Proc. Natl Acad. Sci. USA Vol. 79, pp. 2726-2730, April 1982 Physiological Sciences

Prothoracic glands of the saturniid cynthia ricini possess a circadian clock controlling gut purge timing (circadian rhythm/photoreceptive site/larval-prepupal development/ecdysone release timing) AKIRA MIZOGUCHI AND HIRONORI ISHIZAKI Biological Institute, Faculty of Science, Nagoya University, Nagoya, 464, Japan Communicated by Colin S. Pittendrigh, January 25, 1982

ABSTRACT In the moth ricini, timing of the 12-hr dark photoperiod (LD 12:12) unless otherwise specified. release ofprothoracicotropic hormone is controlled by a circadian Final (fifth) instar larvae under these conditions cease to eat and clock in the cephalic organ (brain); while this hormone release is purge their gut content 5 or 6 days after the last larval ecdysis. necessary for gut purge, the final timing ofthis event is controlled Detailed accounts of gut purge and other accompanying be- by a circadian clock in the prothoracic glands that gates release haviors have been given elsewhere (5). The time of gut purge of ecdysone. The photoreceptor of the prothoracic gland clock(s) was established by observation under a safe dim red light (6). is extraocular and evidently in the glands themselves. Demon- General Strategy. Results from two types of experiments stration that the clock and its photoreceptor are in the prothoracic have led to the conclusion that the prothoracic glands contain glands is based on a mixture of localized illuminations and the their own, autonomous, circadian clock and associated (extra- transplantation of glands from one larva to another. ocular) photoreceptor. One ofthese involves transplantation of prothoracic glands, the significance ofwhich is self-evident. The The molting and eclosion behaviors ofmany are subject other uses localized light applications to define the anatomical to circadian timing, which has been analyzed at the neuroen- site of the photoreceptor. The rationale for these experiments docrine level in several cases involving adult eclosion (1), lar- is as follows. val-larval molting (2, 3), and larval-prepupal development (4, When larvae that have been reared under LD 12:12 are trans- 5). Release oftwo neurosecretory hormones, eclosion hormone ferred to constant darkness (DD) and constant temperature 4 (1) and prothoracicotropic hormone (PTTH) (2-5), involved in days after the last larval ecdysis, gut purge occurs in a gate either triggering these events is under strict temporal control by a 14-18 hr (gate 1) or 38-42 hr (gate 2) after the onset of DD. circadian oscillation (or oscillations). Several experimental tech- Clearly, the circadian system timing gut purge free runs in con- niques, especially transplantation, have localized the oscilla- stant darkness. Our unpublished data show that its pacemaker tions (or clocks) timing eclosion hormone and PTTH release to has a typical phase-response curve for brief but intense light the brain. pulses: such pulses cause phase delays when applied in the first As in most holometabolous insects, the last instar larvae of few hours (early subjective night) after entry into DD. A 15-min Samia cynthia ricini, when fully grown, undergo a remarkable illumination (1 klx) 3 hr after the onset of DD brings about a series of morphological and behavioral changes that are prep- phase shift of -4 hr in the gates, during which gut purge is aratory to pupation. One of these, the so-called gut purge, is permitted. In the experiments reported here, observation is a massive excretion offluid feces from the gut. The onset ofthis limited to gut purges in gate 2: very few larvae exploit gate 1, purge is abrupt and clear-cut, hence easy to assay. Circadian and the data we have indicate that its delay is less than that of control of gut purge timing has been demonstrated (unpub- gate 2, suggesting that it is subject to the transients that char- lished observations). We have characterized the response ofthe acterize the gating system in Drosophila (7). When whole larvae system to normal and abnormal light cycles and to brief pulses are illuminated 3 hr after entry into DD, the gate 2 peak ofgut of light and found it similar to those of other insect circadian purge activity is delayed from 38-42 to 43-47 hr after entry into systems. Ligature experiments show that PTTH release is in- DD. By applying light pulses locally in this system, we were volved in the timing of gut purge (5), as it is in the earlier lar- able to locate the photoreceptor that is associated with the gut val-larval molts (2, 3). Evidently, a circadian oscillator in the purge clock; the photoreceptor was deduced to be present in brain is again involved, but other observations (to be reported the illuminated area when the peak was delayed and to be ab- fully elsewhere) suggested that gated PTTH release was not the sent when the peak remained unchanged. Three types of lo- sole factor involved in circadian timing of gut purge. The ex- calized illumination were used: periments reported here show that that is indeed the case: the (i) Anterior vs. posterior local illumination. Larvae were prothoracic glands themselves contain a circadian pacemaker transversely partitioned into anterior and posterior halves at that has its own (extraocular) photoreceptor that is additional various levels ofthe body and one-halfwas illuminated by using to the brain pacemaker that gates PTTH release. The circadian a light-tight box with windowsjust large enough to insert a larva. clock in the prothoracic glands is the immediate timer of gut Each larva was placed on the edge ofthe window at the segment purge. to be partitioned, and a small plate with an indentation fitting the larval body was placed against the box wall and slid so that MATERIALS AND METHODS the indented part squeezed the body to prevent light leakage. . Larvae of Samia cynthia ricini were reared on an The plate was then fixed in place with adhesive tape and the artificial diet as described (6) at 25 + 0.50C under a 12-hr light/ part outside the box was illuminated with a fluorescent lamp (daylight type; Toshiba, Tokyo, Japan) at 1 klx for 15 min. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: PTTH, prothoracicotropic hormone; DD, constant dark; ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. LD 12:12, 12-hr light/12-hr dark photoperiod. 2726 Downloaded by guest on September 27, 2021 Physiological Sciences: Mizoguchi and Ishizaki Proc. Natl. Acad. Sci. USA 79 (1982) 2727 (ii) Dorsal vs. ventral and spot illumination. The head of a larva to be treated was fixed on a plate with taut threads; the A f10 posterior end ofthe insect was pulled, by hand, so that the fully ,-Al stretched body was tight to the plate either face up or face down. B Care was taken to ensure a bilaterally symmetrical reproducible posture of the treated larva. This is particularly important, in. evaluating the results of localized light whose location is dic- tated by external landmarks but whose target is some internal C 13~~~1 structure (e.g., the prothoracic gland). For dorsal and ventral ~F-h illumination, overhead light for 10 sec was produced by a slide D IIf~~~h projector equipped with a 300-W tungsten lamp. Various light intensities were obtained by placing larvae at various distances E from the light source. For localized illumination, a 2-mm light spot was produced by a glass capillary light guide connected to F the 300-W slide projector, the tip ofthe light guide being lightly attached to the body surface. Light intensity at the tip of the light guide was 100 klx. Heat from the light source was removed G by a water cell. When multiple sites were to be illuminated, 1St~~~~11 localized lighting was used successively. H (iii) Illumination oflarvae with implanted prothoracic glands. Larvae with implanted prothoracic glands and other (control) organs were pulse illuminated on the ventral surface with the I 300-W slide projector for 10 sec at 300 klx. When the regions with the implants were to be shielded from light, the part be- hind the second abdominal segment was wrapped with a sheet of aluminum foil and its anterior edge was squeezed to prevent 0 36 9 33 36 39 42 45 48 light leakage. All procedures preceding illumination were car- Time after entry into DD, hr ried out under a dim red light and no anesthesia was used in any of the illumination procedures. FIG. 1. Effects of anterior vs. posterior localized illumination on Transplantation of Prothoracic Glands. The prothoracic gut purge timing. Fifth instar larvae were transferred from LD 12:12 glands and other organs to be transplanted as controls were to DD 4 days after the last larval ecdysis and illuminated for 15 min dissected out of 2-day-old fifth instar larvae in lepidopteran with 1-klx light 3 hr later. Light-tight partitions were placed at the physiological saline (8) and transplanted into larvae of the same neck (C andG), pro/mesothorax (D and H), meso/metathorax (E and I), and metathorax/abdomen (F and J).a, Area exposed to the light; age under ether anesthesia. The organs were implanted through g, shielded area. The histograms represent the number of larvae that an incision at the intersegmental region between the fourth and underwent gut purge in successive 1-hr periods. fifth abdominal segments slightly lateral to the ventral midline. The prothoracic glands from three donor larvae were trans- terested in is not contained in the head but resides in a rather planted into a single host larva. Control organs were kept in the broad area of the thorax. saline for 40 min, which is the time required for three pairs of Dorsal vs. Ventral Illumination. Larvae fixed on a plate prothoracic glands to be dissected out, owing to their highly either dorsal or ventral side up were pulse illuminated at three diffuse architecture, before being transplanted. different intensities for 10 see (Fig. 2). At 1 klx, neither dorsal nor ventral illumination produced any phase delay in gut purge RESULTS timing whereas, at 25 klx, both elicited a full delay. When an Anterior vs. Posterior LocalIllumination. To locate roughly 1 neklx -l_; 11010 the site of the photoreceptor associated with the gut purge U~rsdl stue ANTI_ I rhythm, transverse partitionings at various levels of the body 1 klx were made and, 3 hr after entry into DD, either the anterior Ventral side or the posterior part was illuminated with 1-klx light for 15 min. rule The resulting changes in gut purge timing are shown in Fig. 1. 5 klx When the head alone (C) or the head and the prothorax (D) was Dorsal side illuminated, there was no evident gut purge delay compared 5 klx with the control in which the whole body was kept in DD (A), Ventral side _ AL A although a slight scatter of the peak was observed. The gut 25 klx purge delay became evident when the partition was placed be- Dorsal side F1-1. tween the mesothorax and the metathorax (E) and a full delay, comparable with that after whole body exposure (B), was ob- 25 klx served when the head and the whole thorax were illuminated Ventral side (F). The reverse relationship between the partitioning location and the response to light was obtained in the series of posterior 0 3 6 9 33 36 39 42 45 48 illuminations; when the whole body except the head was ex- Time after entry into DD, hr posed (G) full delay was observed, and the delay became less as the partition moved posteriorly (H-J). Clearly, no matter how FIG. 2. Effects of dorsal and ventral illumination on gut purge localized the external site of illumination, some light scattering timing. Fifth instar larvae were transferred from LD 12:12 to DD 4 days after the last larval ecdysis and, 3 hr later, were illuminated must occur within the body but, in spite of this scattering, the with 1-, 5-, or25-klx light for 10 sec from either the dorsal or the ventral data in Fig. 1 leave no doubt that the photoreceptor we are in- side. Downloaded by guest on September 27, 2021 2728 Physiological Sciences: Mizoguchi and Ishizaki Proc. Natl. Acad. Sci. USA 79 (1982) intermediate light of 5 klx was given, both illuminations pro- thorax. The duration ofthe light pulses at each site varied from duced a delaying scatter ofthe peak but it (the delay) was slightly 5 to 60 sec and the pulses were given singly or to multiple sites more pronounced after ventral illumination. This difference, in succession. The responses (delay ofthe gut purge peak) were though slight, raises the possibility that the photoreceptor is compared with (i) those of DD controls and (ii) those of insects ventrally distributed (e.g., nerve cord) and prompted the fol- whose entire body was exposed to sufficient light. lowing experiments, in which more definitive localization was When 20-sec light pulses were given singly to the midline attempted by using localized illumination. (Fig. 3C), there was little delay in the phase of the gut purge Localized Illumination. To locate the photoreceptive site peak (Fig. 3A); 60-sec exposures at the same sites resulted in more precisely, various sites were pulse illuminated with a 2- a slight delaying scatter of the peaks. When two symmetrical mm light spot (Fig. 3). The light pulses were targeted to the lateral sites at the same levels were illuminated for either 10 or ventral midline or symmetrical lateral sites at the levels of pos- 30 sec-i.e., a total 20 or 60 sec (Fig. 3D)-the results were terior prothorax, anterior mesothorax, and posterior meso- almost the same as those from midline illuminations. Obviously delayed scattering was seen when the pulses were given to four 4I lateral sites for 20, 40, or 60 sec in total, but the exposures to A two medial sites for the identical durations were clearly less B effective (Fig. 3E). When three midline points or six lateral points were illuminated (Fig. 3F), gut purge delay became even more prominent, but again lateral illumination was more effec- tive. It is worth noting that, in the cases in which the delaying scatter was enhanced (e.g., 60-sec lateral illumination in Fig. 3 E and F, 60-sec midline illumination in Fig. 3 F), an appre- ciable number of larvae underwent gut purge even later than C any in the delayed peak following whole-body exposure (Fig. 3B). In summary these results imply that (i) the photoreceptor is not localized, at least exclusively, in the ganglia of the ventral nerve cord, since illumination of the ventral midline was gen- erally less effective than lateral illumination in delaying gut purge, and (ii) the photoreceptor is distributed diffusely over a wide area of the thorax, since the greater the area (number of sites) illuminated, the greater the delay in gut purge timing. Light/Dark Effects on Transplanted Prothoracic Glands. D The prothoracic glands of Samia are paired highly diffuse or- gans, each ofwhich is composed of -200 large polyploid cells. Individual cells and cell clusters are connected one to another by a highly developed network of connective strands and are distributed over a wide area from the prothorax to the anterior region of the abdomen, as described frequently for lepidopter- ans and hymenopterans (9). An impressive coincidence is evi- dent, therefore, between the distribution of the prothoracic glands and that of the photoreceptive sites as revealed by lo- calized illumination. The following transplantation experiments were therefore undertaken to test the possibility that the pho- E tosensitive circadian clock that controls gut purge timing resides in the prothoracic glands themselves. Larvae raised in LD 12:12 served as donors; their prothoracic glands were dissected out 39-42 hr after ecdysis to the fifth in- star and transplanted into the abdomen of larvae of the same age. In this experiment, slowly growing small larvae were se- lected for use, that were expected to exploit gate 2 (third day ofthe fifth instar) in secreting PTTH (5). By using these larvae, both indigenous and transplanted prothoracic glands were ex- F pected to receive PTTH simultaneously on the first day after transplantation. Two days after transplantation, the larvae were transferred to DD and, 3 hr later, they were pulse illuminated for 10 sec. If the prothoracic glands contain a photosensitive 3 6 36 3942 4548 51 circadian clock controlling gut purge timing, the following two Time after into hr results are expected. First, when the anterior part alone is il- entry DD, luminated, the clock in the in situ prothoracic glands should FIG. 3. Effects of localized light pulses on gut purge timing. Four undergo a phase delay whereas the clock of the transplanted days after the last larval ecdysis, larvae were transferred to DD and, prothoracic glands should remain unshifted. As a result, the gut 3 hr later, they were pulse illuminated with a 2-mm light spot at 100 purge should occurwithout delay as ifthe whole body were-kept klx. (A) DD control. (B) Whole-body-illumination control (25 klx for 10 in DD. Second, a in gut should result sec). (C) Single exposures by the light spot on the ventral midline. (D) phase-delay purge timing in Double exposures on lateral sites. (E) Double exposures on the midline from whole-body illumination if the clock the transplanted and four exposures on lateral sites. (F) Three exposures on the midline prothoracic glands is capable of phase resetting in response to and six exposures on lateral sites. light. Downloaded by guest on September 27, 2021 Physiological Sciences: Mizoguchi and Ishizaki Proc. Natl. Acad. Sci. USA 79 (1982) 2729

As shown in Fig. 4, light pulses to the anterior part of larvae the effects they produce; stimulus-patterning effects could be with implanted prothoracic glands (C) did not induce gut purge involved, depending on the nature of the light-effector system delay in most cases whereas whole-body illumination (D) (e.g., saturation of and adaptation to light stimulus). Further- brought about an obvious delay. Anterior or whole body illu- more, there remains the possibility that a cephalic clock is mination to larvae that had been implanted with three brains entrained by light/dark information received by a thoracic (E and H), three mesothoracic ganglia (F), or the fat body in an photoreceptor. amount approximately equal to three pairs ofprothoracic glands The idea that the prothoracic gland contains both the clock (G) elicited essentially the same response as intact larvae. Sup- and the photoreceptor that controls gut purge timing was then plementary experiments were carried out in which the time tested and confirmed by transplantation experiments: when between DD onset and the light pulse was increased to 5 hr to transplanted prothoracic glands are shielded from light that re- make manifestation of the gut purge delay stronger (I and J). sets (delays) indigenous prothoracic glands, the time of gut The results are the same as those in Fig. 4 B and C but clearer: purge remains unshifted. The timing must be dictated by the when the transplanted prothoracic glands are not exposed to implanted prothoracic glands and the glands must therefore light, there is no delay in the host's gut purge. contain their own free running circadian pacemakers. Futher- more, because whole-body illumination does phase shift them, DISCUSSION they must contain their own (extraocular) photoreceptor. The prothoracic glands of most insects are richly innervated and we Experiments in which localized light pulses were used sug- cannot totally exclude the possibility that our implanted pro- gested that the clock immediately responsible for gut purge tim- thoracic glands reestablish, by regeneration, some neural con- ing was associated with (or in) the prothoracic glands. The light nections with the central nervous system. Given the distance pulses most effective in phase shifting the clock were not those separating the implants from their usual location, that possi- directed to the head or the midline of the thorax (ventral nerve bility is, however, unlikely in the extreme. Clearly, no neuronal cord); the most effective pulses were those directed to theven- pathway transmits light information to the implanted protho- trolateral sections of the thorax where the prothoracic glands racic glands (Fig. 4 C andJ) nor can any regenerated nervous are dispersed. Decisive conclusion should not be drawn, how- connection explain retention of the unshifted phase in these ever, from illumination experiments alone for the following rea- implants. Likewise, we cannot totally exclude the possibility sons. Localized light pulses given singly or successively at mul- that the prothoracic glands interact humorally with some other tiple sites in equalized total duration may not be equivalent in organ. Complete proof of our hypothesis that the prothoracic glands contain a circadian pacemaker and a photoreceptor would require in vitro culture experiments. We regard it as =~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~certain that the implanted prothoracic glands exert control of gut purge timing by a humoral agent and that that agent is al- B most certainly ecdysone. In fact, heart exposure, one of the prodromal signs of pupation in Manduca sexta, has been shown to be induced by ecdysterone (10). Our previous study (5), in whichligations between head and thorax were used showed that PTTH, released by a circadian gating system on the 2nd or 3rd day of the fifth larval instar, is D involved in the eventual, much later, timing of gut purge, as 1~~~~~~~~~~~~~~~~~ it is in larval-larval molts (2, 3). Clearly, the clock controlling PTTH release is in the cephalic organ and quite distinct from E that which we have here shown in the prothoracic gland, pre- sumably gating the release of ecdysone. F --flm Inmost cases of localized illumination, the delay in gut purge timing appeared as a delaying scatter rather than as well-defined G fully delayed peaks such as those obtained when the whole body was pulse illuminated. Of particular interest is the fact that the H 1.S A~~~F gut purge delay was occasionally greater than the fully delayed peaks following whole-body illumination; that excludes the pos- ,11 sibility that failure to fully delay the entire peak was simply due oarThrfnTh I I , to insufficient light. A tentative explanation for these facts can be given by assuming the presence ofa clock in each prothoracic gland cell. Thus, localized light may cause a full phase delay in the clocks of those cells that were sufficiently illuminated but fail to do so in those clocks that received subthreshold illumi- 0 3 6 9 33 36 39 42 454851 nation. Asynchronous ecdysone release among prothoracic Time after entry into DD, hr gland cells then ensues, resulting in a lower titer of hemolymph ecdysteroids at any moment and, accordingly, a slower increase

FIG. 4. Effects of light on prothoracic gland-transplanted larvae. in the titer. The time course of hemolymph ecdysteroid in- Larvae that had been implanted with three pairs of prothoracic glands crease will vary, depending on the proportion of prothoracic (C, D, and J) or control organs [three brains (E and H), three meso- gland cells that have been phase shifted by the localized light, thoracic ganglia (F), or fat body (G)] in the abdomen 39-43 hr after and the mixture of shifted and unshifted clocks may be respon- last larval ecydsis were transferred from LD 12:12 to DD 4 days after sible for the nearly continual occurrence of gut purge in cases ecdysis and, 3 (A-H) or 5(I and J) hr later, were illuminated for 10 sec such as that shown in Fig. The slow increase in with 300-klx light. El, Area was exposed to light; RM, shielded area; 3F. ecdysteroid *, prothoracic glands; o, transplanted control organs. (A, B, and titer may induce gut purge even later than the fully delayed Control operations, no implants. peaks after whole-body illumination, which causes a full but Downloaded by guest on September 27, 2021 2730 Physiological Sciences: Mizoguchi and Ishizaki Proc. Nat. Acad. Sci. USA 79 (1982)

synchronous delay in all clocks to yield a steep increase in the April 1971 (Centre for Agricultural Publishing and Documenta- titer. It should be noted that, in the induction of heart exposure tion, Wageningen), pp. 111-135. in Manduca, a suprathreshold titer ofecdysterone must be pres- 2. Truman, J. W. (1972) J. Exp# Biol 57, 805-.820. 3. Fujishita, M. & Ishizaki, H. (1981)J. Insect Physiol. 27, 121-128. ent for a certain minimum duration oftime for the physiological 4. Truman, J. W. & Riddiford, L. M. (1974) J. Exp. Biol. 60, response to become manifest (10). 371-382. 5. Fujishita, M. & Ishizaki, H. (1982)J. Insect Physiol 28, 77-84. We thank Prof. C. S. Pittendrigh for critical reading of the manu- 6. Ishizaki, H. (1980) ZooL Mag. 89, 277-282. script. This work was partly supported by a grant-in-aid for Special Proj- 7. Pittendvigh, C. S. (1965) in Circadian Clocks, ed. Aschoff, J. ect Research on Mechanisms of Behavior from the Ministry of (North-Holland, Amsterdam), pp. 277-297. Education, Science, and Culture, Japan. 8. Jungreis, A. M., Jatlow, P. & Wyatt, G. R. (1973)J. Insect Phys- iol. 19, 225-233. 1. Truman, J. W. (1972) in Circadian Rhythmicity, Proc. Int. Symp. 9. Herman, W. S. (1967) Int. Rev. Cytol 22, 269-347. Circadian Rhythmicity, Wageningen, The Netherlands, 26-29 10. Nijhout, H. F. (1976)J. Insect Physiol. 22, 453-463. Downloaded by guest on September 27, 2021