Key words: Bombyx - Silk-gland - Starvation - Development - Electron microscopy. During the last larval instar, the posterior part of the silk gland of Bombyx mori.
Cell and Tissue Research
Cell Tissue Res. 213, 311-324 (1980)
9 by Springer-Verlag 1980
The Programming of Silk-Gland Development in Bombyx mori I. Effects of Experimental Starvation on Growth, Silk Production, and Autolysis During the Fifth Larval Instar Studied by Electron Microscopy* Nelly Blaes, Pierre Couble, and Jean Claude Prudhomme D+partement de BiologicG6n~rale et Appliqu~e, Laboratoire associ6 au CNRS (LA 92), Universit6 Claude Bernard, Villeurbanne, France
Summary. The cytological development of the silk gland has been studied by light and electron microscopy in silkworms experimentally starved at different periods of the natural feeding stage during the fifth instar. When newly molted animals are not provided with food, no sign of growth is observed. Starvation initiated early during the obligatory feeding period, stops cell growth and development of the organelles involved in protein synthesis and secretion, whereas it induces the appearance of organelles concerned with autolysis. These effects are reversible if starvation is not prolonged beyond two days. Starvation during the facultative feeding period, at the time of massive fibroin production, results in quantitative and qualitative modifications of organelles related to the decrease of fibroin production and the onset of autolysis. Rough endoplasmic reticulum, responsible for fibroin synthesis, forms transitory whorls. Fibroin transport via the Golgi apparatus and secretion of the protein into the gland lumen decrease parallel to fibroin synthesis, so that no fibroin storage can be detected in any organelle. After food deprivation, autophagosomes and secondary lysosomes rapidly develop in the cytoplasm, and if starvation continues portions of the cytoplasm are sequestered and completely destroyed. If animals are refed, fibroin production is resumed and autolysis declines. These ultrastructural alterations of the silk gland during experimental starvation are very similar to those observed during the periods of physiological starvation (molt and cocoon spinning) and generally considered to be under hormonal control. Our results raise the question of the nature of interactions between alimentary and hormonal factors which control silk-gland development. Send offprint requests to: Dr. NellyBlaes,INSERM, Unit~63, 22Avenuedu DoyenL~pine,69500Bron,
France * This study was supported by a grant from the Centre National de la RechercheScientifique(A.T.P., Contract n~ 1472)and was partly carried out in the "Centre de MicroscopieElectronique,CMEABG, Universit6ClaudeBernard, Lyon" and the "Laboratoire d'Histologie,Servicede Quantim~trie,Facult6 de M6dicine, Universit+Claude Bernard, Lyon"
0302-766X/80/0213/0311/$02.80
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N. Blaes et al. Key words: B o m b y x microscopy.
- Silk-gland - Starvation - Development - Electron
During the last larval instar, the posterior part of the silk gland of B o m b y x m o r i produces a high amount of fibroin, the major protein of the cocoon; during spinning the gland degenerates and finally disappears some h after the nymphal molt. Whereas silk production occurs continuously during the instar, the overall development of the silk-gland cell shows three successive phases: a growth period during the five days following the fourth larval molt, a secretion period from the fifth day till the spinning of the cocoon, a period of autolysis during cocoon spinning till the complete destruction of the organ. M a n y observations have been made on the ultrastructure of the silk-gland cell throughout development (Akai 1965; Matsuura et al. 1968; Morimoto et al. 1968; Tashiro et al. 1968; Matsuura et al. 1976; Tashiro et al. 1976; Couble et al. 1977), but few experimental studies have been undertaken and the programming of the different phases remains unknown. On view of the known participation of hormonal and nutritional factors in the regulation of the molting cycle of the organism (Fukuda 1944; Legay 1960), it has been supposed that the same factors may be involved in the control of silk-gland development. Some experiments have been attempted with the use of insect hormones (ecdysone, Chinzei 1975; juvenile hormone, Kawai 1976; Kurata and Daillie 1978), but experimental studies of the nutritional conditions of development are limited to biochemical parameters directly involved in fibroin production (Chavancy and Fournier 1979). The aim of the present paper is to describe the effect of starvation on the micromorphology of the silk-gland cell during the normal feeding period. It shows that starvation stops growth and development, inhibits the synthesis of fibroin, and induces the development of the lysosomal apparatus involved in autolysis. The comparison of the effects of experimental starvation with the cytological events occurring during the physiological periods of starvation at molt and at cocoon spinning raises the question of the interactions between alimentary and hormonal control factors.
Materials and Methods
Animals'
European hybrids, 200 x 300 of Bombyx mori were reared at 21-22~ and normally fed with flesh mulberry leaves provided four times daily. The animals were fed on autumn leaves;therefore, spinning of the cocoon started on the tenth day of the fifth instar, the first meal after the fourth ecdysis being considered as zero time (Fig. 1). Experimental Procedures
Morphological observation was sheduled in insects in a variety of nutritional states. Experimental starvation started at three different stages of the fifth instar: (1) immediatelyafter the fourth molt (no food at all givento the animals after ecdysis);(2) during the growth stage (food removed3, 6 or 24 h after
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the first meal); (3) during the phase of massive fibroin production (96 h after the first meal). Animals starved for 48 h were refed for 24 h. Control animals were fed ad libitum.
Light and Electron Microscopy Posterior parts of the silk glands were carefully dissected and immediately fixed for 1 h at 4~C in sodium phosphate buffer (pH 7,4) containing 4% glutaraldehyde. After being rinsed in the sodium phosphate buffer, tissues were post-fixed with 4% osmium tetroxide in barbital-sodium acetate buffer (pH 7,4) and embedded in araldite. For routine light microscopy, 1 lam thick sections were stained with methylene blue/azure II acording to Richardson (1960). For electron microscopy, thin sections were contrasted with uranium and lead and observed in a Philips EM 300 or Hitachi HU 12 A electron microscope.
Histochemistry Acid phosphatase was demonstrated by the method of Miller and Palade (1964) with the use of sodium/~-glycerophosphate as substratum and sodium fluororure as inhibitor.
Sampling and Morphometric Evaluation For each experimental condition one gland was taken from two animals in the area between the 3rd and 4th tracheal trunks. On sections perpendicular to the long axis of the gland the relative cytoplasmic and nuclear areas and those occupied by whorls of RER were estimated with a quantimet 720 Imanco on methylene/azure II stained samples. Evaluation of the relative areas occupied by the other cytoplasmic organelles was performed on electron micrographs by means of stereologic point counting procedures (Loud 1968). Since silk-gland cells are of giant size (diameter 40 to 100 ~rn), and since cell polarity is taken into account, three regions were selected in the section through the cell, apical (A), median (M) and basal (B). In each of these three areas, three electron micrographs were taken at a magnification of 3300. The quantitative analysis was then carried out on prints with a final magnification of 10,000.
Results The ultrastructure of the posterior silk gland has been studied in fifth instar larvae starved either in the phase of growth or in the phase of massive fibroin production,
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Fig. 2. Changes in cell area, in normally fed A, starved ~, and refed 9 animals Fig. 3. Changes in nucleo-cytoplasmic ratio in normally fed A, starved A, and refed 9 animals
when growth is almost achieved. These two phases correspond respectively to the periods of obligatory and facultative alimentation defined by the ability of animals to enter nymphal stage (Bounhiol 1938; Legay 1955). In both cases starvation resulted in modifications of the general organization of the cell which is related to its functional polarity and changes of the morphology, size, and spatial distribution of the organelles. Since our approach is basically quantitative we shall first describe the effects of starvation on cell size.
1. Cell Size Growth of the control animals is characterized by an exponential increase of silk-gland weight (Fraisse 1958), length, and diameter (Legay 1949). Cell area measured on sections perpendicular to the long axis of the gland shows a similar growth curve (Fig. 2). The increase of cell size is accompanied by a decrease of the nuclear-cytoplasmic ratio measured on the same sections (Fig. 3). As shown in Fig. 2, starvation resulted in cessation of growth followed by a decrease of cellular size. Early in the instar, when the nuclear-cytoplasmic ratio varies rapidly, starvation elicited an important increase of this ratio, because the nuclear area decreased only slowly compared to that of the cytoplasm.
2. Effects of Starvation During the Phase of Fibroin Production Animals have been deprived of food on the fifth day of the last instar. At that time, growth was not completely achieved, but most of the cell's activity was still oriented toward fibroin production (Prudhomme and Couble 1979). The organization of the secreting cell has been previously described (Akai 1965; Tashiro et al. 1968) and is depicted on Fig. 4.
Fig. 4. Median area of cytoplasm 144 h after first meal (f.m.), control animal; ri ribosomes, go Golgi apparatus, f g fibroin globule, rer rough endoplasmic reticulum, mi mitochondria, mt (arrow) microtubule, x 10,200. Insert: mitochondrion (orthodox form), x 24,000 Fig. 5. Same area 144h after f.m,, 48 h starved animal; wh whorl of rer, ly lysosome, li lipid droplet, go Golgi apparatus, ph autophagosome, arrow transitory vesicles, x 10,200. lnsert: mitochondrion (condensed form), x 27,000 Fig. 6. Portion of transverse section of silk-gland cell, 144 h after f.m., 48 h starved animal. Note whorls of rer (wh), lu gland lumen, nu nuclear lobe. x 1750 Fig. 7. Same area, 144 h after f.m., control animal. Note A apical part, M median part, B basal part of cell. lu gland lumen, nu nuclear lobe. • 1250
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General Organization of the Cell. When growth is achieved, the rough endoplasmic reticulum (RER) and the Golgi apparatus have developped and occupy most of the cytoplasm (Fig. 4). At this time, silk-gland cells display a typical radial striation clearly visible in light microscopy (Fig. 7) which corresponds to the so-called canal system (Sasaki and Tashiro 1976). It consists of radial zones of cytoplasm filled with microtubules, elongated mitochondria, and fibroin globules running between areas of densely packed RER and Golgi complexes (Fig. 4). Starvation altered this organization. The radial striation was no more visible after 48 h of food deprivation (Figs. 5, 6) so that the RER was evenly distributed. Refeeding of animals after a 48 h starvation period for 24h was, however, sufficient to restore the normal morphology of the radial system. In relation to the polarization of the cell, the organelles are distributed differently in its apical and basal parts. Morphometric data showed that this unequal distribution was modified during starvation and recovered in refed animals (Table 1). Rough Endoplasmic Reticulum. A 24 h starvation induced part of the tubulovesicular saccules of the RER to give rise to lamellar profiles. When prolonged, it resulted in the accumulation of concentrically arranged packed membranes, or whorls (Fig. 5). No transitory vesicles can be observed between these membranes and the Golgi complexes. After 24 h starvation, most of the elements of the RER fused to give rise to lamellar profiles (Fig. 11); numerous whorls can be seen. Thus, it may be suggested that the whorls develop by curving and fusion of packed lamellae (Figs. 11, 12). In the course of starvation, whorls increased in relative area (Table 1, Fig. 6). After 96 h starvation the RER completely disappeared from the apical cytoplasm but still persisted in the basal area of the cell, in the form of whorls and saccules. Thus, the relative area occupied by RER decreased dramatically. Refeeding for 24 h following 96 h starvation led to complete "unfolding" of residual whorls and to recovery of the tubulo-vesicular structure of the RER, comparable to the situation in controls. Golgi Apparatus. The Golgi apparatus insuring the intracellular transport and secretion of the protein (Couble et al. 1977) shows complexes of rounded vesicles of variable diameter (Fig. 4) and isolated fibroin globules arising from the complexes. Starvation reduced the relative abundance of these organelles (Table 1) primarily in the apical cytoplasm, (compare Figs. 8, 9) mainly through the rapid elimination of fibroin globules which disappeared completely when starvation was maintained for 96 h. Food deprivation also reduced the number of Golgi complexes per surface unit (Fig. 22) and the diameter of their vesicles (Table 1). Golgi complexes showed transitory vesicles with neighbouring RER saccules up to 48 h of starvation (Fig. 5), but not thereafter. Refeeding animals starved for 48 h resulted in an increase of the relative area of the Golgi apparatus; fibroin globules accumulated again at the apex and their diameter increased (Table 1).
Mitochondria. Mitochondria (Fig. 4, insert) of control animals with narrow intermembrane spaces and thin straight crests in a clear matrix are of the orthodox form according to the classification of Hackenbrock (1966). They are evenly
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Fig. 22. Density changes (number per 1000~t2 of cell area) of Golgi complexes 9 and isolated Golgi vesicles(fibroin globules) e, changes of relative surface of lysosomal apparatus (autophagicvacuoles and lysosomes) o during starvation after the fourth day of the instar
distributed in the cytoplasm (Table 1). In contrast, mitochondria of starved animals had the condensed morphology, characterized by enlarged intermembrane spaces and twisted crests; they accumulated progressively in the apical region of the cytoplasm (Table 1 ; Fig. 5, insert).
Lysosomes and Autophagosomes. After 24 h starvation, secondary lysosomes and autophagosomes which are absent in control animals appeared and increased in relative area (Table 1; Fig. 5). The autophagosomes, formed by paired RER lamellae encircling cytoplasmic areas (Figs. 13, 14) engulfed RER and free ribosomes, but no other organelles. Afterwards these autophagosomes fused with primary or secondary lysosomes (Fig. 15) and their content was degraded giving rise to residual bodies. Acid phosphatase activity was detected in lysosomes and in some of the Golgi complexes (Fig. 15) but never in autophagosomes or whorls. After 96 h starvation, autolytic processes were particularly intense at the cell's apex (Fig. 10). Following 48 h starvation refeeding of the animals for 24 h was sufficient to eliminate all the autophagosomes and lysosomes from the cytoplasm (Table 1).
3. Effect of Starvation in the Cellular Growth Phase Growth of the silk gland extends over the first five days of the fifth instar. The particular feature is the increasing proportion of cytoplasm occupied by RER saccules and by vesicles of the Golgi apparatus (Table 1).
Effect of Early Starvation. When animals remained without food for 24h after ecdysis, no development of the Golgi apparatus and RER was observed. However, whorling of RER lamellae and increase in the number of lysosomes occurred. Similarly, when animals were starved 6 h after the first meal (Fig. 14), transition from the lamellar to the tubular structure of the RER and enlargment of Golgi
Starvation and Silk-glandUltrastructure
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vesicles stopped, whereas a whorling of RER lamellae and an increase of lipid droplets and small lysosomes occurred. Thus food intake is a necessary requirement for the initiation and the continuation of the development of the silk-gland cell.
Effect of Starvation 24 h After the First Meal. Starvation of growing larvae inhibited the development of the RER and the Golgi apparatus (Table 1) and led to similar alterations of their morphology (Fig. 19) as those observed when fibroin producing larvae were deprived of food (decrease of Golgi-vesicle size and formation of RER whorls). Starvation for 48 h elicited also the spreading of autophagosomes and lysosomes and the switch in mitochondrial form. Lipid droplets, visible in controls just after ecdysis but disappearing thereafter, accumulated during starvation (Table 1). Refeeding of animals starved for 48 h resulted in the disappearance of the alterations induced by starvation and in the recovery of the growth of Golgi apparatus and RER. Glycogen generally not visible in control silk glands at any time then appeared in the vicinity of the nuclear lobes (staining of Cardno and Steiner 1965; data not shown). At the apical border, portions of degenerating cytoplasm protruding into the lumen have often been observed (Fig. 21).
Discussion
Experimental starvation results in cytological modifications of the silk-gland cell either during the obligatory or the facultative feeding period of the fifth instar. When applied near the end of the growth phase, starvation stops growth and leads to a reduction of cellular size. At that time the silk-gland cell is well equipped for massive fibroin production so that the effects of starvation on cell organelles are more spectacular than those in the case of early starvation. The cytological changes observed seem to be due to the decrease and cessation of silk synthesis (Prudhomme and Couble 1979). The appearance of RER whorls correlated with a decrease in the amount of ribosomes that progressively loose their ability to form polysomes (Prudhomme and Couble 1979) suggests that whorls are inactive in protein synthesis. Similar compaction of RER in whorls has been described in resting insect secretory cells (Bonnanfant-Jais 1975; Hecker et al. 1977), vertebrate cells (Lazarus 1965; Slot et al. 1979) and plant cells (Dereuddre 1971). In all theses cases whorls can be interpreted as a transitory storage form of RER. The reappearance of vesicular RER in refed animals seems to result from unfolding and not from destruction of the whorl membranes. Fibroin still produced during the first day of starvation is normally secreted and not stored because secretion is blocked. The number of globules decreases with the same kinetics as silk synthesis (Table 2). Lack of storage of synthesized proteins has been observed in a few instances of decreased protein synthesis (Ratcliffe and King 1969; Bonnanfant-Jais 1975). In other cell types, synthesized proteins can be stored at any step of the secretory process: in the RER (Reid 1973), the Golgi apparatus (Weisblum et al. 1962), the secretory granules during transport (Awashi 1972) or at
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Table2. Number of isolated Golgivesiclesand proteosynthetic activity(fromPrudhommeand Couble, 1979)in percentof the number and activityobservedat the time of food removal Hours starvation
Number ofisola~d Golgivesicles
Proteosynthetic activity
0 24 48
100 46 13
100 50 12
the apical part of the cell (Staeubli et al. 1966). In the silk-gland cell the lack of storage during starvation can be interpreted as indicating that the rate of protein synthesis controls the steps in the chain of secretory events (see also Bosquet 1979). However, the mechanism which maintains equilibrium between the organelles involved in the secretory process of silk in starved animals remains to be clarifed. One of the most striking effects of starvation is the induction of autophagocytosis. In natural starvation (Morimoto et al. 1968; Matsuura et al. 1968, 1976) or experimental starvation, autophagosomes formed by RER membranes surround exclusively ribosomes. Such selectiveness in the autophagic process has been reported for other insects tissues (Beaulaton 1967; Rinterknecht et al. 1972; Bonnanfant-Jais 1975; Dean et al. 1978). Biochemical measurements have shown that acid phosphatase is produced all along the 5th instar (Matsuura et al. 1976); however, lysosomes appear only during spinning, when animals have stopped feeding. We have also demonstrated acid phosphatase during starvation. After 24 h starvation the Golgi apparatus seems active, although only few fibroin globules are produced; it seems to become involved primarily in the production of primary lysosomes during starvation. This switch of activity is reversible after refeeding. The effects of 48 h starvation on organelles are reversible. The great need for food or reserves for the high growth rate of the silk gland explains the effects of longer starvation periods. Starvation effects on silk-gland growth, fibroin synthesis, and cell autolysis can be interpreted as mechanisms by which the larva resists starvation. Since silk represents 5 5 - 6 5 ~ of total nitrogen in the well fed larva and only 40~o in the animal starved during the facultative alimentary period (Allegret 1956), cessation of silk production in starving animals saves important nutrients for the organism, and the early autolysis of the silk gland may represent recycling of stored metabotites. This does permit survival; animals can develop after ablation of the silkglands (Amanieu 1954). The ultrastructural modifications reported show a close parallelism with those during cocoon spinning when the animal starves spontaneously. In this latter case, this period corresponds to a peak of ecdysone production (Shaaya and Karlson 1965; Calvez et al. 1976) and the respective ultrastructural changes have been related to hormonal signals (Matsuura et al. 1976). Their presumed role needs further investigation, also in experimentally starved animals. References
Akai H (1965) Studies on the ultrastructure of the silkgland in the silkworm Bombyx mori L. Bull Sericicult Exp St 19:375-384
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Allegret P (1956) Etude des glandes srricigrnes des larves de 16pidoptrres. Leur rble dans la physiologic du drveloppement. Thrse Doctorat Paris Amanieu M (1954) Effets de la section des srriciductes pratiqure sur des vers ~t soie (Bombyx morrO. Compt Rend Soc Biol 148:679 Awashi VB (1972) Studies on the neurosecretory cells of the brain of normal and starved red cotton bugs, Dysdercus Koenigff. Austr Zool 17:24-25 Beaulaton J (1967) Localisation d'activitrs lytiques dans la glande prothoracique du verh soie du chine (Antheraea pernyiguer) au stade prrnymphal. II. Les vacuoles autolytiques (cytolysosomes). J Microsc 6:349-370 Bonnanfant-Jais ML (1975) Morphologie de la glande lactre d'une glossine, Glossina austeni Newst, au cours du cycle de gestation. II. Aspects ultrastructuraux en prriode de repos et au cours des gestations successives J. Microsc Biol Cell 19:295-314 Bosquet G (1979)Jefine et rrgulation du mrtabolisme protrosynth&ique chez les larves de Bombyx mori et Philosamia Cynthia walkeri (Lepidoptrres). Th+se Doctorat Lyon Bounhiol JJ (1938) Recherches exprrimentales sur le drterminisme de la mrtarmorphose chez les lrpidoptrres. Bull Biol F r e t Belg (Thrse) suppl 24, Paris Calvez B, Hirn M, De Reggi M (1976) Ecdysone changes in the haemolymph of the silkworms (Bombyx mori and Phylosamia Cynthia) during larval and pupal development. FEBS Lett 71:57-61 Cardno SS, Steiner JW (1965) Improvement of staining technic for thin sections of epoxy embedding tissue. Am J Clin Pathol 43:1-8 Chavancy G, Fournier A (1979) Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and amino acyl - tRNA synthetase activities in the posterior silk gland of Bombyx rnori L. Biochimie 61: 229-243 Chinzei Y (1975) Induction of autolysis by ecdysterone in vitro: breakdown on anterior silkgland in silkworm Bombyx mori: Applied Ent Zool 10:136-138 Couble P, Prudhomme JC, Daillie J (1977) The biosynthesis of fibroin. III Involvement of the Golgi apparatus in transport and secretion. An electron microscope autoradiographic study. Exp Cell Res 109:139-150 Dean RL (1978) The induction of autophagy in isolated fat body by B-ecdysone. J Insect Physiol 24:439-447 Dereuddre J (1971) Sur la prrsence de groupes de saccules appartenant au rrticulum endoplasmique dans les cellules des 6bauches foliaires en vie ralentie de Betula verucosa ehul. C R Acad Sci Paris 273:2239-2242 Fraisse R (1958) Etude de quelques aspects de ralimentation, de la croissance et de la srcr&ion de la sole chez Bombyx mori L. Variations des caractrres du cocon et du fil de sole. Thrse Doctorat Paris Fukuda S (1944) The hormonal mechanism of larval molting and metamorphosis in the silkworm, Bombyx mori. J Fac Sci Tokyo Imp Univ 6:477-532 Hackenbroek CR (1966) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol 30:345-358 Hecker H (1977) Structure and function of midgut epithelial cells in Culicidae Mosquitoes (Insecta, Diptera). Cell Tissue Res 184:321-341 Kawai N (1976) Hormonal effects on carotenoid uptake by the silkgland in the silkworm, Bombyx mori. J Insect Physiol 22:207-216 Kurata K, Daillie J (1978) Effect of exogenous juvenoid on growth of the silkgland and synthesis of nucleic acids and silkprotein in the silkgland of Bombyx mori. Bull Sericult Exp Stat (Tokyo) 27: 506530 Lazarus S (1965) Ultrastructure and acid phosphatase distribution in the pancreas of rabbit. Arch Pathol 80:135-147 Legay JM (1949) Contribution ~ rrtude biomrtrique de la croissance des glandes srricigrnes chez Bombyx rnori. Revue Ver ~ Soie 1:27-32 Legay JM (1955) La prise de nourriture chez lever ~ soie. Th+se Doctorat Paris Legay JM (1960) Physiologic du ver ~t soie. INRA. Paris Loud AV (1968) A quantitative stereological description of the ultrastructure of normal rat liver parenchymal cell. J Cell Biol 37:37-45 Matsuura S, Morimoto T, Nagata S, Tashiro Y (1968) Studies on the posterior silkgland of silkworm, Bombyx rnori. II Cytolytic processes of the posterior silkgland cells during the metamorphosis from larva to pupa. J Cell Biol 38:589-603
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Matsuura S, Shimadzu T, Tashiro Y (1976) Lysosomes and related structures in the posterior silkgland cells of Bombyx mori. II In prepupal and early pupal stadium. Cell Struct Function 1:223-235 Miller F, Palade GE (1964) Lytic activities in renal protein absorption droplets. An electron microscopical study. J Cell Biol 23:519-552 Morimoto T, Matsuura S, Nagata S, Tashiro Y (1968) Studies on the posterior silkgland of silkworm, Bombyx mori. III Ultrastructural changes of the posterior silkgland cells in the fourth larval instar. J Cell Biol 38:604-614 Prudhomme JC, Couble P (1979) The adaptation of the silkgland cell to the production of fibroin in Bornbyx mori L. Biochimie 61 : 215-227 Ratcliffe RA, King PE (1969) The effect of starvation on the fine structure of the venom system in Nasonia vitripenis. J Insect Physiol 16:885-903 Reid IM (1973) An ultrastructural and morphometric study of the liver of the lactating cow in starvation ketosis. Exp Mol Pathol 18:316-330 Richardson KC, Jarett L, Finke EH (1969) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35: 313-319 Rinterknecht E, Porte A, Joly P (1972) Modifications ultrastructurales des oenocytes sous l'effet du jefine chez Locusta migratoria. C R Acad Sci Paris 275:1063-1066 Sasaki S, Tashiro Y (1976) Studies on the posterior silkgland of the silkworm Bornbyx rnori. VI Distribution of microtubules in the posterior silkgland cells. J Cell Biol 71 : 565-574 Shaaya E, Karlson P (1965) Der Ecdysontiter w/ihrend der Insektentwicklung. IV Die Entwicklung der Lepidopteren Bombyx mori L. und Cerura vinula L. Dev Biol 11:424-432 Slot JN, Strous GJA, Geuze JJ (1979) Effect of fasting and feeding on synthesis and intracellular transport of proteins in the frog exocrine pancreas. J Cell Biol 80:708-715 Staeubli W, Freyvogel TA, Suter J (1966) Structural modifications of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. J Microsc 5:189-204 Tashiro Y, Morimoto T, Matsuura S, Nagata S (1978) Studies on the posterior silkgland of silkworm Bombyx mori. I Growth of the posterior silkgland cells and biosynthesis of fibroin during the fifth larval instar. J Cell Biol 38:574-588 Tashiro Y, Shimadzu T, Matsuura S (1976) Lysosomes and related structures in the posterior silkgland cells of Bombyx mori. I In late larval stadium. Cell Struct Function 1 : 205-222 Weisblum B, Herman L, Fitzgerald PJ (1962) Changes in pancreatic cells during protein deprivation. J Cell Biol 12:313-327
Accepted August 10, 1980
