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Ultrastructure and cytochemistry of an extensive plexiform surface coat on the midgut cells of a fulgorid insect
© 1970 by Academic Press, Inc.
J. ULTRASTRtlCTURERESEARCH33, 161--172 (1970)
161
Ultrastructure and Cytochemistry of an Extensive Plexiform Surface Coat on the Midgut Cells o f a Fulgorid Insect A. T. MARSHALL1 AND W . W . K . CHEUNG 1
Department of Zoology, University of Hong Kong, and Department of Zoology, University of Hull, England Received November 14, 1969 and in revised form March 26, 1970 The midgut epithelial cells of Fulgora candelaria are covered by a unique plexiform surface coat, 2-4 ~ thick. It has two layers. The outer layer consists of saccules of membranes, each saccule containing a membranous vesicle from which radiates a "fuzz" of filaments and granules. The inner layer is formed from flattened sacs of membrane. The coat is cytochemically defined as a PASpositive acid mucopolysaccharide. The membranes of the coat have a striking similarity in dimensions and morphology to the plasma membrane of the underlying microvilli of the midgut epithelial cells.
It is generally recognized that the microvilli of absorptive and secretory cells are covered with a "fuzzy" or filamentous coat or glycocalyx which is regarded as an integral part of the plasma membrane (10, 11, 16, 25, 27). The coat consists of filaments of material extending from the outer layer of the triple-layered plasma membrane and may be up to 0.5 # thick. Histochemical studies show it to contain mucopolysaccharide (3, 16) and in some instances enzymes (17, 18, 22). In the vertebrate intestine mucous secretions from goblet cells may form a coating external to the surface coat or glycocalyx. This is usually said to have a protective or lubricating function. In the insect gut the peritrophic membrane is thought to be an analogous structure. It is formed in two basic ways (30), as a backwardly secreted tube from an anterior annular press or by delamination from the surface of the gut epithelium. Intermediates between these types exist. In at least one insect (Calliphora) the membrane is a complex multilayered structure, the different layers being produced as secretions by different types of cells (25). The chemical composition of some peritrophic membranes has been determined by De Mets and Jeugniaux (6). They contain protein, chitin, and other mucopolysaccharides. 1 Present address: Department of Zoology, La Trobe University, Victoria, Australia. 11 -- 701830 J. UltrastructureResearch
162
MARSHALL AND CHEUNG
D u r i n g the course of u l t r a s t r u c t u r a l studies o n h o m o p t e r a n digestive systems we have observed a n u n u s u a l surface coat o n the m i d g u t epithelial cells of Fulgora
¢andeIaria. This coat is unlike a n y previously described. It is developed into a plexif o r m or reticulate structure which almost occludes the gut l u m e n . It has some of the characteristics of a glycocalyx a n d topographically resembles a peritrophic m e m b r a n e .
MATERIALS A N D METHODS Adults of Fulgora candelaria L. (Homoptera) were collected from Lychee groves in the New Territories of Hong Kong Colony. Dissections were made in phosphate buffer pH 7.6, and pieces of midgut were removed and fixed in glutaraldehyde in phosphate buffer pH 7.6 for 2 hours and postfixed in osmium tetroxide in phosphate buffer pH 7.6 for 1 hour. Tissues were embedded in Araldite. Sections were stained in uranyl acetate and lead citrate and observed in a JEM-TA electron microscope. Thick sections for light microscopy were stained in 1% toluidine blue in 1% borax. For histological observations material was fixed in Bouin's fluid, embedded in Paraplast and sectioned at 5 #. Sections were routinely stained in hematoxylin and eosin. Material for cytochemical tests was fixed in the appropriate fixative (Table I) embedded in Paraplast, sectioned at 5 #, and subjected to standard cytochemical tests (Table I). Frozen sections were also cut for enzyme investigations. OBSERVATIONS The m i d g u t of Fulgora candelaria is a coiled knotlike mass. I n its architecture it shows little change along its length. The observations i n this article were made o n the anterior region b u t hold, with little variation, for the whole midgut. A detailed description of the structure of the gut will be given elsewhere. The m i d g u t cells are binucleate c o l u m n a r cells with a conical apical region f r o m which a threadlike structure extends into the gut l u m e n (Figs. 1 a n d 3). I n Araldite FIG. 1. Transverse section of anterior midgut. Light micrograph of paraffin wax section. Hematoxylin and eosin stained. Note the threadlike structures (t) extending into the gut lumen (l). x 150. Fro. 2. Longitudinal section of apical region of epithelial cell, corresponding to region indicated by arrow in Fig. I. Note the well developed endoplasmic reticulum (er), numerous Golgi bodies (g), lyric bodies (ly), microvilli (my) with fibrillar cores (c) extending into cytoplasm, cell membrane (cm), and nucleus (n). x 9 500. FIo. 3. Transverse section of anterior midgut. Light micrograph of Araldite section. Toluidine blue stained. Showing brush border (bb) of epithelial cells covered with an extensive surface coat (sc). Note the extensions of this coat as threadlike structures (t) into the gut lumen, and interspersed between the threads ringlike structures (r). Nuclei (n) and lytic bodies (ly) are visible, x 360. F~G. 4. Showing ends of threadlike structures in gut lumen. The field is similar to that indicated by an arrow in Fig. 3. The threads consist of polygonal saccules (c) containing dark vesicles (fz). Around the edges of the threads are flattened sacs (s) and between them are seen "myelin figures" (mf) corresponding to the ringlike structures (r) seen in Fig. 3. x 12 000. FIG. 5. Transverse section through apical region of epithelial cells. Showing the thick surface coat (sc) extending from the microvilli (my). It consists of polygonal saccules (c) containing vesicles (fz) and flattened sacs (s). × 8 000.
Z
Z
PLEXIFORM SURFACE COAT IN INSECT MIDGUT
165
166
MARSHALL AND CHEUNG
sections, the threadlike structures are wider and pack the gut lumen more than in paraffin wax sections (Fig. 3). They are extensions of a coat which overlays, and is distinct from, the brush border (Fig. 3).
Ultrastructure The midgut cells (Fig. 2) have a well developed rough endoplasmic reticulum, abundant Golgi bodies and mitochondria. Large lysosome-like bodies are present. The microvilli have fibrillar cores which extend into the subjacent cytoplasm. There is no sign of secretory material passing into or between the microvilli. The long microvilli are remarkable in that they frequently appear to have two or more triple-layered plasma membranes, 40-60 A thick (Figs. 6-8). It is not possible to see whether each layer of plasma membrane completely envelops each microvillus, but transverse sections (Fig. 8) suggest that they may nearly do so. The tips of the microvilli are covered with a "fuzz" of filaments and granules (Figs. 6 and 7). Double plasma membranes frequently cover the tips and the outer one sometimes appears to be separated from the inner one (Fig. 7). Between the microvilli are small membrane vesicles and sheets of membrane that extend out into the lumen beyond the microvilli tips (Figs. 6, 7, 9, and 11). These membranes are triple-layered and 40-60 A thick. Superjacent to the microvilli is a plexiform surface coat 2-4 # thick (Fig. 5). This coat extends into the gut lumen (Fig. 4) as the threadlike structures seen in the light micrographs. This coat has two layers, an outer layer of saccules and an inner layer of flattened saclike structures (Fig. 5). The former consists of polygonal saccules of membranes (Figs. 11 and 12) which contain vesicles (Figs. 7, 11, and 12) from which a "fuzz" of filaments and granules radiates to fill a saccule (Fig. 12). The inner layer of flattened saclike structures also consists of membranes (Fig. 10). These ultimately appear to form aggregated whorls or "myelin figures" (Figs. 3 and 4) which correspond to the ring structures seen in Fig. 3. The membranes of the saccules, flattened sacs, and "myelin figures" appear to exhibit a triple-layered and a globular structure (Figs. 10 and 12).
Cytochemistry Cytochemical tests were carried out on sections observed in the light microscope. The results are summarized in Table I. FIo. 6. Longitudinal section of microvilli partially ensheathed by double plasma membranes (arrows). The tip of each microvillus bears a "fuzz" of granules and fine filaments. Between the microvilli are sheets (rn) and small vesicles (v) of membrane, x 160 000. FIG. 7. Longitudinal section of microvilli showing separation of double plasma membranes at a microvillus tip (arrow). A fuzz-bearing vesicle (fz) having the appearance of a "nipped-off" microvillus tip is seen. x 160 000. FIo. 8. Transverse section of microvilli showing double plasma membranes, x 120 000.
o >H
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o
168
MARSHALL AND CHEUNG
TABLE I CYTOCHEMICAL TESTS ON THE INTESTINAL EPITHELIAL CELLS OF
Surface coat
Test
Alcian blue Toluidine blue Gentian violet Mucicarmine Bismarck brown Hale's test PAS Acid sol©chrome cyanine Mercury-bromophenol blue Sudan Black B Alkaline phosphatase c~-Glucosidase Leucylaminopeptidase
(17) (20) (12) (12) (12) (12)
Microvilli
+ + + ++++ ~ ++++ + + + + + + ++++
+ + + ++++b ++++ + + + + + + ++++
Fulgora candelaria ~
Apical cytoplasm
Basal cytoplasm
+ O o o O ©
o o o o O ©
Fixative
(20)
+ + +
+ + + +
+ +
+
(20)
+
+ + + +
+ + +
+
Formol saline Formolsaline Formolsaline Formol saline Formol saline Formolsaline Bouin Formol calcium
(20) (17) (20) (1) (3)
+ + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + +
+ + o o +
Formol calcium Neutral formalin 95 % alcohol, 4°C Neutral formalin, 4°C Acetone, 4°C
a + positive reaction, the number of +'s being proportional to the intensity of the reaction; o, a negative reaction. Numbers in parentheses refer to references. b 7-Metachromasia. DISCUSSION T h e p l e x i f o r m s u r f a c e c o a t o n t h e m i d g u t e p i t h e l i a l cells of Fulgora candelaria a p p e a r s to be u n i q u e . It is c o m p o s e d of a n e t w o r k of m e m b r a n e saccules, c o n t a i n i n g vesicles, a n d f l a t t e n e d m e m b r a n e sacs. T h e o r i g i n of t h e d i f f e r e n t t y p e s of s t r u c t u r e s seen in t h e b r u s h b o r d e r a n d s u r f a c e c o a t is d i f f i c u l t to e x p l a i n . I t s e e m s likely t h a t t h e f u z z - b e a r i n g vesicles c o u l d arise as "nipped-off"
m i c r o v i l l i tips since t h e r e is a s t r o n g m o r p h o l o g i c a l r e s e m b l a n c e be-
t w e e n t h e s e t w o entities in e l e c t r o n m i c r o g r a p h s . T h e i n n e r , l u m i n a l , l a y e r of f l a t t e n e d sacs c o u l d b e a r e s u l t of t h e m e m b r a n e s a c c u l e s l o s i n g t h e i r c o n t e n t s . H o w e v e r , t h e m o d e of o r i g i n of t h e m e m b r a n e
saccules themselves and the membranes
found
b e t w e e n a n d c l o s e l y i n v e s t i n g t h e m i c r o v i l l i is d i f f i c u l t to e n v i s a g e . A d o u b l e m e m b r a n e is r e g u l a r l y seen a r o u n d e a c h m i c r o v i l l u s . T h i s c a n be explained by suggesting that the microvilli project into the overlying surface coat and FIG. 9. Longitudinal section of microvilli showing membranes (m), between the microvilli, extending out into the gut lumen and apparently enclosing microvilli tips and fuzz-bearing vesicles (fz). x 42 000. FIG. 10. Membranes of flattened sacs and part of a "myelin figure" showing triple-layered structure and a suggestion of globular structure (arrows). Slightly underfocused, x 165 000. Fro. 11. Similar to Fig. 9. x 80 000. Fia. 12. Polygonal saccules of surface coat containing vesicles from which radiate filaments and granules. The membranes show a triple-layered structure and globular structure (arrows). × 165 000.
C~
170
MARSHALL AND CHEUNG
that they are "clothed" by the membranes of the surface coat. A similar topological situation has been described in the chaetognathan photoreceptor cell by Eakin and Westfall (7). This, however, still does not explain whence the surface coat membranes originate. Several explanations seem to be possible: (a) Whole or parts of microvilli are "nipped-off" and the contents dispersed to leave "ghosts." The "nipping-off" of parts of microvilli is not a novel phenomenon and has been previously observed in gryllid and grasshopper Malpighian tubules (2, 4). (b) The membranes arise by coalescence of membrane vesicles extruded from the microvilli. Extrusion of vesicles from microvilli has also been reported in insect Malpighian tubules, e.g. Wigglesworth and Salpeter (31). (c) The surface coat membranes are delaminated from or sloughed off the microvilli. The latter is a somewhat radical suggestion, but the notion of plasma membrane delamination has been previously advanced in the lamellar apposition theory of xylem cell wall formation (28). The present study has not produced evidence which favors unambiguously any one of these theories, and the theories themselves must be viewed as being entirely speculatory. There is a suggestion of globular structure in the membranes of the plexiform coat which is reminiscent of the globular structures of mitochondrial and ~-cytomembranes described by Sj~Sstrand (24). We cannot claim in this study to have unequivocally established such structure as real and not artifactual. However, the membranes of the microvilli and plexiform coat are of the same order of thickness as Sj~Sstrand's mitochondrial membranes and ~-cytomembranes in which globular structure occurs, and they are thinner than the average plasma membrane in which globular structure does not occur. Cytochemically, the plexiform coat has the same characteristics as the usual glycocalyx. It appears to be definable as a PAS-positive acid mucopolysaccharide. This could certainly be accounted for if it is assumed that the fuzz of the vesicles has its origin as glycocalyx on the tips of the microvilli. Traces of enzyme activity are found in the plexiform coat. It is striking, however, that activity is considerably lower than in the microvilli, particularly in view of the contention of some authors (9, 16) that at least some enzymes are located in the plasma membrane of microvilli, and of others (10, 17, 18, 22) that enzymes may be associated with the glycocalyx. The function of the plexiform surface coat can hardly be guessed at. Indeed, no clear-cut evidence is available which is indicative of either the function of surface coats or insect peritrophic membranes in general. Insect peritrophic membranes are usually regarded as protective devices (30) although their porous nature (21, 29) is suggestive of a sievelike function. Peritrophic membranes are usually said to be absent in homopteran insects (13); indeed, their absence in these sap-sucking insects is frequently given as evidence for their protective function in other insects. However,
PLEXIFORM SURFACE COAT IN INSECT MIDGUT
171
in the midgut of one homopteran insect, a PAS-positive peritrophic membrane has been described (14) which consists of "filaments and fibrils." This membrane appears to have similar cytochemical properties to the plexiform surface coat described here. Moreover, Gouranton and Maillet (14) point out the similarity between the filamentous component and the glycocalyx of m a m m a l i a n intestine. It is not possible to see in their micrographs whether the "fibrils" are membranes; there are, however, structures visible which appear to be similar to our fuzz-bearing vesicles. It is tempting to suggest that this type of membrane may be a variation of the plexiform coat described by us. Similarly, regions of the midgut of cercopids (19) and other closely related homopteran insects (Cheung and Marshall, unpublished data) have a mucopolysaccharide coating, and a PAS-positive coating is secreted by the crop epithelium of aphids (8). Threadlike structures similar to those in the fulgorid gut have been described in some heteropteran insects (12). It seems that there may be some correlation between the presence of a mucopolysaccharide coating and sap-sucking and that such coatings, although perhaps produced in a variety of ways, may all ultimately fall within a loose definition of the insect peritrophic membrane. This work was facilitated by the use of a field vehicle donated to the Zoology Department, University of Hong Kong, by Mr. Ma Ying. We are indebted to Miss J. Halder, Zoology Department, University of Hull for her skillful photographic assistance. One of us, W. W. K. C., gratefully acknowledges the receipt of a Commonwealth Scholarship (U. K. award), during the tenure of which part of this work was done.
REFERENCES
1. BARKA,T. and ANDERSON,P. J., Histochemistry: Theory, Practice, and Bibliography. Hoeber, London, 1963. 2. BEAMS, H. W., TAHMISIAN,T. N. and DEVINE, R. L., J. Biophys. Biochem. Cytol. 1, 197 (1955). 3. BENNETT,H. S., J. Histochem. Cytochem. 11, 14 (1963). 4. BERKALOFF,A., C. R. Acad. Sci. 250, 2609 (1960). 5. BURSTONE,M. S. and FOLK, J. E., J. Histochem. Cytochem. 4, 217 (1956). 6. DE METS, R. and JEUGNIAUX,C., Arch. Int. Physiol. Biochim. 70, 93 (1962). 7. EAKIN, R. M. and WESTFALL,J. A., J. Cell Biol. 21, 115 (1964). 8. EDWARDS, J. S., J. Insect Physiol. 12, 31 (1966). 9. ElCnOLZ, A. and CRANe, R. K., J. Cell Biol. 26, 687 (1965). 10. FAWCETT,D. W., J. Histochem. Cytochem. 13, 75 (1965). 11. - An Atlas of Fine Structure. The Cell. Saunders, Philadelphia, 1966. 12. GOODCHILD,A. J. P., Trans. Roy. Entornol. Soc. London 115, 217 (1963). 13. - Biol. Rev. Cambridge Phil. Soc. 41, 97 (1966).
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14. GOURANTON,J. and MAILLET,P. L., C. R. Acad. Sci. 261, 1102 (1965). 15. GURR, G., Methods of Analytical Histology and Histochemistry. Leonard Hill, London, 1958. 16. ITO, S., J. Cell BioL 27, 475 (1965). 17. JOHNSON,C. F., J. Lab. Clin. Med. 68, 883 (1966). 18. - in Ryozi, U. (Ed.), Electron Microscopy, Vol. II, p. 389. Maruzen, Tokyo, 1966. 19. MARSHALL,A. T., J. Insect Physiol. 12, 635 (1966). 20. MCMANuS, J. F. A. and MOWRY, R. W., Staining methods, Histologic and Histochemical. Hoeber, London, 1960. 21. MERCER, E. H. and DAY, M. F., Biol. Bull. 103, 384 (1952). 22. ODA, T. and SEKI, S., in RYozI, U. (Ed.), Electron Microscopy, Vol. II, p. 387. Maruzen, Tokyo, 1966. 23. PEARSE, A. G. E., Histochemistry, Theoretical and Applied. Churchill, London, 1960. 24. SJ6STRAND, F. S., in DALTON, A. J. and HAGENAU, F. (Eds.), p. 151. The Membranes, Academic Press, London, 1968. 25. SMITH, D. S., Insect Cells. Their Structure and Function. Oliver & Boyd, Edinburgh, 1968. 26. STEEDMAN,H. F., Section Cutting in Microscopy. BlackweU, Oxford, 1968. 27. TRIER, J. S., Fed. Proc. 26, 1391 (1967). 28. WARDROP,A. B., in C6t6, W. A. (Ed.), Cellular Ultrastructure of Woody Plants. Syracuse Univ. Press, New York, 1965. 29. WATERHOUSE,D. F. and WRIGHT, M., J. Inseet Physiol. 5, 230 (1960). 30. WIGGLESWORTH,V. B., The Principles of Insect Physiology, 6th ed. Methuen, London, 1965. 31. WIGGLESWORTH,V. B. and SALPETER,M. M., J. Inseet Physiol. 8, 299 (1962).
J. ULTRASTRtlCTURERESEARCH33, 161--172 (1970)
161
Ultrastructure and Cytochemistry of an Extensive Plexiform Surface Coat on the Midgut Cells o f a Fulgorid Insect A. T. MARSHALL1 AND W . W . K . CHEUNG 1
Department of Zoology, University of Hong Kong, and Department of Zoology, University of Hull, England Received November 14, 1969 and in revised form March 26, 1970 The midgut epithelial cells of Fulgora candelaria are covered by a unique plexiform surface coat, 2-4 ~ thick. It has two layers. The outer layer consists of saccules of membranes, each saccule containing a membranous vesicle from which radiates a "fuzz" of filaments and granules. The inner layer is formed from flattened sacs of membrane. The coat is cytochemically defined as a PASpositive acid mucopolysaccharide. The membranes of the coat have a striking similarity in dimensions and morphology to the plasma membrane of the underlying microvilli of the midgut epithelial cells.
It is generally recognized that the microvilli of absorptive and secretory cells are covered with a "fuzzy" or filamentous coat or glycocalyx which is regarded as an integral part of the plasma membrane (10, 11, 16, 25, 27). The coat consists of filaments of material extending from the outer layer of the triple-layered plasma membrane and may be up to 0.5 # thick. Histochemical studies show it to contain mucopolysaccharide (3, 16) and in some instances enzymes (17, 18, 22). In the vertebrate intestine mucous secretions from goblet cells may form a coating external to the surface coat or glycocalyx. This is usually said to have a protective or lubricating function. In the insect gut the peritrophic membrane is thought to be an analogous structure. It is formed in two basic ways (30), as a backwardly secreted tube from an anterior annular press or by delamination from the surface of the gut epithelium. Intermediates between these types exist. In at least one insect (Calliphora) the membrane is a complex multilayered structure, the different layers being produced as secretions by different types of cells (25). The chemical composition of some peritrophic membranes has been determined by De Mets and Jeugniaux (6). They contain protein, chitin, and other mucopolysaccharides. 1 Present address: Department of Zoology, La Trobe University, Victoria, Australia. 11 -- 701830 J. UltrastructureResearch
162
MARSHALL AND CHEUNG
D u r i n g the course of u l t r a s t r u c t u r a l studies o n h o m o p t e r a n digestive systems we have observed a n u n u s u a l surface coat o n the m i d g u t epithelial cells of Fulgora
¢andeIaria. This coat is unlike a n y previously described. It is developed into a plexif o r m or reticulate structure which almost occludes the gut l u m e n . It has some of the characteristics of a glycocalyx a n d topographically resembles a peritrophic m e m b r a n e .
MATERIALS A N D METHODS Adults of Fulgora candelaria L. (Homoptera) were collected from Lychee groves in the New Territories of Hong Kong Colony. Dissections were made in phosphate buffer pH 7.6, and pieces of midgut were removed and fixed in glutaraldehyde in phosphate buffer pH 7.6 for 2 hours and postfixed in osmium tetroxide in phosphate buffer pH 7.6 for 1 hour. Tissues were embedded in Araldite. Sections were stained in uranyl acetate and lead citrate and observed in a JEM-TA electron microscope. Thick sections for light microscopy were stained in 1% toluidine blue in 1% borax. For histological observations material was fixed in Bouin's fluid, embedded in Paraplast and sectioned at 5 #. Sections were routinely stained in hematoxylin and eosin. Material for cytochemical tests was fixed in the appropriate fixative (Table I) embedded in Paraplast, sectioned at 5 #, and subjected to standard cytochemical tests (Table I). Frozen sections were also cut for enzyme investigations. OBSERVATIONS The m i d g u t of Fulgora candelaria is a coiled knotlike mass. I n its architecture it shows little change along its length. The observations i n this article were made o n the anterior region b u t hold, with little variation, for the whole midgut. A detailed description of the structure of the gut will be given elsewhere. The m i d g u t cells are binucleate c o l u m n a r cells with a conical apical region f r o m which a threadlike structure extends into the gut l u m e n (Figs. 1 a n d 3). I n Araldite FIG. 1. Transverse section of anterior midgut. Light micrograph of paraffin wax section. Hematoxylin and eosin stained. Note the threadlike structures (t) extending into the gut lumen (l). x 150. Fro. 2. Longitudinal section of apical region of epithelial cell, corresponding to region indicated by arrow in Fig. I. Note the well developed endoplasmic reticulum (er), numerous Golgi bodies (g), lyric bodies (ly), microvilli (my) with fibrillar cores (c) extending into cytoplasm, cell membrane (cm), and nucleus (n). x 9 500. FIo. 3. Transverse section of anterior midgut. Light micrograph of Araldite section. Toluidine blue stained. Showing brush border (bb) of epithelial cells covered with an extensive surface coat (sc). Note the extensions of this coat as threadlike structures (t) into the gut lumen, and interspersed between the threads ringlike structures (r). Nuclei (n) and lytic bodies (ly) are visible, x 360. F~G. 4. Showing ends of threadlike structures in gut lumen. The field is similar to that indicated by an arrow in Fig. 3. The threads consist of polygonal saccules (c) containing dark vesicles (fz). Around the edges of the threads are flattened sacs (s) and between them are seen "myelin figures" (mf) corresponding to the ringlike structures (r) seen in Fig. 3. x 12 000. FIG. 5. Transverse section through apical region of epithelial cells. Showing the thick surface coat (sc) extending from the microvilli (my). It consists of polygonal saccules (c) containing vesicles (fz) and flattened sacs (s). × 8 000.
Z
Z
PLEXIFORM SURFACE COAT IN INSECT MIDGUT
165
166
MARSHALL AND CHEUNG
sections, the threadlike structures are wider and pack the gut lumen more than in paraffin wax sections (Fig. 3). They are extensions of a coat which overlays, and is distinct from, the brush border (Fig. 3).
Ultrastructure The midgut cells (Fig. 2) have a well developed rough endoplasmic reticulum, abundant Golgi bodies and mitochondria. Large lysosome-like bodies are present. The microvilli have fibrillar cores which extend into the subjacent cytoplasm. There is no sign of secretory material passing into or between the microvilli. The long microvilli are remarkable in that they frequently appear to have two or more triple-layered plasma membranes, 40-60 A thick (Figs. 6-8). It is not possible to see whether each layer of plasma membrane completely envelops each microvillus, but transverse sections (Fig. 8) suggest that they may nearly do so. The tips of the microvilli are covered with a "fuzz" of filaments and granules (Figs. 6 and 7). Double plasma membranes frequently cover the tips and the outer one sometimes appears to be separated from the inner one (Fig. 7). Between the microvilli are small membrane vesicles and sheets of membrane that extend out into the lumen beyond the microvilli tips (Figs. 6, 7, 9, and 11). These membranes are triple-layered and 40-60 A thick. Superjacent to the microvilli is a plexiform surface coat 2-4 # thick (Fig. 5). This coat extends into the gut lumen (Fig. 4) as the threadlike structures seen in the light micrographs. This coat has two layers, an outer layer of saccules and an inner layer of flattened saclike structures (Fig. 5). The former consists of polygonal saccules of membranes (Figs. 11 and 12) which contain vesicles (Figs. 7, 11, and 12) from which a "fuzz" of filaments and granules radiates to fill a saccule (Fig. 12). The inner layer of flattened saclike structures also consists of membranes (Fig. 10). These ultimately appear to form aggregated whorls or "myelin figures" (Figs. 3 and 4) which correspond to the ring structures seen in Fig. 3. The membranes of the saccules, flattened sacs, and "myelin figures" appear to exhibit a triple-layered and a globular structure (Figs. 10 and 12).
Cytochemistry Cytochemical tests were carried out on sections observed in the light microscope. The results are summarized in Table I. FIo. 6. Longitudinal section of microvilli partially ensheathed by double plasma membranes (arrows). The tip of each microvillus bears a "fuzz" of granules and fine filaments. Between the microvilli are sheets (rn) and small vesicles (v) of membrane, x 160 000. FIG. 7. Longitudinal section of microvilli showing separation of double plasma membranes at a microvillus tip (arrow). A fuzz-bearing vesicle (fz) having the appearance of a "nipped-off" microvillus tip is seen. x 160 000. FIo. 8. Transverse section of microvilli showing double plasma membranes, x 120 000.
o >H
>c~
o
168
MARSHALL AND CHEUNG
TABLE I CYTOCHEMICAL TESTS ON THE INTESTINAL EPITHELIAL CELLS OF
Surface coat
Test
Alcian blue Toluidine blue Gentian violet Mucicarmine Bismarck brown Hale's test PAS Acid sol©chrome cyanine Mercury-bromophenol blue Sudan Black B Alkaline phosphatase c~-Glucosidase Leucylaminopeptidase
(17) (20) (12) (12) (12) (12)
Microvilli
+ + + ++++ ~ ++++ + + + + + + ++++
+ + + ++++b ++++ + + + + + + ++++
Fulgora candelaria ~
Apical cytoplasm
Basal cytoplasm
+ O o o O ©
o o o o O ©
Fixative
(20)
+ + +
+ + + +
+ +
+
(20)
+
+ + + +
+ + +
+
Formol saline Formolsaline Formolsaline Formol saline Formol saline Formolsaline Bouin Formol calcium
(20) (17) (20) (1) (3)
+ + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + +
+ + o o +
Formol calcium Neutral formalin 95 % alcohol, 4°C Neutral formalin, 4°C Acetone, 4°C
a + positive reaction, the number of +'s being proportional to the intensity of the reaction; o, a negative reaction. Numbers in parentheses refer to references. b 7-Metachromasia. DISCUSSION T h e p l e x i f o r m s u r f a c e c o a t o n t h e m i d g u t e p i t h e l i a l cells of Fulgora candelaria a p p e a r s to be u n i q u e . It is c o m p o s e d of a n e t w o r k of m e m b r a n e saccules, c o n t a i n i n g vesicles, a n d f l a t t e n e d m e m b r a n e sacs. T h e o r i g i n of t h e d i f f e r e n t t y p e s of s t r u c t u r e s seen in t h e b r u s h b o r d e r a n d s u r f a c e c o a t is d i f f i c u l t to e x p l a i n . I t s e e m s likely t h a t t h e f u z z - b e a r i n g vesicles c o u l d arise as "nipped-off"
m i c r o v i l l i tips since t h e r e is a s t r o n g m o r p h o l o g i c a l r e s e m b l a n c e be-
t w e e n t h e s e t w o entities in e l e c t r o n m i c r o g r a p h s . T h e i n n e r , l u m i n a l , l a y e r of f l a t t e n e d sacs c o u l d b e a r e s u l t of t h e m e m b r a n e s a c c u l e s l o s i n g t h e i r c o n t e n t s . H o w e v e r , t h e m o d e of o r i g i n of t h e m e m b r a n e
saccules themselves and the membranes
found
b e t w e e n a n d c l o s e l y i n v e s t i n g t h e m i c r o v i l l i is d i f f i c u l t to e n v i s a g e . A d o u b l e m e m b r a n e is r e g u l a r l y seen a r o u n d e a c h m i c r o v i l l u s . T h i s c a n be explained by suggesting that the microvilli project into the overlying surface coat and FIG. 9. Longitudinal section of microvilli showing membranes (m), between the microvilli, extending out into the gut lumen and apparently enclosing microvilli tips and fuzz-bearing vesicles (fz). x 42 000. FIG. 10. Membranes of flattened sacs and part of a "myelin figure" showing triple-layered structure and a suggestion of globular structure (arrows). Slightly underfocused, x 165 000. Fro. 11. Similar to Fig. 9. x 80 000. Fia. 12. Polygonal saccules of surface coat containing vesicles from which radiate filaments and granules. The membranes show a triple-layered structure and globular structure (arrows). × 165 000.
C~
170
MARSHALL AND CHEUNG
that they are "clothed" by the membranes of the surface coat. A similar topological situation has been described in the chaetognathan photoreceptor cell by Eakin and Westfall (7). This, however, still does not explain whence the surface coat membranes originate. Several explanations seem to be possible: (a) Whole or parts of microvilli are "nipped-off" and the contents dispersed to leave "ghosts." The "nipping-off" of parts of microvilli is not a novel phenomenon and has been previously observed in gryllid and grasshopper Malpighian tubules (2, 4). (b) The membranes arise by coalescence of membrane vesicles extruded from the microvilli. Extrusion of vesicles from microvilli has also been reported in insect Malpighian tubules, e.g. Wigglesworth and Salpeter (31). (c) The surface coat membranes are delaminated from or sloughed off the microvilli. The latter is a somewhat radical suggestion, but the notion of plasma membrane delamination has been previously advanced in the lamellar apposition theory of xylem cell wall formation (28). The present study has not produced evidence which favors unambiguously any one of these theories, and the theories themselves must be viewed as being entirely speculatory. There is a suggestion of globular structure in the membranes of the plexiform coat which is reminiscent of the globular structures of mitochondrial and ~-cytomembranes described by Sj~Sstrand (24). We cannot claim in this study to have unequivocally established such structure as real and not artifactual. However, the membranes of the microvilli and plexiform coat are of the same order of thickness as Sj~Sstrand's mitochondrial membranes and ~-cytomembranes in which globular structure occurs, and they are thinner than the average plasma membrane in which globular structure does not occur. Cytochemically, the plexiform coat has the same characteristics as the usual glycocalyx. It appears to be definable as a PAS-positive acid mucopolysaccharide. This could certainly be accounted for if it is assumed that the fuzz of the vesicles has its origin as glycocalyx on the tips of the microvilli. Traces of enzyme activity are found in the plexiform coat. It is striking, however, that activity is considerably lower than in the microvilli, particularly in view of the contention of some authors (9, 16) that at least some enzymes are located in the plasma membrane of microvilli, and of others (10, 17, 18, 22) that enzymes may be associated with the glycocalyx. The function of the plexiform surface coat can hardly be guessed at. Indeed, no clear-cut evidence is available which is indicative of either the function of surface coats or insect peritrophic membranes in general. Insect peritrophic membranes are usually regarded as protective devices (30) although their porous nature (21, 29) is suggestive of a sievelike function. Peritrophic membranes are usually said to be absent in homopteran insects (13); indeed, their absence in these sap-sucking insects is frequently given as evidence for their protective function in other insects. However,
PLEXIFORM SURFACE COAT IN INSECT MIDGUT
171
in the midgut of one homopteran insect, a PAS-positive peritrophic membrane has been described (14) which consists of "filaments and fibrils." This membrane appears to have similar cytochemical properties to the plexiform surface coat described here. Moreover, Gouranton and Maillet (14) point out the similarity between the filamentous component and the glycocalyx of m a m m a l i a n intestine. It is not possible to see in their micrographs whether the "fibrils" are membranes; there are, however, structures visible which appear to be similar to our fuzz-bearing vesicles. It is tempting to suggest that this type of membrane may be a variation of the plexiform coat described by us. Similarly, regions of the midgut of cercopids (19) and other closely related homopteran insects (Cheung and Marshall, unpublished data) have a mucopolysaccharide coating, and a PAS-positive coating is secreted by the crop epithelium of aphids (8). Threadlike structures similar to those in the fulgorid gut have been described in some heteropteran insects (12). It seems that there may be some correlation between the presence of a mucopolysaccharide coating and sap-sucking and that such coatings, although perhaps produced in a variety of ways, may all ultimately fall within a loose definition of the insect peritrophic membrane. This work was facilitated by the use of a field vehicle donated to the Zoology Department, University of Hong Kong, by Mr. Ma Ying. We are indebted to Miss J. Halder, Zoology Department, University of Hull for her skillful photographic assistance. One of us, W. W. K. C., gratefully acknowledges the receipt of a Commonwealth Scholarship (U. K. award), during the tenure of which part of this work was done.
REFERENCES
1. BARKA,T. and ANDERSON,P. J., Histochemistry: Theory, Practice, and Bibliography. Hoeber, London, 1963. 2. BEAMS, H. W., TAHMISIAN,T. N. and DEVINE, R. L., J. Biophys. Biochem. Cytol. 1, 197 (1955). 3. BENNETT,H. S., J. Histochem. Cytochem. 11, 14 (1963). 4. BERKALOFF,A., C. R. Acad. Sci. 250, 2609 (1960). 5. BURSTONE,M. S. and FOLK, J. E., J. Histochem. Cytochem. 4, 217 (1956). 6. DE METS, R. and JEUGNIAUX,C., Arch. Int. Physiol. Biochim. 70, 93 (1962). 7. EAKIN, R. M. and WESTFALL,J. A., J. Cell Biol. 21, 115 (1964). 8. EDWARDS, J. S., J. Insect Physiol. 12, 31 (1966). 9. ElCnOLZ, A. and CRANe, R. K., J. Cell Biol. 26, 687 (1965). 10. FAWCETT,D. W., J. Histochem. Cytochem. 13, 75 (1965). 11. - An Atlas of Fine Structure. The Cell. Saunders, Philadelphia, 1966. 12. GOODCHILD,A. J. P., Trans. Roy. Entornol. Soc. London 115, 217 (1963). 13. - Biol. Rev. Cambridge Phil. Soc. 41, 97 (1966).
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14. GOURANTON,J. and MAILLET,P. L., C. R. Acad. Sci. 261, 1102 (1965). 15. GURR, G., Methods of Analytical Histology and Histochemistry. Leonard Hill, London, 1958. 16. ITO, S., J. Cell BioL 27, 475 (1965). 17. JOHNSON,C. F., J. Lab. Clin. Med. 68, 883 (1966). 18. - in Ryozi, U. (Ed.), Electron Microscopy, Vol. II, p. 389. Maruzen, Tokyo, 1966. 19. MARSHALL,A. T., J. Insect Physiol. 12, 635 (1966). 20. MCMANuS, J. F. A. and MOWRY, R. W., Staining methods, Histologic and Histochemical. Hoeber, London, 1960. 21. MERCER, E. H. and DAY, M. F., Biol. Bull. 103, 384 (1952). 22. ODA, T. and SEKI, S., in RYozI, U. (Ed.), Electron Microscopy, Vol. II, p. 387. Maruzen, Tokyo, 1966. 23. PEARSE, A. G. E., Histochemistry, Theoretical and Applied. Churchill, London, 1960. 24. SJ6STRAND, F. S., in DALTON, A. J. and HAGENAU, F. (Eds.), p. 151. The Membranes, Academic Press, London, 1968. 25. SMITH, D. S., Insect Cells. Their Structure and Function. Oliver & Boyd, Edinburgh, 1968. 26. STEEDMAN,H. F., Section Cutting in Microscopy. BlackweU, Oxford, 1968. 27. TRIER, J. S., Fed. Proc. 26, 1391 (1967). 28. WARDROP,A. B., in C6t6, W. A. (Ed.), Cellular Ultrastructure of Woody Plants. Syracuse Univ. Press, New York, 1965. 29. WATERHOUSE,D. F. and WRIGHT, M., J. Inseet Physiol. 5, 230 (1960). 30. WIGGLESWORTH,V. B., The Principles of Insect Physiology, 6th ed. Methuen, London, 1965. 31. WIGGLESWORTH,V. B. and SALPETER,M. M., J. Inseet Physiol. 8, 299 (1962).