1982) Insect clocks, 2nd edn. Pergamon Press, Oxford. Saunders D. S. and Lewis R. D. (1987) A damped circadian oscillator model of an insect photoperiodic clock. II. Stimulation of the shapes of the photoperiodic response curves. J. theor. Biot. 128, 61-71. Spieth H. R. (1985) Die Anpassung des Entwicklungszyklus an
PHOTOPERIODIC unterschiedlich lange Vegetationsperioden beim Wanderfalter Pieris brussicae L. (Lepidoptera: Pieridae). Zool. Jber. Cyst. 112, 3569. Spieth H. R. and Sauer K. P. (1991) Quantitative measurement of photoperiods and its significance for the induction of diapause in Pieris brassicae (Lepidoptera, Pieridae). J. Insect Physiol. 37, 231-238. Vaz Nunes M. and Veerman A. (1982) Photoperiodic time measurement in the spider mite Tetranychus urticae: a novel concept. J. Insect Physiol. 2.8, 1041-1053. Vaz Nunes M., Lewis R. D. and Saunders D. S. (1991a) A coupled oscillator feedback system as a model for the photoperiodic clock in insects and mites. I. The basic control system as a model for circadian rhythms. J. theor. Biol. 152, 287-298. Vaz Nunes M., Saunders D. S. and Lewis R. D. (1991b) A coupled oscillator feedback system as a model for the photoperiodic clock in insects and mites. II. Simulation of photoperiodic responses. J. theor. Biol. 152, 299-311. Veerman A. (1992) Diapause in phytoseiid mites: a review. Expl Appl. Acarol. 14, 140. Veerman A., Beekman M. and Veenendaal R. L. (1988) Photoperiodic induction of diapause in the large white butterfly, Pieris brassicae:
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evidence for hourglass time measurement. J. Insect Physiol. 34, 1063-1069. Zaslavski V. A. (1988) Insect Development: Photoperiodic and Temperature Control. Springer, Berlin. Zaslavski V. A. (1992) Light-break experiments with emphasis on the quantitative perception of nightlength in the aphid Megoura viciae. J. Insect Physiol. 38, 717-725. Zaslavski V. A. and Fomenko R. B. (1980) New data on the photoperiodic reaction of the knotgrass moth Acronycta rumicis L. (Lepidoptera, Noctuidae). Ent Rev. 59, 9-16. Zaslavski V. A. and Fomenko R. B. (1983) Quantitative photoperiodic perception in the aphid Megoura viciae Buckt. (Homoptera, Aphididae). Enc. Rev. 62, l-10. Zaslavski V. A. and Fomenko R. B. (1991) An experimental study of the induction of photoperiodism in the aphid Megoura viciae Buckt. (Homoptera, Aphididae). Ent. Rev. 70, 25-35.
Acknowledgements-The author would like to thank Dr Horst Schwarz, Dr Bob Kosier and Ralf Cordes for discussion and commenting on the manuscript.
0022-1910(94)00072-7
J. Insect Physiol. Vol. 41, No. 1, pp. 77-83, 1995 Copyright 0 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved oozz-1910/95 S9.50 + 0.00
Change in Photoperiodic Sensitivity During Larval Development of Pieris brassicae HUBERT
R. SPIETH*
Received I1 March 1994; revised 14 June 1994
Sensitivity to light/dark cycles in Pieris brussicae starts with the second larval stage at a low level, reaches a maximum l-2 days after the moult to the last larval stage, and then decreases rapidly to zero just before pupation. This peak in photoperiodic sensitivity is an invariable character of the species. It is not affected by temperature or by the strength of the photoperiodic signal, “or does it vary between different populations. This age-dependent sensitivity is a scarcely noticed quantitative component in arthropod time measurement. This study discusses, whether the change in photoperiodic sensitivity is an adaptation to minimize the risk of taking the wrong developmental pathway in late summer. Pieris brassicae
Photoperiodic
sensitivity
Quantitative
INTRODUCTION
time measurement
Fomenko, 1983, 1991; Hardie, 1990; Zaslavski, 1992), in different Drosophila species (Kimura, 1990), and in the large white butterfly, Pieris brussicue (Spieth and Sauer, 1991). These results suggest that the photoperiodic response of an individual not only depends on the mere frequency of a light/dark regime, but also on its inductive strength. Therefore, the concept of the photoperiodic counter has changed over the last years. In the species mentioned above, it seems to be clear now that the counter does not count qualitative signals, but accumulates the quantitative information generated by the clock. It is supposed__that the information is stored as an unknown “diapatise inducing substance”. The daily synthesized amount of this substance depends on both duration of the scotophase (or photophase) and temperature. Diapause is induced when the titre of the accumulated substance, called “induction sum” (Vaz Nunes and Veerman, 1982), exceeds a certain threshold. Recent models of the photoperiodic clock include the quantitative measurement of photoperiodic events to a varying degree (Lewis and Saunders, 1987; Saunders and Lewis, 1987; Zaslavski, 1988; Vaz Nunes et al., 1991a, b). In the model presented by Lewis and Saunders (1987) it is pointed out that the production of the induction sum is not only affected by the number of photocycles and by temperature, but also by the agl of the individual. Until now, this factor was rarely considered. Previous examinations on the large white butterfly could not detect any significant change of photoperiodic sensitivity during larval development (David and Gardiner, 1962; Danilevsky, 1965). Biinning and Jiirrens (1960) mentioned that in P. brussicue the photoperiodic sensitivity was higher after the third
In diapause research, the term “sensitive period” describes a distinct phase in an organism’s life cycle, during which, in contrast to other phases, light/dark regimes affect its photoperiodic response. Until now, the reason for the restriction of this sensitivity to a specific developmental phase has never been examined. Without knowing the underlying physiological mechanisms, experiments dealing with the photoperiodic response in arthropods show that daily measurement of the photoperiodic clock is recorded and stored only during an individuals sensitive period. Saunders (1971, 1981) developed the generally accepted concept of the “photoperiodic counter”. According to this concept, the photoperiodic clock defines a certain light/dark regime as either long-night or short-night, and the photoperiodic counter adds up the number of such qualitative events. The mode of development then depends on whether the number of short-nights (or long-nights), called the “required day number”, exceeds a certain internal threshold. Recent papers on diapause behaviour in arthropods showed that the photoperiodic clock does not classify a certain photocycle as either long-night or short-night in all cases, but rather measures the absolute length of the photo- or scotophase. Evidence for this quantitative measurement of photocycles was found in the knotgrass moth, Acronycta rumicis (Zaslavski and Fomenko, 1980), in the vetch aphid, Megoura viciue (Zaslavski and
*Lehrstuhl fiir Evolutionsforschung, Fakultlt fiir Biologie, Universitlt Bielefeld, UniversitltsstraDe 25, 33615 Bielefeld, Germany. 77
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HUBERT
moult, but did not provide the experimental evidence to support this statement. Claret (1972) later confirmed this in a detailed investigation and stated that the sensitive period started after the third mouft. However, his results also indicated that the photoperiodic sensitivity is not strictly bound to certain larval stages, but that there may be a gradual change in the effectivity of a diurnal light/dark regime. Veerman et al. (1988) emphasized the change in photoperiodic sensitivity, and claimed that the inductive value of a long-night cycle varies in a quantitative manner with the age of the larvae. This paper examines how the diapause inducing effect of diurnal long-night or short-night regimes varies during the larval development of P. brassicae. I also investigate, whether photoperiodic sensitivity changes with temperature or between individuals of different geographical origin.
MATERIALS
AND METHODS
For the experiments, caterpillars from four laboratory stocks of different geographical origin (Horsens in Denmark, 56”N/ 1O”O; Flensburg in Germany, 55”N/lO”O; Anglian Heights in England, .52”N/O”O; and Banyuls in France, 42.5”N/3”0) were used. Eggs and caterpillars were kept in plastic boxes (10 x 10 x 7 cm) with gaze lids under controlled light regimes at constant temperature. Each box contained about 20 individuals. The whole batch of larvae tested under a certain light/dark regime comprised descendants of at least 20 females. Once a day the larvae were fed fresh cauliflower leaves. Pupae were stored in the dark at room temperature until the non-diapause individuals had emerged. The photoperiodic sensitive stage in P. bra&cue is the caterpillar. Unless otherwise noted, the larvae were initially reared under a long-night regime. To induce a non-diapause response in a proportion of the individuals, the long-night background was interrupted by a few short-nights (19 h light-5 h dark). This “short-night sequence” was inserted into the long-night background at different larval stages. With this experimental set-up a change of photoperiodic sensitivity should be indicated as a change in diapause response. A change of photoperiodic sensitivity can be most precisely determined if the short-night sequence consists of one or only a few photocycles with high inductive strength. In P. brassicae, the inductive strength of a short-night is enhanced if the strength of the long-nights, which were used as a background regime, is weakened (Spieth and Sauer, 1991). Long-nights with long photophases have a weak diapause inductive strength. Therefore, in each experiment long-nights with photophases of maximum length were used. This particular photophase at which diapause is induced in all individuals of a population was determined by the photoperiodic response of that population. In this way, the effect of the short-night sequence could be optimized.
R. SPIETH
RESULTS
The sensitive period After eclosion, caterpillars of the Flensburg-population were reared in a L/D regime of 14 h 20 min/9 h 40 min at 20°C. Under this regime less than 1% of the animals (1 of 125) responded with non-diapause. This long-night background was interrupted by two (2SN), three (3SN), and four (4SN) short-nights of L/D 19 h/5 h. As can be seen in Fig. 1, the 2 SN experiment shows that a non-diapause response started with the fourth larval stage and ended just before the prepupa. It was restricted to the 4th and 5th larval stage (L4 and L5). Furthermore, it turned out that the response attained a maximum of 43% non-diapause 1 day after moulting to L5. This indicates a very short ontogenetic phase of maximum photoperiodic sensitivity, which gradually declines to both sides without reaching a phase of saturation at the chosen conditions. This applied to 43% of the individuals. For the remaining 57%, two short-nights are a too weak stimulus for a non-diapause (Spieth and Sauer, 1991). If the larvae were exposed to three short-nights (Fig. 1, 3SN), the position of highest sensitivity remained unchanged at 1-2 days after the moult to L5. The percentage of non-diapause at this phase was about twice as high (89%) as with two short-nights. After L5/2 the photoperiodic sensitivity decreased rapidly and ended, as was the case with two short-nights, just before the prepupal stage. But the course of sensitivity before the L5 stage was different because the sensitivity started earlier. Some sensitivity was even visible in L2 and was obvious with 17% non-diapause in L3. With three short-nights, 11% still responded with diapause at the phase of highest sensitivity. These individuals needed a stronger stimulus of at least four short-nights to develop without diapause. The result (Fig. 1, 4SN) indicates an obvious photoperiodic sensitivity in the L2 stage. A sensitivity in the Ll stage is not proved by this measurement, but can be expected in face of the course of the 4SN curve. An induction of diapause in the first larval stages may only be prevented with a stronger stimulus, that is a higher number of short-night cycles. But in the Ll stage more cycles would be required than this stage actually lasts. From these experiments we can deduce that P. brassicae is photoperiodic sensitive during its whole larval life. Photoperiodic
sensitivity
to long-night
cycles
To test the effect of long-nights an experiment under inverse conditions was carried out. Larvae were reared in a short-night regime interrupted by a long-night sequence. In order to strengthen the effect of long-nights, the photophase of the background (L/D 15 h 45 min/8 h 15 min) was selected to just prevent diapause in all individuals (120 of 122). The long-night sequence comprised 4 cycles of L/D 10 h/14 h. The results are shown in Fig. 2, where the response is directly compared with the 3SN curve of the inverse
PHOTOPERIODIC
0
4
2
t Ll/O
t wo
6
SENSITIVITY
6
t L3/0
lo
79
12:
14
16
t L5lO
t L4lO
16
t t PP P
Larval Development [Days] FIGURE 1. Photoperiodic sensitivity in P. brassicae to short-night cycles. The sensitivity is measured as change of the non-diapause response of individuals which were reared in a diapause inducing regime of L/D 14 h 20 min/9 h 40 min, interrupted by two (2 SN), three (3 SN), and four (4 SN) short-nights (L/D 19 h/5 h) during different stages of development. As in all subsequent figures, the measured values are depicted midway through the short-night sequence, e.g. by a 2 short-night sequence after 1 day. The abscissa indicates the duration of larval development and the moulting stages (Ll/&L5/0 = larval stages; PP = prepupa; P = pupa) are drawn. n = 118-232 for each point.
0
t LllO
4
2
t wo
6
6
t L3/0
10
t L4lO
12:
t L5/0
14
16
16
t t PP P
Larval Development [Days] FIGURE 2. Photoperiodic sensitivity in P. brussicae to non-diapause response of individuals which were reared interrupted by four long-nights (L/D 10 h/4 h) during short-night response (SN), also depicted in Fig.
long-night cycles (LN). The sensitivity is measured as change of the in a non-diapause inducing regime of L/D 15 h 45 min/l h 15 min, different stages of development. The curve is compared with the 1. For the abscissa see Fig. 1. n = 122-170 for each point.
80
HUBERT
R. SPIETH
experiment depicted in Fig. 1. Though the courses are inverse, the configuration of both curves is to a large extent identical. The maximum photoperiodic sensitivity appeared at the L5/1 stage. This indicates that the change in photoperiodic sensitivity during the larval development is not affected by the quality of L/D regimes, which may be experienced by individuals in nature. Geographical
comparison
of photoperiodic
short-nights caused a response that is more or less identical with that of the other populations (Fig. 3). The eflect of temperature Some populations were examined for their photoperiodic sensitivity at 15°C a temperature at which the developmental time of the larvae was 33 days (Fig. 4) which is nearly twice as long as at 20°C. The scotophase of the long-night background was adapted for each population according to its respective photoperiodic response at 15°C as already mentioned (Flensburg: 14 h 30 min; Banyuls: 12 h 55 min; Horsens: 15 h 05 min). All these regimes induce more than 99% diapause. The short-night sequence comprised 6 cycles of L/D 19 h/5 h. The results are depicted in Fig. 4. The change of photoperiodic sensitivity showed the same pattern in all populations, confirming the results at 20°C (Fig. 3). The maximum sensitivity was identical in all three cases and was at the same position on the developmental axis as at 20°C. From these results we can state that temperature, within a range often experienced by individuals in nature, does not influence the course of photoperiodic sensitivity. A difference in the intensity of a non-diapause response among the populations was obvious (Fig. 4). The Flensburg-population showed the strongest response (80% non-diapause). The other two responded about as half as strongly. The reasons for this behaviour were not examined. It may be that the Horsenspopulation needs a stronger short-night stimulus, as already elaborated above. In the Banyuls-population the
sensitivity
The conclusions deduced from the previously described experiments are valid for the Flensburg-population. But no general statement can be made for the species as a whole. Therefore, individuals of different geographical origin were tested. The photophase of the long-night background for the three populations (Anglian Heights: 14 h; Flensburg: 14 h 20 min; Horsens: 14 h 55 min) was determined by their photoperiodic response, as described above. The background regime was interrupted by two short-nights of L/D 19 h/5 h. Figure 3 shows that there were no differences in the peak sensitivity between the populations from England and Germany. Also the configuration of the curves and their position on the horizontal axis are identical. Non-diapause development invariably starts with the fourth and ends with the last larval stage. The population from Denmark behaved differently in that only a few individuals responded to two shortnights with non-diapause. But when the inducing signal was strengthened by an increase in the number of cycles, an appreciable response occurred. A doubled number of
m
0
4
2
Flensburg
B
England
6
6
t
t
WO
L3lO
10
t
L4lO
Larval Development
m
12:
Horsens
14
t L5lO
16
16
t
t
PP P
[Days]
FIGURE 3. Comparison of the photoperiodic sensitivity in populations of P. brassicae from different geographical origins. The non-diapause response induced by two (Anglian Heights = England; Flensburg) and four (Horsens) short-nights (L/D 19 h/5 h) was recorded in individuals which were reared in a long-night regime with population specific photophases (see text). For the abscissa see Fig. 1. n = 83-241 for each point.
PHOTOPERIODIC
0
4
8
12
81
SENSITIVITY
16
20
i
24
28
32
I
+ LiYO
4 L3lO
+ Lao
Larval Development FIGURE 4. Photoperiodic sensitivity origin are compared (H = Horsens; F 19 h/5 h) was recorded in individuals text).
long-night background may adapted to the photoperiodic
4 L5lO
t PP
I
4 P
[Days]
in P. brass&e at a lowered temperature of 15°C. Populations of different geographical = Flensburg; B = Banyuls). The non-diapause response induced by six short-nights (L/D which were reared in a long-night regime specifically adapted for each population (see For the abscissa see Fig. 1. n = 93-175 for each point.
not have been optimally response at 15°C.
DISCUSSION
Individuals of the large white butterfly, P. brassicae, reared under a long-night regime responded to a shortnight sequence, which was shifted through the different with changing proportions of nonlarval stages, diapause. The response started at 0% in newly emerged larvae, increased gradually reaching a maximum l-2 days after the moult to the 5th larval stage, and then decreased again to 0% just before pupation. These results show for the first time that short-nights in the second stage lead to non-diapause in a proportion of the tested individuals. The course of the non-diapause response can be recorded as a gradual change of photoperiodic sensitivity, with a maximum l-2 days after the moult to the L5 stage. The phase of highest photoperiodic sensitivity corresponds to a great extent to Claret’s (1972) conclusions. However, the results did not confirm his statement that no inducing effect of photoperiods could be measured before the third moult of a larva. Rather, the experiments support the findings of an earlier paper (Spieth and Sauer, 1991) that photoperiodic events experienced by larvae of P. brassicae during their first three stages effect the mode of development decisively. If a non-diapause response was induced at the beginning of larval development, then it was not cancelled by long-nights, even if experienced in the phase of highest photoperiodic sensitivity. In their paper on the mechanism of the photoperiodic
clock Veerman et al. (1988) suggested for the first time that in P. brassicae an unchanged long-night cycle does not have the same inductive strength during larval development even in the phase of high photoperiodic sensitivity. The described results confirm this presumption. There is no saturation, but a definite peak of maximum sensitivity (Figs 1 and 3). The inductive strength of a photoperiodic event changed gradually during all larval stages. To prevent a diapause response in the first larval stages or at the end of larval development the caterpillars require a stronger stimulus than in the phase of high photoperiodic sensitivity, e.g. a higher number of short-night cycles. The temporal pattern of the change of photoperiodic sensitivity in P. brassicae is very constant. The response in each geographical population tested at 20 or 15°C was very similar. A separate sensitivity and different maxima for the alternative regimes of short-night and long-night, as mentioned by Claret (1972, 1983), could not be found. According to the present results, the temporal pattern of photoperiodic sensitivity is not variable, but a character of the species. The changing photoperiodic sensitivity is very important for the design of models to describe photoperiodic responses. In recently developed models it is postulated that diapause is induced by the accumulation of an unknown substance. This diapause-inducing substance is synthesized during the scotophase of a photoperiodic cycle and subsequently passed to an internal storage. The daily synthesized amount of the substance determines the inductive strength of a 24 h light/dark cycle. More substance is produced with in-
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HUBERT
creasing duration of the scotophase (Vaz Nunes et al., 199 1b; Spieth and Sauer, 199 1) and decreasing temperature (Lewis and Saunders, 1987). Both factors are thought to alter properties of the photoperiodic clock, so that its output changes quantitatively. The required day number, often seen as a separate response inducing factor, is only a dependent variable of this output. Besides temperature and duration of the scotophase, an age-dependent change of photoperiodic sensitivity, as shown in this paper, is a further process which influences the photoperiodic response in a quantitative way. According to the discussed clock model, the gradual change of sensitivity is equivalent to a change of the inductive strength of a L/D cycle. Until now we did not know which level of the measuring system, the receptor, the clock, or a process subsequent to the clock, is affected by age. Zaslavski (1988, 1992) suggests that if the clock is exposed to a constant photoperiodic stimulus, it generates a constant signal independent of the larval stage. If this is true, then the photosensitivity of an individual (in the sense of receptivity to light or dark) does not really change with age. Some evidence also comes from a comparison of the temperature experiments (Fig. 4). As the peak sensitivity at 15°C as well as 20°C is achieved in the first days after the moult to L5, irrespective of the large difference in duration of larval development at both temperatures, the change of sensitivity does not appear to be triggered by a clock mechanism, but seems rather to be a stage specific character. If not the clock but one of the processes subsequent to the clock, such as those involved in transition and storing of the generated clock signals, are modified or temporarily inhibited with increasing age, then the photoperiodic sensitivity has nothing to do with a sensitivity to light or dark. It merely describes the age-dependent processing of an unchanged light/dark generated photoperiodic clock signal. An interesting question is, why does photoperiodic sensitivity in P. brassicae change at all? Are there physiological constraints, or is it a matter of adaptation to certain life cycles? In arthropod species examined to date the course of photoperiodic sensitivity is so heterogeneous (reviews in: Beck, 1980; Saunders, 1982; Danks, 1987; Veerman, 1992) that physiological contraints cannot be an explanation. On the other hand, the heterogenity also does not allow a general statement about the adaptive value. Therefore, it is necessary to formulate a separate hypothesis for each case (Danks, 1987). In the following this was tried for the adaptive value in P. brassicae. The large white butterfly belongs to a group of butterflies passing through a facultative diapause which is triggered by daylength. The diapause is very strong; it lasts in the field about 7 months, and is terminated by winter temperatures (Feltwell, 1982; Spieth, 1985). In central Europe, caterpillars hatching in August have a developmental time of about 3540 days under normal climatic conditions. During this time they have two choices: either they overwinter as diapausing pupae or they use the remaining food supply in the field
R. SPIETH
and reproduce again before winter. The latter provides the change to propagate its genes in the spring generation as multiple copies, but on the other hand it includes the risk that unfavourable climatic conditions could slow down the development of the progeny and they could either starve or be killed by frost. The decision to hibernate or not, is determined by daylength but modified to a large extent by the highly unpredictable weather conditions. Low temperature prolongs the development decisively so that caterpillars may experience much shorter daylengths at the peak sensitivity than at high temperature. If the phase of highest photoperiodic sensitivity occurs long before the pupal stage, subsequent unfavourable changes of external factors would have no or only minor effect on the mode of development. Against this background, the peak sensitivity during a phase just before the diapause stage makes sense. With a long lasting larval development a relatively late decision can diminish the risk to take the wrong path of development corresponding to season and climate.
REFERENCES Beck S. D. (1980) Insect Photoperiodism, 2nd edn. Academic Press, New York. Biinning E. and Joerrens G. (1960) Tagesperiodische antagonistische Schwankungen der Blauviolettund Gelbrot-Empfindlichkeit als Grundlage der photoperiodischen Diapause-Induktion bei Pieris brassicae. 2. Naturf. 15b, 205-213. Claret J. (1972) Period de sensibilitl des chenilles de Pieris brassicae i la photoptriode controlant la diapause. C. R. hebd. Sane. Acad. Sci., Paris 274, 1055-1058. Claret J. (1983) Signification differentielle des photoperiodes pour l’horloge biologique de Pieris brassicae (Lepidoptera). J. interdiscipl. Cycle Res. 14, 63-73. Danilevsky A. S. (1965) Photoperiodism and Seasonal Development of Insects. Oliver & Boyd, London. Danks H. V. (1987) Insect Dormancy: An Ecological Perspective. Biological Survey of Canada, Ottawa. David W. A. L. and Gardiner B. 0. C. (1962) Observations on the larvae and pupae of Pieris brassicae (L.) in a laboratory culture. Bull. ent. Res. 53, 417436. Feltwell J. (1982) Large White Butterfly: The Biology, Biochemistry, and Physiology of Pieris brassicae (Linnaeus). Dr W. Junk Publishers, The Hague. Hardie J. (1990) The photoperiodic counter, quantitative day-length effects and scotophase timing in the vetch aphid Megoura viciae. J. Insect Physiol. 36, 939-949. Kimura M. T. (1990) Quantitative response to photoperiod during reproductive diapause in the Drosophila auraria species-complex. J. Insect Physiol. 36, 147-152. Lewis R. D. and Saunders D. S. (1987) A damped circadian oscillator model of an insect photoperiodic clock. I. Description of the model based on a feedback control system. J. theor. Biol. 128, 47-59. Saunders D. S. (1971) The temperature-compensated photoperiodic clock ‘programming’ development and pupal diapause in the fleshfly, Sarcophaga argyrostoma. J. Insect Physiol. 17, 801-812. Saunders D. S. (1981) Insect photoperiodism-the clock and the counter: a review. Physiol. Ent. 6, 99-l 16. Saunders D. S. (1982) Insect clocks, 2nd edn. Pergamon Press, Oxford. Saunders D. S. and Lewis R. D. (1987) A damped circadian oscillator model of an insect photoperiodic clock. II. Stimulation of the shapes of the photoperiodic response curves. J. theor. Biot. 128, 61-71. Spieth H. R. (1985) Die Anpassung des Entwicklungszyklus an
PHOTOPERIODIC unterschiedlich lange Vegetationsperioden beim Wanderfalter Pieris brussicae L. (Lepidoptera: Pieridae). Zool. Jber. Cyst. 112, 3569. Spieth H. R. and Sauer K. P. (1991) Quantitative measurement of photoperiods and its significance for the induction of diapause in Pieris brassicae (Lepidoptera, Pieridae). J. Insect Physiol. 37, 231-238. Vaz Nunes M. and Veerman A. (1982) Photoperiodic time measurement in the spider mite Tetranychus urticae: a novel concept. J. Insect Physiol. 2.8, 1041-1053. Vaz Nunes M., Lewis R. D. and Saunders D. S. (1991a) A coupled oscillator feedback system as a model for the photoperiodic clock in insects and mites. I. The basic control system as a model for circadian rhythms. J. theor. Biol. 152, 287-298. Vaz Nunes M., Saunders D. S. and Lewis R. D. (1991b) A coupled oscillator feedback system as a model for the photoperiodic clock in insects and mites. II. Simulation of photoperiodic responses. J. theor. Biol. 152, 299-311. Veerman A. (1992) Diapause in phytoseiid mites: a review. Expl Appl. Acarol. 14, 140. Veerman A., Beekman M. and Veenendaal R. L. (1988) Photoperiodic induction of diapause in the large white butterfly, Pieris brassicae:
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evidence for hourglass time measurement. J. Insect Physiol. 34, 1063-1069. Zaslavski V. A. (1988) Insect Development: Photoperiodic and Temperature Control. Springer, Berlin. Zaslavski V. A. (1992) Light-break experiments with emphasis on the quantitative perception of nightlength in the aphid Megoura viciae. J. Insect Physiol. 38, 717-725. Zaslavski V. A. and Fomenko R. B. (1980) New data on the photoperiodic reaction of the knotgrass moth Acronycta rumicis L. (Lepidoptera, Noctuidae). Ent Rev. 59, 9-16. Zaslavski V. A. and Fomenko R. B. (1983) Quantitative photoperiodic perception in the aphid Megoura viciae Buckt. (Homoptera, Aphididae). Enc. Rev. 62, l-10. Zaslavski V. A. and Fomenko R. B. (1991) An experimental study of the induction of photoperiodism in the aphid Megoura viciae Buckt. (Homoptera, Aphididae). Ent. Rev. 70, 25-35.
Acknowledgements-The author would like to thank Dr Horst Schwarz, Dr Bob Kosier and Ralf Cordes for discussion and commenting on the manuscript.