Fine structure of the Malpighian tubules of chironomus larva in relation to glycogen storage and fate of hemoglobin

TISSUE & CELL 1988 20 (3) 355-380 0 1988 Longman Group UK Ltd

MOHINDER S. JARIAL

FINE STRUCTURE OF THE MALPIGHIAN TUBULES OF CHIRONOMUS LARVA IN RELATION TO GLYCOGEN STORAGE AND OF HEMOGLOBIN

FATE

Keywords: Cell types, basal infoldings, microvilli, vesicles, glycogen, hemoglobin ABSTRACT. The larval Malpighian tubules of Chironorn~~~lentam were studied using tight and electron microscopy. The tubules are composed of two cell types: primary and stellate cells. Both cell types lack muscles, tracheoles, and laminate crystals in the cytoplasm and mitochondria in the microvilli. The primary cells exhibit long, wide basal membrane infoldings associated with mitochondria. They have a number of canaliculi and long, closely packed microvilli. The stellate cells possess shorter interconnecting basal infoldings and shorter, well-spaced microvilli. Both cell types are linked by septate and gap junctions. They have cytoplasmic processes and pedicels which enclose narrow slits between them and that are apposed to a basal lamella. In the ‘fed larva, the cells are stuffed with glycogen which is depleted in the ‘starved’ larva. Both cell types are involved in the vesicular transport of biliverdin. The presence of coated vesicles, tubular elements and various forms of lysosomes in the primary cells suggests they transport and break down functional hemoglobin. Structural modification of basal infoldings, canaliculi and microvilli is strongly correlated with increased secretory activity of the Malpighian tubules in ‘fed’ versus ‘starved’ larva

Introduction

In the last 25 years, ultrastructural features of the Malpighian tubules of various larval and adult insects have been described in detail (Smith and Littau, 1960; Wigglesworth and Salpeter, 1962; Wessing, 1965; Berridge and Oschman, 1969; Jarial and Scudder, 1970; Taylor, 1971a, b; Sohal, 1974; Wall et al. , 1975; Green, 1979; Ryerse, 1979; Bradley et al., 1982)) but so far no detailed ultrastructural study has been carried out on the Malpighian tubules of those insects which contain the respiratory pigment hemoglobin in their hemolymph. Chironomus larvae, aquatic forms which occur in streams and stagnant pools, are among the very few insects which contain free hemoglobin in their hemolymph that is not capsulated in blood cells (Wigglesworth, 1972). The presence of hemoglobin in the hemolymph of chironomid larvae gives them their red color and hence they are popularly known as bloodworms. These larvae inhabit mud and decaying organic matter at the bottom of pools where dissolved oxygen is scarce (Maill, 1895). It has been suggested that the free hemoglobin stores oxygen for use during

The Malpighian tubules of insects are terminally closed tubular extensions of the digestive tract located at the junction of the hindgut with the midgut. They extend into the body cavity where they are bathed directly by the hemolymph. The number of Malpighian tubules is highly variable and in some insects, like springtails and aphids, they are completely absent. Functionally they are concerned with the elimination of nitrogenous waste products and regulation of ionic composition of the hemolymph (Wigglesworth, 1972). There is a remarkable morphological segmentation in the Malpighian tubules in a number of insects which is probably related to differences in function. (Wigglesworth and Salpeter, 1962; Wessing, 1965; Smith, 1968; Jarial and Scudder, 1970; Berridge and Oschman, 1972; Szibbo and Scudder, 1979; Bradley, 1985). Muncie Center for Medical Education, Indiana University School of Medicine at Ball State University, Muncie, Indiana 47306, U.S.A. Received 15 March 1988 355

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anaerobic conditions to keep the larvae active (Mail1 and Hammond, 1900). The objective of the present study was to elucidate the fine structure of the Malpighian tubules of full-grown Chironomus tentans larva as it relates to the scarcity of dissolved oxygen in their habitat and to the tubules’ role in handling hemoglobin and its hreakdown products in which they are bathed.

Materials and Methods Full-grown larvae of Chironomus tentans were collected during summer from Boitano Lake in the Cariboo region of the province of British Columbia. Canada. The larvae were transported to the laboratory in l-gal thermos jugs with mud, pebbles and lake water. They were kept unfed and were fixed within a few days. The Malpighian tubules of the larvae were dissected out in insect Ringer solution (Hoyle, 1953). They were fixed for 15 min at room temperature in the following mixture: 1 part 5% osmium tetroxide. 1 part 10% glutaraldehyde, and two parts 0.2 M phosphate buffer (pH 7.2). After fixation the material was washed in two changes of phosphate buffer, cut into small pieces, dehydrated and embeddedin Epon 812 (Luft, 1961). Sections were cut with a Porter-Blum MT2 microtome and stained with uranyl acetate and lead citrate. The sections were viewed in a Hitachi HU-1lA electron microscope. Material similarly embedded was also cut at l-2 pm and stained with Azure II and viewed with the light microscope.

Results General morphology and histological observations The larvae of C’hironomus tentans possess four fairly large Malpighian tubules which open into the dilated portion of the small intestine or ileum. Each Malpighian tubule is lined by a single layer of flattened epithelial cells with large nuclei (Figs l-3). The tubules vary from 70 to 1OSpl.min diameter and exhibit no morphological segmentation. In comparison with the Malpighian tubules of insects studied so far. the tubules here are devoid of tracheal supply and muscles. In cross-sections, two cell types can be distinguished in the tubular epithelium. These cells are called primary cells and stellate cells (Figs 2. 21) since they bear resemblance in structural detail to similarly named cell types in Cafliphora (Berridge and Oschman, 1969); Periplaneta (Wall et al., 1975) and the saltwater Aedes larva (Bradley et al.. 1982). The primary cells form the bulk of the tubular wall and they often project into the lumen of the tubule (Fig. 2). The large. round. centrally placed nuclei contain chromosomes and welldeveloped nucleoli. and the cytoplasm is devoid of crystals (Fig. 3). The stellate cells arc fewer in number and smaller in size. They are found scattered amongst the primary cells and also lack crystals (Figs 2, 21). Both cell types appear ‘dark’ in some larvae while in other larvae they appear ‘light’. Although no distinction could be made between the different larvae at the time of collection or during dissection, the structural details indicate that the Malpighian tubules with dark

Fig. 1. Diagrammatic representation of the proposed transport by endocytosis and exocytoals of the free hemoglobin and biliverdin through a primary cell (P) of the larval Malpighian tubules of Chironomur fenfans. (H) hemocoel; (Hb) hemoglobin; (BVN) biliverdin; (BL) basal lamella: (PD) pedicel; (p) cytoplasmic process; (BMI) basal membrane infolding: (EC) extracellular channel; (M) mitochondrion; (V) vesicles; (CV) coated vesicles; (TE) tubular elements; (BCV) basal condensing vacuoles; (G) Golgi complex; (ER) rough endoplasmic reticulum; (GLY) glycogen; (PL) primary lysosome; (SL) secondary lysosome: (RB) residual body; (MV) microvilli; (L) lumen; (SJ) septate junction; (B) blister; (GP) gap iunction; (1’2) intercellular channel: (C) canaliculi; (D) desmosome; (S) slit

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Fig. 2. Light micrographs of a thick section of the Malpighian tubule of a fed Chironomuc

tentam larva showing a primary cell(P) and a stellate cell (S). The cells are stuffed with glycogen; hence they appear dark. Note a clear zone at the periphery of the tubule (* *). A number of canaliculi (C) and a brush border (BB) facing the tubular lumen (L) can be seen. x775. Fig. 3. Light micrograph of a thick section of the Malpighian tubule of a starved Chironomuv

tentamlarva showing a primary cell (P). Due to depletion of glycogen the primary cell appears light. Note the absence of a clear peripheral zone and a shorter brush border (BB). (N) nucleus: (NU) nucleolus; (Ch) chromosomes; (C) canaliculus; (BB) brush border; (L) lumen. x560

cells (Fig. 2) belong to the naturally ‘fed’ larvae and the tubules with light cells (Figs 3, 21) belong to ‘starved’ larvae, and they will be so described in this paper. Thick sections of the Malpighian tubules of the fed larvae exhibit a well-developed brush border, canaliculi and a clear peripheral zone (Fig. 2) which at the EM level corresponds to the labyrinth of basal extracellular channels. The tubules of the starved larva have a large diameter (105 vs. 70 pm) and a narrow brush border’ (Fig. 3). Their light .primary cells occasionally contain a large, clear intracellular space (Fig. 21). Ultrastructural observations

Both primary

cells and stellate

cells are

ensheathed (Figs 4,5,8, 1I, 23) by a 150 nm thick basal lamelia (a recent and more appropriate term for basement membrane, Bradley, 1985). The basal lamella consists of an external granular layer and an internal filamentous layer (Fig. 15). There are no tracheoles associated with the basal lamella or interior of the cells. Both cell types are devoid of laminate crystals in the cytoplasm. Primary cells

The basal plasma membrane of the primary cells is extensively infolded to form a complex labyrinth of fairly wide channels which extend about 5 pm deep into the cytoplasm thereby providing an extensive surface area on the peritubular side (Figs 4, 5, 8). The

Fig. 4. An electron micrograph of a section of a Malpighian tubule of a fed Chironomus tentam larva showing a stellate cell (S) and parts of two primary cells (P). (H) hemocoel; (BL) basal lamella; (p) cytoplasmic process; (PD) pedicel; (S) slit; (EC) extracellular channel; (GLY) glycogen; (PL) primary lysosome; (N) nucleus; (MV) microvilli; (BVN) biliverdin: (L) lumen. x 10,ooo.

Fig. 5. Electron micrograph of a section of a primary cell of the Malpighian tubule of a fed larva. The basal membrane infoldings (BMI) which are closely associated with mitochondria (M) form a labyrinth of extracellular channels (EC). Note that the cytoplasm is packed with glycogen (GLY) which makes the cell appear dark. Microvilli (MV) lack mitochondria. (BL) basal lamella; (V) vesicle; (P) cytoplasmicprocess; (PD) pedicel; (SL) secondary lysosome containing a tubular element; (G) Golgi complex; (L) lumen; (BVN) biliverdin. X11,ooO.

Fig. 6. Electron micrograph of a section of a primary cell of the Malpighian tubule of a fed larva showing a canaliculus (C) extending deep into the cell. Near the basal extracellular channels (EC) note the presence of vesicles (V), tubular elements (TE) in cross section, and primary lysosome (PL). (BVN) biliverdin; (MV) microvilli; (M) mitochondrion; (L) lumen. x8000. Fig. 7. Another field of canaliculus (C) and adjoining cytoplasm showing different stages (1-5) of biliverdin (BVN) secretion. (M) mitochondrion; (L) lumen. Xll.ooO.

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basal membrane infoldings are closely associated with large, elongated mitochondria (Figs 4, 5, 8, 9), indicating these are sites of active ion transport. The narrow cytoplasmic processes have contents similar to the rest of the cell (Figs 5, 8,9); where they rest on the basal lamella, they exhibit densities (Fig. 14). A number of pedicels are apposed to the basal lamella (Figs 5, 15, 19) which appear strcturally similar to the pedicels that have been described in the antenna1 gland of the amphipode Gamarrts (Kummel, 1973) and in the labial nephridia of the collembolan Orchesella (Verhoef et al., 1979). They also resemble the podocyte foot processes of the vertebrate glomerulus (Cormack, 1987). The basal slits between the cytoplasmic processes and pedicels are about 50 nm wide and they lack filtration diaphragms. They lead into extracellular channels that are lined by fuzzy glycocalyx and contain particulate material (Figs 5, 8, 9, 18). The extracellular channels have an average width of 100 nm in the fed larvae and about 90 nm in the starved larvae. The cytoplasm contains rough endoplasmic reticulum, Golgi complexes and many mitochondria which appear to be evenly distributed (Figs 5, 10 B-20). The brush border of the luminal surface of the primary cells is composed of closely packed microvilli which are bounded by the apical membrane. The microvilli contain loosely

organized microfilaments (Fig. 12 insert) but are devoid of membrane-bound organelles like endoplasmic reticulum and mitochondria (Figs 5, 6, 12, 16, 18). In the fed larva the microvilli are about 1.4,~rn long and 0.1 ,um in diameter while in the starved larva they are about 0.8 pm long and 0.08 pm in diameter. An interesting feature of the primary cells is the presence of a number of intracellular canaliculi, each connecting with the general tubular lumen and extending deep into the cell (Figs 1,2, 6,7). The canaliculi in the fed larvae are deeper (Figs 2, 6, 7), than in the starved larvae (Figs 3, 16). Occasionally in the starved larva the primary cells exhibit a large crescent-shaped intracellular space (Figs 21, 22) which contains very fine particulate material (Fig. 22). Stellate cells

The small stellate cells are surrounded by the primary cells (Fig. 4). The basal membrane infoldings line a network of interconnecting channels which contain filamentous and fine particulate material. The channels do not extend as deeply into the cytoplasm as those of the primary cells and fewer are associated with mitochondria (Figs 4, 11,23). The stellate cells have cytoplasmic processes and pedicels with narrow slits between them (Figs 4, 11.23). The cytoplasmic processes exhibit densities where they rest on the basal lamella

Fig. 8. Basal region of a primary cell of the tubule of a fed larva showing basal membrane infoldings (BMI) closely associated with large mitochondria (M). Note fuzzy glycocalyx (*), vesicles (V) and particulate material in the extracellular channels (EC). (H) hemocoel; (BL) basal lamella; (P) cytoplasmic process; (S) slit; (V) vesicle; (BCV) basal condensing vacuoles; (GLY) glycogen. ~39,ooO. Fig. 9. Electron micrograph of the basal region of a primary cell of the tubule of a fed larva showing dense tubular elements (TE) in cross-section; vesicles (V) and primary lysosome (PL) near the tips of extracellular channels (EC). (BMI) basal membrane infolding; (M) mitochondria; (P) cytoplasmic process; (G) Golgi complex; (GLY) glycogen. ~21,000. Insert: secondary lysosome (SL) near the tips of extracellular channels. ~21,000. Fig. 10. Cytoplasmic detail of the primary cell of the tubule of the fed larva. Note the filamentous glycocalyx (*) lining the wall of extracellular channel (EC) and rosettes of glycogen (GLY). (M) mitochondrion; (G) Golgi complex. x61,COO. Fig. 11. Electron micrograph of a section of a stellate cell of the Malpighian tubule of a fed larva. Note the abundance of rough endoplasmic reticulum (rER), free ribosomes (R) and glycogen (GLY) in the cytoplasm. The basal extracellular spaces (EC) are continuous with each other and contain particulate material and vesicles (V) in them. (P) cytoplasmic process; (PD) pedicel; (S) slit; (BL) basal lamella; (M) mitochondrion; (MV) microvilli. ~48,000.

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(Fig. 23 insert). The cytoplasm is rich in free ribosomes, vesicles, condensing vacuoles, lysosomes, and small mitochondria (Figs 11, 23). The stellate cells contain abundant rough endoplasmic reticulum (Figs 11, 12). In the starved larva, the cytoplasm appears less dense because of loss of glycogen (Fig. 23). The centrally placed nucleus is somewhat flat and has an irregular periphery (Fig. 4). The microvilli of the stellate cells are shorter and spaced farther apart than those of the primary cells. They are devoid of mitochondria and endoplasmic reticulum and are about 0.8 pm long and 0.07 pm in diameter. In the starved larva, the stellate cells have widely spaced microvilli (Fig. 23). Cell junctions

The lateral membranes of the primary cells are linked by a desmosome, followed by a long pleated septate junction with an interposed gap junction. The pleated septate junction extends from the luminal surface of the primary cells about three-fourths of the way to the base up to the desmosome beyond which the two lateral membranes are separated to enclose an intercellular channel, about 2.6 pm in length and about 65 nm in width, containing fine particulate material (Fig. 18). Near the lumen the pleated septate junction exhibits small blisters (Fig. 18). The

stellate and primary cells are joined for about one-third of the way from the lumen by a pleated septate junction and for the rest of the way to the base by a smooth septate (continuous) junction (Figs 12, 13). Glycogen storage The most spectacular feature of the cells of the Malpighian tubules of the fed larva is that they are completely filled with glycogen (Figs 5-10,13). The electron-dense particles of glycogen are arranged in rosettes which resemble storage glycogen in the hepatocytes of vertebrate liver (Cormack, 1987). In insects, such a marked accumulation of glycogen has been reported only in muscles, the fat body and midgut (Wigglesworth, 1972; Smith. 1968; Filshie et al., 1971; Dean et al., 1985). In the starved larva most of the glycogen is depleted from the cells making them appear light (Figs 14-23). Fate of hemoglobin

The particulate material bound to the glycocalyx radiating from the walls of the extracellular channels of the primary and stellate cells presumably represents biliverdin and/or functional hemoglobin (Figs 8, 11-13, 18, 23). Small pinocytic vesicles containing biliverdin are observed at the tips of the extracellular channels and in the general

Fig. 12. Pleated septatejunction (SJ) between primary cell (P) and stellate cell (S) of a fed larva, adiacent to the tubular lumen (L). (EC) extracellular cbanpel; (M) mitochondrion; (V) vesicle; (;ER) rough endoplasmic reticulum; (MV) microvilli. X.&,ooo. Insert: cross-section of microvilli (MV) containing microfilaments. (BVN) biliverdin. X82,c%fl. Fig. 13. Smooth septate junction (SSJ) between the primary cell (P) and stellate cell (S) of the tubule of a fed larva. Note filamentous and particulate material (*) in the extracellular channels (EC) of the primary cell. (GLY) glycogen; (M) mitochondrion. x85,ooO. Figs 14-17. Electron micrographs of portions of primary cells of the tubules of a starved larva. Note the absence of glycogen in the cells. Fig. 14. Section showing densities (D) at the base of cytoplasmic processes (P) as they rest on the basal lamella (BL). (BMI) basal membrane infoldings; (M) mitochondrion. x45.(MJO Fig. 15. Detail of the basal lamella (BL). The outer layer is granular and the inner layer is filamentous. (BMI) basal membrane infolding; (M) mitochondrion; (PD) pediccl. x26,ooO. Fig. 16. Luminal side showing a shallow canaliculus (C); (SL) secondary (MV) microvilli; (BVN) biliverdin; (L) lumen. ~30,000.

lysosome;

(M)

mitochondrion;

Fig. 17. Nuclear zone of the primary cell. Note reduction in the size of mitochondria and microvilli (MV). (N) nucleus; (CH) chromatin; (PL) primary lysosome; (SL) secondary lysosome; (L) lumen. X17,OW.

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cytoplasm (Figs 1, 5-9, 11, 12, 23). At the brush border, the vesicles coalesce with the apical membrane in between the microvilli. Small globs of biliverdin are observed between the microvilli and in the tubular lumen along with large droplets (Figs 1,4-7, 12, 16). The coated pits and coated vesicles, precarrying sumably trapped functional hemoglobin, are pinched off from the walls of the extracellular channels and appear in the adjoining cytoplasm (Figs 18, 23). Condensing vacuoles are observed in the basal cytoplasm in close proximity to the primary lysosomes (Figs 1,8, 18,23). The cytoplasm also contains secondary lysosomes (Figs 1, 9 insert, 16, 20) and residual bodies (Fig. 20) which may be involved in the breakdown of hemoglobin and release of catabolic products into the tubular lumen (Fig. 1). Another interesting feature of the primary cells is the presence of dense tubular ele-

ments (65 nm diameter) near the basal membrane infoldings (Figs 6,9,18,19). They bear resemblance to the tubular elements that Sohal (1974) has described in the Type I cells in the Malpighian tubules of Musca domestica. In cross-sections, the tubular elements exhibit electron-lucent cores similar to those demonstrated in the protein transporting pericardial cells of the aphid (Bowers, 1964). Profiles of the tubular elements are observed in close proximity to primary lysosomes (Fig. 9) and inside the secondary lysosomes (Fig. 9 insert). Discussion Ceil types The ultrastructural

features of the primary and stellate cells of Chironomus tentans larval Malpighian tubules revealed by this study are essentially similar to those of dipteran adult blowfly Calliphora erythrocephala (Berridge

Fig. 18. Electron micrograph of portions of the two primary cells of the Malpighian tubule of the starved larva that are linked by a single basal desmosome (D), a long pleated scptate junction (SJ) and an interposing gap junction (GJ). Near the luminal border, blisters (B) are present in the septate junction. Both extracellular channels (EC) and intercellular channels (IC) contain particulate material coated vesicles (CV) and dense tubular elements (TE) are found near the basal membrane infoldings (BMI) which alsoexhibit coated pits(CP). Note that theglycogen has been depleted from the cells, which now appear light. (BL) basal lamella; (BVC) basal condensing vacuole; (PL) primary lysosome; (M) mitochondrion: (rER) rough endoplasmic riticulum; (MV) microvilli; (L) lumen. x53,tXJO. Figs 19, 20. Electron micrographs of the primary cell of the tubule of a starved larva. Fig. 19. Basal zone of a primary cell showing pedicels (PD) beneath the basal lamella (BL) and dense tubular elements (TE) near the tips of basal membrane infoldings (BMI). A few mitochondria (M) have fused to form a strange configuration. (rER) rough endoplasmic reticulum; (PL) primary lysosome. X 19,OCO. Fig. 20. Various forms of lysosomes near the luminal border of the primary cells. (PL) primary lysosome; (SL) secondary lysosome; (RB) residual body; (rER) rough endoplasmic reticulum; (MV) microvilli; (L) lumen. ~18,ooO. Fig. 21. Light micrographof a cross-section of a Malpighian tubule of astarved larva showing a clear space (s) in the primary cell (P), (S) stellate cell; (L) lumen. x340. Fig. 22. Electron micrograph of the primary cell depicted in Fig. 21, showing that the large intracellular space (S) containing fine particulate material is not an artefact but is created by the accumulation of fluid which the starved larva is unable to drive towards the lumen. (H) hemocoel. ~16,000. Fig. 23. Electron micrograph of a section of a stellate cell of the Malpighian tubule of a starved larva. Note mtmerous vesicles (V), coated vesicles (CV), basal condensing vacuoles (BCV) and mitochondria (M) in the cytoplasm. The short microvilh (MV) are widely spaced. Extracellular spaces (EC) contain particulate material. (BMI) basal membrane infoldings; (BL) basal lamella; (P) cytoplasmic process; (PD) pedicel; (S) slit; (L) lumen; (H) hemocoel. ~23,000. Insert: Densities (D) at the bases of cytoplasmic processes resting on the basal lamella (BL). (BMI) basal membrane infoldings. ~45,000.

H

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1969), housefly Musca Oschman, (Sohal, 1974) and saline-water mosquito Aedes taeniorhynchus (Bradley et al., 1982). The differences are the absence of tracheal supply related to the presence of functional hemoglobin in the hemolymph, the lack of laminate crystals and the presence of microvilli devoid of mitochondria. Additionally, the tubule cells in the Chironomus larva are involved in the uptake of biliverdin and hemoglobin from the hemolymph. Based on ultrastructural characteristics and numerical predominance, the primary cells appear to be the major ion-transporting cells in the tubules of the Chironomus larva. The thin basal lamella, extensive apical and basal infoldings of the plasma membrane and the associated mitochondria in the primary cells are significant features suggestive of active ion transport, which lend support to the ‘standing gradient’ hypothesis of fluid secretion (Diamond and Bossert, 1967, 1968). According to this hypothesis, osmotic gradients are established in the basal channels and the microvilli of the Malpighian tubules by active transport of small solutes followed by passive movement of water from the hemolymph into the tubular lumen (Berridge and Oschman, 1969). The close association of basal membrane infoldings with large mitochondria suggests the involvement of potassium pumps in creating an osmotic gradient in the tubule cells (Maddrell, 1971). It should be noted here that the basal channels remain relatively clear and are not occluded by biliverdin or hemoglobin, which would adversely affect normal fluid secretion. Absence of mitochondria in the microvilli indicates that the brush border of both primary and stellate cells is not involved in the active transport mechanisms which require expenditure of energy. The microvilli here may function to sweep biliverdin away from the tubular lumen as it is released at the apical membrane. The canaliculi of primary cells are structurally similar to the canaliculi observed in the Malpighian tubules of Microsteles (Smith and Littau, 1960), Calliphora (Berridge and Oschman , 1969)) Calpodes (Ryerse , 1979)) the New Zealand glowworm Arachnocampa (Green, 1979) and mayfly Ecdyonaurti (Nicholls, 1983), and in the salivary glands of Calliphora (Oschman and Berridge, 1970), goblet cells in the larval midgut of Mandusa and

domestica

(Coiffi, 1979) and gastric parietal cells of the bat (Ito and Winchester, 1963). It appears that the presence of canaliculi is a regular feature of potassium-transporting epithelia. The deep extension of the canaliculi in the primary cells apparently reduces the distance between the basal channels and the lumen which facilitates the rapid flow of fluid. The presence of abundant rough endoplasmic reticulum in the cytoplasm of stellate cells suggests that they are involved in protein synthesis. In the Malpighian tubules of Carausius morosus, Type 2 cells which resemble the stellate cells and are rich in rough endoplasmic reticulum have been implicated in the secretion of mucus (Taylor, 1971b). Berridge and Oschman (1969), on the other hand, have proposed that stellate cells may have a role in ion absorption. Wessing (1965) points out that the function of the Malpighian tubules largely depends upon ecological factors. In the present study, there is clear evidence of a correlation between structural modification and increased functional activity of the Malpighian tubules which in turn depends upon the availability of food in the environment. In the fed larvae, when the fluid transport is presumably accelerated, the basal slits, extracellular channels, canaliculi, and microvilli increase in size in comparison to starved larvae in which fluid secretion is relatively slow. Maddrell (1971) reported a similar correlation between shorter microvilli and slower fluid rate in insect epithelia. Also, it has been shown that microvilli are shorter and more widely spaced in the lower absorptive segment of Rhodnius Malpighian tubules (Wigglesworth and Salpeter, 1962). Cell junctions The lateral membranes

of the primary cells are linked by a desmosome followed by a pleated junction and interposed gap junction, while the primary and stellate cells are joined by pleated and smooth septate (continuous) junctions. Desmosomes have been demonstrated in the tubules of the stick insect Carausius (Taylor, 1971a, b) and cockroach Blatella (Meyran, 1982), but their function in the Malpighian tubules is not clearly understood. The pleated septate junctions which exhibit closely placed septa are well developed in the insect transporting epithelia; They have been clearly demonstrated

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in the Malpighian tubules of grasshopper (Tsubo and Brandt, 1962); Chironomus thummi larva (Bullivant and Loewenstein, 1968); Calliphora (Berridge and Oschman, 1969); cockroach Peripluneta (Wall et al., 1975) and other insect tissues (Lane and Skaer, 1980; Noirot-Timothee and Noirot, 1980; Bradley, 1985). In spite of their common occurrence in the Malpighian tubules, the function of the pleated septate junctions still remains unclear. It has been suggested that in conjunction with desmosomes, they play a role in intercellular adhesion and constitute a permeability barrier (Berridge and Oschman, 1972; Noirot-Timothee and Noirot, 1980; Bradley, 1985). In support of this idea, Lord and diBona (1976) demonstrated the formation of ‘blisters’ between septa of planarian epidermal septate junctions subjected to osmotic stress, in a manner similar to ‘blister’ formation previously shown in vertebrate epithelial tight junctions. They concluded that both septate and tight junctions were ‘limited junctions’ which acted as barriers to paracellular flow of water and small solutes. In the present study, the presence of blisters in pleated septate junctions on the luminal side of the primary cells of the starved larval tubules also lends support to their role in intercellular adhesion. In the starved larvae, due to the lack of available energy, the fluid containing organic and inorganic molecules is not secreted fast enough, but rather tends to accumulate in the cells, thereby increasing the intracellular osmolality. This tends to draw water presumably from the lumen into the cells, causing blistering of the septate junction. The smooth septate junctions, which have fewer septa, have been described in the Malpighian tubules of Musca and Rhodniu (Skaer et al., 1979) and saltwater Aedes larva (Bradley et al., 1982). In the upper segment of Rhodnius tubules these junctions appear to offer little resistance to organic molecules like sucrose and inulin which could easily cross into the cells (Maddrell and Gardiner, 1974). In the present study, the presence of a long smooth septate junction joining the primary and stellate cells suggests that paracellular transport may be occurring between these cells. The gap junctions are considered communication junctions in terms of ion and chemical coupling between cells. They con-

stitute ‘low-resistance pathways between cells through which direct exchange of substances can occur’ (Lane and Skaer, 1980). In the present study, coexistence of small gap junctions with pleated septate junctions suggests that the gap junctions play an important role in functional coordination between the primary cells. This view is strongly supported by the work of Loewenstein et al. (1965) which revealed that the lateral cell membranes of the Malpighian tubules of Chironomus thummi larva had a very low electrical resistance as compared with the external non-junctional cell membranes. This was due to far greater permeability at the intercellular junctions than at the basal cell surface. Glycogen storage

The present study reveals that a large amount of glycogen is stored in the tubule cells when the larvae are feeding and this reserve all but disappears in the starved larvae. Coones et al. (1987) recently showed an increase in glycogen particles after feeding, in the Malpighian tubules of the dog tick Dermacenter variabilis. Also it has been shown that the fat body cells become loaded with glycogen when the starved larva of mosquito Aedes is fed on carbohydrates alone. In a fully starved larva of Rhodnius, the fat body cells were not only deprived of glycogen reserves but also the mitochondria probably were removed by autophagy (Wigglesworth, 1972). It is interesting to speculate how glycogen reserve is efficently utilized by the Malpighian tubule cells to meet their metabolic demand in an oxygen-deficient environment. It is known that the chironomid larvae inhabit ditches and dirty streams and feed on decaying vegetable matter at the bottom where dissolved oxygen is scarce (Maill, 1895). In such an environment, the hemoglobin in their hemolymph serves as an efficient store of oxygen (Mail1 and Hammond, 1900). Chironomus tentans larvae used in the present study were collected from Boitano Lake, which has no inflowing or outflowing streams, lacks a fish population and is subjected to pollution by cattle dung (Scudder, 1969). These ecological factors would make Boitano Lake bottom water more static and hence more deficient in dissolved oxygen. In such an environment, a

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OF CHIRONOMUS

LARVA

starved Chironomus tentans larva has no choice but to utilize the glycogen reserve in its Malpighian tubules to provide, by anaerobic glycolysis, the energy necessary for them to function properly. It has been reported that only when glycogen is the substrate in anaerobic glycolysis is there a net gain in energy (Gilmore, 1965). When glycogen reserves, as a gainful energy source, are depleted in the starved larvae, they are unable to drive secretory fluid towards the lumen, and the fluid then tends to accumulate in large intracellular spaces observed in the primary cells. Trehalose (mol. wt 342.31) is a sugar, made up of two glucose units, which characteristically is present in large amounts (IO-50 mM) in the hemolymph of all insects (Florkin and Jeuniaux, 1974). Maddrell and Gardiner (1974) have demonstrated that Rhodnius Malpighian tubules are penetrated by various organic molecules, being more permeable to small molecules, e.g. glucose (mol. wt 360) than large molecules, e.g. inulin (mol. wt 5200). However, it has also been suggested that the low permeability of insect Malpighian tubules to organic molecules may have contributed to the unusually high levels of sugars like trehalose in the insect hemolymph (Maddrell, lY81). Knowles (1975) has shown that, when isolated Malpighian tubules of Calliphora vomitoria were bathed in varying concentrations of different sugars, the concentration of trehalose and glucose in the tubular fluid was much lower in comparison with other sugars of the same size. This suggests that these sugars are retained inside the tubular cells and possibly converted into glycogen. The present study strongly suggests that trehalose from the hemolymph enters and accumulates in the tubular cells. where by an unknown process it is converted into glycogen which in the fed larvae completely fills the cytoplasm. Fate of’ hemoglobin

It is known that the functional hemoglobin (mol. wt 31,400) in the hernolymph of Chironomus plumosus is broken down and the resultant pigments bilirubin and biliverdin are taken up and stored in the fat body (Wigglesworth, 1972). In Rhodnius, after injection of ‘laked’ blood into the hemolymph, the hemoglobin is broken down and the blood pigment, biliverdin (mol. wt

377

582.63) is taken up and excreted by the Malpighian tubules. In addition some breakdown of the injected (blood) hemoglobin (mol. wt 64,000) takes place in the Malpighian tubules (Wigglesworth, 1943). The Malpighian tubules of Drosophila larva also have been shown to transport injected hemoglobin to the lumen (Wessing, 1965). In the dragonfly, Libellula horseradish peroxidase (mol. wt 44,000), after being injected into the hemolymph, penetrates the basement membrane and is found in all basal membrane infoldings of the tubule cells and in small vesicles (Kessel, 1970). These observations indicate that the Malpighian tubules of some insects at least can transport proteins. The present study clearly shows that the primary cells and possibly the stellate cells of the Malpighian tubules of Chironomuy tentans larva are involved in the uptake, transport and excretion of the pigment biliverdin and the naturally occurring hemoglobin. The sequence of biliverdin excretion appears similar to that documented by Wigglesworth (1943) in Rhodnius tubules and to a great extent follows the ‘vesicular transport hypothesis’ of Wessing and Eichelberg (1969) which envisions the removal of water from small vesicles passing through the cytoplasm and the extrusion of their contents into the tubular lumen. In the present study, biliverdin appears to penetrate the basal lamella and reach the tips of extracellular channels where small pinocytic vesicles are pinched off. These vesicles containing biliverdin travel through the cytoplasm to the brush border where they coalesce with the apical membrane in between the microvilli and release biliverdin in small globs into the tubular lumen where they fuse to form large droplets. The basal slits between the cytoplasmic processes and pedicels of the Malpighian tubule cells, which resemble the podocyte foot processes of vertebrate glomerulus, are large enough to allow molecules of functional hemoglobin to enter the wide extracellular channels which are not occluded by hemoglobin or biliverdin. They are comparable in size with slits and intercellular channels of the millipede Glomeris marginata which can excrete low molecular weight dextran (16,OOClY,OOOm.w.) but allow only limited passage to high-molecular weight dextran

378

JARIAL

(60,000-90,000 m.w.) (Farquharson, 1974~; Maddrell, 1981). Small coated pits and vesicles, pinched off from the glycocalyx-lined extracellular channels, contain particulate material and tend to fuse with each other to form large condensing vacuoles. The condensing vacuoles move towards the centre of the cells where they coalesce with primary lysosomes to form secondary lysosomes in which hydrolysis of hemoglobin seems to take place. The residual bodies presumably liberate, by exocytosis, the breakdown products of hemoglobin at the brush border into the tubular lumen. The presence of a number of dense tubular elements at the tips of the extracellular channels suggest that they have a role in the uptake and transport of hemoglobin by the Malpighian tubules of the Chironomw tentans larva. Similar tubular elements have been observed in large number in the proteintransporting nephrocytes of Calliphora (Crossley, 1972, 1985) and in the proximal convoluted tubule of the vertebrate nephron (Tisher et al., 1969, and personal observations) . The coated vesicles and dense tubular elements observed in the Malpighian tubule cells of the Chironomus tentans larva would appear to have a role in the uptake and transport of proteins. This view is supported by the work of Miller (1960) and Maunsbach (1966) related to the uptake of hemoglobin and ferritin by the dense tubules and small vesicles in the proximal convoluted cells of the mouse and rat kidneys. Summary

Even though they transport fluid in opposite directions, there are impressive structural

similarities between the primary cells of the Chironomus tentans larval Malpighian tubules and the proximal convoluted tubule cells of the mammalian nephron. These include: (1) The lack of tracheal supply in these Malpighian tubules, since their function of delivering oxygen to the cells has been taken over by the functional hemoglobin in the hemolymph. (2) Presence of a thin basal lamella (basement membrane) and a labyrinth of membrane infoldings associated with mitochondria. (3) Absence of laminate crystals in the cytoplasm. (4) Absence of mitochondria in the luminal microvilli. (5) The presence of coated vesicles, dense tubucondensing vacuoles and lar elements, various forms of lysosomes, which are all characteristic features of protein-transporting cells. The pedicels, protoplasmic processes and the slits between them resemble podocyte foot processes and filtration slits of the mammalian glomerulus. Also the rosettes of glycogen stored in the tubule cells bear close resemblance to the glycogen (a-particles) in the hepatocytes of mammalian liver. Acknowledgements

The author is indebted to Dr G. G. E. Scudder, Department of Zoology, University of British Columbia, Vancouver, Canada, for providing laboratory facilities for fixation of the material. Thanks are due to Dr M. S. Topping for his help in collecting the larvae. It is a pleasure to thank my colleagues Drs Duncan Kennedy and Thomas Lesh for their critical reading of the manuscript and making constructive suggestions. The author is grateful to MS Glenna Hedge for her excellent secretarial assistance.

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