Fine structure and X-ray microanalysis of mineralized concretions in the Malpighian tubules of the housefly, Musca domestica

TISSUE & CELL 1976 8 (3) 447-458 Published by Longman Group Ltd. Printed in Great Britain

R. S. SOHAL,”

FINE STRUCTURE MICROANALYSIS CONCRETIONS IN TUBULES OF THE DOMESTICA

P. D. PETERS=f$ and T. A. HALLj-$

AND X-RAY OF MINERALIZED THE MALPIGHIAN HOUSEFLY, MUSCA

ABSTRACT. The epithelium and the lumen of the Malpighian tubules of the housefly contains mineralized dense bodies called concretions. The morphological characteristics, mode of origin, nature of the sequestered elements and the age-associated changes in the distribution of concretions are reported. There are three types of concretions in the cytoplasm, which have been designated as type A, type B, and type C. Type A concretions are membrane-bound spherical structures which may arise by the gradual intravacuolar accumulation of dense material. Type B concretions appear to be related to multivesicular bodies. Type C concretions are heteromorphic and morphologically resemble the residual bodies. They show a positive localization of acid phosphatase reaction product. X-ray microanalysis of intracytoplasmic and intraluminal concretions revealed the presence of phosphorus, sulphur, chlorine, calcium, iron, zinc and copper. There was no evidence suggesting the extrusion of the intracytoplasmic concretions into the lumen of the Malpighian tubules. There is an age-associated increase in the distribution of type C concretions. It is hypothesized that the sequestration of metal ions within the concretions may provide a means for the effective excretion of these elements.

1965; Wessing and Eichelberg, 1975). It has been suggested that the concretions may be involved in the transepithelial movement of substances (Wessing and Eichelberg, 1973, but their specific role in the excretory process remains obscure. In order to understand the physiological significance of the concretions it is imperative to know their origin, chemical composition, and eventual fate. The present study is concerned with transmission electron microscopic examination and X-ray microanalysis of the concretions in the Malpighian tubules of adult houseflies of different ages. The specific objectives of this investigation were to study the structural characteristics, possible mode of genesis, elemental composition of the sequestered material, and the age-associated distribution of the concretions. In the present study, the term concretion refers to mineralized structures.

Introduction A MAJOR function of the Malpighian tubules in insects is the excretion of nitrogenous and other metabolic wastes. The tubules secrete fluid into the hindgut where certain materials are resorbed and are then transported into the hemolymph (for references see Maddrell, 1971). The epithelium as well as the lumen of the Malpighian tubules often contains mineralized dense bodies heteromorphic, referred to as “concretions” (Wigglesworth, * Department of Biology, Southern Methodist University, Dallas, Texas. Microscopy Section, Cavendish t Electron Laboratory, University of Cambridge, England. $ Present address: Department of Botany, The University of Cambridge. B Present address: Department of Zoology, The University of Cambridge. Received

10 March

1976. 447

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448

Materials and Methods

Larvae of the housefly were raised on Chemical Specialties Manufacturers’ Association medium obtained from Ralston Purina Company, Richmond, Indiana. Adult flies were maintained on a mixture of sucrose, dry milk and egg yolk. Malpighian tubules were obtained from adult female flies ranging from I to 40 days of age. Transmission electron microscopy

Malpighian tubules were fixed in phosphatebuffered 4% glutaraldehyde, pH 7.3, for I h, washed in phosphate buffer and refixed in phosphate-buffered 1% osmium tetroxide for 45 min. Following dehydration in an ascending series of ethanol concentrations, tissues were embedded in Maraglas. Thin sections were stained with uranyl acetate and !ead citrate. Acid phosphatase localization

Acid phosphatase activity was localized by the procedure of Couch and Mills (1968) using /3-glycerophosphate as a substrate. Details of the procedure have been reported elsewhere (Sohal and Sharma, 1972).

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tus Ltd, Manchester, England). The theory and practice of X-ray microanalysis as applied to biological material is now well known (Hall, 1971; Hall et al., 1974). Analyses were made using energy dispersive, as well as wavelength dispersive, spectrometers. The energy dispersive spectra shown on the display screen were photographed after 30 set analysis. The X-ray microanalyses were done at an accelerating voltage of 40 kV and a magnification of 16,000. The spot diameter of the probe was about 200 nm with a beam current of 200 nA. Results of the microanalysis are presented as R values which represent a measure of the relative elemental concentrations. R values were determined by using the following formula:

R= W$_W II where S is background-corrected characteristic count, W is the total ‘white’ or continuum X-ray count and Wb is the correction for the supporting film. Results

X-ray microanalysis

General description sf’ the Malpighian tubule epithelium

Malpighian tubules were fixed in 2.5% glutaraldehyde buffered with 0.2 M Scollidine for 20 min, washed in buffer and then rapidly dehydrated through an ascending series of ethanol concentrations. Tubules were embedded in Araldite and sectioned at a thickness of about 200 nm. Sections were mounted on nickel grids and analyzed unstained using an EMMA-4 analytical electron microscope (AEI Scientific Appara-

There are two main Malpighian tubules in the housefly, each of which consists of two segments. Towards the proximal end the two segments fuse to form a common collecting duct which joins the alimentary tract at the junction of the mid- and hind-gut (see Hewitt, 1914, p. 38). The lumen of the Malpighian tubules contains numerous dense, spherical granules which radiate needle-like crystalline material (Fig. I). Some

Fig. 1. Section through the Malpighian tubule of a 7-day-old fly. The basal surface of the cell facing the hemolymph is surrounded by a basement membrane (BM). Basal plasma membrane (BPM) shows deep imaginations into the cytoplasm. Microvilli (MV) occur on the luminal surface. Concretions (C) can be seen in the lumen. V, vacuoles; M, mitochondria; MVB, multivesicular body. x 26,000. Fig. 2. Section through type I cell of a 7-day-old fly showing the possible formation of type A concretions. Vacuoles labelled l-5 show a progressive increase in the electron density of their lumina. Structures like the dense vacuole labelled 5 are considered to constitute type A concretions. x 35.000.

450

SOHAL,

of the granules exhibit radial striations. These granules constitute the intraluminal concretions. A single layer of epithelial cells surrounds the lumen. The epithelium of the distal segments of the Malpighian tubules is formed by three types of cells while a fourth cell type is restricted to the proximal collecting duct. In order to avoid nomenclatural confusion, the distinguishing features of each cell type are described briefly (see also Sohal, 1974). Arbitrarily, cells of the distal segments have been named type I, type II, and type III cells. Type IV cells are not included in the present study. Type I cells are most numerous. Their fine structural organization is essentially similar to that of the secretory cells in the Malpighian tubules of other insects (Berridge and Oschman, 1969). The basal surface of these cells is highly infolded whereas the apical cell surface evaginates into microvilli (Fig. I). Slender and elongated mitochondria extend into the microvilli. The cytoplasm is well populated with mitochondria, free polysomes and cisternae of granular endoplasmic reticulum. Type I1 or stellate cells are relatively few in number. They are characterized by the presence of dilated extracellular channels on their basal surface and the absence of mitochondria within the apical microvilli. Type III cells contain para-

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crystalline arrays of tubular filaments in the lumen of the cisternae of granular endoplasmic reticulum. Concretions are frequently observed in type I and type III cells, but rarely in type II cells. Fine structure of’ the concretions The epithelial cells contain a number of concretions in the cytoplasm which exhibit considerable variation in their structural organization. A common feature of all the concretions, however, is that they contain foci of highly electron-dense material. On the basis of morphological criteria, at least three classes of concretions can be recognized. For the sake of convenience, the concretions are arbitrarily termed as type A, type B, and type C concretions. It should be mentioned that in some instances, particular concretions could not be confidently included in any of these categories. Type A concretions Several membrane-limited vacuolar structures, about 0.5 pm in diameter, containing variable amounts of dense material in their luminae are present in the central zone of the cells. Some of the vacuoles are electron-lucid, while others exhibit an electron-dense matrix (Fig. 2). The density of their matrix appears to depend upon the compactness of the enclosed material. In the dense vacuoles

Fig. 3. A multivesicular

body (MVB) is seen near a Golgi complex

Fig. 4. A multivesicular

body showing

dense granules.

(G).

x 57,000.

x 48,000.

Fig. 5. A type B concretion (B) containing an aggregate of dense material. A MVB (arrow) containing the characteristic vesicles as well as membrane fragments is also seen. x 48,000. Fig. 6. A type C concretion from type 111 cell of a 30-day-old fly. Note several aggregates of dense material and membrane whorls in the concretion. Arrow indicates the presence of paracrystalline material in the cisternae of granular endoplasmic reticulum. x 50,000. Fig. 7. A type C concretion from a 25day-old fly showing 13;;yce;;phosphatase in the form of electron-dense precipitate

reaction product of of lead phosphate.

Fig. 8. Section through type I cell of a 5-day-old fly showing residual bodies which contain dense material (arrows). Such residual bodies appear to derive from the multivesicular bodies. x 33,000.

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452

the matrix appears homogeneous and compact. Vacuoles with a partially filled lumen have a granular matrix. The variations in the density of the vacuoles suggest a progressive increase in the concentration of the material. The highly dense vacuoles with a homogeneous matrix (structure 5, Fig. 2) are considered to constitute type A concretions. Type B concretions

These concretions are spherical in contour, about 0.5-l pm in diameter, and are surrounded by a single peripheral membrane (Fig. 5). Their matrix contains an irregular mass of dense material which is surrounded by a substance of considerably less density. Profiles of single membranes or stacks of membranes are often present in the translucent region of the matrix. Some frequently observed profiles in the cytoplasm suggest that type B concretions may be derived from multivesicular bodies (MVBs). It should, however, be stated that the actual sequence of transformation cannot clearly be demonstrated on the basis of static electron micrographs. The suggested sequence of alterations presented in Figs 3-5, therefore, remains speculative. The relative amount of dense material within the transitional forms has been used as a criterion for the proposed scheme of sequential stages involved in the transformation of MVBs into concretions. Typically, the MVBs are located in the vicinity of a Golgi complex and consist of a limiting membrane which encloses a number of small vesicles embedded

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in a matrix of variable density (Fig. 3). In other profiles considered to represent a transitional stage, some of the vesicles within an MVB are substituted by dense granules (Fig. 4). The size and distribution of these dense granules suggests that they may be derived from the vesicles. In a presumably subsequent stage presented in Fig. 5, the dense material in the matrix forms a large aggregation. Such profiles showing a mass of dense material and relatively few or no vesicles in the matrix are considered to be the type B concretions. Type C concretions

Type C concretions vary greatly in size (l-12 pm) and structural complexity. They consist of a membrane-bound mass of dense, homogeneous or finely granular material associated with a complex of membranes (Figs. 6, 12). There is a close morphological similarity between type C concretions and the secondary lysosomes of the residual body variety which have been characterized in other tissues of the housefly (Sohal and Sharma, 1972; Sohal, 1973). Type C concretions show a positive localization for the reaction product of /3_glycerophosphatase activity (Fig. 7) which suggests their lysosomal nature. The mode of formation of type C concretions could not be clearly demonstrated due to the limitations imposed by the static nature of the electron micrographs. Various profiles, however, strongly suggest that type C concretions represent residual bodies derived from MVBs as well as cytolysomes.

Fig. 9. A cytolysome containing relatively hian tubules of a 5-day-old fly. x 48,000. Fig. IO. A residual body from a 5-day-old material (arrow). x 35,000.

well-preserved fly showing

organelles

in the Malpig-

dense as well as membranous

Fig. Il. A cytolysomc (CY) and a residual body (R) show the presence of an aggregate of electron-dense material. x 40,000. Fig. 12. Section through several type C concretions

a type I cell from a 35-day-old (arrow). x 21,000.

Fig. 13. Type III cell from a 35-day-old C concretions (arrow). x 18,000.

fly showing

fly showing

the presence of

the presence of numerous

type

454

SOHAL,

Cytolysomes consist of membrane-limited areas of the cytoplasm containing different organelles which show varying degrees of degradative changes (Fig. 9). Clumps of dense material and membranous configurations are often seen within cytolysomes and the residual bodies which may derive from them (Figs. 10, 11). Fig. 8 includes profiles which suggest that type C concretions may also derive from the transformation of structures resembling type B concretions.

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The intraluminal granules contain the greatest variety of elements, e.g. phosphorus, sulphur, chlorine, potassium, calcium, iron, copper and zinc. The intracytoplasmic concretions have a somewhat similar pattern of elements. There is considerable variation, however, in the intensity of the signals between the structures believed to belong to the same category. These differences may be due to the plane of section, or may reflect the differences in the stages of development of the concretions. Cytoplasmic regions free of the concretions show relatively low concentrations of phosphorus and sulphur. The results of the energy dispersive X-ray analyses also show the presence of the various elements mentioned in Table 1. Typical energy dispersive spectra of the luminal, type A, type B, and type C concretions are presented in Figs. 14-17.

Age-associated changes in concretions

Fine structural examination of the Malpighian tubules from flies of various ages ranging from 1 to 40 days of age reveal an age-associated increase in the number and volume of type C concretions. Relatively large areas of the cytoplasm in the old flies (2540 days old) are occupied by the type C concretions (Figs. 12, 13). The average size of the individual type C concretions is also greater in the older flies. Some concretions become quite enlarged containing several isolated foci of dense material and multiple whorls of membranes (Fig. 6). It appears that the large type C concretions may arise by the fusion of the smaller ones.

Discussion The results of this study reveal the presence of mineralized bodies or concretions in the cytoplasm as well as the lumen of the Malpighian tubule of the housefly. Concretions are apparently formed by the of materials within sequestration the membrane-bound vacuoles, multivesicular bodies, and the secondary lysosomes or residual bodies. Furthermore, the concretions progressively accumulate in the

X-ray microanalysis of concretions

Twenty-six concretions were analyzed and some typical results are given in Table 1.

Table 1. X-ray microanalysisdata* 102 R Type of spot

concretion

Cl

K

Ca

Fe

cu

Zn

18

5 Trace

36

88 234

8 17

Trace? Trace

13 Trace

152 34 31

9 I 6

-

3

12 67

Trace 7

Trace Trace

9 20

13 48

34 23

11 8

-

Trace 12

-

4 44

336 371

123 Trace

Trace Trace

Trace 27

P

S

Luminal concretion 1 2

106 300

-

Type A concretion 3 4 5 Type B concretion 6 7

-

Type C concretion 8 9

111 24

52 16

* Only representative examples of the analysed spots are presented. t Elements detected in relatively small concentration.

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TUBULES

IN

THE

HOUSEFLY

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Figs. 14-17 are photographs of X-ray spectra in the I-10 keV range following 30 set analyses using Kevex Si/Li analyser. The keV range of the different elements is indicated in parentheses. Silicon (1.74), phosphorus (2.01), sulphur (2.3), chlorine (2.62), potassium (3.31), calcium (3.69), titanium (4,51), iron (6.4), nickel (7.47), copper (8.04), zinc (8.63). The peaks of silicon and titanium in the spectra are due to the materials in the instrument. The peak for nickel is due to the use of nickel grids. Chlorine and sulphur in Araldite may make small contributions to the peaks of these elements. Fig. 14. Spectrum

of an intraluminal

Fig. 15. Spectrum

of a type A concretion.

concretion.

Fig. 16. Spectrum

of a type B concretion.

Fig. 17. Spectrum

of a type C concretion

cytoplasm with advancing age of the organisms. Intracytoplasmic concretions in the Malpighian tubules have been observed by electron microscopy in Gryllus by Berkaloff (1958, 1959), Macrosteles by Smith and Littau (1960), Rhodnius by Wigglesworth and Salpeter (1962), Melipond by Mello and BOZZO(1969), Drosphila larvae by Wessing and Eichelberg (1969), Cenocorixa by Jarial

and Scudder (1970), and Periplaneta by Wall et al. (1975). Because the chemical nature and the physiological significance of the intracytoplasmic concretions is poorly understood, different authors have assigned different names to the concretions, e.g. laminated spheres (Wigglesworth and Salpeter, 1962), spherical granules (Jarial and Scudder, 1970), excretory globules (Mello and Bozzo, 1969). A single generic term,

456

even though highly desirable, has not as yet been adopted. One of the major reasons for the poor understanding of the chemical nature of the concretions is their tendency to dissolve in acidic as well as some other fluids (Turbeck, 1974). Another limitation is the relative lack of available staining techniques which specifically localize different metals (Pearse, 1972). Our current knowledge of the chemical of the concretions albeit composition meager is primarily based on histochemical studies. Uric acid, calcium urate and possibly phosphate have been reported in the in Gryllus intracytoplasmic concretions (Berkaloff, 1958). Similar particles in Rhodnius, however, were considered to consist of minerals rather than urates (Wigglesworth, 1965). In the larvae of the honey bee Meligranules are pona, the concretion-like apparently composed of a gylceride core surrounded by a lipoprotein or phospholipid layer. Minerals and urates were not detected (Mello and Bozzo, 1969). Mucopolysaccharides, kynurenin, sodium, and potassium have been localized in the concretions of Drosophilia larvae (Wessing and Eichelberg, 1972, 1975). According to these authors, mucopolysaccharides may be responsible for binding positively charged ions. In this regard, it is interesting to note that of all the sulphur is most consistently elements, detected in the intracytoplasmic concretions of Musca Malpighian tubules. It is possible that some highly sulphated molecules may be involved in the binding of cations. Conversely, the presence of sulphur may be due to its nutritional and metabolic excess. Relatively little is known about the origin of the concretions. According to Wigglesworth and Salpeter (1962), concretions may arise by the mineralization of mitochondria. In Musca, however, concretions appear to be of diverse origin. Type A bodies are formed by the accumulation of materials in the membrane-bound vacuoles. The source of the vacuoles themselves is unknown. Similar vacuoles in Drosophila have been reported to arise from the vesicles pinching off the basal plasma membranes (Eichelberg and Wessing, 1971; Wessing and Eichelberg, 1975). Multivesicular bodies in the Malpighian tubules appear to be engaged in the of dense material which sequestration eventually results in their transformation

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into concretionary structures. The functional role of the MVBs in various types of cells is as yet not clearly understood. In mammalian tissues the MVBs have often been considered to function as digestive vacuoles or phagolysosomes for the endocytized proteins (De Duve and Wattiaux, 1966). However, experimental studies by Friend (1969) on the rat epididymal cells indicated that the vesicles within the MVBs are not involved in protein uptake. Instead, his studies suggested that the vesicles of the MVBs arise from the Golgi complex. The proximity of the MVBs to the Golgi complex in the Malpighian tubules of Musca may be suggestive of a similarity between the vesicles of the two organelles. An interesting observation of the present study is the sequestration of various cations by the secondary lysosomes (type C bodies). The significance of this finding is as yet unclear since little is known about the role of lysosomes in the accumulation of minerals. Lysosomes in the proximal tubule cells of kidney concentrate the experimentally injected cationic dyes, Fe37 and Cu2- (Koenig, 1963). The component binding the cationic substances was characterized as a lipoidal substance containing aldehyde groups (Barret and Dingle, 1967). An interesting finding was that the injected dyes persisted within the lysosomes for a relatively long period (Dingle and Barret, 1968), suggesting not only a strong binding of the dyes but also a relatively long life span of the lysosomes. Whether a comparable mechanism exists in the Malpighian tubules is unknown. The Malpighian tubules of insects do, however, concentrate many acidic dyes (Lison, 1937). The specific role of the intracytoplasmic mineral concretions in the excretory physiology of insects remains controversial. On the basis of their consistent occurrence it can be argued that the concretions represent an essential component of the excretory process. It has been suggested that the intracytoplasmic concretions act as transitory depots for the concentration of substances removed from the hemolymph. They are then extruded into the lumen by a mechanism similar to merocrine secretion (Berkaloff, 1960; Wessing and Eichelberg, 1975). However, convincing evidence based on electron micrographs showing the actual fusion of the

MALPIGHIAN

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HOUSEFLY

limiting membrane of the concretions with the apical plasma membrane has not been presented. Whether the intraluminal concretions arise by the extrusion of those in the cytoplasm remains doubtful. Wigglesworth (1965) and Taylor (1971) on the other hand, believe that the concretions within the lumen do not arise from similar structures in the cytoplasm. Concretions in the lumen contain uric acid while those in the cytoplasm do not (Wigglesworth, 1965). Wigglesworth believes that the luminal concretions are formed by the precipitation of the excreted materials within the lumen. The age-associated increase in the intracytoplasmic volume of the concretions may be a manifestation of a deposit excretion process, by which the redundant minerals are isolated within the cells. Within certain habitats, the excretion of water soluble wastes by intracellular segregation may be of considerable physiological advantage. The houseflies normally inhabit hot climates where they are constantly exposed to the dangers of dehydration. Furthermore, under natural conditions, houseflies derive a substantial proportion of their nutrition from mammalian feces. Fecal material contains a fairly high concentration of minerals (Green, 1925). The elimination of dietary minerals by the houseflies may thus pose a physiological challenge. The intracellular deposition of minerals may also provide a mechanism for the conservation of water. The observed age-associated increase

457

in the mineral concretions lends support to this hypothesis. Spherical structures approximately 20 pm in diameter called ‘formed bodies’ were observed by Riegel(1966) in the Malpighian tubules. According to Riegel (1971), formed bodies arise from the pinocytotic vesicles and contain hydrolytic enzymes which bring about the digestion of the ingested materials. Subsequently the formed bodies are released from the cytoplasm into the lumen, where they cause an increase in the osmotic pressure of the luminal fluid bringing about the movement of water across the epithelium of the Malpighian tubules. Riegel (1971) contends that the formed bodies serve as the primary means for the movement of solutes across the epithelium. In Musca the structures which may be considered to possess some of the characteristics of the formed bodies are the type C concretions. The type C bodies are lysosomal and contain various minerals. However, these structures were never observed within the lumen. Furthermore, we did not encounter any evidence suggesting the secretion of type C concretions into the lumen. Instead, as mentioned previously, the type C concretions progressively accumulate in the cytoplasm with age. On the basis of the present studies, it appears reasonable to suggest that the type C concretions act as a site for the deposit or storage of minerals. The role of the concretions in the fluid transport by the Malpighian tubules needs further investigation.

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