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Malpighian tubules of adult flesh fly, Sarcophaga ruficornis Fab. (Diptera: Sarcophagidae): An ultrastructural study
Tissue and Cell 45 (2013) 312–317
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Malpighian tubules of adult flesh fly, Sarcophaga ruficornis Fab. (Diptera: Sarcophagidae): An ultrastructural study Ruchita Pal, Krishna Kumar ∗ Department of Zoology, University of Allahabad, Allahabad 211002, India
a r t i c l e
i n f o
Article history: Received 10 January 2013 Received in revised form 9 April 2013 Accepted 9 April 2013 Available online 10 May 2013 Keywords: Malpighian tubules SEM TEM Adult flesh fly Sarcophaga ruficornis
a b s t r a c t The Malpighian tubules of adult flesh fly, Sarcophaga ruficornis consist of principal and stellate cells. The principal cells reveal all the characteristics of transporting epithelia with well developed deep basal membrane infoldings forming a complex of interconnecting labyrinth of canaliculi and luminal microvilli, both of which are associated with mitochondria. The central cytoplasm of the cells contains a well developed nucleus, clear vacuoles or vacuoles filled with secretory material, mineral concretions or spherocrystals, lysosomes and a network of endoplasmic reticulum. The mineral concretions are also observed in the region of luminal microvilli and in the lumen of the tubule suggesting their extrusion into the lumen by exocytosis. Several formed bodies are also observed in the lumen. Stellate cells are characterized by simple membrane infoldings and luminal microvilli devoid of mitochondria. The cells are separated by septate junctions. The Malpighian tubules are richly supplied by tracheae and muscle fibers. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Malpighian tubules have been the subject of considerable interest because of their prodigious capacity for fluid transport (Bradley, 1985; Pannabecker, 1995). The insect Malpighian tubules can be compared with the vertebrate kidney tubules and their main functions are the transportation of organic solutes, breakdown and removal or excretion of toxic substances, maintenance of ionic balance and immune defenses (Dow, 2009; Beyenbach et al., 2010). Thus the Malpighian tubules serve the function of both generating the primary urine and, selectively reabsorbing the desirable solutes, or in the terrestrial insects the filtered fluid or primary urine is passed on to the hindgut and rectum, where this filtered fluid is modified as a result of reabsorption of certain substances, and excretion of others to produce urine (Maddrell and Gardiner, 1974; Maddrell, 1981; Chapman, 2008). In a dipteran fly, Calliphora erythrocephala, Berridge and Oschman (1969) explained the urine formation by applying the standing gradient hypothesis suggesting that standing osmotic gradients are set up in the long narrow channels of basal infoldings and also within the microvilli resulting in passive movement of water. In such cases the transporting cells have deep basal membrane infoldings forming a complex interconnected labyrinth or channels and luminal microvilli, both intimately associated with the mitochondria. However, apart from a few studies on the Malpighian tubules of adult cyclorrhaphous
∗ Corresponding author. Tel.: +91 9452180187. E-mail address: [email protected] (K. Kumar). 0040-8166/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2013.04.002
Diptera such as Calliphora erythocephalla (Berridge and Oschman, 1969), Musca domestica (Sohal, 1974; Sohal et al., 1976) and Drosophila melanogaster (Wessing and Zierold, 1999), adequate attention has not been paid to the study of ultrastructural details of Malpighian tubules of adult cyclorrhaphous Diptera. In the present communication, the ultrastructural features of Malpighian tubules of adult flesh fly, Sarcophaga ruficornis, have been described. 2. Materials and methods Flesh fly, S. ruficornis, were maintained in the laboratory at 27 ± 1 ◦ C and 75 ± 5 RH. The adult flies were reared on honey solution and water. Fresh slices of goat’s liver constituted as a standard protein source and were also used for larviposition and for feeding the larvae. For transmission electron microscopy (TEM), adult flies of different age viz. 0–4 h, 24 h, 48 h, 72 h, and 96 h were dissected in Insect Ringer to remove Malpighian tubules which were fixed in 4% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8 at 4 ◦ C for 1 h to accomplish primary fixation. Following primary fixation, the tubules were rinsed in 0.1 M sodium phosphate buffer for 10–20 min at room temperature and post fixed for 45 min in a solution of 1% OsO4 , prepared with 0.05 M sodium phosphate buffer and 0.1 M sucrose and dehydrated with ethanol series and embedded in Araldite. Sections were cut on ultramicrotome (Reichert ultracut E) and stained with 1% uranyl acetate and lead citrate and viewed and photographed on a Philips CM 10 electron microscope at 100 kV. For scanning electron microscopy (SEM), specimens were fixed in glutaraldehyde solution, as described above, to accomplish primary fixation. The tubules were rinsed twice in phosphate buffer
R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 1. SEM image of the Malpighian tubule of adult flesh fly, S. ruficornis.
313
Fig. 3. Cross-section of Malpighian tubule of 0–4 h old fly showing muscle fiber (Mf), tracheole (Tr) and basal membrane infoldings (Bmi).
and treated with 1% OsO4 for 1 h. Post fixation of tubules was followed by rinsing 2–3 times with phosphate buffer solution and dehydrating with an alcohol series. Specimens were then subjected to critical point drying and viewed with a LEO 435 VP scanning electron microscope. 3. Results In the adult flesh fly two pairs of extremely thin, delicate and yellowish Malpighian tubules are found. The tubules of each side are joined by a common ureter that opens into the alimentary canal at the junction of the midgut and hindgut. Scanning electron microscopy reveals that the tubules exhibit a beaded appearance (Fig. 1) and there is no ultrastructural variations in different parts. The ultrastructure of Malpighian tubule epithelium reveals the presence of two types of cells viz. principal and stellate cells intermingling along the entire length of the tubules. 3.1. Principal cells The outer surface of the cells is covered by a basal lamina. The basal membrane is deeply infolded to form a complex of interconnecting anastomosing labyrinth of canaliculi extending deep into the cells, sometimes penetrating into the central region of the cells. These infoldings are more prominent with the advancement of the age of the flies. The mitochondria, very often filamentous in appearance, are associated with the infoldings of
Fig. 2. Cross-section of Malpighian tubule of 0–4 h old fly showing basal lamina (Bl), basal membrane infoldings, and vacuoles (V).
Fig. 4. Cross-section of Malpighian tubule of 24 h old fly showing muscle fiber (Mf), tracheole (Tr), vacuole (V), autophagic vacuole (Au), microvilli (Mv) and lumen (Lu).
the basal membrane (Figs. 2, 8–11). Outside the basal lamina are well developed tracheoles and muscle fibers (Figs. 3, 4, 9, 14). The central region of the principal cells is characterized by the presence of a few deeply penetrating basal infoldings (Figs. 8 and 10), a large round prominent nucleus (having a clump of chromatin material) bound by a nuclear membrane (Figs. 5 and 15) and a number of cell inclusions like mitochondria
Fig. 5. Cross-section of Malpighian tubule of 24 h old fly showing a large nucleus (N), lysosomes (L), microvilli (Mv) and formed bodies (Fb) in the lumen (Lu).
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R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 6. Cross-section of Malpighian tubule of 24 h old fly showing mitochondria (M).
Fig. 9. Cross-section of Malpighian tubule of 48 h old fly showing tracheole (Tr), mineral concretion (Mc), myelin fiber (Ms) and microvilli (Mv) in the tubule lumen (Lu).
or spherocrystals, (Figs. 10, 12, 16), myelin fiber like structures (Fig. 10), and lysosomes (Figs. 5 and 7). The apical surface of the principal cells in younger flies (up to 48 h) is composed of closely packed luminal microvilli containing mitochondria running almost up to the tips (Figs. 8–10, 13). Luminal microvilli with swollen tips and mineral concretions (Fig. 4) and several luminal formed bodies that are the tubules method of releasing cell inclusions into the lumen are seen (Figs. 5 and 7). The luminal microvilli in older flies (96 h) are exceptionally long with mitochondria running up to only two third of the length of the microvilli. The tips of microvilli are filled with granular cytoplasm and several mineral concretions, spherites or formed bodies have been observed in the region of luminal microvilli and in the tubule lumen showing their release into the lumen by exocytosis (Figs. 17 and 18). 3.2. Stellate cells
(Figs. 6 and 11). There is an extensive network of smooth endoplasmic reticulum (Fig. 16), small or large vacuoles filled with dispersed materials (Figs. 2, 4, 6), lipid droplets, some autophagic vacuoles (Figs. 4 and 12) concentric layered mineral concretions
The outer surface of the stellate cells is covered by a basal lamina. There are a few basal membrane infoldings as compared to wide anastomosing labyrinth of canaliculi found in the principal cells. There are a few cellular inclusions and the luminal microvilli are extremely short and devoid of mitochondria. Mineral concretions, spherites or formed bodies have not been observed in the lumen of stellate cells. A long septate junction separating the cells is visible (Fig. 19).
Fig. 8. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), basal membrane infoldings, microvilli (Mv) and tubule lumen (Lu).
Fig. 10. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), mitochondria (M), mineral concretion (Mc), myelin fiber (Ms) and microvilli (Mv).
Fig. 7. Cross-section of Malpighian tubule of 24 h old fly showing basal lamina (Bl), lysosomes (L), microvilli (Mv), formed bodies (Fb) in the tubule lumen (Lu).
R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 11. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), basal membrane infoldings (Bmi) and several mitochondria (M).
Fig. 12. Cross-section of Malpighian tubule of 48 h old fly showing mineral concretion (Mc), autophagic vacuole (Au) and several mitochondria (M).
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Fig. 14. Cross-section of Malpighian tubule of 72 h old fly showing a striated muscle cell (Mce) and several tracheoles (Tr).
Fig. 15. Cross-section of Malpighian tubule of 72 h old fly showing a large nucleus (N), muscle fiber (Mf) and basal lamina (Bl).
larva (Alkassis and Schoeller-Raccaud, 1984), M. domestica (Sohal, 1974), Aedes taeniorhynchus (Bradley et al., 1982) and Chironomus tentans (Jarial, 1988), the ultrastuctural variations in the tubules, as in the present case, have not been reported. The tubular epithelium consists of two types of cells namely principal and stellate cells. Similar cell types have also been reported in other dipteran insects such as C. erythrocephala adult
Fig. 13. Cross-section of Malpighian tubule of 48 h old fly showing luminal microvilli (Mv) filled with mitochondria (M).
4. Discussion The Malpighian tubules of S. ruficornis, do not reveal ultrastructural variations in different regions as compared to that found in Rhodnius prolixus (Wigglesworth and Salpeter, 1962) and Drosophila (Sozen et al., 1997). In many dipteran insects like C. erythrocephala adult (Berridge and Oschman, 1969), C. erythrocephala
Fig. 16. Cross-section of Malpighian tubule of 72 h old fly showing endoplasmic reticulum (Er) and mineral concretion (Mc).
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R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 17. Cross-section of Malpighian tubule of 72 h old fly showing muscle fiber (Mf), tracheole (Tr), vacuole (V), microvilli (Mv), mitochondria (M), mineral concretion (Mc) in the microvillar region and in the tubule lumen (Lu).
Fig. 18. Cross-section of Malpighian tubule of 96 h old fly showing vacuole (V), long microvilli (Mv) having mitochondria (M), spherites (Sp) in the microvillar region and in the tubule lumen (Lu).
Fig. 19. Cross-section of Malpighian tubule of 48 h old fly showing a stellate cell with extremely short microvilli (Mv) devoid of mitochondria, tubule lumen (Lu), sepate junction (S), basal lamina (Bl).
(Berridge and Oschman, 1969), larval C. erythrocephala (Alkassis and Schoeller-Raccaud, 1984), A. taeniorhynchus (Bradley et al., 1982), C. tentans (Jarial, 1988), Drosophila hydei and D. melanogaster (Wessing et al., 1999) and type I and type II cells (similar to principal and stellate cells respectively) in M. domestica (Sohal, 1974). The ultrastructure of principal cells demonstrates the characteristics of transporting epithelia involved in the ion and water transport. Berridge and Oschman (1969) have applied the standing gradient hypothesis as a model to explain urine formation in the Malpighian tubules where an osmotic gradient is set up within the basal infolds and microvilli of principal cells leading to active transport of small solutes especially potassium followed by passive movement of water from the haemolymph into the tubule lumen. In the principal cells of Sarcophaga, microvilli are not only packed with mitochondria but the later are also associated with basal membrane infoldings. Besides mitochondria are found in the central cytoplasm and at the bases of microvilli. In the mite, Macrocheles muscaedomesticae, the tubule cells possess myelin fiber like mitochondria apart from normal mitochondria that are associated with the basal plasma membrane infoldings (Coons and Axtell, 1971). The distribution of mitochondria in the tubule cells indicates the sites of maximum energy requirements (Wigglesworth and Salpeter, 1962). The mitochondria are recruited from the cell cytoplasm and inserted into microvilli in stimulated Malpighian tubules of Rhodnius which results in maintenance of the mitochondria in the microvilli as long as the stimulation persists (Bradley and Satir, 1977). Moreover, in Calpodes Malpighian tubules, there is close association of mitochondria with basal infolds and microvilli in larval and adult stage flies but a significant change in their number and distribution during pharate adult stage when fluid secretion is switched off (Ryerse, 1979). This suggests the role of mitochondria movement is associated with the energy requirement of the cells. Such a close association of mitochondria with basal infolds and microvilli is found in the cells involved in active fluid secretion or ion absorption and the mitochondria are supplying energy for membrane associated transport pumps (Kukel and Komnick, 1989). It has been observed that with the advancement of age of adult flies, the luminal microvilli become excessively long and several spherites/formed bodies and mineral concretions are observed in the region of luminal microvilli and also in the tubular lumen suggesting their release into the lumen by exocytosis (Wessing and Eichelberg, 1975; Wessing and Zierold, 1999). Thus the secretory activity increases with the advancement of age of flies and this high secretory activity might be associated with consumption of food and water in the adult (Ryerse, 1978). In the tubular lumen of adult flies, spherical structures or formed bodies have been observed and sometimes the lumen is closely packed with these structures. The formed bodies were first observed by Riegel (1966) in the Malpighian tubules and these bodies serve as the tubules method of releasing cell inclusions into the lumen and/or movement of solutes across the epithelium (Riegel, 1971). However, in M. domestica type C, concretions similar to formed bodies have not been observed in the lumen, though these were present in the cytoplasm of the cell (Sohal et al., 1976). In principal cells of Malpighian tubules of Sarcophaga, several mineral concretions or spherocrystals are found in the central cytoplasm, at the bases of microvilli, in the luminal microvillar region and also in the tubule lumen. The spherocrystals are supposed to be storage excretion sites (Bradley, 1985) and a characterstic feature of secretory Malpighian tubule cells. The mineral concretions may contain phosphorus, sulphur, calcium, iron, zinc and copper and these may be intracytoplasmic, not being discharged into the lumen (Sohal et al., 1976) and may act as transitory depots for the concentration of substances removed from the haemolymph or alternatively these are transported to apical plasma membrane and subsequently discharged into the lumen
R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
by the process of exocytosis (Wessing and Zierold, 1999). In the Malpighian tubules of Sarcophaga, the mineral concretions are observed at the bases of apical plasma membrane and also in the tubule lumen suggesting their extrusion by exocytosis. The stellate cells in the Malpighian tubules of adult flesh fly have short microvilli devoid of mitochondria suggesting that these cells are not actively involved in the ion transport. Furthermore unlike the principal cells, mineral concretions or spherocrystals, which are the characteristic features of secretory cells, have not been observed in the stellate cells. These cells, interspersed with principal cells, have been observed in many insects (Martoja and Ballan-Dufrancais, 1984). It has been suggested that the function of stellate cells is reabsorptive and, these cells reabsorb sodium ions from the lumen or directly from the principal cells, and return it to the blood (Berridge and Oschman, 1969). The ultrastructural features of principal cells demonstrate that these are the major ion transporting cells requiring the expenditure of energy, whereas the stellate cells, as compared to principal cells, lack the characteristics of transporting cells and may perform reabsorptive function. References Alkassis, W., Schoeller-Raccaud, J., 1984. Ultrastructure of the Malpighian tubules of blow fly larva, Calliphora erythrocephala Meigen (Diptera: Calliphoridae). Int. J. Insect Morphol. Embryol. 13, 215–231. Berridge, M.J., Oschman, J.L., 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue Cell 1, 247–272. Beyenbach, K.W., Skaer, H., Dow, J.A.T., 2010. The development, molecular, and transport biology of Malpighian tubules. Annu. Rev. Entomol. 55, 351–374. Bradley, T.J., 1985. The excretory system: structure and physiology. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 4. Pergamon Press, Oxford, pp. 421–465. Bradley, T.J., Satir, P., 1977. Microvillar beating and mitochondrial migration in Malpighian tubules. J. Cell Biol. 75, 255a. Bradley, T.J., Stuart, A.M., Satir, P., 1982. The ultrastructure of the larval Malpighian tubules of a saline-water mosquito. Tissue Cell 14, 759–773. Chapman, R.F., 2008. The Insects: Structure and Function, 4th ed. Cambridge University Press, New York, pp. 478–508.
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Coons, L.B., Axtell, R.C., 1971. Ultrastructure of the excretory tubes of the mite Macrocheles muscaedomesticae (Mesostigmata, Macrochelidae) with notes on altered mitochondria. J. Morphol. 133, 319–338. Dow, J.A.T., 2009. Insights into the Malpighian tubule from functional genomics. J. Exp. Biol. 212, 435–445. Jarial, M.S., 1988. Fine structure of the Malpighian tubules of Chironomus larva in relation to glycogen storage and fate of hemoglobin. Tissue Cell 20, 355–380. Kukel, S., Komnick, H., 1989. Development, cytology, lipid storage and motility of the Malpighian tubules of the nymphal dragonfly, Aeshna cyanea (Muller)(Odonata: Aeshnidae). Int. J. Insect Morphol. Embryol. 18, 119–134. Maddrell, S.H.P., 1981. The functional design of the insect excretory system. J. Exp. Biol. 90, 1–15. Maddrell, S.H.P., Gardiner, B.O.C., 1974. The passive permeability of insect Malpighian tubules to organic solutes. J. Exp. Biol. 60, 641–652. Martoja, R., Ballan-Dufrancais, C., 1984. The ultrastucture of the digestive and excretory organs. In: King, R., Akai, H. (Eds.), Insect Ultrastructure, vol. 2. Plenum Press, New York, pp. 199–268. Pannabecker, T., 1995. Physiology of the Malpighian tubule. Ann. Rev. Entomol. 40, 493–510. Riegel, J.A., 1966. Micropuncture studies of formed-body secretion by the excretory organs of the crayfish, frog and stick insect. J. Exp. Biol. 44, 379–385. Riegel, J.A., 1971. Excretion-Arthropoda. In: Florkin, M., Scheer, B.T. (Eds.), Chemical Zoology, vol. 6. Academic Press, New York, pp. 249–277. Ryerse, J.S., 1978. Developmental changes in Malpighian tubule fluid transport. J. Insect Physiol. 24, 315–319. Ryerse, J.S., 1979. Developmental changes in Malpighian tubule cell structure. Tissue Cell 11, 533–551. Sohal, R.S., 1974. Fine structure of the Malpighian tubules in the housefly, Musca domestica. Tissue Cell 6, 719–728. Sohal, R.S., Peters, P.D., Hall, T.A., 1976. Fine structure and X-ray microanalysis of mineralized concretions in the Malpighian tubules of the housefly, Musca domestica. Tissue Cell 8, 447–458. Sozen, M.A., Armstrong, J.D., Yang, M., Kaiser, K., Dow, J.A.T., 1997. Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc. Natl. Acad. Sci. U. S. A. 94, 5207–5212. Wessing, A., Eichelberg, D., 1975. Ultrastructural aspects of transport and accumulation of substances in Malpighian tubules. Fortschr. Zool. 23, 148–172. Wessing, A., Zierold, K., 1999. The formation of type-I concretions in Drosophila Malpighian tubules studied by electron microscopy and X-ray microanalysis. J. Insect Physiol. 45, 39–44. Wessing, A., Zierold, K., Polenz, A., 1999. Stellate cells in the Malpighian tubules of Drosophila hydei and D. melanogaster larvae (Insecta, Diptera). Zoomorphology 119, 63–71. Wigglesworth, V.B., Salpeter, M.M., 1962. Histology of the Malpighian tubules in Rhodnius prolixus. J. Insect Physiol. 8, 299–307.
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Malpighian tubules of adult flesh fly, Sarcophaga ruficornis Fab. (Diptera: Sarcophagidae): An ultrastructural study Ruchita Pal, Krishna Kumar ∗ Department of Zoology, University of Allahabad, Allahabad 211002, India
a r t i c l e
i n f o
Article history: Received 10 January 2013 Received in revised form 9 April 2013 Accepted 9 April 2013 Available online 10 May 2013 Keywords: Malpighian tubules SEM TEM Adult flesh fly Sarcophaga ruficornis
a b s t r a c t The Malpighian tubules of adult flesh fly, Sarcophaga ruficornis consist of principal and stellate cells. The principal cells reveal all the characteristics of transporting epithelia with well developed deep basal membrane infoldings forming a complex of interconnecting labyrinth of canaliculi and luminal microvilli, both of which are associated with mitochondria. The central cytoplasm of the cells contains a well developed nucleus, clear vacuoles or vacuoles filled with secretory material, mineral concretions or spherocrystals, lysosomes and a network of endoplasmic reticulum. The mineral concretions are also observed in the region of luminal microvilli and in the lumen of the tubule suggesting their extrusion into the lumen by exocytosis. Several formed bodies are also observed in the lumen. Stellate cells are characterized by simple membrane infoldings and luminal microvilli devoid of mitochondria. The cells are separated by septate junctions. The Malpighian tubules are richly supplied by tracheae and muscle fibers. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Malpighian tubules have been the subject of considerable interest because of their prodigious capacity for fluid transport (Bradley, 1985; Pannabecker, 1995). The insect Malpighian tubules can be compared with the vertebrate kidney tubules and their main functions are the transportation of organic solutes, breakdown and removal or excretion of toxic substances, maintenance of ionic balance and immune defenses (Dow, 2009; Beyenbach et al., 2010). Thus the Malpighian tubules serve the function of both generating the primary urine and, selectively reabsorbing the desirable solutes, or in the terrestrial insects the filtered fluid or primary urine is passed on to the hindgut and rectum, where this filtered fluid is modified as a result of reabsorption of certain substances, and excretion of others to produce urine (Maddrell and Gardiner, 1974; Maddrell, 1981; Chapman, 2008). In a dipteran fly, Calliphora erythrocephala, Berridge and Oschman (1969) explained the urine formation by applying the standing gradient hypothesis suggesting that standing osmotic gradients are set up in the long narrow channels of basal infoldings and also within the microvilli resulting in passive movement of water. In such cases the transporting cells have deep basal membrane infoldings forming a complex interconnected labyrinth or channels and luminal microvilli, both intimately associated with the mitochondria. However, apart from a few studies on the Malpighian tubules of adult cyclorrhaphous
∗ Corresponding author. Tel.: +91 9452180187. E-mail address: [email protected] (K. Kumar). 0040-8166/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2013.04.002
Diptera such as Calliphora erythocephalla (Berridge and Oschman, 1969), Musca domestica (Sohal, 1974; Sohal et al., 1976) and Drosophila melanogaster (Wessing and Zierold, 1999), adequate attention has not been paid to the study of ultrastructural details of Malpighian tubules of adult cyclorrhaphous Diptera. In the present communication, the ultrastructural features of Malpighian tubules of adult flesh fly, Sarcophaga ruficornis, have been described. 2. Materials and methods Flesh fly, S. ruficornis, were maintained in the laboratory at 27 ± 1 ◦ C and 75 ± 5 RH. The adult flies were reared on honey solution and water. Fresh slices of goat’s liver constituted as a standard protein source and were also used for larviposition and for feeding the larvae. For transmission electron microscopy (TEM), adult flies of different age viz. 0–4 h, 24 h, 48 h, 72 h, and 96 h were dissected in Insect Ringer to remove Malpighian tubules which were fixed in 4% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8 at 4 ◦ C for 1 h to accomplish primary fixation. Following primary fixation, the tubules were rinsed in 0.1 M sodium phosphate buffer for 10–20 min at room temperature and post fixed for 45 min in a solution of 1% OsO4 , prepared with 0.05 M sodium phosphate buffer and 0.1 M sucrose and dehydrated with ethanol series and embedded in Araldite. Sections were cut on ultramicrotome (Reichert ultracut E) and stained with 1% uranyl acetate and lead citrate and viewed and photographed on a Philips CM 10 electron microscope at 100 kV. For scanning electron microscopy (SEM), specimens were fixed in glutaraldehyde solution, as described above, to accomplish primary fixation. The tubules were rinsed twice in phosphate buffer
R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 1. SEM image of the Malpighian tubule of adult flesh fly, S. ruficornis.
313
Fig. 3. Cross-section of Malpighian tubule of 0–4 h old fly showing muscle fiber (Mf), tracheole (Tr) and basal membrane infoldings (Bmi).
and treated with 1% OsO4 for 1 h. Post fixation of tubules was followed by rinsing 2–3 times with phosphate buffer solution and dehydrating with an alcohol series. Specimens were then subjected to critical point drying and viewed with a LEO 435 VP scanning electron microscope. 3. Results In the adult flesh fly two pairs of extremely thin, delicate and yellowish Malpighian tubules are found. The tubules of each side are joined by a common ureter that opens into the alimentary canal at the junction of the midgut and hindgut. Scanning electron microscopy reveals that the tubules exhibit a beaded appearance (Fig. 1) and there is no ultrastructural variations in different parts. The ultrastructure of Malpighian tubule epithelium reveals the presence of two types of cells viz. principal and stellate cells intermingling along the entire length of the tubules. 3.1. Principal cells The outer surface of the cells is covered by a basal lamina. The basal membrane is deeply infolded to form a complex of interconnecting anastomosing labyrinth of canaliculi extending deep into the cells, sometimes penetrating into the central region of the cells. These infoldings are more prominent with the advancement of the age of the flies. The mitochondria, very often filamentous in appearance, are associated with the infoldings of
Fig. 2. Cross-section of Malpighian tubule of 0–4 h old fly showing basal lamina (Bl), basal membrane infoldings, and vacuoles (V).
Fig. 4. Cross-section of Malpighian tubule of 24 h old fly showing muscle fiber (Mf), tracheole (Tr), vacuole (V), autophagic vacuole (Au), microvilli (Mv) and lumen (Lu).
the basal membrane (Figs. 2, 8–11). Outside the basal lamina are well developed tracheoles and muscle fibers (Figs. 3, 4, 9, 14). The central region of the principal cells is characterized by the presence of a few deeply penetrating basal infoldings (Figs. 8 and 10), a large round prominent nucleus (having a clump of chromatin material) bound by a nuclear membrane (Figs. 5 and 15) and a number of cell inclusions like mitochondria
Fig. 5. Cross-section of Malpighian tubule of 24 h old fly showing a large nucleus (N), lysosomes (L), microvilli (Mv) and formed bodies (Fb) in the lumen (Lu).
314
R. Pal, K. Kumar / Tissue and Cell 45 (2013) 312–317
Fig. 6. Cross-section of Malpighian tubule of 24 h old fly showing mitochondria (M).
Fig. 9. Cross-section of Malpighian tubule of 48 h old fly showing tracheole (Tr), mineral concretion (Mc), myelin fiber (Ms) and microvilli (Mv) in the tubule lumen (Lu).
or spherocrystals, (Figs. 10, 12, 16), myelin fiber like structures (Fig. 10), and lysosomes (Figs. 5 and 7). The apical surface of the principal cells in younger flies (up to 48 h) is composed of closely packed luminal microvilli containing mitochondria running almost up to the tips (Figs. 8–10, 13). Luminal microvilli with swollen tips and mineral concretions (Fig. 4) and several luminal formed bodies that are the tubules method of releasing cell inclusions into the lumen are seen (Figs. 5 and 7). The luminal microvilli in older flies (96 h) are exceptionally long with mitochondria running up to only two third of the length of the microvilli. The tips of microvilli are filled with granular cytoplasm and several mineral concretions, spherites or formed bodies have been observed in the region of luminal microvilli and in the tubule lumen showing their release into the lumen by exocytosis (Figs. 17 and 18). 3.2. Stellate cells
(Figs. 6 and 11). There is an extensive network of smooth endoplasmic reticulum (Fig. 16), small or large vacuoles filled with dispersed materials (Figs. 2, 4, 6), lipid droplets, some autophagic vacuoles (Figs. 4 and 12) concentric layered mineral concretions
The outer surface of the stellate cells is covered by a basal lamina. There are a few basal membrane infoldings as compared to wide anastomosing labyrinth of canaliculi found in the principal cells. There are a few cellular inclusions and the luminal microvilli are extremely short and devoid of mitochondria. Mineral concretions, spherites or formed bodies have not been observed in the lumen of stellate cells. A long septate junction separating the cells is visible (Fig. 19).
Fig. 8. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), basal membrane infoldings, microvilli (Mv) and tubule lumen (Lu).
Fig. 10. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), mitochondria (M), mineral concretion (Mc), myelin fiber (Ms) and microvilli (Mv).
Fig. 7. Cross-section of Malpighian tubule of 24 h old fly showing basal lamina (Bl), lysosomes (L), microvilli (Mv), formed bodies (Fb) in the tubule lumen (Lu).
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Fig. 11. Cross-section of Malpighian tubule of 48 h old fly showing basal lamina (Bl), basal membrane infoldings (Bmi) and several mitochondria (M).
Fig. 12. Cross-section of Malpighian tubule of 48 h old fly showing mineral concretion (Mc), autophagic vacuole (Au) and several mitochondria (M).
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Fig. 14. Cross-section of Malpighian tubule of 72 h old fly showing a striated muscle cell (Mce) and several tracheoles (Tr).
Fig. 15. Cross-section of Malpighian tubule of 72 h old fly showing a large nucleus (N), muscle fiber (Mf) and basal lamina (Bl).
larva (Alkassis and Schoeller-Raccaud, 1984), M. domestica (Sohal, 1974), Aedes taeniorhynchus (Bradley et al., 1982) and Chironomus tentans (Jarial, 1988), the ultrastuctural variations in the tubules, as in the present case, have not been reported. The tubular epithelium consists of two types of cells namely principal and stellate cells. Similar cell types have also been reported in other dipteran insects such as C. erythrocephala adult
Fig. 13. Cross-section of Malpighian tubule of 48 h old fly showing luminal microvilli (Mv) filled with mitochondria (M).
4. Discussion The Malpighian tubules of S. ruficornis, do not reveal ultrastructural variations in different regions as compared to that found in Rhodnius prolixus (Wigglesworth and Salpeter, 1962) and Drosophila (Sozen et al., 1997). In many dipteran insects like C. erythrocephala adult (Berridge and Oschman, 1969), C. erythrocephala
Fig. 16. Cross-section of Malpighian tubule of 72 h old fly showing endoplasmic reticulum (Er) and mineral concretion (Mc).
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Fig. 17. Cross-section of Malpighian tubule of 72 h old fly showing muscle fiber (Mf), tracheole (Tr), vacuole (V), microvilli (Mv), mitochondria (M), mineral concretion (Mc) in the microvillar region and in the tubule lumen (Lu).
Fig. 18. Cross-section of Malpighian tubule of 96 h old fly showing vacuole (V), long microvilli (Mv) having mitochondria (M), spherites (Sp) in the microvillar region and in the tubule lumen (Lu).
Fig. 19. Cross-section of Malpighian tubule of 48 h old fly showing a stellate cell with extremely short microvilli (Mv) devoid of mitochondria, tubule lumen (Lu), sepate junction (S), basal lamina (Bl).
(Berridge and Oschman, 1969), larval C. erythrocephala (Alkassis and Schoeller-Raccaud, 1984), A. taeniorhynchus (Bradley et al., 1982), C. tentans (Jarial, 1988), Drosophila hydei and D. melanogaster (Wessing et al., 1999) and type I and type II cells (similar to principal and stellate cells respectively) in M. domestica (Sohal, 1974). The ultrastructure of principal cells demonstrates the characteristics of transporting epithelia involved in the ion and water transport. Berridge and Oschman (1969) have applied the standing gradient hypothesis as a model to explain urine formation in the Malpighian tubules where an osmotic gradient is set up within the basal infolds and microvilli of principal cells leading to active transport of small solutes especially potassium followed by passive movement of water from the haemolymph into the tubule lumen. In the principal cells of Sarcophaga, microvilli are not only packed with mitochondria but the later are also associated with basal membrane infoldings. Besides mitochondria are found in the central cytoplasm and at the bases of microvilli. In the mite, Macrocheles muscaedomesticae, the tubule cells possess myelin fiber like mitochondria apart from normal mitochondria that are associated with the basal plasma membrane infoldings (Coons and Axtell, 1971). The distribution of mitochondria in the tubule cells indicates the sites of maximum energy requirements (Wigglesworth and Salpeter, 1962). The mitochondria are recruited from the cell cytoplasm and inserted into microvilli in stimulated Malpighian tubules of Rhodnius which results in maintenance of the mitochondria in the microvilli as long as the stimulation persists (Bradley and Satir, 1977). Moreover, in Calpodes Malpighian tubules, there is close association of mitochondria with basal infolds and microvilli in larval and adult stage flies but a significant change in their number and distribution during pharate adult stage when fluid secretion is switched off (Ryerse, 1979). This suggests the role of mitochondria movement is associated with the energy requirement of the cells. Such a close association of mitochondria with basal infolds and microvilli is found in the cells involved in active fluid secretion or ion absorption and the mitochondria are supplying energy for membrane associated transport pumps (Kukel and Komnick, 1989). It has been observed that with the advancement of age of adult flies, the luminal microvilli become excessively long and several spherites/formed bodies and mineral concretions are observed in the region of luminal microvilli and also in the tubular lumen suggesting their release into the lumen by exocytosis (Wessing and Eichelberg, 1975; Wessing and Zierold, 1999). Thus the secretory activity increases with the advancement of age of flies and this high secretory activity might be associated with consumption of food and water in the adult (Ryerse, 1978). In the tubular lumen of adult flies, spherical structures or formed bodies have been observed and sometimes the lumen is closely packed with these structures. The formed bodies were first observed by Riegel (1966) in the Malpighian tubules and these bodies serve as the tubules method of releasing cell inclusions into the lumen and/or movement of solutes across the epithelium (Riegel, 1971). However, in M. domestica type C, concretions similar to formed bodies have not been observed in the lumen, though these were present in the cytoplasm of the cell (Sohal et al., 1976). In principal cells of Malpighian tubules of Sarcophaga, several mineral concretions or spherocrystals are found in the central cytoplasm, at the bases of microvilli, in the luminal microvillar region and also in the tubule lumen. The spherocrystals are supposed to be storage excretion sites (Bradley, 1985) and a characterstic feature of secretory Malpighian tubule cells. The mineral concretions may contain phosphorus, sulphur, calcium, iron, zinc and copper and these may be intracytoplasmic, not being discharged into the lumen (Sohal et al., 1976) and may act as transitory depots for the concentration of substances removed from the haemolymph or alternatively these are transported to apical plasma membrane and subsequently discharged into the lumen
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by the process of exocytosis (Wessing and Zierold, 1999). In the Malpighian tubules of Sarcophaga, the mineral concretions are observed at the bases of apical plasma membrane and also in the tubule lumen suggesting their extrusion by exocytosis. The stellate cells in the Malpighian tubules of adult flesh fly have short microvilli devoid of mitochondria suggesting that these cells are not actively involved in the ion transport. Furthermore unlike the principal cells, mineral concretions or spherocrystals, which are the characteristic features of secretory cells, have not been observed in the stellate cells. These cells, interspersed with principal cells, have been observed in many insects (Martoja and Ballan-Dufrancais, 1984). It has been suggested that the function of stellate cells is reabsorptive and, these cells reabsorb sodium ions from the lumen or directly from the principal cells, and return it to the blood (Berridge and Oschman, 1969). The ultrastructural features of principal cells demonstrate that these are the major ion transporting cells requiring the expenditure of energy, whereas the stellate cells, as compared to principal cells, lack the characteristics of transporting cells and may perform reabsorptive function. References Alkassis, W., Schoeller-Raccaud, J., 1984. Ultrastructure of the Malpighian tubules of blow fly larva, Calliphora erythrocephala Meigen (Diptera: Calliphoridae). Int. J. Insect Morphol. Embryol. 13, 215–231. Berridge, M.J., Oschman, J.L., 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue Cell 1, 247–272. Beyenbach, K.W., Skaer, H., Dow, J.A.T., 2010. The development, molecular, and transport biology of Malpighian tubules. Annu. Rev. Entomol. 55, 351–374. Bradley, T.J., 1985. The excretory system: structure and physiology. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 4. Pergamon Press, Oxford, pp. 421–465. Bradley, T.J., Satir, P., 1977. Microvillar beating and mitochondrial migration in Malpighian tubules. J. Cell Biol. 75, 255a. Bradley, T.J., Stuart, A.M., Satir, P., 1982. The ultrastructure of the larval Malpighian tubules of a saline-water mosquito. Tissue Cell 14, 759–773. Chapman, R.F., 2008. The Insects: Structure and Function, 4th ed. Cambridge University Press, New York, pp. 478–508.
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