- Email: [email protected]
Fine structure of the Malpighian tubules in the housefly, Musca domestica
TISSUE & CELL 1974 6 (4) 719-728 Published by Longman Group Ltd. Printed in Great Britain
R. S. SOHAL
FINE STRUCTURE TUBULES IN THE DOMESTICA
OF THE MALPIGHIAN HOUSEFLY, MUSCA
ABSTRACT. The epithelium of the Malpighian tubules in the housefly is comprised of four distinct cellular types. Type I cells are characterized by the presence of intimate associations between infoldings of basal plasma membrane and mitochondria. On the luminal surface, cytoplasm is extended into microvilli which contain mitochondria. Membrane-bound vacuoles in the cytoplasm seem to progressively accumulate granular material. Type II cells have dilated canaliculi. Microvilli lack mitochondria. The Type III cell has not been reported previously in Malpighian tubules. It has very welldeveloped granular endoplasmic reticulum which contains intracisternal bundles of tubules. Cytoplasm contains numerous electron dense bodies. Type IV cells occur in the common duct region of the Malpighian tubules. Mitochondria do not extend into the microvilli.
Introduction
The present study is concerned with the fine structural organization of the Malpighian tubules in the housefly, Musca domestica.
STUDIESon the physiology and ultrastructure of Malpighian tubules in insects indicate a remarkable diversity in their organization in different species, which is probably related to differences in water balance, nutrition, and acquisition of specialized functions (for references see Wigglesworth, 1965; Smith, 1968; Maddrell, 1971). The primary function of Malpighian tubules is the excretion of nitrogenous wastes from the hemolymph. They discharge a nearly isosmotic secretion into the hindgut where this fluid may be modified by the selective absorption of useful substances. Reabsorption of useful substances may also be augmented by the activity of cells located in the Malpighian tubules (Shaw and Stobbart, 1963; Wigglesworth, 1965). Due to their simple tubular shape, convenient size, and easy access, the Malpighian tubules provide a very suitable material for the investigation of functional-morphological relationships in a fluid transporting tissue. Department of Biology, Southern University, Dallas, Texas 75275.
Materials and Methods
Malpighian tubules were obtained from 5 to 10 day old laboratory reared adult female houseflies. Flies were fed a mixture of sucrose, dried milk and powdered egg yolk (6 : 6 : 1). Malpighian tubules were fixed in phosphate buffered 4 % glutaraldehyde containing 4.5% sucrose, pH 7.2 for 13 hr. Following several washes in phosphate buffer, tissues were postfixed in phosphate buffered 1% osmium tetroxide for 1 hr and were embedded in Maraglas. Thin sections were stained with uranyl acetate and lead citrate. The present description is based on the study of Malpighian tubules from about 30 flies and of about 400 electron micrographs. Observations
A single pair of Malpighian tubules is present in the housefly. Each tubule consists of two moniliform segments which fuse proximally, forming a short common duct. This duct
Methodist
Received 4 March 1974. Revised 17 May 1974. 719
SOHAL
joins the alimentary tract at the junction of mid- and hindgut (see Hewitt, 1914, p. 38). The distal, middle, and proximal regions of the main segment, and the common duct region of the Malpighian tubule were examined. Three types of cells intermingle to form the main segment, while a fourth cellular type is exclusively observed in the common duct. Arbitrarily, these cell types are designated as Type I, Type II, Type III and Type IV cells. Type I cell. These cells occur most frequently along the length of the main segment. On their basal surface, plasma membrane is extensively infolded, penetrating more than halfway through the thickness of the cell (Fig. 1). The infolded membranes follow a tortuous path and anastomose, resulting in a network of canalicular spaces. Canaliculi have low electron density and open into the space facing the basement membrane. Diameter of the canaliculi is variable. Midway between the basal surface and the center of the cell, infolded plasma membranes or canaliculi show a characteristic association with mitochondria. Two or more mitochondria become closely apposed enclosing
portions of the canaliculi (Figs. 2, 3). Cristae of these mitochondria are usually longitudinally oriented. These profiles resemble the ‘mitochondrial pump’ configurations described by Copeland (1964) in the anal papillae of Culex quinquefasciatus larvae. On the luminal surface, the cytoplasm is evaginated forming closely packed microvilli (Fig. 1). Mitochondria extend into most of the microvilli. Occasional pinocytotic vesicles are associated with the luminal plasma membrane. The cytoplasm is well populated with granular endoplasmic reticulum (GER) and free polyribosomes. The length of GER cisternae is variable, but their diameter is relatively constant (250 A)). Several dense tubuli with diameters similar to those of the GER cisternae are interspersed in the perinuclear region (Fig. 4). Similar structures have been observed in the epithelial cells of midgut in Musca (Sohal, unpublished) and in Culliphora (Priester, 1971). Smooth endoplasmic reticulum is difficult to identify in these cells due to its morphological similarity to the profiles of infolded plasma membrane. Many clear and granulated vacuoles occur in the cytoplasm. They are surrounded by a
Fig. 1. Transverse section through the Malpighian tubule showmg Type 1 cell. Microvilli (MV) on the luminal surface are closely packed together and contain mitochondria. Inflections of the basal plasma membrane (arrows) anastomose to form a network of canaliculi. Several membrane bound vacuoles (V) are seen in the cytoplasm. x 21,000. Fig. 2. Type I cell showing close approximations between infoldings of basal plasma membrane (arrow). x 26,000.
mitochondria
(M) and
Fig. 3. A high magnification view of mitochondria-canaliculi association. Canalicular membranes appear thicker than the outer membrane of mitochondria. x 43,000. Fig. 4. Dense tubuli (arrow) in the perinuclear Fig. 5. Membrane-bound material. Profiles labelled material. x 30,000.
region of Type I cell. x 45,000.
vacuoles in Type I cell showing varying amounts of dense l-5 seem to indicate progressive accumulation of dense
Fig. 6. Some of the mitochondria cristae and the presence of granular
(arrows) in Type I cell showing material. x 30,000.
Fig. 7. Type 11 cell. Note that the canaliculi (arrows) do not extend into the microvilli (MV). x 39,000.
a loss of their
are dilated and mitochondria
MALPIGHIAN
TUBULES
IN THE
HOUSEFLY
limiting membrane and contain variable amounts of granular or amorphous material. Profiles which may represent progressive accumulation of granular material in the membrane-bound vacuoles are shown in Fig. 5. These vacuoles do not appear to be identical to the transversely sectioned canalicular spaces for the following reasons. The diameter of the vacuoles is usually much greater than the longitudinally sectioned canaliculi. Profiles indicating unequivocal continuations between membranes of the vacuoles and plasma membrane are never observed. Furthermore, materials of detectable electron density are not seen in the canaliculi. Mitochondria are predominantly discoidal in shape and tend to align at right angles to the longitudinal axis of the Malpighian tubules. Several mitochondria exhibit a reduction in the number of their cristae or even a complete loss of cristae and contain a fine granular or dense material (Fig. 6). These profiles suggest degeneration of mitochondria, which have also been observed in Malpighian tubules of Rhodnius (Wigglesworth and Salpeter, 1962) and Macrosteles (Smith and Littau, 1960). The Golgi material is present in the perinuclear region and is not as well developed as that found in protein secreting cells. Type II cell. These cells are much smaller in diameter than Type I cells and can be easily recognized in cross-sections of the Malpighian tubules as forming a relatively thin wall around the lumen. They occur throughout the main segment of the tubule, but are less frequent than Type I cells. The basal plasma membrane is extensively infolded. The canaliculi are noticeably wider than in the Type I cell and extend relatively deeper into the cell (Fig. 7). Mitochondria do not extend into the microvilli and are relatively fewer in number than in Type I cells. Granulation of mitochondria is also observed in these cells. Vacuoles occur only rarely. GER is relatively poorly developed. These cells frequently adjoin Type I cells forming septate desmosomes along a portion of their intercellular junction. Type IZi cell. The major difference between Type III and Type I cells is that GER is very well developed and occupies a large
723
proportion of the cytoplasm in the former cells (Figs. 8, 9). Cisternae of GER are quite distended and contain spirally oriented aggregates of tubuli in their lumina (Fig. 9). The number of individual bundles within a single cisterna as well as their diameter is variable. The diameter of a single tubule is approximately 100 A. The GER cisternae branch profusely forming a vast anastomosing network of intracisternal spaces in the central region of the cell. The size of these cells is similar to that of the Type I cell. Plasma membrane on the basal surface is extensively infolded, but the characteristic associations between the mitochondria and the infoldings of plasma membrane such as those observed in Type I cells are less frequent. Microvilli on the luminal surface contain elongated mitochondria. Another interesting feature of this cell type is the presence of many uniformly dense and rounded structures in the cytoplasm (Fig. 10). It is often difficult to resolve the inner structure or the presence of a limiting membrane around these dense bodies, although in some instances electron lucent vesicles occur in their cortical regions (Fig. 10). Structures with a similar morphological appearance have been identified as ommochrome pigments in the ‘red’ mutant of Drosophila melanogaster (Wessing and Bonse, 1966). It is also possible that such dense bodies are composed of highly unsaturated lipid droplets (Fawcett, 1966). Vacuoles with an electron lucent interior as well as those containing dense granular material are also present in these cells. Whether the above-mentioned intensely dense bodies arise from the accumulation of materials in the vacuoles is uncertain. Occasionally, a few dense bodies are also seen in Type I cells. Mitochondria are mostly aggregated in the basal and luminal region of the cell. It is noteworthy that some mitochondria contain an array of tubules in their matrices which are about 150 A in diameter (Fig. 11). The center-to-center spacing of these tubules is of the order of 250 A. Type IV cell. These cells form the epithelial lining of the common duct in its entirety. They resemble Type I cells in their cytoarchitecture except that mitochondria do not extend into the microvilli (Fig. 12).
SOHAL
124
Clusters of undifferentiated regenerative cells occur in the basal region of the common duct. Undifferentiated cells do not extend up to the lumen. Muscle fibers with morphological features similar to those of visceral muscles of other insects (Smith et al., 1966) lie on the outer surface of the common duct. Neural innervation of these muscle fibers was not observed. The lumen of the Malpighian tubule in the main segment as well as in the common duct contains numerous spherical masses which are composed of needle-like material. Radially concentric spherical bodies within the lumen (Smith, 1968) are seen only rarely. Discussion Results of the present study indicate that the main segments of the Malpighian tubules in the housefly are composed of three distinct types of randomly distributed cells and do not show any regional structural differences along their length. Cells in the common duct constitute a fourth type of cell. In contrast, ultrastructural variations in different linear regions of the Malpighian tubules have been reported in MacrosteIes (Homoptera) (Smith and Littau, 1960), Rhodnias (Hemiptera) (Wigglesworth and Salpeter, 1962), Dams (Diptera) (Baccetti et al., 1963; Mazzi and Baccetti, 1963), Drosophila (Diptera) (Wessing and Eichelberg, 1969), Cenocorixa (Hemiptera) (Jarial and Scudder,
1970), and Carausius (Orthoptera) (Taylor, 1971a, b). At the phsyiological level, regional differentiation of the Malpighian tubules has been demonstrated in Rhodnius (Wigglesworth, 1931) and Dixippus (Ramsay, 1955). The distal region of the Malpighian tubule in these insects transports water and solutes from the hemolymph to the lumen, whereas in the proximal region, some of the substances are reabsorbed from the lumen. An ultrastructural comparison of distal and proximal regions of the tubule in Rhodnius indicates that the microvilli in the distal region are closely packed together, whereas they are more widely spaced in the proximal region (Wigglesworth and Salpeter, 1962). Otherwise, the cells in the two regions are structurally quite similar. It therefore seems unfeasible at present to identify the specific structural mechanisms which may underlie the physiological transport of solutes in the Malpighian tubules. Type I cells in the housefly are essentially similar in structure to those in the distal region of the Malpighian tubule in Rhodnius and the ‘primary’ cells in Calhphora (Berridge and Oschman, 1969) and may be secretory in nature. It is relatively well established that potassium ions are transported from the hemolymph into the lumen against a concentration gradient and are essential for the normal fluid secretion by the Malpighian tubules (Maddrell, 1971). It is tempting to speculate that the mitochondrial associations
Fig. 8. Type III cell. Granular endoplasmic reticulum is very well developed. Rods of dense material (arrows) are seen in the lumen of GER cisternae. Several highly dense bodies (D) are seen in the cytoplasm. x 20,000. A high magnification view showing dense material in the lumen of GER cisternae is presented in the inset. x 71,000. Fig. 9. A high magnification view of GER in a Type III cell. The intracisternal material is comprised of slender tubules (arrow). x 64,000. Fig. 10. Dense bodies from Type III ceil. Note the presence vesicles (arrow) in the cortex of a dense body. x 48,000.
of electron
Fig. Il. A mitochondrion in the matrix. x 59,000.
of tubules
from Type III cell showing
an array
Fig. 12. Luminal region of a Type IV cell. Note the absence of mitochondria microvilli (MV). x 16,000.
lucent (arrow) in the
MALPIGHIAN
TUBULES
IN THE
HOUSEFLY
with the plasma membrane infoldings in the basal region of Type I cells may represent possible sites for the active transfer of ions. The spherical or ovoid granules within the epithelial cells of the Malpighian tubules have been observed in several insects such as Rhodnius (Wigglesworth and Salpeter, 1962), GryIZus (Berkaloff, 1958, 1959, 1960); Cenocorixa (Jarial and Scudder, 1970) and Drosophila (Wessing and Eichelberg, 1969). In Rhodnius, they have been considered to be mineralized deposits which do not seem to contain any urates. Spherical bodies referred to as ‘concretions’ accumulate in epithelial cells of midgut in cercopid larvae and adults by storage excretion (Gouranton, 1968). Cytochemical and biochemical studies indicate that they are composed of various minerals, proteins and mucopolysaccharides. In Musca, the accumulation of materials in the vacuoles resulting in the formation of granular bodies may also indicate storage of excretory material since profiles indicating extrusion of these bodies into the lumen were not encountered. Furthermore, large quantities of dense bodies which are somewhat similar in structure to the storage excretion granules observed by Gouranton occur in Malpighian tubules of aged houseflies (Sohal, unpublished). Cytochemical studies on the nature of this material in old flies are currently in progress. There is considerable confusion regarding the mode of origin of the vacuoles, accumulation of contents within the membrane-bound structures, as well as the mechanism of secretion of materials into the lumen of the Malpighian tubules. Wigglesworth and Salpeter (1962) have suggested that granular bodies arise from degenerating mitochondria. On the other hand, Wessing and his associates have suggested various ways by which materials could be transported from the hemolymph to the lumen of the Malpighian tubules in the larvae of D. meianogaster. Following concentration of materials in the canaliculi, the materials are segregated into
727
vacuoles and vesicles which pinch off the infolds of the basal plasma membrane. The contents are then released at the luminal surface by a mechanism similar to merocrine secretion (Wessing, 1968; Eichelberg and Wessing, 1971; Wessing and Eichelberg, 1969, 1972). In Musca, some of the granular bodies arise from the mitochondria; however, more frequently, granular bodies seem to arise from gradual accumulation of material within the vacuoles. As has been mentioned above, profiles suggesting plasma membrane origin of vacuoles or the release of electron dense material into the lumen are not observed in the housefly. Type II cells in Musca which lack mitochondria in the microvilli and have wide canalicular spaces resemble ‘stellate cells’ in Malpighian tubules of Calliphora (Berridge and Oschman, 1969). These authors have suggested that stellate cells may be concerned with the reabsorption of sodium from the luminal medium or from the primary cells via septate desmosomes. In this regard, it is interesting to note that the canalicular spaces in the rectal papillae of Aedes aegypti also dilate following a blood meal presumably due to the accumulation of absorbed material before its drainage into the hemolymph (Hopkins, 1967). To the best of my knowledge, Type III cells have not been previously reported at the fine structural level. Although the chemical nature of the intracisternal precipitate is unknown, on the basis of the generally recognized function of GER it can be suggested that this material is proteinous in nature. Crystalline rods of ‘proteinous’ material have been reported in GER cisternae in hepatocytes of salamander (Fawcett, 1966) and in a clone of cells in lamina propria of man (Sane1 and Lepore, 1968). Mucocomplexes are known to be produced in the Malpighian tubules of a number of insect species and possibly function as a lubricant or a surfactant (Gabe, 1962; Marshall, 1966).
References BACCETTI,B., MAZZI, V. and MASSIMELLO,G. 1963. Richerche istochimiche e al microscopico elettronico sui tubi Malpighiani di Dacus oleae. Gmel II. L’adulto. Redia, 48, 47-68. BERKALOFF,A. 1958. Les grains de secretion des tubes Malpighi de GryNus domesticus (Orthoptire, Gryllidae). C. r. hebd. Skmc. Acad. Sri., Paris, 246, 2807-2809.
728
SOHAL
BERKALOFF,A. 1959. Variations de I’ultrastructure des tubes de Malpighi et leur functionement chez GQ&(J domesticus (Orthoptere, Gryllidae). C. r. hebd. SPanc. Acad. Sci., Paris, 248, 466469. BERKALOFF,A. 1960. Contribution a l’etude des tubes de Malpighi et de I’excretion chez les insectes. Observations au microscope electronique. Ann. Sri. Nat. Zoo/. biol. anim., 12, ser. 2, 869-947. BERRIDGE, M. J. and OSCHMAN, J. L. 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue & Cell, 1, 247-272.
COPELAND, D. E. 1964. A mitochondrial
pump in the cells of the anal papillae
of mosquito
larvae. /. Cc/l
Bit&, 23, 253-263.
EICHELBERG,D. and WESSING,A. 1971. Elektronenoptische Untersuchungen an den Nirentubuli (Malpighische Gefasse) von Drosophila mekmogaster. 11. Transzellulare membrangebundene Stofftransportmechanismen. Z. Zellforsch. mikrosk. Anat., 121, 127-l 52. FAWCET~, D. W. 1966. The CeN. Saunders, Philadelphia. GABE, M. 1962. In Handbuch der Histochemie, Band II. Polysaccharide (eds. W. Graumann and K. G. Neumann). Fischer Verlag, Stuttgart. GOURANTON,J. 1968. Composition, structure, et mode de formation de concretions minerales dans l’intestine moyen des homopteres cercopides. J. Cell Biol., 37, 3 16-328. HEWITT, C. G. 1914. The Housefly. University Press, Cambridge. HOPKINS, C. R. 1967. The fine-structural changes observed in the rectal papillae of the mosquito A&s aegypti, L. and their relation to the epithelial transport of water and inorganic ions. J. R. microsc. Sot.. 86, 235-252. JARIAL, M. S. and SCUDDER, G. G. E. 1970. The morphology and ultrastructure of the Malpighian tubules and hindgut in Cenocorixa bifido (Hung.) (Hemiptera, Corixidae). Z. Morph. iikol. Tiere, 68, 269-299. MADDRELL, S. H. P. 1971. The mechanisms of insect excretory systems. In Adv. Insect Physiol. (eds. J. W. L. Beament, I. E. Treherne and V. B. Wigglesworth), Vol. 8, pp. 199-33 1. Academic Press, London. MARSHALL, A. T. 1966. Histochemical studies on a mucocomplex in the Malpighian tubules of cercopid larvae. J. Insect Physiol., 12, 925-932. MAZZI, V. and BACCETTI, B. 1963. Richerche istochimiche e al microscopio elettronico sui tubi Malpighiani di Dot-us olew. Gmel. I. La larva. Z. Zel(for.wh. mikrosk. Anal., 59,47-70. PRIESTER, W. D. 1971. Ultrastructure of the midgut epithelial cells in the fly Calliphora er>lthrocephaln. J. Ultrastruct.
Rex., 36, 783-805.
RAMSAY,J. A. 1955. The excretion of sodium, potassium and water by the Malpighian tubules of the stick insect Diwippus morons (Orthoptera : Phasmidae). J. exp. Biol., 32, 200-216. SANEL, T. and LEPORE, M. I. 1968. Granular and crystalline deposits in perinuclear and ergastoplasmic cisternae of human lamina propria. Expl. molec. Path., 9, 110-124. SHAW, J. and STOBBART,R. H. 1963. Osmotic and ion regulation in insects. In Adv. fnswt Physiol. teds. J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth), Vol. I, pp. 315-399. Academic Press, London. SMITH, D. S. 1968. Insecf Ceils, Their Structure and Fwrctiot~ Oliver and Boyd, Edinburgh. SMITH, D. S. and LITTAU, V. C. 1960. Cellular specializations in the excretory epithelia of an insect, Mrwosteles,fascifrons St&l (Homoptera). J. biophys. biochcm. Cytol., 8, 103-I 33. SMITH, D. S., GUPTA, B. L. and SMITH, U. 1966. The organization and myofilament array of insect visceral muscles. J. Cell Sri., 1, 49-57. TAYLOR, H. H. 197la. Water and solute transport by the Malpighian tubules of stick insect, Carurrsius morosus. The normal ultrastructure of type I cells. 2. Zehforsch. mikrosk. Anat., 118, 333-368. TAYLOR, H. H. 1971 b. The fine structure of the type II cells in the Malpighian tubules of the stick insect, Carausius morosus. Z. Zellforsch.
mikrosk.
Anat., 122, 41 l-424.
WESSING, A. 1960. Funktionsmorphologie von Exkretionsorganen bei insekten. Zoo/. AK., 31, suppl.. 633-681. WESSING, A. and BONSE. A. 1966. Natur und Bildung des roten Farbstoffes in den Nierentubuli der mutante ‘red’ von Drosophila melanogaster. Z. Naturf., 21b, 1219-1223. WESSING, A. and EICHELBERG,D. 1969. Elekronenoptische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster. I. Regionale Gliederung der Tubuli. Z. Zel(forsch. mikrosk. Anat., 101, 285-322.
WESSING,A. and EICHELBERG,D. 1972. Elekronenmikropische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster. IV. Die Lokalisation von Natrium und Chloridionen. 2. ZeUforsch.
mikrosk.
An&.,
131, 269-286.
WIGGLESWORTH,V. B. 1931. The physiology of excretion in a blood-sucking insect, Rhodmu prolix-us Stal (Hemiptera, Reduviidae). III. The mechanism of uric acid secretion. J. up. Biol., 8, 443-451. WIGGLESWORTH,V. B. 1965. The Principles of § Physiology, 6th edn. Methuen, London. WIGGLESWORTH,V. B. and SALPETER, M. M. 1962. Histology of the Malpighian tubules in Rhodnirts proli.vrfs Stll (Hemiptera). 1. fnsect Physiol., 8, 299-307.
R. S. SOHAL
FINE STRUCTURE TUBULES IN THE DOMESTICA
OF THE MALPIGHIAN HOUSEFLY, MUSCA
ABSTRACT. The epithelium of the Malpighian tubules in the housefly is comprised of four distinct cellular types. Type I cells are characterized by the presence of intimate associations between infoldings of basal plasma membrane and mitochondria. On the luminal surface, cytoplasm is extended into microvilli which contain mitochondria. Membrane-bound vacuoles in the cytoplasm seem to progressively accumulate granular material. Type II cells have dilated canaliculi. Microvilli lack mitochondria. The Type III cell has not been reported previously in Malpighian tubules. It has very welldeveloped granular endoplasmic reticulum which contains intracisternal bundles of tubules. Cytoplasm contains numerous electron dense bodies. Type IV cells occur in the common duct region of the Malpighian tubules. Mitochondria do not extend into the microvilli.
Introduction
The present study is concerned with the fine structural organization of the Malpighian tubules in the housefly, Musca domestica.
STUDIESon the physiology and ultrastructure of Malpighian tubules in insects indicate a remarkable diversity in their organization in different species, which is probably related to differences in water balance, nutrition, and acquisition of specialized functions (for references see Wigglesworth, 1965; Smith, 1968; Maddrell, 1971). The primary function of Malpighian tubules is the excretion of nitrogenous wastes from the hemolymph. They discharge a nearly isosmotic secretion into the hindgut where this fluid may be modified by the selective absorption of useful substances. Reabsorption of useful substances may also be augmented by the activity of cells located in the Malpighian tubules (Shaw and Stobbart, 1963; Wigglesworth, 1965). Due to their simple tubular shape, convenient size, and easy access, the Malpighian tubules provide a very suitable material for the investigation of functional-morphological relationships in a fluid transporting tissue. Department of Biology, Southern University, Dallas, Texas 75275.
Materials and Methods
Malpighian tubules were obtained from 5 to 10 day old laboratory reared adult female houseflies. Flies were fed a mixture of sucrose, dried milk and powdered egg yolk (6 : 6 : 1). Malpighian tubules were fixed in phosphate buffered 4 % glutaraldehyde containing 4.5% sucrose, pH 7.2 for 13 hr. Following several washes in phosphate buffer, tissues were postfixed in phosphate buffered 1% osmium tetroxide for 1 hr and were embedded in Maraglas. Thin sections were stained with uranyl acetate and lead citrate. The present description is based on the study of Malpighian tubules from about 30 flies and of about 400 electron micrographs. Observations
A single pair of Malpighian tubules is present in the housefly. Each tubule consists of two moniliform segments which fuse proximally, forming a short common duct. This duct
Methodist
Received 4 March 1974. Revised 17 May 1974. 719
SOHAL
joins the alimentary tract at the junction of mid- and hindgut (see Hewitt, 1914, p. 38). The distal, middle, and proximal regions of the main segment, and the common duct region of the Malpighian tubule were examined. Three types of cells intermingle to form the main segment, while a fourth cellular type is exclusively observed in the common duct. Arbitrarily, these cell types are designated as Type I, Type II, Type III and Type IV cells. Type I cell. These cells occur most frequently along the length of the main segment. On their basal surface, plasma membrane is extensively infolded, penetrating more than halfway through the thickness of the cell (Fig. 1). The infolded membranes follow a tortuous path and anastomose, resulting in a network of canalicular spaces. Canaliculi have low electron density and open into the space facing the basement membrane. Diameter of the canaliculi is variable. Midway between the basal surface and the center of the cell, infolded plasma membranes or canaliculi show a characteristic association with mitochondria. Two or more mitochondria become closely apposed enclosing
portions of the canaliculi (Figs. 2, 3). Cristae of these mitochondria are usually longitudinally oriented. These profiles resemble the ‘mitochondrial pump’ configurations described by Copeland (1964) in the anal papillae of Culex quinquefasciatus larvae. On the luminal surface, the cytoplasm is evaginated forming closely packed microvilli (Fig. 1). Mitochondria extend into most of the microvilli. Occasional pinocytotic vesicles are associated with the luminal plasma membrane. The cytoplasm is well populated with granular endoplasmic reticulum (GER) and free polyribosomes. The length of GER cisternae is variable, but their diameter is relatively constant (250 A)). Several dense tubuli with diameters similar to those of the GER cisternae are interspersed in the perinuclear region (Fig. 4). Similar structures have been observed in the epithelial cells of midgut in Musca (Sohal, unpublished) and in Culliphora (Priester, 1971). Smooth endoplasmic reticulum is difficult to identify in these cells due to its morphological similarity to the profiles of infolded plasma membrane. Many clear and granulated vacuoles occur in the cytoplasm. They are surrounded by a
Fig. 1. Transverse section through the Malpighian tubule showmg Type 1 cell. Microvilli (MV) on the luminal surface are closely packed together and contain mitochondria. Inflections of the basal plasma membrane (arrows) anastomose to form a network of canaliculi. Several membrane bound vacuoles (V) are seen in the cytoplasm. x 21,000. Fig. 2. Type I cell showing close approximations between infoldings of basal plasma membrane (arrow). x 26,000.
mitochondria
(M) and
Fig. 3. A high magnification view of mitochondria-canaliculi association. Canalicular membranes appear thicker than the outer membrane of mitochondria. x 43,000. Fig. 4. Dense tubuli (arrow) in the perinuclear Fig. 5. Membrane-bound material. Profiles labelled material. x 30,000.
region of Type I cell. x 45,000.
vacuoles in Type I cell showing varying amounts of dense l-5 seem to indicate progressive accumulation of dense
Fig. 6. Some of the mitochondria cristae and the presence of granular
(arrows) in Type I cell showing material. x 30,000.
Fig. 7. Type 11 cell. Note that the canaliculi (arrows) do not extend into the microvilli (MV). x 39,000.
a loss of their
are dilated and mitochondria
MALPIGHIAN
TUBULES
IN THE
HOUSEFLY
limiting membrane and contain variable amounts of granular or amorphous material. Profiles which may represent progressive accumulation of granular material in the membrane-bound vacuoles are shown in Fig. 5. These vacuoles do not appear to be identical to the transversely sectioned canalicular spaces for the following reasons. The diameter of the vacuoles is usually much greater than the longitudinally sectioned canaliculi. Profiles indicating unequivocal continuations between membranes of the vacuoles and plasma membrane are never observed. Furthermore, materials of detectable electron density are not seen in the canaliculi. Mitochondria are predominantly discoidal in shape and tend to align at right angles to the longitudinal axis of the Malpighian tubules. Several mitochondria exhibit a reduction in the number of their cristae or even a complete loss of cristae and contain a fine granular or dense material (Fig. 6). These profiles suggest degeneration of mitochondria, which have also been observed in Malpighian tubules of Rhodnius (Wigglesworth and Salpeter, 1962) and Macrosteles (Smith and Littau, 1960). The Golgi material is present in the perinuclear region and is not as well developed as that found in protein secreting cells. Type II cell. These cells are much smaller in diameter than Type I cells and can be easily recognized in cross-sections of the Malpighian tubules as forming a relatively thin wall around the lumen. They occur throughout the main segment of the tubule, but are less frequent than Type I cells. The basal plasma membrane is extensively infolded. The canaliculi are noticeably wider than in the Type I cell and extend relatively deeper into the cell (Fig. 7). Mitochondria do not extend into the microvilli and are relatively fewer in number than in Type I cells. Granulation of mitochondria is also observed in these cells. Vacuoles occur only rarely. GER is relatively poorly developed. These cells frequently adjoin Type I cells forming septate desmosomes along a portion of their intercellular junction. Type IZi cell. The major difference between Type III and Type I cells is that GER is very well developed and occupies a large
723
proportion of the cytoplasm in the former cells (Figs. 8, 9). Cisternae of GER are quite distended and contain spirally oriented aggregates of tubuli in their lumina (Fig. 9). The number of individual bundles within a single cisterna as well as their diameter is variable. The diameter of a single tubule is approximately 100 A. The GER cisternae branch profusely forming a vast anastomosing network of intracisternal spaces in the central region of the cell. The size of these cells is similar to that of the Type I cell. Plasma membrane on the basal surface is extensively infolded, but the characteristic associations between the mitochondria and the infoldings of plasma membrane such as those observed in Type I cells are less frequent. Microvilli on the luminal surface contain elongated mitochondria. Another interesting feature of this cell type is the presence of many uniformly dense and rounded structures in the cytoplasm (Fig. 10). It is often difficult to resolve the inner structure or the presence of a limiting membrane around these dense bodies, although in some instances electron lucent vesicles occur in their cortical regions (Fig. 10). Structures with a similar morphological appearance have been identified as ommochrome pigments in the ‘red’ mutant of Drosophila melanogaster (Wessing and Bonse, 1966). It is also possible that such dense bodies are composed of highly unsaturated lipid droplets (Fawcett, 1966). Vacuoles with an electron lucent interior as well as those containing dense granular material are also present in these cells. Whether the above-mentioned intensely dense bodies arise from the accumulation of materials in the vacuoles is uncertain. Occasionally, a few dense bodies are also seen in Type I cells. Mitochondria are mostly aggregated in the basal and luminal region of the cell. It is noteworthy that some mitochondria contain an array of tubules in their matrices which are about 150 A in diameter (Fig. 11). The center-to-center spacing of these tubules is of the order of 250 A. Type IV cell. These cells form the epithelial lining of the common duct in its entirety. They resemble Type I cells in their cytoarchitecture except that mitochondria do not extend into the microvilli (Fig. 12).
SOHAL
124
Clusters of undifferentiated regenerative cells occur in the basal region of the common duct. Undifferentiated cells do not extend up to the lumen. Muscle fibers with morphological features similar to those of visceral muscles of other insects (Smith et al., 1966) lie on the outer surface of the common duct. Neural innervation of these muscle fibers was not observed. The lumen of the Malpighian tubule in the main segment as well as in the common duct contains numerous spherical masses which are composed of needle-like material. Radially concentric spherical bodies within the lumen (Smith, 1968) are seen only rarely. Discussion Results of the present study indicate that the main segments of the Malpighian tubules in the housefly are composed of three distinct types of randomly distributed cells and do not show any regional structural differences along their length. Cells in the common duct constitute a fourth type of cell. In contrast, ultrastructural variations in different linear regions of the Malpighian tubules have been reported in MacrosteIes (Homoptera) (Smith and Littau, 1960), Rhodnias (Hemiptera) (Wigglesworth and Salpeter, 1962), Dams (Diptera) (Baccetti et al., 1963; Mazzi and Baccetti, 1963), Drosophila (Diptera) (Wessing and Eichelberg, 1969), Cenocorixa (Hemiptera) (Jarial and Scudder,
1970), and Carausius (Orthoptera) (Taylor, 1971a, b). At the phsyiological level, regional differentiation of the Malpighian tubules has been demonstrated in Rhodnius (Wigglesworth, 1931) and Dixippus (Ramsay, 1955). The distal region of the Malpighian tubule in these insects transports water and solutes from the hemolymph to the lumen, whereas in the proximal region, some of the substances are reabsorbed from the lumen. An ultrastructural comparison of distal and proximal regions of the tubule in Rhodnius indicates that the microvilli in the distal region are closely packed together, whereas they are more widely spaced in the proximal region (Wigglesworth and Salpeter, 1962). Otherwise, the cells in the two regions are structurally quite similar. It therefore seems unfeasible at present to identify the specific structural mechanisms which may underlie the physiological transport of solutes in the Malpighian tubules. Type I cells in the housefly are essentially similar in structure to those in the distal region of the Malpighian tubule in Rhodnius and the ‘primary’ cells in Calhphora (Berridge and Oschman, 1969) and may be secretory in nature. It is relatively well established that potassium ions are transported from the hemolymph into the lumen against a concentration gradient and are essential for the normal fluid secretion by the Malpighian tubules (Maddrell, 1971). It is tempting to speculate that the mitochondrial associations
Fig. 8. Type III cell. Granular endoplasmic reticulum is very well developed. Rods of dense material (arrows) are seen in the lumen of GER cisternae. Several highly dense bodies (D) are seen in the cytoplasm. x 20,000. A high magnification view showing dense material in the lumen of GER cisternae is presented in the inset. x 71,000. Fig. 9. A high magnification view of GER in a Type III cell. The intracisternal material is comprised of slender tubules (arrow). x 64,000. Fig. 10. Dense bodies from Type III ceil. Note the presence vesicles (arrow) in the cortex of a dense body. x 48,000.
of electron
Fig. Il. A mitochondrion in the matrix. x 59,000.
of tubules
from Type III cell showing
an array
Fig. 12. Luminal region of a Type IV cell. Note the absence of mitochondria microvilli (MV). x 16,000.
lucent (arrow) in the
MALPIGHIAN
TUBULES
IN THE
HOUSEFLY
with the plasma membrane infoldings in the basal region of Type I cells may represent possible sites for the active transfer of ions. The spherical or ovoid granules within the epithelial cells of the Malpighian tubules have been observed in several insects such as Rhodnius (Wigglesworth and Salpeter, 1962), GryIZus (Berkaloff, 1958, 1959, 1960); Cenocorixa (Jarial and Scudder, 1970) and Drosophila (Wessing and Eichelberg, 1969). In Rhodnius, they have been considered to be mineralized deposits which do not seem to contain any urates. Spherical bodies referred to as ‘concretions’ accumulate in epithelial cells of midgut in cercopid larvae and adults by storage excretion (Gouranton, 1968). Cytochemical and biochemical studies indicate that they are composed of various minerals, proteins and mucopolysaccharides. In Musca, the accumulation of materials in the vacuoles resulting in the formation of granular bodies may also indicate storage of excretory material since profiles indicating extrusion of these bodies into the lumen were not encountered. Furthermore, large quantities of dense bodies which are somewhat similar in structure to the storage excretion granules observed by Gouranton occur in Malpighian tubules of aged houseflies (Sohal, unpublished). Cytochemical studies on the nature of this material in old flies are currently in progress. There is considerable confusion regarding the mode of origin of the vacuoles, accumulation of contents within the membrane-bound structures, as well as the mechanism of secretion of materials into the lumen of the Malpighian tubules. Wigglesworth and Salpeter (1962) have suggested that granular bodies arise from degenerating mitochondria. On the other hand, Wessing and his associates have suggested various ways by which materials could be transported from the hemolymph to the lumen of the Malpighian tubules in the larvae of D. meianogaster. Following concentration of materials in the canaliculi, the materials are segregated into
727
vacuoles and vesicles which pinch off the infolds of the basal plasma membrane. The contents are then released at the luminal surface by a mechanism similar to merocrine secretion (Wessing, 1968; Eichelberg and Wessing, 1971; Wessing and Eichelberg, 1969, 1972). In Musca, some of the granular bodies arise from the mitochondria; however, more frequently, granular bodies seem to arise from gradual accumulation of material within the vacuoles. As has been mentioned above, profiles suggesting plasma membrane origin of vacuoles or the release of electron dense material into the lumen are not observed in the housefly. Type II cells in Musca which lack mitochondria in the microvilli and have wide canalicular spaces resemble ‘stellate cells’ in Malpighian tubules of Calliphora (Berridge and Oschman, 1969). These authors have suggested that stellate cells may be concerned with the reabsorption of sodium from the luminal medium or from the primary cells via septate desmosomes. In this regard, it is interesting to note that the canalicular spaces in the rectal papillae of Aedes aegypti also dilate following a blood meal presumably due to the accumulation of absorbed material before its drainage into the hemolymph (Hopkins, 1967). To the best of my knowledge, Type III cells have not been previously reported at the fine structural level. Although the chemical nature of the intracisternal precipitate is unknown, on the basis of the generally recognized function of GER it can be suggested that this material is proteinous in nature. Crystalline rods of ‘proteinous’ material have been reported in GER cisternae in hepatocytes of salamander (Fawcett, 1966) and in a clone of cells in lamina propria of man (Sane1 and Lepore, 1968). Mucocomplexes are known to be produced in the Malpighian tubules of a number of insect species and possibly function as a lubricant or a surfactant (Gabe, 1962; Marshall, 1966).
References BACCETTI,B., MAZZI, V. and MASSIMELLO,G. 1963. Richerche istochimiche e al microscopico elettronico sui tubi Malpighiani di Dacus oleae. Gmel II. L’adulto. Redia, 48, 47-68. BERKALOFF,A. 1958. Les grains de secretion des tubes Malpighi de GryNus domesticus (Orthoptire, Gryllidae). C. r. hebd. Skmc. Acad. Sri., Paris, 246, 2807-2809.
728
SOHAL
BERKALOFF,A. 1959. Variations de I’ultrastructure des tubes de Malpighi et leur functionement chez GQ&(J domesticus (Orthoptere, Gryllidae). C. r. hebd. SPanc. Acad. Sci., Paris, 248, 466469. BERKALOFF,A. 1960. Contribution a l’etude des tubes de Malpighi et de I’excretion chez les insectes. Observations au microscope electronique. Ann. Sri. Nat. Zoo/. biol. anim., 12, ser. 2, 869-947. BERRIDGE, M. J. and OSCHMAN, J. L. 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue & Cell, 1, 247-272.
COPELAND, D. E. 1964. A mitochondrial
pump in the cells of the anal papillae
of mosquito
larvae. /. Cc/l
Bit&, 23, 253-263.
EICHELBERG,D. and WESSING,A. 1971. Elektronenoptische Untersuchungen an den Nirentubuli (Malpighische Gefasse) von Drosophila mekmogaster. 11. Transzellulare membrangebundene Stofftransportmechanismen. Z. Zellforsch. mikrosk. Anat., 121, 127-l 52. FAWCET~, D. W. 1966. The CeN. Saunders, Philadelphia. GABE, M. 1962. In Handbuch der Histochemie, Band II. Polysaccharide (eds. W. Graumann and K. G. Neumann). Fischer Verlag, Stuttgart. GOURANTON,J. 1968. Composition, structure, et mode de formation de concretions minerales dans l’intestine moyen des homopteres cercopides. J. Cell Biol., 37, 3 16-328. HEWITT, C. G. 1914. The Housefly. University Press, Cambridge. HOPKINS, C. R. 1967. The fine-structural changes observed in the rectal papillae of the mosquito A&s aegypti, L. and their relation to the epithelial transport of water and inorganic ions. J. R. microsc. Sot.. 86, 235-252. JARIAL, M. S. and SCUDDER, G. G. E. 1970. The morphology and ultrastructure of the Malpighian tubules and hindgut in Cenocorixa bifido (Hung.) (Hemiptera, Corixidae). Z. Morph. iikol. Tiere, 68, 269-299. MADDRELL, S. H. P. 1971. The mechanisms of insect excretory systems. In Adv. Insect Physiol. (eds. J. W. L. Beament, I. E. Treherne and V. B. Wigglesworth), Vol. 8, pp. 199-33 1. Academic Press, London. MARSHALL, A. T. 1966. Histochemical studies on a mucocomplex in the Malpighian tubules of cercopid larvae. J. Insect Physiol., 12, 925-932. MAZZI, V. and BACCETTI, B. 1963. Richerche istochimiche e al microscopio elettronico sui tubi Malpighiani di Dot-us olew. Gmel. I. La larva. Z. Zel(for.wh. mikrosk. Anal., 59,47-70. PRIESTER, W. D. 1971. Ultrastructure of the midgut epithelial cells in the fly Calliphora er>lthrocephaln. J. Ultrastruct.
Rex., 36, 783-805.
RAMSAY,J. A. 1955. The excretion of sodium, potassium and water by the Malpighian tubules of the stick insect Diwippus morons (Orthoptera : Phasmidae). J. exp. Biol., 32, 200-216. SANEL, T. and LEPORE, M. I. 1968. Granular and crystalline deposits in perinuclear and ergastoplasmic cisternae of human lamina propria. Expl. molec. Path., 9, 110-124. SHAW, J. and STOBBART,R. H. 1963. Osmotic and ion regulation in insects. In Adv. fnswt Physiol. teds. J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth), Vol. I, pp. 315-399. Academic Press, London. SMITH, D. S. 1968. Insecf Ceils, Their Structure and Fwrctiot~ Oliver and Boyd, Edinburgh. SMITH, D. S. and LITTAU, V. C. 1960. Cellular specializations in the excretory epithelia of an insect, Mrwosteles,fascifrons St&l (Homoptera). J. biophys. biochcm. Cytol., 8, 103-I 33. SMITH, D. S., GUPTA, B. L. and SMITH, U. 1966. The organization and myofilament array of insect visceral muscles. J. Cell Sri., 1, 49-57. TAYLOR, H. H. 197la. Water and solute transport by the Malpighian tubules of stick insect, Carurrsius morosus. The normal ultrastructure of type I cells. 2. Zehforsch. mikrosk. Anat., 118, 333-368. TAYLOR, H. H. 1971 b. The fine structure of the type II cells in the Malpighian tubules of the stick insect, Carausius morosus. Z. Zellforsch.
mikrosk.
Anat., 122, 41 l-424.
WESSING, A. 1960. Funktionsmorphologie von Exkretionsorganen bei insekten. Zoo/. AK., 31, suppl.. 633-681. WESSING, A. and BONSE. A. 1966. Natur und Bildung des roten Farbstoffes in den Nierentubuli der mutante ‘red’ von Drosophila melanogaster. Z. Naturf., 21b, 1219-1223. WESSING, A. and EICHELBERG,D. 1969. Elekronenoptische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster. I. Regionale Gliederung der Tubuli. Z. Zel(forsch. mikrosk. Anat., 101, 285-322.
WESSING,A. and EICHELBERG,D. 1972. Elekronenmikropische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster. IV. Die Lokalisation von Natrium und Chloridionen. 2. ZeUforsch.
mikrosk.
An&.,
131, 269-286.
WIGGLESWORTH,V. B. 1931. The physiology of excretion in a blood-sucking insect, Rhodmu prolix-us Stal (Hemiptera, Reduviidae). III. The mechanism of uric acid secretion. J. up. Biol., 8, 443-451. WIGGLESWORTH,V. B. 1965. The Principles of § Physiology, 6th edn. Methuen, London. WIGGLESWORTH,V. B. and SALPETER, M. M. 1962. Histology of the Malpighian tubules in Rhodnirts proli.vrfs Stll (Hemiptera). 1. fnsect Physiol., 8, 299-307.