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Protein synthesis and secretion by the Malpighian tubules of cercopoid larvae (Homoptera)
J. Insect Physiol,, 1973, Vol. 19, pp. 2317 to 2326. Pergamon Press. Printed in Great Britain
PROTEIN SYNTHESIS AND SECRETION BY THE MALPIGHIAN TUBULES OF CERCOPOID LARVAE (HOMOPTERA) A. T. MARSHALL Department of Zoology, La Trobe University, Melbourne, Victoria 3083, Australia (Received 3 April 1973) Abstract-The Malpighian tubules of cercopoid larvae are anatomically differentiated. The cells of the distal segments have an ultrastructure which is typical of cells synthesizing and exporting protein. They give intense positive reactions with cytochemical tests for protein and contain large quantities of RNA. Secretory activity is not synchronized in different cells. The protein secretion forms a component of machaerotid dwelling-tubes and cercopid foti but may also function as an excretory product. INTRODUCTION
THE MALPIGHIANtubules of insects are primarily concerned with the excretion of the waste products of metabolism and the regulation of ion and water balance. However, in a variety of insects they appear to carry out accessory functions which are quite unrelated to excretion or osmoregulation (WIGGLESWORTH, 1965). This is particularly true of cercopoid larvae. MARSHALL (1964a, b, 1965, 1966a, b, 1968) has shown that the proximal segment of the Malpighian tubules of cercopoid larvae is concerned with the synthesis and secretion of mucopolysaccharides. These are associated with the peculiar modes of larval life. The distal segment of the Malpighian tubules has not been previously examined and it will be shown that it is highly specialized for the synthesis and secretion of protein. MATERIALS
AND METHODS
The insects examined included the machaerotids Machaerota coronata Maa, Chaetophyes compacta Walk., Pectinariophyes stalii Spongberg, and the cercopids Cosmoscarta abdominalis Don. and Philaenus spumarius L. For electron microscopy larvae were dissected and the Malpighian tubules fixed in 1% osmium tetroxide in Verona1 acetate buffer pH 7.3 or in 2.5% glutaraldehyde in phsophate buffer pH 7.2 and post osmicated in 1% osmium tetroxide in phosphate buffer. Th e material was embedded in Araldite. Sections were stained in uranyl acetate and lead citrate. For cytochemical tests Malpighian tubules were fixed in formaldehyde-calcium, dehydrated in tetrahydrofuran (SALTHOUSE, 1958), embedded in 55°C paraffin wax, 2317
A. T. MARSHALL
2318
and sectioned at 7 pm. Cytochemical tests included Hg-bromophenol blue, acid solochrome cyanine, Millon’s reaction, and ribonuclease extractions (PEARSE,1960). For electrophoresis, urine was collected from 13 dwelling-tubes of M. cormata, pooled, centrifuged, and the supernatant retained. The distal segments of the Malpighian tubules of 12 larvae were dissected out in insect saline, homogenized in 0.1 ml of distilled water, centrifuged, and the supernatant retained. Electrophoresis was carried out on 10 cm strips of buffered cellulose acetate paper. Samples of 5 ~1 were applied to the papers as narrow bands. For each buffer a current of 2 n&/2*5 cm width of strip was applied. After drying at 105°C the strips were stained overnight in 0.002% nigrosin (SMITH, 1960). The buffer solutions used were: (1) 0.04 M sodium diethylbarbiturate-HCl, pH 8.6, (2) 0.04 M sodium diethylbarbiturate-sodium acetate-HCl, pH 5.0, (3) O-1 M Na,HPO,-NaHsPO,, pH 7.0, and (4) 0.05 M borax-NaOH, pH 10.5. OBSERVATIONS
The gross anatomical appearance of the Malpighian tubules is shown in Fig. 1. In fresh dissections and in sections the cells are seen to contain a branching system of intracellular canaliculi continuous with the tubule lumen. This is schematically shown in Fig. 2.
DS
FIG. 1. Diagram showing gut and Malpighian tubules. PS, Proximal segment; DS, distal segment; FC, filter chamber; R, rectum.
Ultrastructure The cells of the Malpighian tubule distal segments of all the species examined are very similar in fine structure. They are characterized by the presence of large numbers of membrane bounded granules ranging from 0.5 to 2-O pm in diameter. These differ in electron density and homogeneity in the different species (Figs. 3,6, 7). The rough endoplasmic reticulum (RER) is extensively developed and its appearance varies with the granule content of the cells. Where the granule population is relatively small the RER is developed into enormous whorls of concentric
THE
MALPIGHIAN
TUBULES
OF CERCOPOJD
LARVAE
2319
FIG. 2. Diagram of distal segment cells of Malpighian tubule in longitudinal se&on. N, Nucleus; C, intracellular canaliculi; L, lumen.
cisternae (Fig. 7). In cells with high populations of granules the RER is compressed between the granules (Fig. 6). Material of low and high electron density is seen within the cisternae of the RER (Figs. 5, 6). Numerous Golgi bodies occur within the cells. Transition vesicles are present at the forming face and electron dense material appears to be condensing at the maturing face (Figs. 4,5). The cells are penetrated by an extensively arborized system of canaliculi which are lined with microvilii (Figs. 3, 6). Extrusion of granules into the canaliculi appears to occur without fusion of the bounding granule membrane with the plasma membrane. The granule contents then appear to go into solution leaving profiles of membrane within the lumina of the canaliculi (Fig. 8). In adult cercopoids no granules appear in the distal segment cells. The cytoplasm is packed with mitochondria and the canaliculi are lined with microvilli which are considerably longer than in the larvae. Cytochemistry The distal segment cells stain very intensely with Hg-bromophenol blue and They also give an intense positive reaction with acid solochrome cyanine. Millon’s reagent. These results suggest the presence of large amounts of protein. Sections incubated in ribonuclease exhibit a considerable decrease in basophilia compared with control sections, indicating the presence of large amounts of cytoplasmic ribosenucleic acid (RNA). Electrophoresis Only urine was run with sodium diethylbarbiturate-HCl buffer at pH 8.6. After a 3 hr run no movement from the origin was observed. AlI other results are shown in Fig. 9. There appear to be two proteins in urine which have their isoelectric points around pH 8.6. Three proteins are present in the water extracts
A. T. MARSHALL
2320
”
I
-
-_-+ --
~H5.0
QH7.Q
1.45hr Q
6.0hr
0
ziim A
FIG. 9. Tracings
of electrophoretic patterns from Malpighian tubule segment extract (D) and urine (A) of M. coronata larvae.
distal
from the distal segments. One of these appears to be electrophoretically identical to one of the urine proteins. The pH of il4. coronata urine is pH 8.2 and that of C. compacta is pH 8.1. DISCUSSION
The fine structure of the distal segment cells of the Malpighian tubules of cercopoid larvae is typical of cells which synthesize and export proteins. Cell features such as secretion granules, extensive rough endoplasmic reticulum, and numerous Golgi bodies are all characteristic of protein secreting cells as demonstrated in the classical studies by CARO and PALADE(1964) and JAMIESONand PALADE(1967a, b). Cytochemically, the distal segment cells are rich in protein and cytoplasmic RNA and there can be no doubt that they synthesize and secrete protein in the form of granules. The ultrastructure of the cells suggests that as in pancreas, protein is synthesized in the cisternae of the RRR and packaged into granules in the Golgi bodies. The granules appear to be extruded into the intracellular canaliculi complete with their bounding membranes. Here they seem to go into solution. This may be facilitated by the osmoregulatory flow of ions and water into the tubule lumen which is believed to occur in addition to secretory activity (MARSHALL and CHEUNG,in press). The secretory cycle has not so far been investigated in detail but preliminary studies indicate that secretory activity is not synchronized in different cells in the same Malpighian tubule. There must therefore be a continuous release of small quantities of protein from each of the Malpighian tubules. The function of this protein secretion is not fully understood. Machaerotid larvae build and live within dwelling-tubes (MARSHALL and MARSHALL, 1966) and protein is a component of these tubes (MARSHALL, 1968). The electrophoresis results suggest that there is at least one protein in the urine filling the dwelling-tubes which originates in the distal segments of the Malpighian tubules. As the urine evaporates this protein will be rapidly precipitated,
2321
FIG. 3. Distal segment cell of M. coronata. P, Protein granules; G, Golgi bodies; C, canaliculi ; ER, endoplasmic reticulum.
2322
FIGS. 4 and 5. Golgi bodies in distal segment cells of M. corona&. T, Transition vacuoles ; C, condensing vacuoles ; arrows, dense material jn endoplasmic reticulum cisternae.
2323
FIG. 6. Distal segment cells of C. abdominalis. Arrows, Material within cisternae of endoplasmic reticulum. FIG. 7. Distal segment cells of C. compacta showing whorls of rough endoplasmic reticulum. FIG. 8. Distal segment lumen of C. abdominalis containing membrane profiles and dissolving granules.
THE MALPIGHIANTUBULESOF CERCOPOID LARVAE
2325
since its isoelectric point occurs at about the pH of the urine, and incorporated into the dwelling-tube as matrix protein. Cercopoid larvae live within a foam or spittle mass. Protein has been detected in this by ZIEGLER and ZIEGLER(1958) and MARSHALL(1966a). Protein in foam may contribute to bubble stability by bonding to mucopolysaccharide (MARSHALL, 1966a). Protein synthesis and secretion is certainly a function of the larval Malpighian tubules and ceases in both machaerotid and cercopid adults. It therefore seems likely that it is related to the peculiar modes of larval life. However, it must be borne in mind that the feeding site of the adults appears to be different to that of the larvae (HAGLEY, 1965; Marshall, unpublished) and it is possible that the protein functions as an excretory product in the larval stage but not in the adult. SMITH and LITTAU (1960) have suggested that the secretion of protein in the form of brochosomes from the Malpighian tubules of cicadellids is a possible excretory mechanism. Similarly it has been suggested by SPIEGLER(1962) that the silk and adhesive secretion of the Malpighian tubules of Ch~ysopa larvae are products of nitrogenous excretion. However, the silk does not appear to be a protein but a form of nylon (RUDALL and KFZNCHINGTON, 1971). Silks are also secreted by the Malpighian tubules of several coleoptera larvae (e.g. SILVESTRI,1904) but the nature of the silks does not appear to have been investigated. Acknowledgements-I wish to record my thanks to Dr. D. F. WATERHOUSE and Dr. B. FILSHIE for allowing me to use the electron microscopy facilities in the Division of Entomology, C.S.I.R.O., Canberra, where part of this work was carried out. Part of this study was also done in the Zoology Departments of the Universities of Hong Kong and Hull, REFERENCES CARO L. G. and PALADE G. E. (1964) Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic study. y. Cell Biol. 20, 473-495. HAGLEY E. A. C. (196.5) Site of feeding of the froghopper. Rep. Tate and Lyle cent. agric. Res. Stn TGaidad pp. 408-413. JAMIESONJ. D. and PALADEG. E. (1967a) Intracellular transport of secretory proteins in the pancreatic exocrine cell-I. Role of the peripheral elements of the Golgi complex. y. Cell Biol. 34, 577-596. JAMIESONJ. D. and PALADEG. E. (1967b) Intracellular transport of secretory proteins in the pancreatic exocrine cell-II. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34, 597-615. MARSHALL A. T. (1964a) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-1. The cytology of the Malpighian tubules of spittle-bug nymphs. Quart. J. micr. Sci. 105, 257-262. MARSHALL A. T. (1964b) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-2. The cytology and function of the granule zone of the Malpighian tubules of tube-building nymphs. Quart. J. micr. Sci. 105,415-422. MARSHALL A. T. (1965) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-3. The cytology and function of the fibril zone of the Malpighian tubules of tube-building nymphs. Quart. _‘f.micr. Sci. 106, 37-44. MARSHALLA. T. (1966a) Spittle-production and tube-building by cercopoid larvae (Homoptera)--4. Mucopolysaccharide associated with spittle-production. 2. Insect Physiol. 12, 635-644.
2326
A. T. MARSHALL
MARSHALLA. T. (1966b) Histochemical studies on a mucocomplex in the Malpighian tubules of cercopid larvae. J. Insect Physiol. 12, 925-932. MARSHALLA. T. (1968) The chemical nature of Malpighian tubule mucofibrils in cercopoid dwelling-tubes. J. Insect Physiol. 14, 1435-1444. MARSHALLA. T. and MASHALL P. M. (1966) Life history of a tube-dwelling cercopoid: Machaerota coronata Maa (Homoptera: Machaerotidae). Proc. R. ent. Sot. Lond. (A) 41,17-20. PEARSEA. G. E. (1960) Histochemistry, Theoretical and Applied. Churchill, London. RUDALL K. M. and KENCHINGTON W. (1971) Arthropod silks: the problem of fibrous proteins in animal tissues. A. Rev. Ent. 16, 73-96. SALTHOUSET. N. (1958) Tetrahydrofuran and its use in insect histology. Can. Ent. 90, 555-557. SILVESTRIF. (1904) Contribuzione alla conocenza della metamorfosi. Redia 2, 68-84. SMITH I. (1960) Chromatographic and Electrophoretic Techniques. Heinemann, London. SMITH D. S. and LITTAU V. C. (1960) Cellular specialization in the excretory epithelia of an insect Macrosteles fascifrons Stal (Homoptera). J. biophys. biochem. Cytol. 8, 103-133. SPIEGLERP. E. (1962) The origin and nature of the adhesive substance in the larvae of the genus Chrysopa (Neuroptera: Chrysopidae). Ann. ent. Sot. Am. 55,69-77. WIGGLESWORTH V. B. (1965) Prilaciples of Insect Physiology. Methuen, London. ZIEGLER H. and ZIEGLER I. (1958) Uber die Zusammensetzung des Zikadenschaumes. Z. vergl. Physiot. 40, 549-555.
PROTEIN SYNTHESIS AND SECRETION BY THE MALPIGHIAN TUBULES OF CERCOPOID LARVAE (HOMOPTERA) A. T. MARSHALL Department of Zoology, La Trobe University, Melbourne, Victoria 3083, Australia (Received 3 April 1973) Abstract-The Malpighian tubules of cercopoid larvae are anatomically differentiated. The cells of the distal segments have an ultrastructure which is typical of cells synthesizing and exporting protein. They give intense positive reactions with cytochemical tests for protein and contain large quantities of RNA. Secretory activity is not synchronized in different cells. The protein secretion forms a component of machaerotid dwelling-tubes and cercopid foti but may also function as an excretory product. INTRODUCTION
THE MALPIGHIANtubules of insects are primarily concerned with the excretion of the waste products of metabolism and the regulation of ion and water balance. However, in a variety of insects they appear to carry out accessory functions which are quite unrelated to excretion or osmoregulation (WIGGLESWORTH, 1965). This is particularly true of cercopoid larvae. MARSHALL (1964a, b, 1965, 1966a, b, 1968) has shown that the proximal segment of the Malpighian tubules of cercopoid larvae is concerned with the synthesis and secretion of mucopolysaccharides. These are associated with the peculiar modes of larval life. The distal segment of the Malpighian tubules has not been previously examined and it will be shown that it is highly specialized for the synthesis and secretion of protein. MATERIALS
AND METHODS
The insects examined included the machaerotids Machaerota coronata Maa, Chaetophyes compacta Walk., Pectinariophyes stalii Spongberg, and the cercopids Cosmoscarta abdominalis Don. and Philaenus spumarius L. For electron microscopy larvae were dissected and the Malpighian tubules fixed in 1% osmium tetroxide in Verona1 acetate buffer pH 7.3 or in 2.5% glutaraldehyde in phsophate buffer pH 7.2 and post osmicated in 1% osmium tetroxide in phosphate buffer. Th e material was embedded in Araldite. Sections were stained in uranyl acetate and lead citrate. For cytochemical tests Malpighian tubules were fixed in formaldehyde-calcium, dehydrated in tetrahydrofuran (SALTHOUSE, 1958), embedded in 55°C paraffin wax, 2317
A. T. MARSHALL
2318
and sectioned at 7 pm. Cytochemical tests included Hg-bromophenol blue, acid solochrome cyanine, Millon’s reaction, and ribonuclease extractions (PEARSE,1960). For electrophoresis, urine was collected from 13 dwelling-tubes of M. cormata, pooled, centrifuged, and the supernatant retained. The distal segments of the Malpighian tubules of 12 larvae were dissected out in insect saline, homogenized in 0.1 ml of distilled water, centrifuged, and the supernatant retained. Electrophoresis was carried out on 10 cm strips of buffered cellulose acetate paper. Samples of 5 ~1 were applied to the papers as narrow bands. For each buffer a current of 2 n&/2*5 cm width of strip was applied. After drying at 105°C the strips were stained overnight in 0.002% nigrosin (SMITH, 1960). The buffer solutions used were: (1) 0.04 M sodium diethylbarbiturate-HCl, pH 8.6, (2) 0.04 M sodium diethylbarbiturate-sodium acetate-HCl, pH 5.0, (3) O-1 M Na,HPO,-NaHsPO,, pH 7.0, and (4) 0.05 M borax-NaOH, pH 10.5. OBSERVATIONS
The gross anatomical appearance of the Malpighian tubules is shown in Fig. 1. In fresh dissections and in sections the cells are seen to contain a branching system of intracellular canaliculi continuous with the tubule lumen. This is schematically shown in Fig. 2.
DS
FIG. 1. Diagram showing gut and Malpighian tubules. PS, Proximal segment; DS, distal segment; FC, filter chamber; R, rectum.
Ultrastructure The cells of the Malpighian tubule distal segments of all the species examined are very similar in fine structure. They are characterized by the presence of large numbers of membrane bounded granules ranging from 0.5 to 2-O pm in diameter. These differ in electron density and homogeneity in the different species (Figs. 3,6, 7). The rough endoplasmic reticulum (RER) is extensively developed and its appearance varies with the granule content of the cells. Where the granule population is relatively small the RER is developed into enormous whorls of concentric
THE
MALPIGHIAN
TUBULES
OF CERCOPOJD
LARVAE
2319
FIG. 2. Diagram of distal segment cells of Malpighian tubule in longitudinal se&on. N, Nucleus; C, intracellular canaliculi; L, lumen.
cisternae (Fig. 7). In cells with high populations of granules the RER is compressed between the granules (Fig. 6). Material of low and high electron density is seen within the cisternae of the RER (Figs. 5, 6). Numerous Golgi bodies occur within the cells. Transition vesicles are present at the forming face and electron dense material appears to be condensing at the maturing face (Figs. 4,5). The cells are penetrated by an extensively arborized system of canaliculi which are lined with microvilii (Figs. 3, 6). Extrusion of granules into the canaliculi appears to occur without fusion of the bounding granule membrane with the plasma membrane. The granule contents then appear to go into solution leaving profiles of membrane within the lumina of the canaliculi (Fig. 8). In adult cercopoids no granules appear in the distal segment cells. The cytoplasm is packed with mitochondria and the canaliculi are lined with microvilli which are considerably longer than in the larvae. Cytochemistry The distal segment cells stain very intensely with Hg-bromophenol blue and They also give an intense positive reaction with acid solochrome cyanine. Millon’s reagent. These results suggest the presence of large amounts of protein. Sections incubated in ribonuclease exhibit a considerable decrease in basophilia compared with control sections, indicating the presence of large amounts of cytoplasmic ribosenucleic acid (RNA). Electrophoresis Only urine was run with sodium diethylbarbiturate-HCl buffer at pH 8.6. After a 3 hr run no movement from the origin was observed. AlI other results are shown in Fig. 9. There appear to be two proteins in urine which have their isoelectric points around pH 8.6. Three proteins are present in the water extracts
A. T. MARSHALL
2320
”
I
-
-_-+ --
~H5.0
QH7.Q
1.45hr Q
6.0hr
0
ziim A
FIG. 9. Tracings
of electrophoretic patterns from Malpighian tubule segment extract (D) and urine (A) of M. coronata larvae.
distal
from the distal segments. One of these appears to be electrophoretically identical to one of the urine proteins. The pH of il4. coronata urine is pH 8.2 and that of C. compacta is pH 8.1. DISCUSSION
The fine structure of the distal segment cells of the Malpighian tubules of cercopoid larvae is typical of cells which synthesize and export proteins. Cell features such as secretion granules, extensive rough endoplasmic reticulum, and numerous Golgi bodies are all characteristic of protein secreting cells as demonstrated in the classical studies by CARO and PALADE(1964) and JAMIESONand PALADE(1967a, b). Cytochemically, the distal segment cells are rich in protein and cytoplasmic RNA and there can be no doubt that they synthesize and secrete protein in the form of granules. The ultrastructure of the cells suggests that as in pancreas, protein is synthesized in the cisternae of the RRR and packaged into granules in the Golgi bodies. The granules appear to be extruded into the intracellular canaliculi complete with their bounding membranes. Here they seem to go into solution. This may be facilitated by the osmoregulatory flow of ions and water into the tubule lumen which is believed to occur in addition to secretory activity (MARSHALL and CHEUNG,in press). The secretory cycle has not so far been investigated in detail but preliminary studies indicate that secretory activity is not synchronized in different cells in the same Malpighian tubule. There must therefore be a continuous release of small quantities of protein from each of the Malpighian tubules. The function of this protein secretion is not fully understood. Machaerotid larvae build and live within dwelling-tubes (MARSHALL and MARSHALL, 1966) and protein is a component of these tubes (MARSHALL, 1968). The electrophoresis results suggest that there is at least one protein in the urine filling the dwelling-tubes which originates in the distal segments of the Malpighian tubules. As the urine evaporates this protein will be rapidly precipitated,
2321
FIG. 3. Distal segment cell of M. coronata. P, Protein granules; G, Golgi bodies; C, canaliculi ; ER, endoplasmic reticulum.
2322
FIGS. 4 and 5. Golgi bodies in distal segment cells of M. corona&. T, Transition vacuoles ; C, condensing vacuoles ; arrows, dense material jn endoplasmic reticulum cisternae.
2323
FIG. 6. Distal segment cells of C. abdominalis. Arrows, Material within cisternae of endoplasmic reticulum. FIG. 7. Distal segment cells of C. compacta showing whorls of rough endoplasmic reticulum. FIG. 8. Distal segment lumen of C. abdominalis containing membrane profiles and dissolving granules.
THE MALPIGHIANTUBULESOF CERCOPOID LARVAE
2325
since its isoelectric point occurs at about the pH of the urine, and incorporated into the dwelling-tube as matrix protein. Cercopoid larvae live within a foam or spittle mass. Protein has been detected in this by ZIEGLER and ZIEGLER(1958) and MARSHALL(1966a). Protein in foam may contribute to bubble stability by bonding to mucopolysaccharide (MARSHALL, 1966a). Protein synthesis and secretion is certainly a function of the larval Malpighian tubules and ceases in both machaerotid and cercopid adults. It therefore seems likely that it is related to the peculiar modes of larval life. However, it must be borne in mind that the feeding site of the adults appears to be different to that of the larvae (HAGLEY, 1965; Marshall, unpublished) and it is possible that the protein functions as an excretory product in the larval stage but not in the adult. SMITH and LITTAU (1960) have suggested that the secretion of protein in the form of brochosomes from the Malpighian tubules of cicadellids is a possible excretory mechanism. Similarly it has been suggested by SPIEGLER(1962) that the silk and adhesive secretion of the Malpighian tubules of Ch~ysopa larvae are products of nitrogenous excretion. However, the silk does not appear to be a protein but a form of nylon (RUDALL and KFZNCHINGTON, 1971). Silks are also secreted by the Malpighian tubules of several coleoptera larvae (e.g. SILVESTRI,1904) but the nature of the silks does not appear to have been investigated. Acknowledgements-I wish to record my thanks to Dr. D. F. WATERHOUSE and Dr. B. FILSHIE for allowing me to use the electron microscopy facilities in the Division of Entomology, C.S.I.R.O., Canberra, where part of this work was carried out. Part of this study was also done in the Zoology Departments of the Universities of Hong Kong and Hull, REFERENCES CARO L. G. and PALADE G. E. (1964) Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic study. y. Cell Biol. 20, 473-495. HAGLEY E. A. C. (196.5) Site of feeding of the froghopper. Rep. Tate and Lyle cent. agric. Res. Stn TGaidad pp. 408-413. JAMIESONJ. D. and PALADEG. E. (1967a) Intracellular transport of secretory proteins in the pancreatic exocrine cell-I. Role of the peripheral elements of the Golgi complex. y. Cell Biol. 34, 577-596. JAMIESONJ. D. and PALADEG. E. (1967b) Intracellular transport of secretory proteins in the pancreatic exocrine cell-II. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34, 597-615. MARSHALL A. T. (1964a) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-1. The cytology of the Malpighian tubules of spittle-bug nymphs. Quart. J. micr. Sci. 105, 257-262. MARSHALL A. T. (1964b) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-2. The cytology and function of the granule zone of the Malpighian tubules of tube-building nymphs. Quart. J. micr. Sci. 105,415-422. MARSHALL A. T. (1965) Spittle-production and tube-building by cercopoid nymphs (Homoptera)-3. The cytology and function of the fibril zone of the Malpighian tubules of tube-building nymphs. Quart. _‘f.micr. Sci. 106, 37-44. MARSHALLA. T. (1966a) Spittle-production and tube-building by cercopoid larvae (Homoptera)--4. Mucopolysaccharide associated with spittle-production. 2. Insect Physiol. 12, 635-644.
2326
A. T. MARSHALL
MARSHALLA. T. (1966b) Histochemical studies on a mucocomplex in the Malpighian tubules of cercopid larvae. J. Insect Physiol. 12, 925-932. MARSHALLA. T. (1968) The chemical nature of Malpighian tubule mucofibrils in cercopoid dwelling-tubes. J. Insect Physiol. 14, 1435-1444. MARSHALLA. T. and MASHALL P. M. (1966) Life history of a tube-dwelling cercopoid: Machaerota coronata Maa (Homoptera: Machaerotidae). Proc. R. ent. Sot. Lond. (A) 41,17-20. PEARSEA. G. E. (1960) Histochemistry, Theoretical and Applied. Churchill, London. RUDALL K. M. and KENCHINGTON W. (1971) Arthropod silks: the problem of fibrous proteins in animal tissues. A. Rev. Ent. 16, 73-96. SALTHOUSET. N. (1958) Tetrahydrofuran and its use in insect histology. Can. Ent. 90, 555-557. SILVESTRIF. (1904) Contribuzione alla conocenza della metamorfosi. Redia 2, 68-84. SMITH I. (1960) Chromatographic and Electrophoretic Techniques. Heinemann, London. SMITH D. S. and LITTAU V. C. (1960) Cellular specialization in the excretory epithelia of an insect Macrosteles fascifrons Stal (Homoptera). J. biophys. biochem. Cytol. 8, 103-133. SPIEGLERP. E. (1962) The origin and nature of the adhesive substance in the larvae of the genus Chrysopa (Neuroptera: Chrysopidae). Ann. ent. Sot. Am. 55,69-77. WIGGLESWORTH V. B. (1965) Prilaciples of Insect Physiology. Methuen, London. ZIEGLER H. and ZIEGLER I. (1958) Uber die Zusammensetzung des Zikadenschaumes. Z. vergl. Physiot. 40, 549-555.