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Ultrastructure of the posterior silk gland of the ‘Naked pupa’ silkworm, Bombyx mori
J. Insect Physiol., 1972, Vol. 18, pp. 2055 to 2063. Pergamon Press. Printed in Great Britain
ULTRASTRUCTURE OF THE POSTERIOR SILK GLAND OF THE ‘NAKED PUPA’ SILKWORM, BOlMBYX MORI T. IIJIMA Laboratory of Sericulture, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan (Received
18 February
1972)
Abstract-The posterior silk gland of the silkworm, Bombyx mori, produces the silk protein, fibroin. But the silkworms with the hereditary trait ‘Naked pupa (Nd)’ spin the silk thread without fibroin. The ultrastructure of the gland cells of the posterior silk gland of three Nd mutants was examined by electron microscopy. The cytoplasm of the gland cells of these Nd mutants was occupied by the endoplasmic reticulum of which cisternae were remarkably distended, the Golgi complexes consisting of poorly developed flattened saccules and vesicles, and mitochondria with poor cristae. In the gland lumen, cytoplasmic fragments and residues were observed. Two steps of membrane fission-fusion, intracellular transport and reverse pinocytosis, seem to be defective in the posterior silk gland of the Nd silkworm.
INTRODUCTION
SECRETIONis a very widespread
phenomenon
among
many relevant reviews and reports have been published
various
animal
cells,
and
(DALTON, 1961; KUROSUMI, 1961; CAROand PALADE,1964; JAMIESONand PALADE,1968 ; FAVARD,1969). In the silkworm, Bom&yyxmod, it has been well established that the silk gland secretes two kinds of exportable proteins, fibroin and sericin; the former from the posterior silk gland, and the latter from the middle silk gland. The ‘Naked pupa’ (symbolized as Nd) mutant of the silkworm was initially reported by NAKANO (1951). The term ‘naked pupa’ means a pupa without a cocoon. The larva of the Nd silkworm does not spin a cocoon or spins a very poor cocoon. Ninety-nine per cent or more of the silk protein of the thread spun by the Nd silkworm is sericin. Also the posterior silk gland of the Nd silkworm is extremely degenerate. There is, however, little difference in the function and morphology of the middle silk gland. Recently, based on a morphological difference of the silk gland, MACHIDA(1970) subdivided the Nd silkworm into two types; Nd, of which the larva has a strikingly underdeveloped posterior silk gland, and Nd, having a rather well-developed posterior silk gland. A genetical study on another Nd strain, Nd-s, confirmed that the gene responsible for the character was located on the fourteenth chromosome (unpublished data by Horiuchi and Chikushi). The homozygous offspring for the Nd-s gene has an Nd, type silk gland, but larva heterogeneous for the gene has an Nd, type silk gland. 20.55
2056
TADAHIKOIIJIMA
The mechanism by which the secretory function of fibroin in the Nd silk gland is distributed has been studied by numerous authors. Although there are many reports on the Nd silkworm, no clear conclusion on this problem has been made. Ultrastructural observations on the cells of the Nd silk gland may provide not only valuable information concerning the abnormal function of the gland cells, but is also important for understanding the mechanisms of secretion of protein. Thus, the author carried out an electron microscopic study of the posterior silk gland of the Nd silkworm. On the basis of the observations, a possible relationship between the defect in the fission-fusion process of intracellular membranes and that of the secretory functions of the Nd silk gland is discussed. MATERIALS
AND
METHODS
The strains of silkworm used in this study are b 40 (Nd,), b 41 (Nd,), b 50 (Nd-s), and p 22 (normal). The larvae were reared at 24°C and matured within 140 hr after the fourth ecdysis. The larvae were sacrificed at the ninetieth hour after the fourth ecdysis. The posterior silk glands were fixed with 625% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.3. They were then postfixed with 1% osmium tetraoxide in 0.14 M Verona1 acetate buffer at pH 7.4. Tissues were dehydrated with a graded concentration of ethanol and embedded in Epon 812. Sections were stained with both uranyl acetate and lead citrate. OBSERVATIONS
In the normal silk gland, it is considered that fibroin in the cisternae of roughsurfaced endoplasmic reticulum (ER) migrates into the secretory granules through the Golgi complexes and that the granules are finally secreted into the gland lumen by reverse pinocytosis (AIM, 1963; TASHIRO et al., 1968). The cytoplasm of the normal silk gland is filled with well-developed ER, Golgi complexes, mitochondria, and free ribosomes (Fig. 1). The ER are mostly vesicular or of the tubular type of which the intracisternal spaces are moderately distended. The main component of the Golgi complex is a vacuole, ranging in diameter from O-3 to O-5 CL. The Golgi vacuole may change into a secretory granule as a result of the condensation of a substance which is the same as in the pancreatic exocrine cells (DALTON, 1961; CARO and PALADE, 1964). The mitochondria have well-developed cristae. The apical cytoplasm and gland lumen of the normal silk gland are represented in Figs. 2 and 3. A number of secretory granules containing filamentous materials are observed in the apical cytoplasm, and similar fibrils are extruded in the lumen. It is very likely that fibroin is secreted by a reverse pinocytosis, for the mass of extruded fibrils is relatively homogeneous and it contains no debris or residue of membranes. There was little difference in the ultrastructure of the gland cells between the three Nd mutants. As shown in Figs. 4 to 6, the cytoplasm of the Nd silk gland is occupied largely by the ER in most of which the cisternae are remarkably distended. The distended cisternae of the ER are filled with filamentous materials similar to fibroin fibrils in the secretory granules which were observed in the normal silk
FIG. 1. A portion of the cytoplasm (G), and mitochondria
of normal silk gland. The ER, Golgi complexes (M) are well developed. Scale = 1 CL.
2058
FIGS. 2 and 3. Apical portion of the cytoplasm and gland lumen of normal silk gland. A number of secretory granules (SG) can be seen in the apical cytoplasm. These granules contain filamentous materials, and similar fibrils (F) are observed in the gland lumen. A mass of extruded fibrils is homogeneous and contains no debris or residue of the membranes. G, Golgi complex; M, mitochondrion. Scale = 1 I*.
2059
FIGS. 4 to 6. A portion of cytoplasm of Nd silk gland. The cisternae of the ER are remarkably distended forming vacuoles and are filled with filamentous materials. A large vacuole containing membranes (arrows) which divide the vacuole into several cells can often be seen in the cytoplasm (Fig. 6). The Golgi complexes (G) and mitochondria (M) are underdeveloped. Scale = 1 /.L.
2060
FIGS. 7 to 9. An apical portion of the cytoplasm and gland lumen of Nd silk gland. The secretory granules cannot be seen in the apical cytoplasm. The cytoplasmic fragment (CF), distended ER, and cytoplasmic residue or debris (D) in the gland lumen are visible. An arrow indicates direct continuity between the cytoplasmic fragment and the microvilli. Scale = 1 p_
ULTRASTRUCTURE
OF THE
SILK
GLAND
OF
Nd
SILKWORM
2061
gland. The cisternae of the ER distend forming vacuoles, ranging in diameter from 1 to 10 CL,and these vacuoles appear to coalesce to form the larger vacuoles, ranging in diameter from 20 to 30 p. There can be seen in Fig. 6 such a large vacuole containing membranes which divide the vacuole into several cells. The Golgi complexes consist of poorly developed flattened saccules and vesicles. The large round vacuole which is a major component of the organelle in the normal silk gland cannot be seen. The mitochondria are also underdeveloped; they are small in size and have poorly developed cristae. The apical cytoplasm and gland lumen of the Nd silk gland are shown in Figs. 7 to 9. The secretory granules in the apical cytoplasm are absent. In the gland lumen, cytoplasmic fragments and residues of membranes are observed. Occasionally one observes a direct continuity between the fragment of cytoplasm and the microvilli, indicating that the microapocrine secretion (KUROSUMI,1961) participates to some extent in the extrusion of protein in the Nd silk gland. DISCUSSION It has not been established whether fibroin biosynthesis takes place in the Nd silk gland or not. MACHIDA(1970) h as observed fibroin-like masses in the cytoplasm of the Nd silk gland under a light microscope, and has also detected an intense incorporation of 14C glycine, which is a major amino acid of fibroin, in the Nd silk gland (private communication). MUKAI et al. (1962) have studied an elution pattern of RNA’s prepared by the phenol method from the posterior silk gland on a fibroin powder column, and a clear difference was observed between the RNA’s from normal and those from the Nd silkworm. This may indicate that a template RNA for the fibroin of the Nd mutant differs from that of normal fibroin. In other words, the true fibroin may not be synthesized in the Nd silk gland. Recently, Gamo (private communication) demonstrated that a fibroin fraction of Nd mutant differed in mobility on polyacrylamide-gel electrophoresis from the fibroin of normal larva. Although there is the question whether the protein is a true fibroin or not, it at least seems certain that the fibroin-like protein is synthesized in the Nd silk gland. And it seems also safe to describe the filamentous materials observed in both the dilated cisternae of the ER and the vacuoles in the cytoplasm of the Nd silk gland as a fibroin-like protein. It has been emphasized that in the pancreatic exocrine cells of the guinea pig the mitochondria supply the energy for the intracellular transport of secretory proteins from the cisternae of the ER to the Golgi complexes (JAMIESONand PALADE,1968). A similar suggestion was also made for the silk gland of Bombyx larva (IIJIMA, 1971a, b). In the Nd silk gland, the Golgi complexes and mitochondria are poorly developed. The Golgi complexes consist of flattened saccules and vesicles. When a Bombyx larva is starved for a few days, the Golgi complexes of the silk gland cells show a similar feature (unpublished data). Consequently, such a feature of the Golgi complexes seems to suggest an inferior activity of the organelle. The mitochondria of the Nd silk gland are small in size and have few cristae. HILL and
2062
TADAHIKOIIJIMA
ANDERSON (1969)
h ave reported that a loss of mitochondrial cristae in acriflavinetreated Crithidia fasciculata was accompanied with a decrease of mitochondrial enzyme activity. A distinct decrease of mitochondrial cristae in the Nd silk gland may indicate a decreased function of respiration in the gland cells. Thus, a remarkable dilatation of ER cisternae accompanied by an underdevelopment of both the Golgi complexes and the mitochondria in the Nd silk gland may indicate that the intracellular transport is defective in the gland cells. The intracellular transport of secretory protein involves membrane fissionfusion, that is, budding of the Golgi vesicles from the membranes of the ER and fusing of these vesicles into the Golgi vacuoles. Reverse pinocytosis is another step involved in membrane fission-fusion. In the lumen of the Nd silk gland there are many cytoplasmic fragments which associate directly with the microvilli. These fragments contain ER which is dilated with filamentous materials. It is very likely, therefore, that the microapocrine secretion participates in the secretory cycle of the Nd silk gland. This participation of apocrine secretion has been suggested to occur in silk gland cells during oxygen deficiency (IIJIMA, 1971a). It seems likely that apocrine secretion is a more efficient mode for the extrusion of secretory products than reverse pinocytosis in respect of the membrane fissionfusion or of the energy requirements. The present results indicate that this defect of the membrane fission-fusion steps may be one of the decisive factors for the abnormal secretory cycle in the Nd silk gland. Acknowledgement-The author wishes to express his deep obligation to Professor H. CHIKUSHIof Kyushu University for many helpful suggestions throughout the course of the present work. REFERENCES AKAI H. (1963) Electron microscopical observation on the secretion of fibroin in the silk gland of the silkworm, Bombyx mori. J. Sericult. Sci. Japan 32, 22-24. CARO L. G. and PALADE G. E. (1964) Protein synthesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20, 473-495. DALTONS. J. (1961) Golgi apparatus and secretion granule. In The Cell (Ed. by BRACHETJ. and MIRSKY A. E.) 2, 603-619. Academic Press, New York. FAVARDP. (1969) The Golgi apparatus. In Handbook of Molecular Cytology (Ed. by LIMADE-FARIA A.) 15, 1131-1155. North-Holland, Amsterdam. HILL G. C. and ANDERSONW. A. (1969) Effect of acriflavine on the mitochondria and kinetoplast of Crithidia fasciczdata. Correlation of fine structure changes with decreased mitochondrial enzyme activity. J. Cell Biol. 41, 547-561. IIJIMA T. (1971a) Cytological changes of the fine structure of the silk gland in Bombyx larva under the in vitro condition. J. Sericult. Sci. Japan 40, 181-191 IIJIMA T. (1971b) Ultrastructures of silk gland cells in the silkworm, Bombyx mori L., injected with actinomycin D. J. Sericult. Sci.Japan 40, 357-367. JAMIESONJ. D. and PALADEG. E. (1968) Intracellular transport of secretory proteins in the pancreatic exocrine cell-IV. Metabolic requirements. J. Cell Biol. 39, 589-603. KUROSUMIK. (1961) Electron microscopic analysis of the secretion mechanism. Znt. Rev. Cytol. 11, l-124.
ULTRASTRUCTURE OF THE SILK GLAND
OF
Nd SILKWORM
2063
MACHIDA Y. (1970) Studies on the silkglands of silkworm Bombyx mori L.-II. The singularity of the silkglands in hereditary trait, Naked pupa (Nd) in the silkworm-l. Bull. Fukuoka Women’sJunior College 3, 1-21. In Japanese. MUKAI J. K., KOMATSU K., and AKUNE S. (1962) Studies on the isolation of a specific ribonucleic acid-II. Chromatographic interaction between silkgland RNA and a fibroin powder column. Sci. Bull. Fat. Agri. Kyushu Univ. 19, 273-278. In Japanese. NAKANOY. (1951) Physiological, anatomical and genetical studies on the ‘Naked’ silkworm pupa. ‘j. Sericult. Sci. Japan 20, 232-248. In Japanese. TA~HIRO Y., MORIMOTO T., MATSUURAS., and NAGATAS. (1968) Studies on the posterior Growth of posterior silk gland cells and silk gland of the silkworm, Bombyx mori-I. biosynthesis of fibroin during the fifth larval instar. J. Cell Biol. 38, 574-588.
ULTRASTRUCTURE OF THE POSTERIOR SILK GLAND OF THE ‘NAKED PUPA’ SILKWORM, BOlMBYX MORI T. IIJIMA Laboratory of Sericulture, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan (Received
18 February
1972)
Abstract-The posterior silk gland of the silkworm, Bombyx mori, produces the silk protein, fibroin. But the silkworms with the hereditary trait ‘Naked pupa (Nd)’ spin the silk thread without fibroin. The ultrastructure of the gland cells of the posterior silk gland of three Nd mutants was examined by electron microscopy. The cytoplasm of the gland cells of these Nd mutants was occupied by the endoplasmic reticulum of which cisternae were remarkably distended, the Golgi complexes consisting of poorly developed flattened saccules and vesicles, and mitochondria with poor cristae. In the gland lumen, cytoplasmic fragments and residues were observed. Two steps of membrane fission-fusion, intracellular transport and reverse pinocytosis, seem to be defective in the posterior silk gland of the Nd silkworm.
INTRODUCTION
SECRETIONis a very widespread
phenomenon
among
many relevant reviews and reports have been published
various
animal
cells,
and
(DALTON, 1961; KUROSUMI, 1961; CAROand PALADE,1964; JAMIESONand PALADE,1968 ; FAVARD,1969). In the silkworm, Bom&yyxmod, it has been well established that the silk gland secretes two kinds of exportable proteins, fibroin and sericin; the former from the posterior silk gland, and the latter from the middle silk gland. The ‘Naked pupa’ (symbolized as Nd) mutant of the silkworm was initially reported by NAKANO (1951). The term ‘naked pupa’ means a pupa without a cocoon. The larva of the Nd silkworm does not spin a cocoon or spins a very poor cocoon. Ninety-nine per cent or more of the silk protein of the thread spun by the Nd silkworm is sericin. Also the posterior silk gland of the Nd silkworm is extremely degenerate. There is, however, little difference in the function and morphology of the middle silk gland. Recently, based on a morphological difference of the silk gland, MACHIDA(1970) subdivided the Nd silkworm into two types; Nd, of which the larva has a strikingly underdeveloped posterior silk gland, and Nd, having a rather well-developed posterior silk gland. A genetical study on another Nd strain, Nd-s, confirmed that the gene responsible for the character was located on the fourteenth chromosome (unpublished data by Horiuchi and Chikushi). The homozygous offspring for the Nd-s gene has an Nd, type silk gland, but larva heterogeneous for the gene has an Nd, type silk gland. 20.55
2056
TADAHIKOIIJIMA
The mechanism by which the secretory function of fibroin in the Nd silk gland is distributed has been studied by numerous authors. Although there are many reports on the Nd silkworm, no clear conclusion on this problem has been made. Ultrastructural observations on the cells of the Nd silk gland may provide not only valuable information concerning the abnormal function of the gland cells, but is also important for understanding the mechanisms of secretion of protein. Thus, the author carried out an electron microscopic study of the posterior silk gland of the Nd silkworm. On the basis of the observations, a possible relationship between the defect in the fission-fusion process of intracellular membranes and that of the secretory functions of the Nd silk gland is discussed. MATERIALS
AND
METHODS
The strains of silkworm used in this study are b 40 (Nd,), b 41 (Nd,), b 50 (Nd-s), and p 22 (normal). The larvae were reared at 24°C and matured within 140 hr after the fourth ecdysis. The larvae were sacrificed at the ninetieth hour after the fourth ecdysis. The posterior silk glands were fixed with 625% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.3. They were then postfixed with 1% osmium tetraoxide in 0.14 M Verona1 acetate buffer at pH 7.4. Tissues were dehydrated with a graded concentration of ethanol and embedded in Epon 812. Sections were stained with both uranyl acetate and lead citrate. OBSERVATIONS
In the normal silk gland, it is considered that fibroin in the cisternae of roughsurfaced endoplasmic reticulum (ER) migrates into the secretory granules through the Golgi complexes and that the granules are finally secreted into the gland lumen by reverse pinocytosis (AIM, 1963; TASHIRO et al., 1968). The cytoplasm of the normal silk gland is filled with well-developed ER, Golgi complexes, mitochondria, and free ribosomes (Fig. 1). The ER are mostly vesicular or of the tubular type of which the intracisternal spaces are moderately distended. The main component of the Golgi complex is a vacuole, ranging in diameter from O-3 to O-5 CL. The Golgi vacuole may change into a secretory granule as a result of the condensation of a substance which is the same as in the pancreatic exocrine cells (DALTON, 1961; CARO and PALADE, 1964). The mitochondria have well-developed cristae. The apical cytoplasm and gland lumen of the normal silk gland are represented in Figs. 2 and 3. A number of secretory granules containing filamentous materials are observed in the apical cytoplasm, and similar fibrils are extruded in the lumen. It is very likely that fibroin is secreted by a reverse pinocytosis, for the mass of extruded fibrils is relatively homogeneous and it contains no debris or residue of membranes. There was little difference in the ultrastructure of the gland cells between the three Nd mutants. As shown in Figs. 4 to 6, the cytoplasm of the Nd silk gland is occupied largely by the ER in most of which the cisternae are remarkably distended. The distended cisternae of the ER are filled with filamentous materials similar to fibroin fibrils in the secretory granules which were observed in the normal silk
FIG. 1. A portion of the cytoplasm (G), and mitochondria
of normal silk gland. The ER, Golgi complexes (M) are well developed. Scale = 1 CL.
2058
FIGS. 2 and 3. Apical portion of the cytoplasm and gland lumen of normal silk gland. A number of secretory granules (SG) can be seen in the apical cytoplasm. These granules contain filamentous materials, and similar fibrils (F) are observed in the gland lumen. A mass of extruded fibrils is homogeneous and contains no debris or residue of the membranes. G, Golgi complex; M, mitochondrion. Scale = 1 I*.
2059
FIGS. 4 to 6. A portion of cytoplasm of Nd silk gland. The cisternae of the ER are remarkably distended forming vacuoles and are filled with filamentous materials. A large vacuole containing membranes (arrows) which divide the vacuole into several cells can often be seen in the cytoplasm (Fig. 6). The Golgi complexes (G) and mitochondria (M) are underdeveloped. Scale = 1 /.L.
2060
FIGS. 7 to 9. An apical portion of the cytoplasm and gland lumen of Nd silk gland. The secretory granules cannot be seen in the apical cytoplasm. The cytoplasmic fragment (CF), distended ER, and cytoplasmic residue or debris (D) in the gland lumen are visible. An arrow indicates direct continuity between the cytoplasmic fragment and the microvilli. Scale = 1 p_
ULTRASTRUCTURE
OF THE
SILK
GLAND
OF
Nd
SILKWORM
2061
gland. The cisternae of the ER distend forming vacuoles, ranging in diameter from 1 to 10 CL,and these vacuoles appear to coalesce to form the larger vacuoles, ranging in diameter from 20 to 30 p. There can be seen in Fig. 6 such a large vacuole containing membranes which divide the vacuole into several cells. The Golgi complexes consist of poorly developed flattened saccules and vesicles. The large round vacuole which is a major component of the organelle in the normal silk gland cannot be seen. The mitochondria are also underdeveloped; they are small in size and have poorly developed cristae. The apical cytoplasm and gland lumen of the Nd silk gland are shown in Figs. 7 to 9. The secretory granules in the apical cytoplasm are absent. In the gland lumen, cytoplasmic fragments and residues of membranes are observed. Occasionally one observes a direct continuity between the fragment of cytoplasm and the microvilli, indicating that the microapocrine secretion (KUROSUMI,1961) participates to some extent in the extrusion of protein in the Nd silk gland. DISCUSSION It has not been established whether fibroin biosynthesis takes place in the Nd silk gland or not. MACHIDA(1970) h as observed fibroin-like masses in the cytoplasm of the Nd silk gland under a light microscope, and has also detected an intense incorporation of 14C glycine, which is a major amino acid of fibroin, in the Nd silk gland (private communication). MUKAI et al. (1962) have studied an elution pattern of RNA’s prepared by the phenol method from the posterior silk gland on a fibroin powder column, and a clear difference was observed between the RNA’s from normal and those from the Nd silkworm. This may indicate that a template RNA for the fibroin of the Nd mutant differs from that of normal fibroin. In other words, the true fibroin may not be synthesized in the Nd silk gland. Recently, Gamo (private communication) demonstrated that a fibroin fraction of Nd mutant differed in mobility on polyacrylamide-gel electrophoresis from the fibroin of normal larva. Although there is the question whether the protein is a true fibroin or not, it at least seems certain that the fibroin-like protein is synthesized in the Nd silk gland. And it seems also safe to describe the filamentous materials observed in both the dilated cisternae of the ER and the vacuoles in the cytoplasm of the Nd silk gland as a fibroin-like protein. It has been emphasized that in the pancreatic exocrine cells of the guinea pig the mitochondria supply the energy for the intracellular transport of secretory proteins from the cisternae of the ER to the Golgi complexes (JAMIESONand PALADE,1968). A similar suggestion was also made for the silk gland of Bombyx larva (IIJIMA, 1971a, b). In the Nd silk gland, the Golgi complexes and mitochondria are poorly developed. The Golgi complexes consist of flattened saccules and vesicles. When a Bombyx larva is starved for a few days, the Golgi complexes of the silk gland cells show a similar feature (unpublished data). Consequently, such a feature of the Golgi complexes seems to suggest an inferior activity of the organelle. The mitochondria of the Nd silk gland are small in size and have few cristae. HILL and
2062
TADAHIKOIIJIMA
ANDERSON (1969)
h ave reported that a loss of mitochondrial cristae in acriflavinetreated Crithidia fasciculata was accompanied with a decrease of mitochondrial enzyme activity. A distinct decrease of mitochondrial cristae in the Nd silk gland may indicate a decreased function of respiration in the gland cells. Thus, a remarkable dilatation of ER cisternae accompanied by an underdevelopment of both the Golgi complexes and the mitochondria in the Nd silk gland may indicate that the intracellular transport is defective in the gland cells. The intracellular transport of secretory protein involves membrane fissionfusion, that is, budding of the Golgi vesicles from the membranes of the ER and fusing of these vesicles into the Golgi vacuoles. Reverse pinocytosis is another step involved in membrane fission-fusion. In the lumen of the Nd silk gland there are many cytoplasmic fragments which associate directly with the microvilli. These fragments contain ER which is dilated with filamentous materials. It is very likely, therefore, that the microapocrine secretion participates in the secretory cycle of the Nd silk gland. This participation of apocrine secretion has been suggested to occur in silk gland cells during oxygen deficiency (IIJIMA, 1971a). It seems likely that apocrine secretion is a more efficient mode for the extrusion of secretory products than reverse pinocytosis in respect of the membrane fissionfusion or of the energy requirements. The present results indicate that this defect of the membrane fission-fusion steps may be one of the decisive factors for the abnormal secretory cycle in the Nd silk gland. Acknowledgement-The author wishes to express his deep obligation to Professor H. CHIKUSHIof Kyushu University for many helpful suggestions throughout the course of the present work. REFERENCES AKAI H. (1963) Electron microscopical observation on the secretion of fibroin in the silk gland of the silkworm, Bombyx mori. J. Sericult. Sci. Japan 32, 22-24. CARO L. G. and PALADE G. E. (1964) Protein synthesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20, 473-495. DALTONS. J. (1961) Golgi apparatus and secretion granule. In The Cell (Ed. by BRACHETJ. and MIRSKY A. E.) 2, 603-619. Academic Press, New York. FAVARDP. (1969) The Golgi apparatus. In Handbook of Molecular Cytology (Ed. by LIMADE-FARIA A.) 15, 1131-1155. North-Holland, Amsterdam. HILL G. C. and ANDERSONW. A. (1969) Effect of acriflavine on the mitochondria and kinetoplast of Crithidia fasciczdata. Correlation of fine structure changes with decreased mitochondrial enzyme activity. J. Cell Biol. 41, 547-561. IIJIMA T. (1971a) Cytological changes of the fine structure of the silk gland in Bombyx larva under the in vitro condition. J. Sericult. Sci. Japan 40, 181-191 IIJIMA T. (1971b) Ultrastructures of silk gland cells in the silkworm, Bombyx mori L., injected with actinomycin D. J. Sericult. Sci.Japan 40, 357-367. JAMIESONJ. D. and PALADEG. E. (1968) Intracellular transport of secretory proteins in the pancreatic exocrine cell-IV. Metabolic requirements. J. Cell Biol. 39, 589-603. KUROSUMIK. (1961) Electron microscopic analysis of the secretion mechanism. Znt. Rev. Cytol. 11, l-124.
ULTRASTRUCTURE OF THE SILK GLAND
OF
Nd SILKWORM
2063
MACHIDA Y. (1970) Studies on the silkglands of silkworm Bombyx mori L.-II. The singularity of the silkglands in hereditary trait, Naked pupa (Nd) in the silkworm-l. Bull. Fukuoka Women’sJunior College 3, 1-21. In Japanese. MUKAI J. K., KOMATSU K., and AKUNE S. (1962) Studies on the isolation of a specific ribonucleic acid-II. Chromatographic interaction between silkgland RNA and a fibroin powder column. Sci. Bull. Fat. Agri. Kyushu Univ. 19, 273-278. In Japanese. NAKANOY. (1951) Physiological, anatomical and genetical studies on the ‘Naked’ silkworm pupa. ‘j. Sericult. Sci. Japan 20, 232-248. In Japanese. TA~HIRO Y., MORIMOTO T., MATSUURAS., and NAGATAS. (1968) Studies on the posterior Growth of posterior silk gland cells and silk gland of the silkworm, Bombyx mori-I. biosynthesis of fibroin during the fifth larval instar. J. Cell Biol. 38, 574-588.