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Hormonal control of a seasonal polyphenism in Precis coenia (Lepidoptera: Nymphalidae)
Pergamon
0022-1910(95)00046-l
J. Insect Physiol. Vol. 41, No. 11, pp. 987-992, 1995 Copyright 6 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved
0022.1910/95 $9.50 + 0.00
Hormonal Control of a Seasonal Polyphenism in Precis coenia (Lepidoptera: Nymphalidae) D. B. ROUNTREE,*t Received 22 November
H. F. NIJHOUT$ 1994; revised 23 March
1995
Adults of the buckeye butterfly, Precis coenia, exhibit a seasonal color polyphenism. During the late spring and summer, under high temperatures and long photoperiods, the ventral surface of the hind wing and that of the exposed portion of the fore wing are a light beige (the finea form), while in the autumn, under low temperatures and short photoperiods, these wing surfaces are a dark reddish-brown (the rosa form). Removal of the brain immediately after pupation causes all animals to develop the rosa phenotype, irrespective of environmental conditions. The lima phenotype can be restored in brainless animals by injection of 20-hydroxyecdysone, and this response is dose-dependent. The critical period of sensitivity to ecdysteroids falls between 28 and 48 h after pupation. Measurement of ecdysteroid titers revealed that under long-day conditions ecdysteroid titers begin to rise 20 h after pupation, while under short-day conditions ecdysteroid titers do not begin to rise until 60 h after pupation, well after the ecdysteroid-critical period is over. Precis coenia
Ecdysteroids
Photoperiodism
Seasonal polyphenism
develop
INTRODUCTION The buckeye butterfly, Precis coenia, like all other members of its genus, has a seasonally diphenic pigment pattern. The generations that develop in the summer have a light beige ventral hind wing pigmentation, while the autumn generations have a dark reddish-brown pigmentation. These two color forms are called the Zinea and the rosa morph, respectively. Smith (1991) has shown that the linea morph develops under long-day photoperiods and high temperatures, while development of the rosa morph requires short photoperiods or cool temperatures. As with many polyphenisms, photoperiod and temperature have an interactive effect, so that at high temperatures induction of the rosa morph requires shorter photoperiods than are needed at low temperatures (Beck, 1980; Shapiro, 1976; Smith, 1991; Tauber et al., 1986). In most seasonal polyphenisms studied so far, the photoperiod has its effect via the endocrine system. In some cases, development of one of the morphs is strictly tied to diapause, itself an endocrine-mediated event. In Araschnia ievana, for instance, diapausing pupae *Present address: Department of Biology, University of Louisville, Louisville, Kentucky 40292, U.S.A. tTo whom all correspondence should be addressed. $Department of Zoology, Duke University, Durham, NC 27708-0325, U.S.A. 987
into the brown and orange spring morph while non-diapausing pupae always develop into the black and white summer morph (prorsa). The developmental switch in Araschnia occurs during a critical period that extends from day 3 to day 10 after pupation; if ecdysteroid secretion occurs before this critical period the summer morph develops, whereas if ecdysteroid secretion occurs after this time the spring morph develops. Injection of exogenous ecdysteroids into diapause-destined pupae during the critical period results in morphological intermediates between the two morphs (Koch, 1987; Koch and Biickmann, 1987). In the swallowtail Papilio xuthus, seasonal polyphenism in wing pattern is likewise linked to pupal diapause (Endo and Funatsu, 1985). In other species, such as the anglewing, Polygonia c-aureum, and the small copper, Lycaena phlaeas, seasonal polyphenism is not correlated with pupal diapause and can be continuously modulated by the photoperiod (Fukuda and Endo, 1966; Endo and Kamata, 1985; Endo et al., 1988). In Polygonia the photoperiod exerts its effect via the neuroendocrine system. Apparently the brains of long-day pupae release a neurohormone that induces development of the summer morph (the summer-morph-producing hormone, SMPH). The identity of SMPH has not yet been established. Its critical period falls during the first 2 days of the pupal stage. Masaki et al. (1988) investigated the (levana)
988
D. B. ROUNTREE
possibility that SMPH might not be identical with PTTH. They found that the SMPH from Polygonia had many physical similarities to bombyxin (also referred to as small-PTTH). Furthermore, purified preparations of bombyxin which could terminate diapause of Papilio xuthus pupae also produced the summer morph in Polygonia. These results suggest that SMPH is either identical to, or co-purifies with, small PTTH or bombyxin. Masaki et al. (1988) suggest the possibility that there may actually be several forms of small PTTH which differ slightly in their physical and biological properties, and that SMPH activity is due to one of these. If SMPH is a small PTTH and has its normal action through the stimulation of ecdysteroid secretion, then the polypenism of Polygonia, like that of Araschnia, may be ultimately controlled by the timing of ecdysteroid secretion. Alternatively, SMPH could be a novel neurohormone with unique effects on development of seasonal characters. In Lycaena phlaeas the difference between spring and summer morph development resides, at least in part, in a change in the timing of ecdysteroid secretion, and development of the paler spring morph under short-day conditions is accompanied by an advance in ecdysteroid secretion (Endo and Kamata, 1985). In addition, a putative SMPH appears to be secreted by long-day pupae and may act synergistically with a later secretion of ecdysteroids to stimulate development of the dark-colored summer morph (Endo and Kamata, 1985). The present paper reports on the endocrine control of the rosallinea seasonal polyphenism in P. coenia, a polyphenism not associated with pupal diapause. The results of our studies show that differences in the timing of brain-mediated ecdysteroid secretion are sufficient to account for the differences in developmental response to photoperiod. MATERIALS
and H. F. NIJHOUT
beeswax and lanolin. After surgery, long-day and shortday animals were returned to their respective photoperiod regime and temperature. Ecdysteroid titers were determined according to the method of Warren et al. (1984) using the H-22 antibody and with 20-hydroxyecdysone as a standard. Haemolymph samples were collected from punctures of the dorsal vessel in the pro-and/or meso thorax. Volumes were measured and then mixed with four volumes of ice-cold 100% ethanol, spun for 3 min in a microcentrifuge, after which aliquots of supernatant were assayed for ecdysteroid content. For injections, 20hydroxyecdysone (Sigma Chemical Co.) was dissolved in
AND METHODS
Larvae of P. coenia were raised on an artificial laboratory diet, under controlled temperature and photoperiod conditions. Inductive conditions for the linea morph were long day photoperiods (16L:8D) at 28°C (this combination of photoperiod and temperature is referred to as “long-day” hereafter). Inductive conditions for the rosa morph were short day photoperiods (8L: 16D) at 21°C (referred to as “short-day” conditions). Animals were reared under appropriate inductive conditions for their entire larval life. In order to quantify the response to experimental manipulations we gave animals that had a fully developed Zinea phenotype a score of 1, intermediates a score of 2, and fully rosa animals a score of 3 (Fig. 1). Brain extirpations were done on unanesthetized pupae, through an aperture cut in the dorsal prothorax and head capsule. After surgery a few crystals of a 1: 1 mixture of streptomycin and penicillin (Sigma Chemical Co.) were placed in the wound, the original cuticle was put back in place and the edges sealed with a mixture of
FIGURE 1. Scoring system of the environmentally-induced polyphenism in P. coeniu. Numerical score is listed below each specimen, and is based on the intensity of reddish-brown color on the ventral hind wing and the tip of the ventral fore wing. A score of 1 is given to the pure linea phenotype, a score of 3 to the pure rosa phenotype, as described in text.
HORMONAL
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CONTROL
OF SEASONAL
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12
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48
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FIGURE 2. Developmental response to brain removal at various times after pupation of animal reared under long-day conditions. Each point represents results from 7 to 14 animals. Bars are standard errors of the mean.
lepidopteran ringers, and injected into the abdomen through the intersegmental membrane between segments 6 and 7.
MORPH
illustrates the response of short-day intact animals to injections of 20-hydroxyecdysone at various times after pupation. The 50% response for rescue of the linea morph in these short-day animals occurred at about 48 h after pupation, as it did for long-day animals whose brains had been removed [cf. Fig. 3(A) and Fig. 3(B)]. The time interval between the 50% response points in Fig. 2 and Fig. 3(A), i.e. the period between 28 and 48 h after pupation, can be taken as the estimate of the critical period during which the developmental switch of this seasonal diphenism takes place. In addition to being time dependent, there is also a dose-dependency in the response to injections of 20-hydroxyecdysone: when injected 12 h after pupation, the ED,, for animals whose brains had been removed was 0.09 pg/pupa [Fig. 4(A)], while the EDso was 0.25 pg/pupa for short-day animals [Fig. 4(B)]. The fact that extirpation of the brain induced the rosa morph while an injection of 20-hydroxyecdysone could restore the linea morph in brainless animals suggests that a difference in the timing of ecdysteroid secretion could be the basis for differentiation of the rosa and linea
(A)
RESULTS
When P. coenia are reared under linea-inducing conditions the duration of the pupal and pharate adult stage is 67 days, with >95% of the emerging adults showing the linea phenotype. The remaining animals exhibit varying degrees of intermediacy in pigmentation between the normal linea and rosa phenotypes. Under rosa-inducing conditions animals remain in the pupal and pharate adult stage for 14-16 days, with >90% of adults developing the deeply pigmented rosa phenotype and the rest developing intermediate phenotypes. This percentage of rosas and intermediates is not altered if short-day pupae are transferred to long-day conditions 12-24 h after pupation, although under such conditions the duration of the pupal and pharate adult stage is shortened to 7-8 days. When brains were removed from pupae that had been reared under linea-inductive conditions, many developed into rosa forms. The percentage of rosas that developed depended on the timing of the brain extirpation (Fig. 2). If extirpations were done ~28 h after pupation more than 50% of individuals developed the rosa form. By 40 h after pupation brain extirpation had little or no effect on seasonal morph development. We found that the linea morph could be “rescued” in animals whose brain had been removed 4 h after pupation by injection of 20-hydroxyecdysone. Figure 3(A) shows the effects of 20-hydroxyecdysone injections (4 pg/animal) at various times after pupation. As long as the ecdysteroid was injected <36 h after pupation 100% of individuals developed the normal Zinea morph, even in the absence of their brain. The 50% response occurred about 48 h after pupation, while injections after about 60 h were no longer able to restore the linea phenotype. Figure 3(B)
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FIGURE 3. (A) Response of long-day animals whose brains were removed at 4 h after pupation to injections of 20-hydroxyecdysone. Each point represents results from 10 to 17 animals. (B) Response of short-day animals to injections of 20-hydroxyecdysone. Each point represents the results from 6 to 12 animals. Bars are standard errors of the mean.
990
D. B. ROUNTREE
there are many previous documentations that some insects can molt in the absence of their brain (e.g. Fukuda, 1944; Nijhout, 1994). One possibility is that there are additional sources of PTTH in the ganglia of the ventral nerve cord, as the work of Westbrook et al. (1993) suggests. In any event, the ecdysteroid titers of both brainless and short-day animals begin to rise only after the critical period for Zinea morph induction is past.
(A)
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FIGURE 4. Dose-response curve of 20-hydroxyecdysone injections in long-day animals whose brains had been removed at 4 h after pupation (A), and in short-day animals (B). Open triangles represent the response of saline injected controls. Open squares indicate the response of uninjected controls. Each point represents 8-20 animals. Bars are standard errors of the mean.
morphs. To examine whether there were significant differences in the ecdysteroid profiles of normal animals reared under rosa-and linea-inducing conditions we determined hemolymph ecdysteroid titers throughout the pupal and pharate adult stage (Fig. 5). Under long-day conditions the hemolymph ecdysteroid titer begins to rise about 20 h after pupation, while under short-day conditions this rise is delayed some 40 h and does not begin until about 60 h after pupation. This delay in ecdysteroid secretion corresponds well with the observation that the pupal stage of short-day animals that develop the rosa morph is 1-2 days longer than that of long day animals that develop the Zinea morph, when both are maintained under long-days at a common temperature (28°C). The ecdysteroid titers of animals whose brain had been removed at 4 h after pupation is also shown in Fig. 5. This titer profile closely resembles that of short-day animals, in that the titer remains low for some 48 h before it begins to rise gradually. Exactly why the ecdysteroid titer should begin to rise in the absence of a brain is not at all clear at present, though
The experiments described above demonstrate that the polyphenism of P. coenia is controlled by the brain. When the brain is removed within 12 h after pupation all animals develop into the rosa morph, suggesting that an intact brain is necessary to induce the linea morph. Injection of ecdysteroids can rescue the linea morph in brainless animals, as long as it is done during the first 24 h after pupation. We have defined the critical period for linealrosa morph induction (summarized in Fig. 6) as the interval between the time that the brain is no longer necessary for development of the linea morph (50% point at 28 h after pupation) and the time that ecdysteroid injection can no longer rescue the linea morph (50% point at 48 h after pupation). The choice of this interval is based on the evidence that the brain exerts its effect through the stimulation of ecdysteroid secretion via the release of PTTH (Nijhout, 1994). The difference between a presumptive linea and a presumptive rosa animal lies simply in a shift in the timing of onset of ecdysteroid secretion. Under long day conditions ecdysteroid secretion begins during the critical period (at about 20 h after pupation), while under short-day conditions ecdysteroid secretion does not linealrosa
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FIGURE 5. Ecdysteroid titers in long-day animals destined to develop the linea phenotype (circles), short-day animals destined to develop the rosa phenotype (squares), and long-day animals whose brains had been removed (triangles) and which developed the rosa phenotype. Bars are standard errors of the mean. Grey region represents the extent of the critical period for seasonal morph induction (see Fig. 6).
HORMONAL
CONTROL
Brain extirpation
OF SEASONAL
MORPH
991
IN BUTTERFLIES
Ecdysteroid rescue of brainless animal
50% response
Brain necessary for development of linea
12 FIGURE
24 6. Model of the critical
Brain no longer necessary but ecdystemids can still rescue linea in animals debmined prior to 12 hours
Ecdysteroids can no longer rescue linea
36 48 Hours after pupation period in the physiological control of seasonal morph induction data summarized in Fig. 2, Fig. 3(B), and the text.
begin until the critical period is over (at about 60 h after pupation). The endocrine control over seasonal polyphenism in Precis is thus superficially similar to that found by Endo and Kamata (1985) in Lycaena phlaeas, and by Koch and Biickmann (1987) in Araschnia levana. In these species the seasonal polyphenism is also controlled by differences in the timing of ecdysteroid secretion, but the details of the underlying mechanism appear to be different in each. In Araschnia there is a critical period for ecdysteroid sensitivity that occurs between 3 and 10 days after pupation. Injection of 20-hydroxyecdysone before the onset of this critical period results in development of the summer morph. In Araschnia the shift in the timing of endogenous ecdysteroid secretion is due to the fact that the spring morph normally undergoes diapause. Accordingly, animals that undergo direct development without diapause normally secrete ecdysteroids well before the critical period begins and develop into summer morphs, while animals that undergo a pupal diapause secrete ecdysteroids long after the critical period is past and develop into spring morphs. The rather prolonged critical period in Araschnia occurs during the first week of diapause in the pupa, and may thus be a component of the process of diapause development (Andrewartha, 1952; Beck, 1980). In Lycaena, by contrast, injections of 20-hydroxyecdysone into long-day pupae induced development of the spring (short-day) morph. Unlike Araschnia and Precis, Lycaena pupae reared under short-day conditions release ecdysteroids one to two days before larvae reared under long-day conditions (Endo and Kamata, 1985). Hence, the spring morph has an accelerated development, instead of the delayed development we observe in the spring morph of Araschnia, and the fall morph of Precis. The critical periods for seasonal morph induction differ significantly between these species. In Araschnia it is between 2 and 10 days after pupation, while in JIP4,.111--D
60 in P. coenia,
based on the
Lycaena the critical period probably lies somewhere within the first two days after pupation, though the exact limits have not yet been investigated. In Precis, the critical period lies between 28 and 48 h after pupation (Fig. 6). Endo and Kamata (1985) also report that excision of the brain from long-day pupae induces a slight increase in development of the spring (short-day) morph in Lycaena. This effect cannot be easily reconciled with the results of their ecdysteroid titer and injection experiments except by assuming that we are dealing with a wound effect or, as Endo and Kamata have suggested, that the brain may produce a SMPH. Endo and Kamata propose that development of the summer morph in Lycaena phlaeas requires this putative SMPH, while development of the spring morph requires the absence of SMPH and secretion of ecdysteroids early in the pupal stage. The presumptive SMPH of Lycaena thus has a different function than that of Polygonia, where SMPH appears to function primarily in the stimulation of ecdysteroid secretion (Endo et al., 1988). In Precis we have found no need to postulate the existence of a SMPH. The brain-prothoracic gland axis controls rosallinea seasonal morph development via ecdysteroids, whose secretion is presumably stimulated via the conventional tropic stimulus of PTTH. If a SMPH exists in Precis, then it clearly does not act upstream of the ecdysteroid signal. The possibility that a SMPH may act downstream of the ecdysteroid signal is not addressed by our experiments, nor is it suggested by the prior work of others. Development of the rosa morph of P. coenia can be triggered by temperature as well as by photoperiod. At high temperatures, a short photoperiod can induce a large portion of animals to develop the rosa morph. On the other hand, if temperatures are sufficiently low (e.g. at lSC), the rosa morph develops even under long-day photoperiods (Smith, 1991). In most insects studied so far, there is a synergism between temperature and
992
D. B. ROUNTREE
photoperiod in which low temperatures during the photoperiod-sensitive phase increase the percentage of individuals that respond to a short-day signal (Tauber et al., 1986). Often this is due to the fact that low temperatures slow down development and thus expose the insect to a longer series of inductive photoperiod cycles during its photoperiod-sensitive period. It is uncommon, however, for low temperature to be able to completely substitute for short daylengths, as it does in the seasonal polyphenism of P. coenia (Smith, 1991). One explanation for this effect might be that low temperature delays the onset of ecdysteroid secretion so that it begins after the critical period for lines/rosa morph induction is over.
REFERENCES Andrewartha H. G. (1952) Diapause in relation to the ecology of insects. Biol. Rev. 27, 5&107. Beck S. D. (1980) Insect Photoperiodism. Academic Press, New York. Endo K. and Funatsu S. (1985) Hormonal control of seasonal morph determination in the swallowtail butterfly, Pupilio xuthus L. (Lepidoptera Papilionidae) J. Insect Physiol. 31, 669-674. Endo K. and Kamata Y. (1985) Hormonal control of seasonal-morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz. J. Insect Physiol. 31, 701-706. Endo K., Masaki T. and Kumagai K. (1988) Neuroendocrine regulation of the development of seasonal morphs in the Asian comma butterfly, Polygonia c-aureum L.: difference in activity of the summer-morph-producing hormone from brain-extracts of the long-day and short-day pupae. 2001. Sri. 5, 145152. Fukuda S. (1944) The hormonal mechanism of larval molting and metamorphosis in the silkworm. J. Fat. Sci. Tokyo Univ. 4,477-532.
and H. F. NIJHOUT Fukuda S. and Endo K. (1966) Hormonal control of the development of seasonal forms in the butterfly, Polygonia c-aureum L. Proc. Japan Acad. 42, 1082-1087. Koch P. B. (1987) Die Steuerung der Saisondimorphen Fllgelfarbung von Araschnia levana L. (Nymphalidae, Lepidoptera) durch ecdysteroide. Mitt. Deutsch. Gesell. Allg. Angew. Entomol. 5, 1955197. Koch P. B. and Biickmann D. (1987) Hormonal control of seasonal morphs by the timing of ecdysteroid release in Araschnia levana L. (Nymphalidae: Lepidoptera) J. Insect Physiol. 33, 823-829. Masaki T., Endo K. and Kumagai K. (1988) Neuroendocrine regulation of development of seasonal morphs in the Asian comma butterfly, Polygoniu c-aureum L.: is the factor producing summer morphs (SMPH) identical to the small prothoracicotropic hormone (4K-PTTH)? Zool. Sci. 5, 1051-1057. Nijhout H. F. (1994) Insect Hormones. Princeton University Press, Princeton, NJ. Shapiro A. M. (1976) Seasonal polyphenism. Euol. Biol. 9, 259-333. Smith K. C. (1991) The effects of temperature and daylength on the rosa polyphenism in the buckeye butterfly, Precis coenia (Lepidoptera: Nymphalidae) J. Res. Lep. 30, 2255236. Tauber M. T., Tauber C. A. and Masaki S. (1986) Seasonal Aduptafions of Insects. Oxford University Press, New York. Warren J. T., Smith W. A. and Gilbert L. I. (1984) Simplification of the ecdysteroid radioimmunoassay by the use of protein A from Staphylococcus aureus. Experientia 40, 561-576. Westbrook A., Regan S. A. and Bollenbacher W. E. (1993) Developmental expression of the prothoracicotropic hormone in the CNS of the tobacco hornworm Manduca sexta. J. Comp. Neurol. 327, 1-16. Acknowledgements-We would like to thank Dr Larry Gilbert, University of North Carolina at Chapel Hill, for the use of his laboratory for ecdysteroid radioimmunoassays. This work was supported by Grant IBN-9220211 from the National Science Foundation.
0022-1910(95)00046-l
J. Insect Physiol. Vol. 41, No. 11, pp. 987-992, 1995 Copyright 6 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved
0022.1910/95 $9.50 + 0.00
Hormonal Control of a Seasonal Polyphenism in Precis coenia (Lepidoptera: Nymphalidae) D. B. ROUNTREE,*t Received 22 November
H. F. NIJHOUT$ 1994; revised 23 March
1995
Adults of the buckeye butterfly, Precis coenia, exhibit a seasonal color polyphenism. During the late spring and summer, under high temperatures and long photoperiods, the ventral surface of the hind wing and that of the exposed portion of the fore wing are a light beige (the finea form), while in the autumn, under low temperatures and short photoperiods, these wing surfaces are a dark reddish-brown (the rosa form). Removal of the brain immediately after pupation causes all animals to develop the rosa phenotype, irrespective of environmental conditions. The lima phenotype can be restored in brainless animals by injection of 20-hydroxyecdysone, and this response is dose-dependent. The critical period of sensitivity to ecdysteroids falls between 28 and 48 h after pupation. Measurement of ecdysteroid titers revealed that under long-day conditions ecdysteroid titers begin to rise 20 h after pupation, while under short-day conditions ecdysteroid titers do not begin to rise until 60 h after pupation, well after the ecdysteroid-critical period is over. Precis coenia
Ecdysteroids
Photoperiodism
Seasonal polyphenism
develop
INTRODUCTION The buckeye butterfly, Precis coenia, like all other members of its genus, has a seasonally diphenic pigment pattern. The generations that develop in the summer have a light beige ventral hind wing pigmentation, while the autumn generations have a dark reddish-brown pigmentation. These two color forms are called the Zinea and the rosa morph, respectively. Smith (1991) has shown that the linea morph develops under long-day photoperiods and high temperatures, while development of the rosa morph requires short photoperiods or cool temperatures. As with many polyphenisms, photoperiod and temperature have an interactive effect, so that at high temperatures induction of the rosa morph requires shorter photoperiods than are needed at low temperatures (Beck, 1980; Shapiro, 1976; Smith, 1991; Tauber et al., 1986). In most seasonal polyphenisms studied so far, the photoperiod has its effect via the endocrine system. In some cases, development of one of the morphs is strictly tied to diapause, itself an endocrine-mediated event. In Araschnia ievana, for instance, diapausing pupae *Present address: Department of Biology, University of Louisville, Louisville, Kentucky 40292, U.S.A. tTo whom all correspondence should be addressed. $Department of Zoology, Duke University, Durham, NC 27708-0325, U.S.A. 987
into the brown and orange spring morph while non-diapausing pupae always develop into the black and white summer morph (prorsa). The developmental switch in Araschnia occurs during a critical period that extends from day 3 to day 10 after pupation; if ecdysteroid secretion occurs before this critical period the summer morph develops, whereas if ecdysteroid secretion occurs after this time the spring morph develops. Injection of exogenous ecdysteroids into diapause-destined pupae during the critical period results in morphological intermediates between the two morphs (Koch, 1987; Koch and Biickmann, 1987). In the swallowtail Papilio xuthus, seasonal polyphenism in wing pattern is likewise linked to pupal diapause (Endo and Funatsu, 1985). In other species, such as the anglewing, Polygonia c-aureum, and the small copper, Lycaena phlaeas, seasonal polyphenism is not correlated with pupal diapause and can be continuously modulated by the photoperiod (Fukuda and Endo, 1966; Endo and Kamata, 1985; Endo et al., 1988). In Polygonia the photoperiod exerts its effect via the neuroendocrine system. Apparently the brains of long-day pupae release a neurohormone that induces development of the summer morph (the summer-morph-producing hormone, SMPH). The identity of SMPH has not yet been established. Its critical period falls during the first 2 days of the pupal stage. Masaki et al. (1988) investigated the (levana)
988
D. B. ROUNTREE
possibility that SMPH might not be identical with PTTH. They found that the SMPH from Polygonia had many physical similarities to bombyxin (also referred to as small-PTTH). Furthermore, purified preparations of bombyxin which could terminate diapause of Papilio xuthus pupae also produced the summer morph in Polygonia. These results suggest that SMPH is either identical to, or co-purifies with, small PTTH or bombyxin. Masaki et al. (1988) suggest the possibility that there may actually be several forms of small PTTH which differ slightly in their physical and biological properties, and that SMPH activity is due to one of these. If SMPH is a small PTTH and has its normal action through the stimulation of ecdysteroid secretion, then the polypenism of Polygonia, like that of Araschnia, may be ultimately controlled by the timing of ecdysteroid secretion. Alternatively, SMPH could be a novel neurohormone with unique effects on development of seasonal characters. In Lycaena phlaeas the difference between spring and summer morph development resides, at least in part, in a change in the timing of ecdysteroid secretion, and development of the paler spring morph under short-day conditions is accompanied by an advance in ecdysteroid secretion (Endo and Kamata, 1985). In addition, a putative SMPH appears to be secreted by long-day pupae and may act synergistically with a later secretion of ecdysteroids to stimulate development of the dark-colored summer morph (Endo and Kamata, 1985). The present paper reports on the endocrine control of the rosallinea seasonal polyphenism in P. coenia, a polyphenism not associated with pupal diapause. The results of our studies show that differences in the timing of brain-mediated ecdysteroid secretion are sufficient to account for the differences in developmental response to photoperiod. MATERIALS
and H. F. NIJHOUT
beeswax and lanolin. After surgery, long-day and shortday animals were returned to their respective photoperiod regime and temperature. Ecdysteroid titers were determined according to the method of Warren et al. (1984) using the H-22 antibody and with 20-hydroxyecdysone as a standard. Haemolymph samples were collected from punctures of the dorsal vessel in the pro-and/or meso thorax. Volumes were measured and then mixed with four volumes of ice-cold 100% ethanol, spun for 3 min in a microcentrifuge, after which aliquots of supernatant were assayed for ecdysteroid content. For injections, 20hydroxyecdysone (Sigma Chemical Co.) was dissolved in
AND METHODS
Larvae of P. coenia were raised on an artificial laboratory diet, under controlled temperature and photoperiod conditions. Inductive conditions for the linea morph were long day photoperiods (16L:8D) at 28°C (this combination of photoperiod and temperature is referred to as “long-day” hereafter). Inductive conditions for the rosa morph were short day photoperiods (8L: 16D) at 21°C (referred to as “short-day” conditions). Animals were reared under appropriate inductive conditions for their entire larval life. In order to quantify the response to experimental manipulations we gave animals that had a fully developed Zinea phenotype a score of 1, intermediates a score of 2, and fully rosa animals a score of 3 (Fig. 1). Brain extirpations were done on unanesthetized pupae, through an aperture cut in the dorsal prothorax and head capsule. After surgery a few crystals of a 1: 1 mixture of streptomycin and penicillin (Sigma Chemical Co.) were placed in the wound, the original cuticle was put back in place and the edges sealed with a mixture of
FIGURE 1. Scoring system of the environmentally-induced polyphenism in P. coeniu. Numerical score is listed below each specimen, and is based on the intensity of reddish-brown color on the ventral hind wing and the tip of the ventral fore wing. A score of 1 is given to the pure linea phenotype, a score of 3 to the pure rosa phenotype, as described in text.
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FIGURE 2. Developmental response to brain removal at various times after pupation of animal reared under long-day conditions. Each point represents results from 7 to 14 animals. Bars are standard errors of the mean.
lepidopteran ringers, and injected into the abdomen through the intersegmental membrane between segments 6 and 7.
MORPH
illustrates the response of short-day intact animals to injections of 20-hydroxyecdysone at various times after pupation. The 50% response for rescue of the linea morph in these short-day animals occurred at about 48 h after pupation, as it did for long-day animals whose brains had been removed [cf. Fig. 3(A) and Fig. 3(B)]. The time interval between the 50% response points in Fig. 2 and Fig. 3(A), i.e. the period between 28 and 48 h after pupation, can be taken as the estimate of the critical period during which the developmental switch of this seasonal diphenism takes place. In addition to being time dependent, there is also a dose-dependency in the response to injections of 20-hydroxyecdysone: when injected 12 h after pupation, the ED,, for animals whose brains had been removed was 0.09 pg/pupa [Fig. 4(A)], while the EDso was 0.25 pg/pupa for short-day animals [Fig. 4(B)]. The fact that extirpation of the brain induced the rosa morph while an injection of 20-hydroxyecdysone could restore the linea morph in brainless animals suggests that a difference in the timing of ecdysteroid secretion could be the basis for differentiation of the rosa and linea
(A)
RESULTS
When P. coenia are reared under linea-inducing conditions the duration of the pupal and pharate adult stage is 67 days, with >95% of the emerging adults showing the linea phenotype. The remaining animals exhibit varying degrees of intermediacy in pigmentation between the normal linea and rosa phenotypes. Under rosa-inducing conditions animals remain in the pupal and pharate adult stage for 14-16 days, with >90% of adults developing the deeply pigmented rosa phenotype and the rest developing intermediate phenotypes. This percentage of rosas and intermediates is not altered if short-day pupae are transferred to long-day conditions 12-24 h after pupation, although under such conditions the duration of the pupal and pharate adult stage is shortened to 7-8 days. When brains were removed from pupae that had been reared under linea-inductive conditions, many developed into rosa forms. The percentage of rosas that developed depended on the timing of the brain extirpation (Fig. 2). If extirpations were done ~28 h after pupation more than 50% of individuals developed the rosa form. By 40 h after pupation brain extirpation had little or no effect on seasonal morph development. We found that the linea morph could be “rescued” in animals whose brain had been removed 4 h after pupation by injection of 20-hydroxyecdysone. Figure 3(A) shows the effects of 20-hydroxyecdysone injections (4 pg/animal) at various times after pupation. As long as the ecdysteroid was injected <36 h after pupation 100% of individuals developed the normal Zinea morph, even in the absence of their brain. The 50% response occurred about 48 h after pupation, while injections after about 60 h were no longer able to restore the linea phenotype. Figure 3(B)
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990
D. B. ROUNTREE
there are many previous documentations that some insects can molt in the absence of their brain (e.g. Fukuda, 1944; Nijhout, 1994). One possibility is that there are additional sources of PTTH in the ganglia of the ventral nerve cord, as the work of Westbrook et al. (1993) suggests. In any event, the ecdysteroid titers of both brainless and short-day animals begin to rise only after the critical period for Zinea morph induction is past.
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FIGURE 4. Dose-response curve of 20-hydroxyecdysone injections in long-day animals whose brains had been removed at 4 h after pupation (A), and in short-day animals (B). Open triangles represent the response of saline injected controls. Open squares indicate the response of uninjected controls. Each point represents 8-20 animals. Bars are standard errors of the mean.
morphs. To examine whether there were significant differences in the ecdysteroid profiles of normal animals reared under rosa-and linea-inducing conditions we determined hemolymph ecdysteroid titers throughout the pupal and pharate adult stage (Fig. 5). Under long-day conditions the hemolymph ecdysteroid titer begins to rise about 20 h after pupation, while under short-day conditions this rise is delayed some 40 h and does not begin until about 60 h after pupation. This delay in ecdysteroid secretion corresponds well with the observation that the pupal stage of short-day animals that develop the rosa morph is 1-2 days longer than that of long day animals that develop the Zinea morph, when both are maintained under long-days at a common temperature (28°C). The ecdysteroid titers of animals whose brain had been removed at 4 h after pupation is also shown in Fig. 5. This titer profile closely resembles that of short-day animals, in that the titer remains low for some 48 h before it begins to rise gradually. Exactly why the ecdysteroid titer should begin to rise in the absence of a brain is not at all clear at present, though
The experiments described above demonstrate that the polyphenism of P. coenia is controlled by the brain. When the brain is removed within 12 h after pupation all animals develop into the rosa morph, suggesting that an intact brain is necessary to induce the linea morph. Injection of ecdysteroids can rescue the linea morph in brainless animals, as long as it is done during the first 24 h after pupation. We have defined the critical period for linealrosa morph induction (summarized in Fig. 6) as the interval between the time that the brain is no longer necessary for development of the linea morph (50% point at 28 h after pupation) and the time that ecdysteroid injection can no longer rescue the linea morph (50% point at 48 h after pupation). The choice of this interval is based on the evidence that the brain exerts its effect through the stimulation of ecdysteroid secretion via the release of PTTH (Nijhout, 1994). The difference between a presumptive linea and a presumptive rosa animal lies simply in a shift in the timing of onset of ecdysteroid secretion. Under long day conditions ecdysteroid secretion begins during the critical period (at about 20 h after pupation), while under short-day conditions ecdysteroid secretion does not linealrosa
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FIGURE 5. Ecdysteroid titers in long-day animals destined to develop the linea phenotype (circles), short-day animals destined to develop the rosa phenotype (squares), and long-day animals whose brains had been removed (triangles) and which developed the rosa phenotype. Bars are standard errors of the mean. Grey region represents the extent of the critical period for seasonal morph induction (see Fig. 6).
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begin until the critical period is over (at about 60 h after pupation). The endocrine control over seasonal polyphenism in Precis is thus superficially similar to that found by Endo and Kamata (1985) in Lycaena phlaeas, and by Koch and Biickmann (1987) in Araschnia levana. In these species the seasonal polyphenism is also controlled by differences in the timing of ecdysteroid secretion, but the details of the underlying mechanism appear to be different in each. In Araschnia there is a critical period for ecdysteroid sensitivity that occurs between 3 and 10 days after pupation. Injection of 20-hydroxyecdysone before the onset of this critical period results in development of the summer morph. In Araschnia the shift in the timing of endogenous ecdysteroid secretion is due to the fact that the spring morph normally undergoes diapause. Accordingly, animals that undergo direct development without diapause normally secrete ecdysteroids well before the critical period begins and develop into summer morphs, while animals that undergo a pupal diapause secrete ecdysteroids long after the critical period is past and develop into spring morphs. The rather prolonged critical period in Araschnia occurs during the first week of diapause in the pupa, and may thus be a component of the process of diapause development (Andrewartha, 1952; Beck, 1980). In Lycaena, by contrast, injections of 20-hydroxyecdysone into long-day pupae induced development of the spring (short-day) morph. Unlike Araschnia and Precis, Lycaena pupae reared under short-day conditions release ecdysteroids one to two days before larvae reared under long-day conditions (Endo and Kamata, 1985). Hence, the spring morph has an accelerated development, instead of the delayed development we observe in the spring morph of Araschnia, and the fall morph of Precis. The critical periods for seasonal morph induction differ significantly between these species. In Araschnia it is between 2 and 10 days after pupation, while in JIP4,.111--D
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Lycaena the critical period probably lies somewhere within the first two days after pupation, though the exact limits have not yet been investigated. In Precis, the critical period lies between 28 and 48 h after pupation (Fig. 6). Endo and Kamata (1985) also report that excision of the brain from long-day pupae induces a slight increase in development of the spring (short-day) morph in Lycaena. This effect cannot be easily reconciled with the results of their ecdysteroid titer and injection experiments except by assuming that we are dealing with a wound effect or, as Endo and Kamata have suggested, that the brain may produce a SMPH. Endo and Kamata propose that development of the summer morph in Lycaena phlaeas requires this putative SMPH, while development of the spring morph requires the absence of SMPH and secretion of ecdysteroids early in the pupal stage. The presumptive SMPH of Lycaena thus has a different function than that of Polygonia, where SMPH appears to function primarily in the stimulation of ecdysteroid secretion (Endo et al., 1988). In Precis we have found no need to postulate the existence of a SMPH. The brain-prothoracic gland axis controls rosallinea seasonal morph development via ecdysteroids, whose secretion is presumably stimulated via the conventional tropic stimulus of PTTH. If a SMPH exists in Precis, then it clearly does not act upstream of the ecdysteroid signal. The possibility that a SMPH may act downstream of the ecdysteroid signal is not addressed by our experiments, nor is it suggested by the prior work of others. Development of the rosa morph of P. coenia can be triggered by temperature as well as by photoperiod. At high temperatures, a short photoperiod can induce a large portion of animals to develop the rosa morph. On the other hand, if temperatures are sufficiently low (e.g. at lSC), the rosa morph develops even under long-day photoperiods (Smith, 1991). In most insects studied so far, there is a synergism between temperature and
992
D. B. ROUNTREE
photoperiod in which low temperatures during the photoperiod-sensitive phase increase the percentage of individuals that respond to a short-day signal (Tauber et al., 1986). Often this is due to the fact that low temperatures slow down development and thus expose the insect to a longer series of inductive photoperiod cycles during its photoperiod-sensitive period. It is uncommon, however, for low temperature to be able to completely substitute for short daylengths, as it does in the seasonal polyphenism of P. coenia (Smith, 1991). One explanation for this effect might be that low temperature delays the onset of ecdysteroid secretion so that it begins after the critical period for lines/rosa morph induction is over.
REFERENCES Andrewartha H. G. (1952) Diapause in relation to the ecology of insects. Biol. Rev. 27, 5&107. Beck S. D. (1980) Insect Photoperiodism. Academic Press, New York. Endo K. and Funatsu S. (1985) Hormonal control of seasonal morph determination in the swallowtail butterfly, Pupilio xuthus L. (Lepidoptera Papilionidae) J. Insect Physiol. 31, 669-674. Endo K. and Kamata Y. (1985) Hormonal control of seasonal-morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz. J. Insect Physiol. 31, 701-706. Endo K., Masaki T. and Kumagai K. (1988) Neuroendocrine regulation of the development of seasonal morphs in the Asian comma butterfly, Polygonia c-aureum L.: difference in activity of the summer-morph-producing hormone from brain-extracts of the long-day and short-day pupae. 2001. Sri. 5, 145152. Fukuda S. (1944) The hormonal mechanism of larval molting and metamorphosis in the silkworm. J. Fat. Sci. Tokyo Univ. 4,477-532.
and H. F. NIJHOUT Fukuda S. and Endo K. (1966) Hormonal control of the development of seasonal forms in the butterfly, Polygonia c-aureum L. Proc. Japan Acad. 42, 1082-1087. Koch P. B. (1987) Die Steuerung der Saisondimorphen Fllgelfarbung von Araschnia levana L. (Nymphalidae, Lepidoptera) durch ecdysteroide. Mitt. Deutsch. Gesell. Allg. Angew. Entomol. 5, 1955197. Koch P. B. and Biickmann D. (1987) Hormonal control of seasonal morphs by the timing of ecdysteroid release in Araschnia levana L. (Nymphalidae: Lepidoptera) J. Insect Physiol. 33, 823-829. Masaki T., Endo K. and Kumagai K. (1988) Neuroendocrine regulation of development of seasonal morphs in the Asian comma butterfly, Polygoniu c-aureum L.: is the factor producing summer morphs (SMPH) identical to the small prothoracicotropic hormone (4K-PTTH)? Zool. Sci. 5, 1051-1057. Nijhout H. F. (1994) Insect Hormones. Princeton University Press, Princeton, NJ. Shapiro A. M. (1976) Seasonal polyphenism. Euol. Biol. 9, 259-333. Smith K. C. (1991) The effects of temperature and daylength on the rosa polyphenism in the buckeye butterfly, Precis coenia (Lepidoptera: Nymphalidae) J. Res. Lep. 30, 2255236. Tauber M. T., Tauber C. A. and Masaki S. (1986) Seasonal Aduptafions of Insects. Oxford University Press, New York. Warren J. T., Smith W. A. and Gilbert L. I. (1984) Simplification of the ecdysteroid radioimmunoassay by the use of protein A from Staphylococcus aureus. Experientia 40, 561-576. Westbrook A., Regan S. A. and Bollenbacher W. E. (1993) Developmental expression of the prothoracicotropic hormone in the CNS of the tobacco hornworm Manduca sexta. J. Comp. Neurol. 327, 1-16. Acknowledgements-We would like to thank Dr Larry Gilbert, University of North Carolina at Chapel Hill, for the use of his laboratory for ecdysteroid radioimmunoassays. This work was supported by Grant IBN-9220211 from the National Science Foundation.