J. Insect Physiol., 1976, Vol. 22, pp. 473 to 481. Pergamon Press. Printed in Great Britain.
ULTRASTRUCTURE OF ACTIVE AND INHIBITED PROTHORACIC GLANDS* C. N. McDmm,t
E. JOHNSON,T. SAUM,and S. J. BJXRRY
Wesleyan University Department
of Biology, Middletown,
Connecticut
06457, U.S.A.
(Received 27 August 1975) Abstract-Changes in the cytoplasm of prothoracic gland cells were compared in pharate adults, dauer pupae, and aminophylline inhibited pupae of H. cecropic. For the first 3 to 4 days after transfer from 4 to 22”C, a similar sequence of changes in the cytoplasmic elements was observed. At day 4 the cytoplasm of pharate adults exhibited further differentiation which was consistent with the initiation of secretion, while dauer and inhibited pupae remained at the stage achieved at day 4 and did not advance further even after a substantial lapse of time. These results are interpreted as indicating that the early changes represent a response by the cells to the temperature change, while the initiation of secretion requires the intervention of brain hormone. INTRODUCTION PROTHORACICglands are the source of the insect
moulting hormone, ecdyson (WILLIAMS,1952; KAMBYSELLISand WILLIAMS, 1972; CHINO et al., 1974). In the normal course of events, the prothoracic gland cells (PGC) are activated by neurosecretions released from the brain via the corpus cardiacurn. The brain hormone stimulates PGC to synthesize and secrete ecdyson into the haemolymph, which, in turn, promotes differentiation of adult tissues in the pupa (WILLIAMS, 1952; SCHNEIDERMAN and GILBERT, 1964; WIGGLESWORTH,1970). Light and electron microscopical studies have documented various cytological changes in the prothoracic glands during the course of activation by brain hormone (see review by HERMAN,1967). In studies published to date, the cytological changes associated with activation of PGC have been derived from observations on changes in the glands of intact animals in which initiation of ecdyson synthesis and secretion proceeds in the normal fashion. Studies of normal activation do not distinguish between events triggered by brain hormone, and those which are the result of the activity of the prothoracic glands as a response to environmental change in the absence of brain hormone. In a previous publication (MCDANIEL and BERRY, 1974, we suggested that methylxanthine compounds such as caffeine and theophylline interfere with the release of brain hormone. In this communication we discuss the changes in the cytology of PGC in pharate adults deprived of brains * This work was supported by N.I.H. Career Development Award No. HD50195 and a National Science Foundation Grant (GB 34155). 7 Present address: Dept. of Biology, Rensselaer Polytechnic Institute, Troy, New York, 12181 USA. 473
(dauer pupae) and compare these changes with those observed during the normal course of brain hormone-induced activation and in the presence of inhibitory concentrations of methylxanthine. MAmRL+LS
AND
mTI-IODS
H. cecropia were reared on cherry trees during the summer and maintained at the natural photoperiod for 6 weeks to ensure that all were in diapause. Brains were surgically removed from one group of pupae and a second group received aminophylline (1.0 mg/g live weight), and all animals were held for another month at ambient temperature and natural photoperiod. All pupae were then stored at 4°C for 6 months and then transferred to 22°C and a long day photoperiod (17 hr light, 7 hr dark). Immediately following removal from 4”C, a third group of pupae was arrested with aminophylline. Starting on the day of removal from the cold, prothoracic glands were dissected from at least two animals in each of the four groups (untreated controls, dauer pupae, arrested prior to chilling, arrested after chilling), and fixed for microscopy. Glands were collected daily for the first 8 days after removal from the cold, and at various times after the eighth day. After ‘day 5’ of visible development according to the timetable of SCHNEIDER~MAN and WILLIAMS (1954) which corresponded to 10 days after removal from the cold, prothoracic glands could not be successfully dissected from the untreated controls. Fixation for electron microscopy was accomplished in 4% buffered glutaraldehyde and for light microscopy in Bouin’s solution. Light microscope preparations were stained with Mayer’s hemalum and Orange G while EM sections were post-fixed in 2% buffered osmium tetroxide, and embedded in Epon-Araldite. Staining of sections for EM was
474
C. N. MCDANIEL,E. JOHNSON, T. SAUM,and S. J. BWRY
accomplished using alcoholic uranyl acetate and lead citrate (VENABLEand COGGENSHALL, 1965). RESULTS
scribed are not consistent for all the prothoracic gland cells from a given animal, since some cells lag behind the majority, but the trend of changes is typical of the bulk of the cells.
Prothoracic glands from pharate adults Cells removed from the control group immediately after transfer from 4°C showed a characteristic pattern of vacuolation (Fig. 1). Large spaces were observed in the central cytoplasm of the majority of cells examined. These spaces were not bounded by membrane and contained no electron dense material other than scattered glycogen rosettes. Cytoplasm also contained scattered free ribosomes, microtubules, glycogen rosettes, myelin figures containing lipid droplets, and clumps of elongated mitochondria. Surface microvilli were not extensive and the plasma membrane was closely covered with a heavy basement membrane. A notable cytoplasmic feature was the absence of rough endoplasmic reticulum (RER). The nucleus was cup-shaped and contained patterns of scattered heterochromatin which persisted as long as development was observed. The empty spaces disappeared after 1 or 2 days at 22°C and did not subsequently reappear, while free ribosomes and microtubules increased for the first 3 days. An extensive palisade of microvillae developed on the plasma membrane (Fig. 2) and glycogen deposits increased and then disappeared entirely. During this 3 day period small ring nucleoli (Fig. 3) were frequently observed in the nucleus and the first small cisternae of the rough endoplasmic reticulum appeared, as well as other membrane-derived organelles such as Golgi bodies (Fig. 4), and an occasional annulate lamella (Fig. 5). Myelin figures containing lipid droplets (Fig. 6), present from day 0, were retained. On days 3 to 4 at 22°C small vesicles appeared in the cytoplasm coincident with a substantial increase in the cistemae of rough endoplasmic reticulum (Fig. 7). Long stretches of RER appeared particularly near the cell periphery, and flocculent material appeared in the cistemae. Larger vesicles appeared to form from accumulations of the smaller ones and contained flocculent material. These vesicles were also observed in light micrographs through the pigmented tarsal claw stage, and an increase in cytoplasmic basophilia was noted coincident with the increase in RER. During the fourth and fifth days the vesicles migrated to the plasma membrane where they were released between the microvillae as the thick basement membrane began to break down (Fig. 8). As the basement membrane became further disrupted the microvillae appeared to be resorbed (Fig. 9) until no remnants of membrane remained, and the surface of the cells was relatively smooth (Fig. 10). At this stage the endoplasmic reticulum and vesicle content was considerably reduced. The cytological changes de-
Prothoracic gland from a!auer and arrested pupae Cytoplasmic changes in cells from dauer and arrested pupae were similar to those observed in the course of ‘normal’ activation for the first 3 to 4 days after removal from the cold. The large spaces observed on day 0 (Fig. 11) disappeared, free ribosomes increased, glycogen rosettes increased and then decreased, and small profiles of RER appeared. The cisternae of the RER remained small and contained little flocculent material. Lipid containing myelin figures appeared and were retained as in active cells. No differences were noted in animals arrested with aminophylline before or after chilling, and light microscope preparations showed no further progress for 3 months. A single dauer pupa initiated adult development on transfer to 22°C and was dissected 10 days after transfer from 4°C (day 2 of visible development according to the timetable of SCHNEIDERMAN and WILLIAMS (1954). The prothoracic glands from this animal exhibited the typical cytoplasmic features of glands obtained from pupae with the brain intact. The basement membrane had been extensively disrupted and the cytoplasm was replete with vesicles and RER (Fig. 12). In all other cases the prothoracic gland cytoplasm from dauer pupae (Fig. 13) and aminophylline arrested pupae (Fig. 14) was identical for periods in excess of 2 months to that of activated gland cells 3 to 4 days after transfer from 4°C. DISCUSSION As the source of the moulting hormone, ecdyson, the prothoracic glands assume a critical r81e in the regulation of insect growth and development (WILLIAMS, 1969; CHINOet al., 1974). In a previous communication (MCDANIEL and BERRY, 1974), we described methylxanthine-induced developmental arrest in pharate adults, and suggested that the basis for inhibition might be a failure of release of brain hormone. The absence of brain hormone would result in failure to activate the prothoracic glands. When we examined the ultrastructure of prothoracic gland cells in animals inhibited with methylxanthine (aminophylline) we were surprised to find that certain changes occurred that are often associated with activation by brain hormone. To determine whether these changes were dependent on brain hormone, we examined the sequence of events in animals from which the brain was removed. We had previously determined that the injury attending brain removal could activate prothoracic glands (MCDANIEL and BERRY, 1967, 1974) and,
475
Fig. 1. Pharate adult-day 0. Large characteristic empty vesicles fill the centre of the cell. The loosely organized cytoplasm contains ribosomes, scattered microtubules, and abundant tubular mitochondria. (Bars in lower right indicate 1 /*m in all micrographs.) Fig. 2. Pharate adult-day 2. Cytoplasm contains large amounts of glycogen. The density of free ribosomes and microtubules is greater and Golgi bodies are evident (G).
476
Fig. 3. Pharate adult-day
2. Nuclei characteristically contain scattered heterochromatin or more ring nucleoli are frequently observed.
and one
Fig. 4. Pharate adult-day 2. Cytoplasmic detail shows polysomes and short profiles of developing RER (arrows). Scattered glycogen rosettes (R) and two Golgi bodies (G) with typically flat lamellae are present. Fig. 5. Pharate adult-day
Fig. 6.
Pharate
adult-day
2. Segments of annulate lamellae (AL) are seen in perinuclear in developing cells.
regions
2. Membranous whorls (M) containing many lipid droplets quently found throughout the cytoplasm.
are fre-
477
Fig. 7. Pharate adult-day 6. Cytoplasm of an active PGC cell. Large vesicles in association with RER (arrows) are filled with flocculent material. A microtubular network (MT) runs perpendil cular to the cell border (out of micrograph at upper left).
‘ig. 8. Pharate adult-day 6. Filled vesicles at the cell border just prior to the release of secretory material. The simultaneous breakdown of acellular sheath has begun at the right of the micrograph. Gg. 9. Pharate adult-day 6. Disorganized microvillar border during secretory release. The sheath as been lost but some portions are enclosed within the cell. The cytoplasm still contains many polysomes but few filled vesicles or RER profiles. ‘ig. 10. Pharate adult-day 6. Cell surface at termination of vesicle release. Microvilli are rarely een. No sheath material remains at the surface and the cytoplasm has reverted to its appearance at day 1 or 2.
479
Fig. il.
Dauer pupa-day The cytoplasm
0. Cells have the same characteristic vacuoles as normal day 0 animals. is loosely organized and membranous whorls are present.
Fig. 12. Dauer pupa-day 10. PGC from dauer pupa which initiated adult development. Sheath degeneration and presumably secretory release has occurred. The cytoplasm contains some secretory vesicles (SV), Golgi bodies and RER.
480
Fig.
13.
Fig. 14.
Dauer pupa--4
months at 22°C. Heavy basement
Cytoplasm membrane
contains many ribosomes remains intact.
Arrested pupa-day 8 at 22°C. Scattered free ribosomes, microtubules, myelin figures characterize the cytoplasm which lacks RER.
but little RER.
Golgi bodies, and
Ultrastructure of active and inhibited prothoracic glands to eliminate this possibility, brains were removed from diapausing pupae 1 month before chilling. Parallel changes in early cytoplasmic features in PGC from controls, dauer, and inhibited pupae suggest that the cells respond directly to environmental change and not to brain hormone. Only the later events such as the accumulation of extensive RER and the accumulation and release of secretory material from the cisternae seem to require the presence of brain hormone. These observations support the suggestion that methylxanthines may prevent brain hormone activation of prothoracic gland cells. The sequence of events observed during cytodifferentiation of the control animals is certainly consistent with the synthesis of a carrier protein in addition to ecdyson. We noted the lysosome-like, lipid-containing bodies reported by ISHIZAKI (1969) in Samia Cynthia ricini, but saw no evidence of release of these organelles. Nor did we observe the alterations in the relation of chromatin to nuclear membrane as reported in the same paper. BEAULATON (1968) suggested that synthesis and secretion of ecdyson might involve the fusion of mitochondria with the RER. We were not able to locate either the macromitochondria observed by Beaulaton or any profiles which suggested degeneration of mitochondria or fusion with the endoplasmic reticulum. The escape from diapause by one of approximately 30 dauer pupae is a phenomenon we have encountered in the past (e.g. MCDANIEL and BERRY, 1974) and has been observed in Bombyx mori (ISHIZAKI, 1972) and Manduca sexta (JUDY, 1972) (see also review by JUDY (1974)). The normal ‘active’ appearance of the prothoracic cytoplasm in this animal may indicate that the r61e of brain
hormone involves a general activation and does not qualitatively alter the course of differentiation in the prothoracic glands. REFERENCES BEAULATONJ. (1968) Modifications ultrastructurales des cellules s&r&rices de la glande prothoraciques de vers a soie au cours des deux derniers lges larvairesI. Le chondriome, et ses relations avec le reticulum agranulaire. J. Cell Biol. 39, 501-525.
481
CHINO H., SAKURAI S., OHTAKI T., IKEKAWA N., MIYAZAKI H., ISHIBA~HI M., and A~UKI H. (1974) Biosynthesis of cY-ecdysone by prothoracic glands in vitro. Science, Wash. 183, 529-530. HERMANW. S. (1967) The ecdysial glands of arthropods. ht. Rev. Cytol. 22, 269-347. ISHIZAKIH. (1969) Mode of action of insect brain hormone. An electron microscope study. In Nucleic Acid Metabolism, Cell Dz~erentiation and Cancer Growth (ed. by COWDRY E. V. and SENO S.). Pergamon Press, Oxford. ISHIZAKI H. (1972) Arrest of adult development debrained pupae of the silkmoth, Bombyx mori. J. Insect Physiol. 18, 1621-1627. JUDY K. J. (1972) Diapause termination and metamorphosis in brainless tobacco hornworms (Lepidoptera). Life Sci. 11,605-611. JUDY K. J. (1974) Hormonal control of insect development. In Invertebrate Endocrinology and Hormonal Heterophylly (Ed. by BURDETTE, W. J.), pp. 7-28. Springer, New York. KNV~BYSELLIS M. P. and WILLIAMS C. M. (1972) Spermatogenesis in cultured testes of the Cynthia silkworm: Effects of ecdysone and of prothoracic glands. Science, Wash. 175, 769-770. MCDANIEL C. N. and BERRY S. J. (1967) Activation of the prothoracic glands of Antheraea polyphemus. Nature, Lond. 214, 1032-1034. MCDANIEL C. N. and BERRY S. J. (1974) Effects of caffeine and aminophylline on adult development of the cecropia silkmoth. J. Insect Physiol. 20, 245-252. SCHNEID~RMAN H. A. and GILBERT L. I. (1964) Control of growth and development in insects. Science, Wash. 143, 325-333. SCHNEIDERMAN H. A. and WILLIAMS C. M. (1954) The physiology of insect diapause-IX. The cytochrome oxidase system in relation to the diapause and development of the cecropia silkworm. Biol. Bull., Woods Hole 106.238-252. VFZNABLE J. H. and’CoucENsHALL R. (1965) A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407. WIGGLE~WORTHV. B. (1970) Insect Hormones. Oliver & Boyd, Edinburgh. WILLIAMS C. M. (1952) Physiology of insect diapauseIV. The brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull., Woods Hole 103, 120-138. WILLIAMS C. M. (1969) Nervous and hormonal communication in insect development. 28th Symp. Sot. Dev. Biol. (Ed. by LANG A.). Academic Press, New York.
ULTRASTRUCTURE OF ACTIVE AND INHIBITED PROTHORACIC GLANDS* C. N. McDmm,t
E. JOHNSON,T. SAUM,and S. J. BJXRRY
Wesleyan University Department
of Biology, Middletown,
Connecticut
06457, U.S.A.
(Received 27 August 1975) Abstract-Changes in the cytoplasm of prothoracic gland cells were compared in pharate adults, dauer pupae, and aminophylline inhibited pupae of H. cecropic. For the first 3 to 4 days after transfer from 4 to 22”C, a similar sequence of changes in the cytoplasmic elements was observed. At day 4 the cytoplasm of pharate adults exhibited further differentiation which was consistent with the initiation of secretion, while dauer and inhibited pupae remained at the stage achieved at day 4 and did not advance further even after a substantial lapse of time. These results are interpreted as indicating that the early changes represent a response by the cells to the temperature change, while the initiation of secretion requires the intervention of brain hormone. INTRODUCTION PROTHORACICglands are the source of the insect
moulting hormone, ecdyson (WILLIAMS,1952; KAMBYSELLISand WILLIAMS, 1972; CHINO et al., 1974). In the normal course of events, the prothoracic gland cells (PGC) are activated by neurosecretions released from the brain via the corpus cardiacurn. The brain hormone stimulates PGC to synthesize and secrete ecdyson into the haemolymph, which, in turn, promotes differentiation of adult tissues in the pupa (WILLIAMS, 1952; SCHNEIDERMAN and GILBERT, 1964; WIGGLESWORTH,1970). Light and electron microscopical studies have documented various cytological changes in the prothoracic glands during the course of activation by brain hormone (see review by HERMAN,1967). In studies published to date, the cytological changes associated with activation of PGC have been derived from observations on changes in the glands of intact animals in which initiation of ecdyson synthesis and secretion proceeds in the normal fashion. Studies of normal activation do not distinguish between events triggered by brain hormone, and those which are the result of the activity of the prothoracic glands as a response to environmental change in the absence of brain hormone. In a previous publication (MCDANIEL and BERRY, 1974, we suggested that methylxanthine compounds such as caffeine and theophylline interfere with the release of brain hormone. In this communication we discuss the changes in the cytology of PGC in pharate adults deprived of brains * This work was supported by N.I.H. Career Development Award No. HD50195 and a National Science Foundation Grant (GB 34155). 7 Present address: Dept. of Biology, Rensselaer Polytechnic Institute, Troy, New York, 12181 USA. 473
(dauer pupae) and compare these changes with those observed during the normal course of brain hormone-induced activation and in the presence of inhibitory concentrations of methylxanthine. MAmRL+LS
AND
mTI-IODS
H. cecropia were reared on cherry trees during the summer and maintained at the natural photoperiod for 6 weeks to ensure that all were in diapause. Brains were surgically removed from one group of pupae and a second group received aminophylline (1.0 mg/g live weight), and all animals were held for another month at ambient temperature and natural photoperiod. All pupae were then stored at 4°C for 6 months and then transferred to 22°C and a long day photoperiod (17 hr light, 7 hr dark). Immediately following removal from 4”C, a third group of pupae was arrested with aminophylline. Starting on the day of removal from the cold, prothoracic glands were dissected from at least two animals in each of the four groups (untreated controls, dauer pupae, arrested prior to chilling, arrested after chilling), and fixed for microscopy. Glands were collected daily for the first 8 days after removal from the cold, and at various times after the eighth day. After ‘day 5’ of visible development according to the timetable of SCHNEIDER~MAN and WILLIAMS (1954) which corresponded to 10 days after removal from the cold, prothoracic glands could not be successfully dissected from the untreated controls. Fixation for electron microscopy was accomplished in 4% buffered glutaraldehyde and for light microscopy in Bouin’s solution. Light microscope preparations were stained with Mayer’s hemalum and Orange G while EM sections were post-fixed in 2% buffered osmium tetroxide, and embedded in Epon-Araldite. Staining of sections for EM was
474
C. N. MCDANIEL,E. JOHNSON, T. SAUM,and S. J. BWRY
accomplished using alcoholic uranyl acetate and lead citrate (VENABLEand COGGENSHALL, 1965). RESULTS
scribed are not consistent for all the prothoracic gland cells from a given animal, since some cells lag behind the majority, but the trend of changes is typical of the bulk of the cells.
Prothoracic glands from pharate adults Cells removed from the control group immediately after transfer from 4°C showed a characteristic pattern of vacuolation (Fig. 1). Large spaces were observed in the central cytoplasm of the majority of cells examined. These spaces were not bounded by membrane and contained no electron dense material other than scattered glycogen rosettes. Cytoplasm also contained scattered free ribosomes, microtubules, glycogen rosettes, myelin figures containing lipid droplets, and clumps of elongated mitochondria. Surface microvilli were not extensive and the plasma membrane was closely covered with a heavy basement membrane. A notable cytoplasmic feature was the absence of rough endoplasmic reticulum (RER). The nucleus was cup-shaped and contained patterns of scattered heterochromatin which persisted as long as development was observed. The empty spaces disappeared after 1 or 2 days at 22°C and did not subsequently reappear, while free ribosomes and microtubules increased for the first 3 days. An extensive palisade of microvillae developed on the plasma membrane (Fig. 2) and glycogen deposits increased and then disappeared entirely. During this 3 day period small ring nucleoli (Fig. 3) were frequently observed in the nucleus and the first small cisternae of the rough endoplasmic reticulum appeared, as well as other membrane-derived organelles such as Golgi bodies (Fig. 4), and an occasional annulate lamella (Fig. 5). Myelin figures containing lipid droplets (Fig. 6), present from day 0, were retained. On days 3 to 4 at 22°C small vesicles appeared in the cytoplasm coincident with a substantial increase in the cistemae of rough endoplasmic reticulum (Fig. 7). Long stretches of RER appeared particularly near the cell periphery, and flocculent material appeared in the cistemae. Larger vesicles appeared to form from accumulations of the smaller ones and contained flocculent material. These vesicles were also observed in light micrographs through the pigmented tarsal claw stage, and an increase in cytoplasmic basophilia was noted coincident with the increase in RER. During the fourth and fifth days the vesicles migrated to the plasma membrane where they were released between the microvillae as the thick basement membrane began to break down (Fig. 8). As the basement membrane became further disrupted the microvillae appeared to be resorbed (Fig. 9) until no remnants of membrane remained, and the surface of the cells was relatively smooth (Fig. 10). At this stage the endoplasmic reticulum and vesicle content was considerably reduced. The cytological changes de-
Prothoracic gland from a!auer and arrested pupae Cytoplasmic changes in cells from dauer and arrested pupae were similar to those observed in the course of ‘normal’ activation for the first 3 to 4 days after removal from the cold. The large spaces observed on day 0 (Fig. 11) disappeared, free ribosomes increased, glycogen rosettes increased and then decreased, and small profiles of RER appeared. The cisternae of the RER remained small and contained little flocculent material. Lipid containing myelin figures appeared and were retained as in active cells. No differences were noted in animals arrested with aminophylline before or after chilling, and light microscope preparations showed no further progress for 3 months. A single dauer pupa initiated adult development on transfer to 22°C and was dissected 10 days after transfer from 4°C (day 2 of visible development according to the timetable of SCHNEIDERMAN and WILLIAMS (1954). The prothoracic glands from this animal exhibited the typical cytoplasmic features of glands obtained from pupae with the brain intact. The basement membrane had been extensively disrupted and the cytoplasm was replete with vesicles and RER (Fig. 12). In all other cases the prothoracic gland cytoplasm from dauer pupae (Fig. 13) and aminophylline arrested pupae (Fig. 14) was identical for periods in excess of 2 months to that of activated gland cells 3 to 4 days after transfer from 4°C. DISCUSSION As the source of the moulting hormone, ecdyson, the prothoracic glands assume a critical r81e in the regulation of insect growth and development (WILLIAMS, 1969; CHINOet al., 1974). In a previous communication (MCDANIEL and BERRY, 1974), we described methylxanthine-induced developmental arrest in pharate adults, and suggested that the basis for inhibition might be a failure of release of brain hormone. The absence of brain hormone would result in failure to activate the prothoracic glands. When we examined the ultrastructure of prothoracic gland cells in animals inhibited with methylxanthine (aminophylline) we were surprised to find that certain changes occurred that are often associated with activation by brain hormone. To determine whether these changes were dependent on brain hormone, we examined the sequence of events in animals from which the brain was removed. We had previously determined that the injury attending brain removal could activate prothoracic glands (MCDANIEL and BERRY, 1967, 1974) and,
475
Fig. 1. Pharate adult-day 0. Large characteristic empty vesicles fill the centre of the cell. The loosely organized cytoplasm contains ribosomes, scattered microtubules, and abundant tubular mitochondria. (Bars in lower right indicate 1 /*m in all micrographs.) Fig. 2. Pharate adult-day 2. Cytoplasm contains large amounts of glycogen. The density of free ribosomes and microtubules is greater and Golgi bodies are evident (G).
476
Fig. 3. Pharate adult-day
2. Nuclei characteristically contain scattered heterochromatin or more ring nucleoli are frequently observed.
and one
Fig. 4. Pharate adult-day 2. Cytoplasmic detail shows polysomes and short profiles of developing RER (arrows). Scattered glycogen rosettes (R) and two Golgi bodies (G) with typically flat lamellae are present. Fig. 5. Pharate adult-day
Fig. 6.
Pharate
adult-day
2. Segments of annulate lamellae (AL) are seen in perinuclear in developing cells.
regions
2. Membranous whorls (M) containing many lipid droplets quently found throughout the cytoplasm.
are fre-
477
Fig. 7. Pharate adult-day 6. Cytoplasm of an active PGC cell. Large vesicles in association with RER (arrows) are filled with flocculent material. A microtubular network (MT) runs perpendil cular to the cell border (out of micrograph at upper left).
‘ig. 8. Pharate adult-day 6. Filled vesicles at the cell border just prior to the release of secretory material. The simultaneous breakdown of acellular sheath has begun at the right of the micrograph. Gg. 9. Pharate adult-day 6. Disorganized microvillar border during secretory release. The sheath as been lost but some portions are enclosed within the cell. The cytoplasm still contains many polysomes but few filled vesicles or RER profiles. ‘ig. 10. Pharate adult-day 6. Cell surface at termination of vesicle release. Microvilli are rarely een. No sheath material remains at the surface and the cytoplasm has reverted to its appearance at day 1 or 2.
479
Fig. il.
Dauer pupa-day The cytoplasm
0. Cells have the same characteristic vacuoles as normal day 0 animals. is loosely organized and membranous whorls are present.
Fig. 12. Dauer pupa-day 10. PGC from dauer pupa which initiated adult development. Sheath degeneration and presumably secretory release has occurred. The cytoplasm contains some secretory vesicles (SV), Golgi bodies and RER.
480
Fig.
13.
Fig. 14.
Dauer pupa--4
months at 22°C. Heavy basement
Cytoplasm membrane
contains many ribosomes remains intact.
Arrested pupa-day 8 at 22°C. Scattered free ribosomes, microtubules, myelin figures characterize the cytoplasm which lacks RER.
but little RER.
Golgi bodies, and
Ultrastructure of active and inhibited prothoracic glands to eliminate this possibility, brains were removed from diapausing pupae 1 month before chilling. Parallel changes in early cytoplasmic features in PGC from controls, dauer, and inhibited pupae suggest that the cells respond directly to environmental change and not to brain hormone. Only the later events such as the accumulation of extensive RER and the accumulation and release of secretory material from the cisternae seem to require the presence of brain hormone. These observations support the suggestion that methylxanthines may prevent brain hormone activation of prothoracic gland cells. The sequence of events observed during cytodifferentiation of the control animals is certainly consistent with the synthesis of a carrier protein in addition to ecdyson. We noted the lysosome-like, lipid-containing bodies reported by ISHIZAKI (1969) in Samia Cynthia ricini, but saw no evidence of release of these organelles. Nor did we observe the alterations in the relation of chromatin to nuclear membrane as reported in the same paper. BEAULATON (1968) suggested that synthesis and secretion of ecdyson might involve the fusion of mitochondria with the RER. We were not able to locate either the macromitochondria observed by Beaulaton or any profiles which suggested degeneration of mitochondria or fusion with the endoplasmic reticulum. The escape from diapause by one of approximately 30 dauer pupae is a phenomenon we have encountered in the past (e.g. MCDANIEL and BERRY, 1974) and has been observed in Bombyx mori (ISHIZAKI, 1972) and Manduca sexta (JUDY, 1972) (see also review by JUDY (1974)). The normal ‘active’ appearance of the prothoracic cytoplasm in this animal may indicate that the r61e of brain
hormone involves a general activation and does not qualitatively alter the course of differentiation in the prothoracic glands. REFERENCES BEAULATONJ. (1968) Modifications ultrastructurales des cellules s&r&rices de la glande prothoraciques de vers a soie au cours des deux derniers lges larvairesI. Le chondriome, et ses relations avec le reticulum agranulaire. J. Cell Biol. 39, 501-525.
481
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