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Segmental specializations in the Malpighian tubules of the fire ant Solenopsis saevissima Forel 1904 (Myrmicinae): an electron microscopical study
Arthropod Structure & Development 30 (2002) 281±292
ARTHROPOD STRUCTURE & DEVELOPMENT www.elsevier.com/locate/asd
Segmental specializations in the Malpighian tubules of the ®re ant Solenopsis saevissima Forel 1904 (Myrmicinae): an electron microscopical study Alberto Arab*, FlaÂvio Henrique Caetano Departamento de Biologia, Instituto de BiocieÃncias, Universidade Estadual Paulista, Rio Claro, SaÄo Paulo, CEP 13506-900, Brazil Received 5 October 2001; accepted 8 December 2001
Abstract The Malpighian tubules of workers of the ®re ant Solenopsis saevissima (Myrmicinae) were analyzed by scanning and transmission electron microscopy in order to determine their functional organization and association with the hindgut epithelium. The ants showed six Malpighian tubules with three segments morphologically and structurally different. The proximal segment was long and its cells showed abundant smooth endoplasmic reticulum and lipid droplets, which suggest their role in lipid secretion. The mid segment was long and undulated and it was composed by the cells that showed the typical features of ion transporting epithelia. The distal segment, short and ¯attened, adheres to the rectum wall. The cells of this segment showed the basal lamina fused to that of the rectum, it is probable that this part of the tubule may play a role in ion and water uptake from the feces. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ant; Excretion; Ion uptaking; Malpighian tubules; Rectum
1. Introduction Although ants are among the most widely studied insects, only a few works deal with the ultrastructure of the Malpighian tubules. Malpighian tubules are the main excretory and osmoregulatory organs of insects. They produce an isosmotic ®ltrate from the haemolymph, termed primary urine, which carries excretory wastes and toxic compounds into the hindgut. The ®nal phase of the excretory process commonly occurs in the rectum, from which some ions and water are reabsorbed through the rectal pads. The Malpighian tubules are tubular structures inserted in the region located between the midgut and the hindgut. They are usually free and wave around inside the body cavity, but in some insects the distal end of these tubules are held in contact with the rectal wall by the perinephric membrane, in an arrangement known as cryptonephridial complex (Bradley, 1985; Chapman, 1998). The Malpighian tubules consist of a single layer of one or more types of cells, surrounding a blind-ending lumen. In some insects, the ultrastructure of the cells is uniform throughout the tubules, as in the case of Calpodes ethlius (Lepidoptera) (Ryerse, 1978), Musca domestica (Diptera) * Corresponding author. Tel.: 155-19-526-4131; fax: 155-19-534-0009. E-mail address: [email protected] (A. Arab).
(Sohal, 1974), and Frankliniella occidentalis (Thysanoptera) (Dallai et al., 1991). However, in other insects, the Malpighian tubules are divided into functionally and morphologically distinct segments, showing distinct cell types (Wigglesworth, 1974; Bradley, 1985). Ants of the genus Solenopsis are omnivorous. They are among the most thoroughly studied species because of their enormous medical and agricultural impact. However, it is surprising that very few reports deal with the Malpighian tubules ultrastructure, when such studies are essential in understanding the physiology of excretion of the ant with the objective of ®nding successful control methods. For these reasons, this paper focuses on the ultrastructural and functional organization of the Malpighian tubules and describes the association of the cells of the distal segment with the hindgut epithelium.
2. Materials and methods Major and minor workers of Solenopsis saevissima Forel 1904 were collected from nests located in open areas around the Bioscience Institute of the Universidade Estadual Paulista in Rio Claro, SaÄo Paulo State, Brazil. They were anaesthetized by freezing and dissected in Karnovsky ®xative. Digestive tracts were removed, ®xed in Karnovsky
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®xative, and rinsed in 0.2 M phosphate buffer at pH 7.4 for 2 h at room temperature. Scanning electron microscopy (SEM) was performed in whole mounts. These were prepared using critical point drying and gold sputtering of ®xed digestive tracts and examined with a Jeol JMS-P15 scanning electron microscope. Transmission electron microscopy (TEM) was also carried out on the digestive tracts previously ®xed in Karnovsky. After rinsing, the material was post-®xed in a solution of 1% osmium tetroxide in the same buffer for 1 h at the same temperature. Block staining was carried out in 1% aqueous uranyl acetate for 16±18 h at room temperature. The material was dehydrated in a standard acetone series ending at a concentration of 100%, and was embedded in EPON resin. Ultrathin sections of the Malpighian tubules were cut using a Sorvall Porter Blum MT2 ultramicrotome, then stained with uranyl acetate and lead citrate, and viewed under a Phillips CM-100 transmission electron microscope. 3. Results The workers of S. saevissima have six Malpighian tubules. The tubules are ,1±2 mm in length and open separately into a region located between the midgut and the hindgut (Fig. 1A). When the Malpighian tubules were viewed using SEM, 3 distinct segments were recognized based on their length and shape: the proximal segment (long and poorly undulated) (Fig. 1A), the mid segment (long and undulated) (Fig. 1B), and the distal segment (short and ¯attened) attached to the rectum's wall without penetrating it (Fig. 1C). Two outer parallel muscle ®bers coil along each tubule (Fig. 1B), and they are accompanied by tracheoles, which are numerous in the distal segment of the tubule that is attached to the rectum (Fig. 1C). Each Malpighian tubule is made up of a single epithelium consisting of only one type of cuboidal cell lying on a thin basal lamina. In general, all the cells showed the following features: basal infoldings that extend to about one fourth of the cell's height; the apical cytoplasm with microvilli, which are regular in size and show cisternae of smooth endoplasmic reticulum inside them; mitochondria close to the basal infoldings and microvilli. Nevertheless, some differences of the cell structure can be distinguished in the different segments. 3.1. Proximal segment The cells from the proximal segment present their cytoplasm occupied by a large vacuole of clear content (Fig. 2A). A thin basal lamina covers the outside of the cells. It is comprised of two layers that separate in order to surround the muscle ®bers (Fig. 2A and B). The basal infoldings appear to be associated with mitochondria (Fig. 2B) and the cytoplasm includes abundant smooth endoplasmic reticulum, lipid droplets, a few spherocrystals, and clear
membrane-bounded vesicles (Fig. 2A±C). Some mitochondria show the presence of droplets inside them (Fig. 2D). The central cytoplasm is occupied by a large nucleus with an irregular outline and some nucleoli (Figs. 2A, C and 3A). Rough and smooth endoplasmic reticulum, Golgi complexes and peroxisomes are also found in this region (Fig. 3A). The apical microvilli are packed together. In the lumen, a dense secretion can be observed, together with what appears to be remnants of membrane-bounded vesicles (Fig. 3B). 3.2. Mid segment The cells of this segment of the Malpighian tubule are laying on a narrow basal lamina consisting of two layers that diverge in order to surround the muscle ®bers (Fig. 4A), as was observed in the proximal segment. The basal cytoplasm shows a few mitochondria associated with the infoldings of the plasma membrane (Fig. 4A and B). The cytoplasm contains lipid droplets, spherocrystals, and a large amount of clear membrane-bounded vesicles, some of which are apparently empty (Fig. 4B). The nucleus is large, oval and located centrally (Fig. 4B). Neighboring cells are linked by extensive and very sinuous junctional complexes (Fig. 5A) of septate type (Fig. 5B). The microvilli are densely packed and clear vesicles are occasionally seen between them. The tips of the microvilli are often swollen and hosting dilated smooth reticulum cisternae containing a clear secretion. A dense secretion is observed in the lumen of this segment of the Malpighian tubule (Fig. 5C). 3.3. Distal segment The striking cell features of this segment of the Malpighian tubules are that they are closely adhering to the rectal epithelium (Fig. 6A), from which they are separated by the basal laminae of both the tubule and rectum fused together (Fig. 6B and C). Sometimes, the basal lamina appears separated by muscle bundles (Fig. 7A). In the contact region, the rectal epithelium is narrow and lined by a thin cuticle, with cells showing a few organelles (Fig. 6B). The basal infoldings are associated with a high number of mitochondria (Fig. 7A). The cytoplasm contains peroxisomes, spherocrystals (Fig. 7A) and a large number of clear membrane-bounded vesicles, which are more numerous in the cell region that is connected to the rectal wall (Fig. 6A). The nucleus is elongated and contains some nucleoli (Fig. 7B). The microvilli are similar to those in the previous segments with their tips often swollen. The lumen appears to be occupied by a dense secretion and apparent remnants of membrane bounded vesicles (Fig. 8). 4. Discussion Malpighian tubules show a great diversity in number, size, morphology, and arrangement among ant species.
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Fig. 1. SEM pictures of the Malpighian tubules. (A) The tubules open between the ventriculus (vet) and the ileum (il). (B) General view of the mid segment (ms) of the tubules. (C) Detail of the distal segment (ds) of the tubules attached to the rectum (r). Abbreviations: ps: proximal segment; t: tracheoles; arrow: muscle ®ber. Scale bars: A 30 mm; B and C 20 mm:
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Fig. 2. Cross-section through the proximal segment of the Malpighian tubules. (A) Note the vacuole (v) occupying most part of the cytoplasm. (B) Infoldings (i) associated with mitochondria (m) and clear membrane-bounded vesicles (ve) in the basal cytoplasm. (C) View of the central cytoplasm. (D) Detail of the mitochondria which contains a droplet (big arrow). Abbreviations: bl: basal lamina; f: muscle ®ber; g: Golgi complex; l: lipid droplet; n: nucleus; small arrow: spherocrystal. Scale bars: A 3 mm; B and C 1 mm; D 0:5 mm:
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The number of Malpighian tubules in ants can vary among species: Paraponera clavata and Dinoponera gigantea (Ponerinae) have ,50 tubules (Caetano et al., 1986/1987) while workers of Crematogaster lineolata and Myrmicina americana (Myrmicinae) have only ®ve (Ettershank and Brown, 1964). According to Caetano (1984), some ant species show a variation in the number of tubules in relation to the body size of the different castes; this was observed for leaf cutting ants of the genus Atta (Myrmicinae), in which the soldiers and minor workers possess 16 and 5 tubules, respectively. S. saevissima does not show variation in the number of tubules between major and minor workers. In ants, the muscles associated with the Malpighian tubules consist of one or two ®bers that coil around the tubule extending along them, as has been recorded for S. saevissima, Pachycondyla villosa (Caetano, 1988) and Formica polyctena (Garayoa et al., 1992). However, in Atta sexdens rubropilosa muscle ®bers are absent in the distal segment (Martinelli, 1998). The Malpighian tubules of many insects have been reported to move because of the presence of intrinsic muscles. In the cockroach Periplaneta americana (Dyctioptera), the band disposition of the ®bers allows the shortening and contraction of the tubule (Crowder and Shankland, 1972) and this may be implicated in the transport of the content of the tubules' lumen into the hindgut. Three segments were distinguished in the Malpighian tubules of S. saevissima: proximal, mid, and distal. The distinction is based on the shape and structure of the epithelial cells lining the different segments of the tubules even though the cells of the three segments share certain features. These include basal infoldings of the plasma membrane and microvilli of regular size or sometimes swollen containing smooth endoplasmic reticulum, which could be associated with vesicle transport as proposed by Wessing and Eichelberg (1975). The tubule cells lay on a thin basal lamina, which appear fused with that of the rectal cells in the distal segment. The permeability properties of the basal lamina may be important in determining the composition of the ¯uid transported by the cells of the Malpighian tubules. Thus, the basal lamina provides a size-dependent barrier for proteins in the haemolymph (Berrigde and Oschman, 1969); nevertheless, the basal lamina is not an entirely passive barrier and may play an important role in ion transport (Bradley, 1985). There are no differences among the segments as regards to length, number, and distribution of the basal infoldings and apical microvilli. The latter represent an increase of the cellular surface exchange capacity and they may be responsible for water or solute ¯uxes by an osmotic gradient (Berrigde and Oschman, 1969); thus, they are the place of active mechanisms of solute transport (Nicolson, 1993). The mitochondria in the cells of the Malpighian tubules do not show an even distribution throughout the cytoplasm. These organelles appear to lie closer to the apical microvilli
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or the basal infoldings. This association is common in cells involved in active ¯uid secretion or ion absorption and it has been interpreted as evidence that the mitochondria are supplying energy for membrane-associated transport pumps (Bradley, 1985; Kukel and Komnick, 1989). However, the cells of the Malpighian tubules of S. saevissima do not exhibit mitochondria extending into the microvilli, as occurs in the type I cells of some Diptera (Berrigde and Oschman, 1969; Sohal, 1974) and in the cells of the cryptonephridial system of Tenebrio molitor (Coleoptera) (Meyran, 1982). The existence of several segments, each one with a different cell structure, along the Malpighian tubules of insects has been established in numerous species: Rhodnius prolixus, Hemiptera (Wigglesworth and Salpeter, 1962); Calliphora erythrocephala, Diptera (Berrigde and Oschman, 1969); P. americana and Blatella germanica, Dyctioptera (Wall et al., 1975; Meyran, 1982); T. molitor, Coleoptera (Meyran, 1982); C. ethlius, Lepidoptera (Ryerse, 1979); Melipona quadrifasciata anthidioides, Hymenoptera (CruzLandim, 1988. Moreover, the number of recognized segments is not the same and the cell types observed seem to be often associated with different functions (Berridge and Oschmann, 1969; Meyran, 1982). In the proximal segment of the Malpighian tubules of S. saevissima, which is long and poorly undulated, the most striking feature of the cells is the presence of a large vacuole occupying most of the cytoplasm and mitochondria containing droplets. The vacuole may be the result of a fusion of the clear vesicles found in the cytoplasm. To our knowledge, mitochondria containing droplets have never been reported in insect's Malpighian tubules. Since large amounts of lipids were found inside the Malpighian tubules and mitochondria selectively uptake lipids from the cytoplasm to be metabolized into Acetyl-CoA (Alberts et al., 1994), it is possible that these droplets could be the forms of lipid storage. However, histochemical studies would be needed in order to con®rm this assumption. The tubule cells exhibit abundant smooth endoplasmic reticulum and lipid droplets. This suggests that they are involved in the metabolic process of lipid synthesis and may explain the numerous lipid droplets found in the cytoplasm. Nevertheless, smooth endoplasmic reticulum is also thought to be involved in transcellular transport (Wessing and Eichelberg 1975; Green, 1979). The functional role of lipid synthesis in the Malpighian tubules is unknown. However, in T. molitor and F. polyctena, lipids of the Malpighian tubules are converted into eicosanoids, which regulate the basal ¯uid secretion rates of this organ (Howard et al., 1992; Van Kerkhove et al., 1995). Additionally, in Aeshna cyanea (Odonata), lipid droplets appear in some cells of the tubules of ®rst-instar nymphs, being consumed during metamorphosis (Kukel and Komnick, 1989). Peroxisomes appear regularly in the Malpighian tubules' cells of many insects. The functions of these structures deal with the elimination of several toxic molecules and the break of alkyl chains from fatty acids to be transformed
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Fig. 3. Detail of the central and apical cytoplasm of a cell from the proximal segment. (A) Numerous peroxisomes (p) are observed in the plasma membrane. (B) Densely packed microvilli (mv) border the lumen where both a dense secretion (dse) and remnants of membrane-bounded vesicles (r) can be observed. Abbreviations: n: nucleus; rer: rough endoplasmic reticulum; ser: smooth endoplasmic reticulum. Scale bars: A 1 mm; B 0:4 mm:
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Fig. 4. Cross-section through the mid segment of the Malpighian tubule. (A) View of the basal cytoplasm. Observe the muscle ®ber (f) and the basal infoldings (i) of the plasma membrane. (B) Cross-section of the cell showing the nucleus (n) and numerous mitochondria (m) adjacent to the microvilli (mv). Abbreviations: bl: basal lamina; l: lipid droplet; ve: clear membrane bounded vesicles; arrow: spherocrystal. Scale bars: A 0:2 mm; B 1 mm:
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Fig. 5. General view of the apical cytoplasm of the cells of the mid segment. (A) Junctional complexes (jc) between neighboring cells. (B) Detail of a septate junction. (C) Microvilli (mv) some of which are dilated at the tip contain smooth reticulum cisternae (ser). Abbreviations: dse: dense secretion; lu: tubule's lumen; m: mitochondria. Scale bars: A 0:5 mm; B 0:1 mm; C 0:5 mm:
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Fig. 6. General view of the ultrastructure of the distal segment. (A) Note the numerous clear membrane-bounded vesicles (ve) at the tubule's side in contact with the rectum (r). (B) In the contact region, the rectal epithelium (re) is narrow containing a few organelles. (C) Note that the basal laminae of both the tubule (blt) and rectum (blr) are fused together. Abbreviations: arrowhead: basal laminae fused together; c: cuticle; f: muscle ®ber; i: basal infoldings; m: mitochondria; rlu: rectal lumen; te: tubule epithelium. Scale bars: A 5 mm; B 0:4 mm; C 0:2 mm:
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Fig. 7. (A) Basal infoldings (i) and mitochondria (m) in a cell from the distal segment. (B) Note the elongated nucleus (n) and the mitochondria (m) adjacent to the microvilli (mv). Abbreviations: bl: basal lamina; f: muscle ®ber; nc: nucleoli; p: peroxisomes; ve: clear membrane bounded vesicle; arrow: spherocrystal. Scale bars: A 0:4 mm; B 2 mm:
into Acetyl-CoA (Alberts et al., 1994). Another function of peroxisomes concerns uric acid metabolism. They contain a number of enzymes related to nitrogen metabolism, including amino acid oxidases, uricase and catalase. Peroxisomes are common in the Malpighian tubules of P. americana, where they may be involved in uric acid metabolism, converting it to ammonia, which is the main excretory product of this insect (Wall et al., 1975). The cells from the proximal segment of S. saevissima have certain features in common with cells from the proximal segment of the ants Atta sexdens (Myrmicinae) (Martinelli, 1998) and Dolichoderus decollatus (Dolichoderinae) (Caetano et al., 1990). These features include large quantities of smooth endoplasmic reticulum and the presence of lipid droplets in the cytoplasm. Thus, it is possible that in these ants the cells of the proximal segment of the Malpighian tubules, apart from their excretory functions, could play a role in lipid secretion and storage. The ultrastructure of the cells from the Malpighian tubules' mid segment of S. saevissima is indicative of a transport function. These cells may be involved in the active transport of ions into the tubule's lumen, generating the primary urine. The basal and apical cell surfaces are extensively folded and mitochondria are found below the microvilli.
The most conspicuous feature of the cells from the mid segment is the presence of clear vesicles. These structures probably contained spherocrystals, which can be easily extracted because they are poorly penetrated by the plastic embedding media. The spherocrystals are generally presumed to serve as a form of storage excretion (Bradley, 1985). Thus, some ions can be stored in the cytoplasm as crystals, along the entire insect life cycle; however, there is evidence of extrusion of spherocrystals through the Malpighian tubule's lumen (Wall et al., 1975; Bradley et al., 1982; Wessing and Zierold, 1999). In some insects, the Malpighian tubules are closely associated with the hindgut. This arrangement, known as cryptonephridial system, is present in most larval Lepidoptera and Neuroptera, in some Symphyta and Diptera larvae, and in many larvae and adults of Coleoptera (Bradley, 1985). However, only those of T. molitor, Onymacris plana plana (Coleoptera) and Arachnocampa luminosa (Diptera) have been well documented. In these insects, the distal ends of the tubules lie close to the rectum and the entire complex is covered by a perinephric membrane (Green, 1980; Bradley, 1985). In ants, the adhesion of Malpighian tubules to the hindgut was documented by Ettershank and Brown (1964) with light microscopy in C. lineolata and Trachymyrmex
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Fig. 8. Microvilli (mv) of the distal segment contain smooth endoplasmic reticulum (ser). Note the dense secretion (dse) and remnants of membrane-bounded vesicles (r) in the lumen. Abbreviations: m: mitochondria. Scale bar 1 mm:
septentrionalis (Myrmicinae); however in M. americana (Myrmicinae) only three of the ®ve tubules are associated to the hindgut. Moreover, when this arrangement is found in ants, it does not present the perinephric membrane found in the other cryptonephridial complexes; in ants, the tubules appear fused only by the basal lamina, as was observed in F. occidentalis (Thysanoptera) (Dallai et al., 1991). The cryptonephridial system is known to be involved in water uptake from the rectum of insects that feed on very dry diets or live in dry habitats (Hanrahan and Nicolson, 1987). Thus, in T. molitor, KCl is actively transported into the tubule, increasing the concentration inside the tubule's lumen and drawing out water from the feces through osmosis (Noble-Nesbitt, 1990). However, in most larval Lepidoptera, which feed on succulent leaves, the cryptonephridial system seems to be involved on removing excess ions from the haemolymph. Green (1980) argues that in A. luminosa, the cryptonephridial system may serve primarily as a storage site for nutrients reabsorbed by the rectum and used by the light organ present on the fourth segment of the Malpighian tubule. S. saevissima is frequently found in dry habitats and it is widely known that this ant is capable of exploiting meals with low water content, such as grains and seeds. Then, it is
possible that the distal segment of the Malpighian tubules of this ant is involved in water and ion uptaking from the feces, as it is suggested by the presence of numerous clear membrane-bounded vesicles on the side of contact between the Malpighian tubule and the rectum wall. Thus, the association of the Malpighian tubules with the rectum may be adaptive, serving both the osmoregulatory and metabolic needs of these ants. However, physiological studies as well as histochemical evidence are required to test our observations. Acknowledgements The authors wish to thank Antonio Yabuki and MoÃnika Iamonte for their technical assistance and Johana Rincones for reviewing the grammar and syntaxes of the English language. This work was supported by CAPES and FAPESP. References Alberts, B., Bray, D., Lewis, L., Raff, M., Roberts, K., Watson, J.D., 1994. Molecular Biology of the Cell. Third ed. Garland Publishing, Inc, New York.
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ARTHROPOD STRUCTURE & DEVELOPMENT www.elsevier.com/locate/asd
Segmental specializations in the Malpighian tubules of the ®re ant Solenopsis saevissima Forel 1904 (Myrmicinae): an electron microscopical study Alberto Arab*, FlaÂvio Henrique Caetano Departamento de Biologia, Instituto de BiocieÃncias, Universidade Estadual Paulista, Rio Claro, SaÄo Paulo, CEP 13506-900, Brazil Received 5 October 2001; accepted 8 December 2001
Abstract The Malpighian tubules of workers of the ®re ant Solenopsis saevissima (Myrmicinae) were analyzed by scanning and transmission electron microscopy in order to determine their functional organization and association with the hindgut epithelium. The ants showed six Malpighian tubules with three segments morphologically and structurally different. The proximal segment was long and its cells showed abundant smooth endoplasmic reticulum and lipid droplets, which suggest their role in lipid secretion. The mid segment was long and undulated and it was composed by the cells that showed the typical features of ion transporting epithelia. The distal segment, short and ¯attened, adheres to the rectum wall. The cells of this segment showed the basal lamina fused to that of the rectum, it is probable that this part of the tubule may play a role in ion and water uptake from the feces. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ant; Excretion; Ion uptaking; Malpighian tubules; Rectum
1. Introduction Although ants are among the most widely studied insects, only a few works deal with the ultrastructure of the Malpighian tubules. Malpighian tubules are the main excretory and osmoregulatory organs of insects. They produce an isosmotic ®ltrate from the haemolymph, termed primary urine, which carries excretory wastes and toxic compounds into the hindgut. The ®nal phase of the excretory process commonly occurs in the rectum, from which some ions and water are reabsorbed through the rectal pads. The Malpighian tubules are tubular structures inserted in the region located between the midgut and the hindgut. They are usually free and wave around inside the body cavity, but in some insects the distal end of these tubules are held in contact with the rectal wall by the perinephric membrane, in an arrangement known as cryptonephridial complex (Bradley, 1985; Chapman, 1998). The Malpighian tubules consist of a single layer of one or more types of cells, surrounding a blind-ending lumen. In some insects, the ultrastructure of the cells is uniform throughout the tubules, as in the case of Calpodes ethlius (Lepidoptera) (Ryerse, 1978), Musca domestica (Diptera) * Corresponding author. Tel.: 155-19-526-4131; fax: 155-19-534-0009. E-mail address: [email protected] (A. Arab).
(Sohal, 1974), and Frankliniella occidentalis (Thysanoptera) (Dallai et al., 1991). However, in other insects, the Malpighian tubules are divided into functionally and morphologically distinct segments, showing distinct cell types (Wigglesworth, 1974; Bradley, 1985). Ants of the genus Solenopsis are omnivorous. They are among the most thoroughly studied species because of their enormous medical and agricultural impact. However, it is surprising that very few reports deal with the Malpighian tubules ultrastructure, when such studies are essential in understanding the physiology of excretion of the ant with the objective of ®nding successful control methods. For these reasons, this paper focuses on the ultrastructural and functional organization of the Malpighian tubules and describes the association of the cells of the distal segment with the hindgut epithelium.
2. Materials and methods Major and minor workers of Solenopsis saevissima Forel 1904 were collected from nests located in open areas around the Bioscience Institute of the Universidade Estadual Paulista in Rio Claro, SaÄo Paulo State, Brazil. They were anaesthetized by freezing and dissected in Karnovsky ®xative. Digestive tracts were removed, ®xed in Karnovsky
1467-8039/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 1467-803 9(01)00039-1
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®xative, and rinsed in 0.2 M phosphate buffer at pH 7.4 for 2 h at room temperature. Scanning electron microscopy (SEM) was performed in whole mounts. These were prepared using critical point drying and gold sputtering of ®xed digestive tracts and examined with a Jeol JMS-P15 scanning electron microscope. Transmission electron microscopy (TEM) was also carried out on the digestive tracts previously ®xed in Karnovsky. After rinsing, the material was post-®xed in a solution of 1% osmium tetroxide in the same buffer for 1 h at the same temperature. Block staining was carried out in 1% aqueous uranyl acetate for 16±18 h at room temperature. The material was dehydrated in a standard acetone series ending at a concentration of 100%, and was embedded in EPON resin. Ultrathin sections of the Malpighian tubules were cut using a Sorvall Porter Blum MT2 ultramicrotome, then stained with uranyl acetate and lead citrate, and viewed under a Phillips CM-100 transmission electron microscope. 3. Results The workers of S. saevissima have six Malpighian tubules. The tubules are ,1±2 mm in length and open separately into a region located between the midgut and the hindgut (Fig. 1A). When the Malpighian tubules were viewed using SEM, 3 distinct segments were recognized based on their length and shape: the proximal segment (long and poorly undulated) (Fig. 1A), the mid segment (long and undulated) (Fig. 1B), and the distal segment (short and ¯attened) attached to the rectum's wall without penetrating it (Fig. 1C). Two outer parallel muscle ®bers coil along each tubule (Fig. 1B), and they are accompanied by tracheoles, which are numerous in the distal segment of the tubule that is attached to the rectum (Fig. 1C). Each Malpighian tubule is made up of a single epithelium consisting of only one type of cuboidal cell lying on a thin basal lamina. In general, all the cells showed the following features: basal infoldings that extend to about one fourth of the cell's height; the apical cytoplasm with microvilli, which are regular in size and show cisternae of smooth endoplasmic reticulum inside them; mitochondria close to the basal infoldings and microvilli. Nevertheless, some differences of the cell structure can be distinguished in the different segments. 3.1. Proximal segment The cells from the proximal segment present their cytoplasm occupied by a large vacuole of clear content (Fig. 2A). A thin basal lamina covers the outside of the cells. It is comprised of two layers that separate in order to surround the muscle ®bers (Fig. 2A and B). The basal infoldings appear to be associated with mitochondria (Fig. 2B) and the cytoplasm includes abundant smooth endoplasmic reticulum, lipid droplets, a few spherocrystals, and clear
membrane-bounded vesicles (Fig. 2A±C). Some mitochondria show the presence of droplets inside them (Fig. 2D). The central cytoplasm is occupied by a large nucleus with an irregular outline and some nucleoli (Figs. 2A, C and 3A). Rough and smooth endoplasmic reticulum, Golgi complexes and peroxisomes are also found in this region (Fig. 3A). The apical microvilli are packed together. In the lumen, a dense secretion can be observed, together with what appears to be remnants of membrane-bounded vesicles (Fig. 3B). 3.2. Mid segment The cells of this segment of the Malpighian tubule are laying on a narrow basal lamina consisting of two layers that diverge in order to surround the muscle ®bers (Fig. 4A), as was observed in the proximal segment. The basal cytoplasm shows a few mitochondria associated with the infoldings of the plasma membrane (Fig. 4A and B). The cytoplasm contains lipid droplets, spherocrystals, and a large amount of clear membrane-bounded vesicles, some of which are apparently empty (Fig. 4B). The nucleus is large, oval and located centrally (Fig. 4B). Neighboring cells are linked by extensive and very sinuous junctional complexes (Fig. 5A) of septate type (Fig. 5B). The microvilli are densely packed and clear vesicles are occasionally seen between them. The tips of the microvilli are often swollen and hosting dilated smooth reticulum cisternae containing a clear secretion. A dense secretion is observed in the lumen of this segment of the Malpighian tubule (Fig. 5C). 3.3. Distal segment The striking cell features of this segment of the Malpighian tubules are that they are closely adhering to the rectal epithelium (Fig. 6A), from which they are separated by the basal laminae of both the tubule and rectum fused together (Fig. 6B and C). Sometimes, the basal lamina appears separated by muscle bundles (Fig. 7A). In the contact region, the rectal epithelium is narrow and lined by a thin cuticle, with cells showing a few organelles (Fig. 6B). The basal infoldings are associated with a high number of mitochondria (Fig. 7A). The cytoplasm contains peroxisomes, spherocrystals (Fig. 7A) and a large number of clear membrane-bounded vesicles, which are more numerous in the cell region that is connected to the rectal wall (Fig. 6A). The nucleus is elongated and contains some nucleoli (Fig. 7B). The microvilli are similar to those in the previous segments with their tips often swollen. The lumen appears to be occupied by a dense secretion and apparent remnants of membrane bounded vesicles (Fig. 8). 4. Discussion Malpighian tubules show a great diversity in number, size, morphology, and arrangement among ant species.
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Fig. 1. SEM pictures of the Malpighian tubules. (A) The tubules open between the ventriculus (vet) and the ileum (il). (B) General view of the mid segment (ms) of the tubules. (C) Detail of the distal segment (ds) of the tubules attached to the rectum (r). Abbreviations: ps: proximal segment; t: tracheoles; arrow: muscle ®ber. Scale bars: A 30 mm; B and C 20 mm:
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Fig. 2. Cross-section through the proximal segment of the Malpighian tubules. (A) Note the vacuole (v) occupying most part of the cytoplasm. (B) Infoldings (i) associated with mitochondria (m) and clear membrane-bounded vesicles (ve) in the basal cytoplasm. (C) View of the central cytoplasm. (D) Detail of the mitochondria which contains a droplet (big arrow). Abbreviations: bl: basal lamina; f: muscle ®ber; g: Golgi complex; l: lipid droplet; n: nucleus; small arrow: spherocrystal. Scale bars: A 3 mm; B and C 1 mm; D 0:5 mm:
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The number of Malpighian tubules in ants can vary among species: Paraponera clavata and Dinoponera gigantea (Ponerinae) have ,50 tubules (Caetano et al., 1986/1987) while workers of Crematogaster lineolata and Myrmicina americana (Myrmicinae) have only ®ve (Ettershank and Brown, 1964). According to Caetano (1984), some ant species show a variation in the number of tubules in relation to the body size of the different castes; this was observed for leaf cutting ants of the genus Atta (Myrmicinae), in which the soldiers and minor workers possess 16 and 5 tubules, respectively. S. saevissima does not show variation in the number of tubules between major and minor workers. In ants, the muscles associated with the Malpighian tubules consist of one or two ®bers that coil around the tubule extending along them, as has been recorded for S. saevissima, Pachycondyla villosa (Caetano, 1988) and Formica polyctena (Garayoa et al., 1992). However, in Atta sexdens rubropilosa muscle ®bers are absent in the distal segment (Martinelli, 1998). The Malpighian tubules of many insects have been reported to move because of the presence of intrinsic muscles. In the cockroach Periplaneta americana (Dyctioptera), the band disposition of the ®bers allows the shortening and contraction of the tubule (Crowder and Shankland, 1972) and this may be implicated in the transport of the content of the tubules' lumen into the hindgut. Three segments were distinguished in the Malpighian tubules of S. saevissima: proximal, mid, and distal. The distinction is based on the shape and structure of the epithelial cells lining the different segments of the tubules even though the cells of the three segments share certain features. These include basal infoldings of the plasma membrane and microvilli of regular size or sometimes swollen containing smooth endoplasmic reticulum, which could be associated with vesicle transport as proposed by Wessing and Eichelberg (1975). The tubule cells lay on a thin basal lamina, which appear fused with that of the rectal cells in the distal segment. The permeability properties of the basal lamina may be important in determining the composition of the ¯uid transported by the cells of the Malpighian tubules. Thus, the basal lamina provides a size-dependent barrier for proteins in the haemolymph (Berrigde and Oschman, 1969); nevertheless, the basal lamina is not an entirely passive barrier and may play an important role in ion transport (Bradley, 1985). There are no differences among the segments as regards to length, number, and distribution of the basal infoldings and apical microvilli. The latter represent an increase of the cellular surface exchange capacity and they may be responsible for water or solute ¯uxes by an osmotic gradient (Berrigde and Oschman, 1969); thus, they are the place of active mechanisms of solute transport (Nicolson, 1993). The mitochondria in the cells of the Malpighian tubules do not show an even distribution throughout the cytoplasm. These organelles appear to lie closer to the apical microvilli
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or the basal infoldings. This association is common in cells involved in active ¯uid secretion or ion absorption and it has been interpreted as evidence that the mitochondria are supplying energy for membrane-associated transport pumps (Bradley, 1985; Kukel and Komnick, 1989). However, the cells of the Malpighian tubules of S. saevissima do not exhibit mitochondria extending into the microvilli, as occurs in the type I cells of some Diptera (Berrigde and Oschman, 1969; Sohal, 1974) and in the cells of the cryptonephridial system of Tenebrio molitor (Coleoptera) (Meyran, 1982). The existence of several segments, each one with a different cell structure, along the Malpighian tubules of insects has been established in numerous species: Rhodnius prolixus, Hemiptera (Wigglesworth and Salpeter, 1962); Calliphora erythrocephala, Diptera (Berrigde and Oschman, 1969); P. americana and Blatella germanica, Dyctioptera (Wall et al., 1975; Meyran, 1982); T. molitor, Coleoptera (Meyran, 1982); C. ethlius, Lepidoptera (Ryerse, 1979); Melipona quadrifasciata anthidioides, Hymenoptera (CruzLandim, 1988. Moreover, the number of recognized segments is not the same and the cell types observed seem to be often associated with different functions (Berridge and Oschmann, 1969; Meyran, 1982). In the proximal segment of the Malpighian tubules of S. saevissima, which is long and poorly undulated, the most striking feature of the cells is the presence of a large vacuole occupying most of the cytoplasm and mitochondria containing droplets. The vacuole may be the result of a fusion of the clear vesicles found in the cytoplasm. To our knowledge, mitochondria containing droplets have never been reported in insect's Malpighian tubules. Since large amounts of lipids were found inside the Malpighian tubules and mitochondria selectively uptake lipids from the cytoplasm to be metabolized into Acetyl-CoA (Alberts et al., 1994), it is possible that these droplets could be the forms of lipid storage. However, histochemical studies would be needed in order to con®rm this assumption. The tubule cells exhibit abundant smooth endoplasmic reticulum and lipid droplets. This suggests that they are involved in the metabolic process of lipid synthesis and may explain the numerous lipid droplets found in the cytoplasm. Nevertheless, smooth endoplasmic reticulum is also thought to be involved in transcellular transport (Wessing and Eichelberg 1975; Green, 1979). The functional role of lipid synthesis in the Malpighian tubules is unknown. However, in T. molitor and F. polyctena, lipids of the Malpighian tubules are converted into eicosanoids, which regulate the basal ¯uid secretion rates of this organ (Howard et al., 1992; Van Kerkhove et al., 1995). Additionally, in Aeshna cyanea (Odonata), lipid droplets appear in some cells of the tubules of ®rst-instar nymphs, being consumed during metamorphosis (Kukel and Komnick, 1989). Peroxisomes appear regularly in the Malpighian tubules' cells of many insects. The functions of these structures deal with the elimination of several toxic molecules and the break of alkyl chains from fatty acids to be transformed
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Fig. 3. Detail of the central and apical cytoplasm of a cell from the proximal segment. (A) Numerous peroxisomes (p) are observed in the plasma membrane. (B) Densely packed microvilli (mv) border the lumen where both a dense secretion (dse) and remnants of membrane-bounded vesicles (r) can be observed. Abbreviations: n: nucleus; rer: rough endoplasmic reticulum; ser: smooth endoplasmic reticulum. Scale bars: A 1 mm; B 0:4 mm:
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Fig. 4. Cross-section through the mid segment of the Malpighian tubule. (A) View of the basal cytoplasm. Observe the muscle ®ber (f) and the basal infoldings (i) of the plasma membrane. (B) Cross-section of the cell showing the nucleus (n) and numerous mitochondria (m) adjacent to the microvilli (mv). Abbreviations: bl: basal lamina; l: lipid droplet; ve: clear membrane bounded vesicles; arrow: spherocrystal. Scale bars: A 0:2 mm; B 1 mm:
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Fig. 5. General view of the apical cytoplasm of the cells of the mid segment. (A) Junctional complexes (jc) between neighboring cells. (B) Detail of a septate junction. (C) Microvilli (mv) some of which are dilated at the tip contain smooth reticulum cisternae (ser). Abbreviations: dse: dense secretion; lu: tubule's lumen; m: mitochondria. Scale bars: A 0:5 mm; B 0:1 mm; C 0:5 mm:
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Fig. 6. General view of the ultrastructure of the distal segment. (A) Note the numerous clear membrane-bounded vesicles (ve) at the tubule's side in contact with the rectum (r). (B) In the contact region, the rectal epithelium (re) is narrow containing a few organelles. (C) Note that the basal laminae of both the tubule (blt) and rectum (blr) are fused together. Abbreviations: arrowhead: basal laminae fused together; c: cuticle; f: muscle ®ber; i: basal infoldings; m: mitochondria; rlu: rectal lumen; te: tubule epithelium. Scale bars: A 5 mm; B 0:4 mm; C 0:2 mm:
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Fig. 7. (A) Basal infoldings (i) and mitochondria (m) in a cell from the distal segment. (B) Note the elongated nucleus (n) and the mitochondria (m) adjacent to the microvilli (mv). Abbreviations: bl: basal lamina; f: muscle ®ber; nc: nucleoli; p: peroxisomes; ve: clear membrane bounded vesicle; arrow: spherocrystal. Scale bars: A 0:4 mm; B 2 mm:
into Acetyl-CoA (Alberts et al., 1994). Another function of peroxisomes concerns uric acid metabolism. They contain a number of enzymes related to nitrogen metabolism, including amino acid oxidases, uricase and catalase. Peroxisomes are common in the Malpighian tubules of P. americana, where they may be involved in uric acid metabolism, converting it to ammonia, which is the main excretory product of this insect (Wall et al., 1975). The cells from the proximal segment of S. saevissima have certain features in common with cells from the proximal segment of the ants Atta sexdens (Myrmicinae) (Martinelli, 1998) and Dolichoderus decollatus (Dolichoderinae) (Caetano et al., 1990). These features include large quantities of smooth endoplasmic reticulum and the presence of lipid droplets in the cytoplasm. Thus, it is possible that in these ants the cells of the proximal segment of the Malpighian tubules, apart from their excretory functions, could play a role in lipid secretion and storage. The ultrastructure of the cells from the Malpighian tubules' mid segment of S. saevissima is indicative of a transport function. These cells may be involved in the active transport of ions into the tubule's lumen, generating the primary urine. The basal and apical cell surfaces are extensively folded and mitochondria are found below the microvilli.
The most conspicuous feature of the cells from the mid segment is the presence of clear vesicles. These structures probably contained spherocrystals, which can be easily extracted because they are poorly penetrated by the plastic embedding media. The spherocrystals are generally presumed to serve as a form of storage excretion (Bradley, 1985). Thus, some ions can be stored in the cytoplasm as crystals, along the entire insect life cycle; however, there is evidence of extrusion of spherocrystals through the Malpighian tubule's lumen (Wall et al., 1975; Bradley et al., 1982; Wessing and Zierold, 1999). In some insects, the Malpighian tubules are closely associated with the hindgut. This arrangement, known as cryptonephridial system, is present in most larval Lepidoptera and Neuroptera, in some Symphyta and Diptera larvae, and in many larvae and adults of Coleoptera (Bradley, 1985). However, only those of T. molitor, Onymacris plana plana (Coleoptera) and Arachnocampa luminosa (Diptera) have been well documented. In these insects, the distal ends of the tubules lie close to the rectum and the entire complex is covered by a perinephric membrane (Green, 1980; Bradley, 1985). In ants, the adhesion of Malpighian tubules to the hindgut was documented by Ettershank and Brown (1964) with light microscopy in C. lineolata and Trachymyrmex
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Fig. 8. Microvilli (mv) of the distal segment contain smooth endoplasmic reticulum (ser). Note the dense secretion (dse) and remnants of membrane-bounded vesicles (r) in the lumen. Abbreviations: m: mitochondria. Scale bar 1 mm:
septentrionalis (Myrmicinae); however in M. americana (Myrmicinae) only three of the ®ve tubules are associated to the hindgut. Moreover, when this arrangement is found in ants, it does not present the perinephric membrane found in the other cryptonephridial complexes; in ants, the tubules appear fused only by the basal lamina, as was observed in F. occidentalis (Thysanoptera) (Dallai et al., 1991). The cryptonephridial system is known to be involved in water uptake from the rectum of insects that feed on very dry diets or live in dry habitats (Hanrahan and Nicolson, 1987). Thus, in T. molitor, KCl is actively transported into the tubule, increasing the concentration inside the tubule's lumen and drawing out water from the feces through osmosis (Noble-Nesbitt, 1990). However, in most larval Lepidoptera, which feed on succulent leaves, the cryptonephridial system seems to be involved on removing excess ions from the haemolymph. Green (1980) argues that in A. luminosa, the cryptonephridial system may serve primarily as a storage site for nutrients reabsorbed by the rectum and used by the light organ present on the fourth segment of the Malpighian tubule. S. saevissima is frequently found in dry habitats and it is widely known that this ant is capable of exploiting meals with low water content, such as grains and seeds. Then, it is
possible that the distal segment of the Malpighian tubules of this ant is involved in water and ion uptaking from the feces, as it is suggested by the presence of numerous clear membrane-bounded vesicles on the side of contact between the Malpighian tubule and the rectum wall. Thus, the association of the Malpighian tubules with the rectum may be adaptive, serving both the osmoregulatory and metabolic needs of these ants. However, physiological studies as well as histochemical evidence are required to test our observations. Acknowledgements The authors wish to thank Antonio Yabuki and MoÃnika Iamonte for their technical assistance and Johana Rincones for reviewing the grammar and syntaxes of the English language. This work was supported by CAPES and FAPESP. References Alberts, B., Bray, D., Lewis, L., Raff, M., Roberts, K., Watson, J.D., 1994. Molecular Biology of the Cell. Third ed. Garland Publishing, Inc, New York.
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