The larval midgut of the housefly (Musca domestica): Ultrastructure, fluid fluxes and ion secretion in relation to the organization of digestion

J. Insect Physiol. Vol. 34, No. 6, pp. 463-472, 1988 0022-1910/88 S3.00 + 0.00 Rinkd in Great Britain. All rights reserved Copyright 0 1988 Pergamon Press plc

THE LARVAL MIDGUT OF THE HOUSEFLY (MUSCA DOMESTICA): ULTRASTRUCTURE, FLUID

FLUXES AND ION SECRETION IN RELATION TO THE ORGANIZATION OF DIGESTION

WALTER R. TERRA, F. P. ESPINOZA-FUENTES, ALBERTO F. RIBEIRO* and CLBLIA FERREIRA Departamento de Bioquimica, Instituto de Quimica, Universidade de SBo Paulo, C.P. 20780, 01498, S&o Paulo and *Departamento de Biologia, Instituto de BiociZncias, Universidade de SHo Paulo, C.P. I 1461,

01000, Sgo Paulo, Brazil

(Received 4 August 1987; revised 26 October 1987)

Abstract-The basal infoldings of the cells from the larval caeca, fore- and hind-midgut of the housefly, Murca domestica, may be branched with few apertures to the underlying space (midgut caeca, fore-midgut and anterior hind-midgut) or may be modified into wide channels with many apertures (posterior hind-midgut). The first third of mid-midgut is composed of interstitial and oxyntic cells. Interstitial cells are characterized by basal infoldings modified into long and narrow channels with particles attached to their cytoplasmic side. Oxyntic cells are cup-shaped and display particle studding the cytoplasmic face of their microvillar membranes. The second third of mid-midgut display flat cells without remarkable features, and the last third, cells similar to those in anterior hind-midgut. Anterior hind-midgut and caecal cells display the most remarkable signs of secretory activity. Carbonic anhydrase is most active in the mid-midgut, and fore-midgut, whereas HCO;-ATPase is found only in fore- and hind-midgut cells. The observations suggest that the interstitial cells absorb water and the posterior hind-midgut cells secrete water. This results in a countercurrent flux of fluid, which is responsible for the enzyme recovery from undigested food before it is expelled. Oxyntic cells in mid-midgut are supposed to be responsible for acidifying the food, which is probably neutralized by HCO; pumped from the hind-midgut cells.

Key Word Index: Midgut (ultrastructure), water transport, carbonic anhydrase, HCO;-ATPase, ion pumps, oxyntic cells, interstitial cells, columnar cells

INTRODUCTION

The food ingested by insects usually passes through the foregut and is enclosed by the peritrophic mem- brane in the midgut. There, the food is digested at first by enzymes which penetrate into the endo- peritrophic space (inside the peritrophic membrane), then by enzymes acting on diffused material in the ectoperitrophic space (luminal space outside the peri- trophic membrane) and finally at midgut cell surface (Terra et al., 1985).

The larval midgut in higher Diptera (Diptera Cy clorrhapha) displays three major regions: fore- midgut, mid-midgut and hind-midgut. These di- visions are readily recognized under a dissecting microscope by their characteristic morphologies (Hobson, 1931; Waterhouse and Stay, 1955; Filshie et al., 1971; Dimitriadis and Kastritsis, 1984), and also by their luminal pH values, which are nearly neutral in fore- and hind-midgut and very acidic in mid- midgut (Greenberg 1968). The nature of the sub- stances responsible for buffering the different midgut regions, in spite of the existence of some suggestions (Hobson, 1931), is uncertain.

Based on observations on blowfly (Lucilia sericata) larvae feeding on coloured foods, Hobson (1931) postulated that the mid-midgut absorbs water. Work- ing with housefly (Musca domestica) larvae, Espinoza-Fuentes and Terra (1987) extended the studies of Hobson (1931) and showed that at each passage of food, water is secreted into the gut lumen

in the fore-midgut (1.5 ~1) and in the hind-midgut (3.9 pl), and water is absorbed from the gut lumen in the mid-midgut (2.3 ~1). Despite studies on the ultra- structure of the midgut of cyclorrhaphous flies re- viewed above, sufficient data have not been produced to relate midgut-cell ultrastructural features with actual midgut fluid fluxes.

In this paper, ultrastructural data on the larval midgut of the housefly are presented suggesting the involvement of specific midgut cells in the previously proposed midgut fluid fluxes. In addition, biochem- ical data ace given supporting a hypothesis on the nature of the substances which maintain the pH in the different midgut regions.

Animals MATERIALS AND METHODS

Larvae of Muscu domestica (Diptera, Cyclorr- hapha, Muscidae) were reared in a mixture of fer- mented commercial pig food and rice hull (1: 2, v/v) [Targa and Peres, 19791. The larvae used in this study were actively feeding individuals at third larval instar.

Electron microscopy

Larvae were dissected in their own haemolymph. After its removal, the midgut was divided ;nto several sections. Midgut sections were fixed in 3% glu- taraldehyde in 0.1 M cacodylate buffer pH 7.4 for 2 h at 4°C. After rinsing with 0.2 M sucrose in the same cacodylate buffer, the midgut pieces were post-fixed

463

464 WALTER R. TERRA et al.

in 1% osmium tetroxide in the same cacodylate buffer for 1 h at 4°C and washed in 0.1 M NaCl. En bloc staining was performed in 1% uranyl acetate for 16-18 h. After dehydration in graded ethanol at room temperature, the material was embedded in Polylite 8001 polyster resin (Resana S/A, Brasil; Coiro et al. 1972). Ultra-thin sections were cut using a Porter- Blum MT II ultramicrotome, stained with lead citrate (Reynolds, 1963) and examined in a Siemens El- miskop IA electron microscope operated at 60 KV.

Preparation of samples, hydrolase assays and protein determination

Larvae were dissected in cold 0.3 M sucrose. Mid- gut tissue, free from contents, was divided into appropriated sections which were homogenized in 0.3 M sucrose with the aid of a Potter-Elvehjem homogenizer. The homogenates were passed through a nylon mesh of 45 pm pore size. The filtrates were centrifuged at 6OOg, IO min at 4°C. The resulting supernatants were used in carbonic anhydrase assays and the pellets, in ATPase assays, as detailed below. Plasma-membrane markers, such as y -glutamyl transferase are enriched about ‘I-fold in the men- tioned pellets (Espinoza-Fuentes et al., 1987). Protein was determined according to Bradford (1976) using ovalbumin as a standard. ATPase was assayed by measuring the inorganic phosphate released from ATP according to Baginski et al. (1967). The stan- dard assay medium contained 50 mM Tris-HCl pH 8.0, 1 mM MgSO,,, 1 mM ATP (disodium salt), and either 20 mM KC1 or 25 mM sodium bicar- bonate. In some experiments, 0.1 % olygomycin in ethanol was added to the reaction media to become 0.02mg olygomycin per ml and 2% in ethanol. K+-ATPase and HCOC-ATPase were determined by subtraction of the rate of phosphate release in reac- tion mixtures lacking K+ and HCO;, respectively. Incubations at 30°C were carried out for at least four different periods of time and the initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. A unit of enzyme (u) is defined as the amount that catalyzes the cleavage of one pmol of ATP/min. Carbonic anhydrase activity was determined as the rate of the catalyzed carbon dioxide hydration by a modification of the method of Wilbur and Anderson (1948). The assay solution was composed of 1 ml of 16 mM N- 2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) buffer (pH 8.3) at 4°C and 0.5 ml of enzyme solution to which was added 1 ml of carbon dioxide- saturated water (4°C). The time for the pH change from pH 8.0 to pH 6.5 was recorded. The units of enzymatic activity (U) were calculated according to Wilbur and Anderson (1948) as: U = (to - t-,)/t,,, where t, is the time for the pH to change from pH 8.0 to pH 6.5 in the uncatalyzed reaction and t,, is the time for the same pH change occur in the catalyzed reaction.

larvae (Hobson, 1931; Waterhouse and Stay, 1955) and therefore only a brief description will be given. The gut of the larvae is composed of a short foregut, a large midgut with four anteriorly connected midgut caeca, and a narrow hindgut. A proventriculus occurs between the foregut and midgut and the openings of the four Malpighian tubules mark the junction be- tween the midgut and the hindgut. Three regions are discernible with a stereoscopic microscope in the midgut: fore-, mid- and hind-midgut. The fore- and hind-midgut contain liquid food, whereas the mid- midgut, which is constricted at either end and in between is distended into a small expansion, is filled with a paste-like food. The three regions of the midgut are also distinguished by the pH of their contents. The fore- and hind-midguts are nearly neutral (pH 6.1 and 6.8, respectively), whereas the mid-midgut is acid (pH 3.1) (Espinoza-Fuentes and Terra, 1987).

In order to describe better the differences found in the morphology of the cells along the M. domestica larval midgut, the three above-mentioned midgut regions were subdivided further as follows (see Fig. 7). The fore-midgut was divided into two (regions I and II) and hind-midgut into three (regions I, II and III) sections of approximately the same length. The mid-midgut was divided into three regions (I, II and III): region I, a section anterior to the small expan- sion; region II, a section containing the small expan- sion; region III, a section posterior to the small expansion.

Columnar cells can be recognized in all the midgut regions, in spite of the fact that their morphology can change along the midgut and even within the same region. Cup-shaped cells occur in region I of the mid-midgut and regenerative and endocrine cells seem to be absent from midgut.

The fine structure of M. domestica larval midgut cells is similar to that of other cyclorraphous larvae (Waterhouse and Wright, 1955; Filshie et al., 1971; Dimitriadis and Kastritsis, 1984). Because of this, illustrations will be provided only for the features which were overlooked by previous authors, or which seem to be characteristic for M. domestica.

Fine structure of the midgut caecal and fore-midgut ceils

The apical membrane of the midgut caecal cells is modified into cylindrical microvilli in a compact arrangement. The basal membrane is highly infolded, with few apertures to the underlying space. There is a large number of mitochondria associated with the infoldings, although the mitochondria are also found in the cell apex, at the bases of the microvilli. There are morphological features which are usually associ- ated with secretory activity, such as elements of Golgi, abundant rough endoplasmic reticulum and secretory vesicles (Fig. 7).

RESULTS

The cells along the fore-midgut differ from those of the midgut caeca only in displaying less elaborate basal infoldings with fewer apertures to the under- lying space (Fig. 7).

General morphology of the midgut Fine structure of the mid-midgut cells

The general morphology of M. domestica larval midgut is similar to that of other cyclorrhaphous

Region I of mid-midgut is composed of two cell

types: the interstitial (IC in Fig. 1) and the oxyntic

Fig. I. Base of an interstitial (IC) and an oxyntic (OC) cell from region I of the mid-midgut of 154. domestica larvae. Note, in the oxyntic cell, the apical membrane invaginated into ramified crypts. Other abbrevi- ations used in this and in the other figures: BL, basal lamina; BMI, basal membrane infoldings; G, Golgi complex; L, lumen; LS, Lipid sphere; Mi, mitochondria; MV, microvilh; RER, rough endoplasmic reticulum; SV, secretory vesicle. Bar = 1 pm on this and all following figures, except Fig. 3 and insert on

Fig. 2, in which the bar corresponds to 0.1 pm.

Fig. 2. Detail of the base of an interstitial cell. Note basal-membrane infoldings forming narrow channels (arrowheads) which open into the underlying space (arrows). Inset, detail of the basal-membrane infoldings. Note particles attached to the cytoplasmic side of basal-membrane infoldings (small arrows).

Fig. 3. Detail of the apex of an oxyntic cell. Note particles attached to the cytoplasmic side of the microvillar membranes (arrows).

Fig. 4. General aspect of a cell from region III of the hind-midgut of M. domesticn larvae. Note that basal membrane infoldings extend almost to the apical face.

Fig. 5. Apex of a cell from region III of the hind-midgut of M. dormsfica larvae. Note series of small vesicles (arrows).

Fig. 6. Base of a cell from region III of the hind-midgut of M. domestica larvae. Note basal-membrane infoldings forming wide channels which open to the underlying space (arrows).

Ahh

Larval midgut of the housefly 46-I

(OC in Fig. 1) cells, both of which display lipid spheres in their cytoplasm. Interstitial cells are char- acterized by highly developed basal infoldings (Fig. 2) displaying particles attached to their cytoplasmic side (Fig. 2, inset) and arranged with associated mito- chondria in a parallel array (Fig. 2). Oxyntic cells have an apical membrane invaginated into ramified crypts which are coated with microvilli, and display numerous mitochondria in their cytoplasm (Fig. 1). The cytoplasmic side of oxyntic microvillar mem- branes are studded with small particles (Fig. 3).

Cells in region II of the mid-midgut are flat, without differentiated apical microvilli, and with a cytoplasm rich in lipid spheres and mitochondria but without apparent secretory activity. Cells in region III of the mid-midgut show few developed apical microvilli, and cytoplasm rich in lipid spheres and poor in those features associated with secretory activ- ity. In the base of these cells there are plasma- membrane infoldings with associated mitochondria. The infoldings display apertures to the underlying space (Fig. 7).

Fine structure of the hind-midgut cells

In the anterior region I of the hind-midgut, the cells present a base similar to those of the cells in region III of the mid-midgut, whereas in the cell apex, well developed and numerous microvilli are found. The cytoplasm of those cells contains abundant rough endoplasmic reticulum, Golgi elements and secretory vesicles. The cells of region II of the hind-midgut are intermediate between the cells from the anterior region I and those of the posterior region III of the hind-midgut (Fig. 7).

The cells in region III of the hind-midgut display basal-membrane infoldings forming wide channels which extend almost up to the cell apex (Fig. 4). The channels have associated mitochondria and numer- ous apertures to the underlying space (Fig. 6). The apical membrane is undulated and shows con- spicuous and well developed microvilli (Figs 4 and S), close to which there are series of small vesicles (Fig. 5, arrows). These vesicles are observed in many insects and were shown to correspond to double- membrane vesicles which bud from the microvilli (Nopanitaya and Misch, 1974; Santos et al., 1984). Rough endoplasmic reticulum and, elements of Golgi are scarce, and secretory vesicles are apparently absent, whereas a smooth endoplasmic reticulum is well-developed in the cells in region III of the hind- midgut.

Carbonic anhydrase and ATPase from d$&erent midgut regions

Carbonic anhydrase is more active in the fore- and mid-midgut than in the hind-midgut of M. domestica larvae (Table 1). This is the first quantitative deter- mination of carbonic anhydrase along an insect mid- gut (for a review, see Darlington et al., 1985).

An ATPase activated by HCO; was found in fore- and hind-midguts of M. domestica larvae (Table 1). This enzyme seems to be different from the mito- chondrial ATPase, since it is not affected by oli- gomycin, a known inhibitor of the mitochondrial ATPase (Lardy et al., 1958). As a control, M. domes- tica larval midgut Mg*+-ATPase, which is probably derived mostly from the mitochondria (for a review on mitochondrial ATPases, see Penefsky, 1974), is appreciably inhibited by oligomycin (Table 1). It is possible that the midgut HCO;-ATPase occurs in the microvillar membranes of midgut cells, taking into account that the pellets used to assay the ATPase is enriched in plasma membranes (see Materials and Methods).

Attempts to assay a K+-activated ATPase in M. domestica larval midgut were not successful (legend to Table 1).

DISCUSSION

Fluid fluxes, enzyme secretion and digestion

Except for lysozyme and pepsin, which are more active in the mid-midgut, M. domestica larval di- gestive enzymes are found mainly in the hind-midgut, where the majority of digestion must take place in these insects (Espinoza-Fuentes and Terra, 1987). Otherwise, dye experiments with M. domestica larvae suggest that water is secreted into the gut lumen in the fore-midgut and in the hind-midgut and water is absorbed from the gut lumen in the mid-midgut. This may lead to the existence of a flux of fluid from the fore-midgut into mid-midgut and from the hind- midgut into the mid-midgut. The last-mentioned flux should occur in opposite direction to the movement of the food bolus and seems to be responsible for the recovery of major amounts of digestive enzymes from undigested food (enclosed by the peritrophic mem- brane), thus reducing the amounts of digestive en- zymes which are excreted (Espinoza Fuentes and Terra, 1987). Ultrastructural data presented in this paper provide a cytological basis for the above- mentioned fluid fluxes as detailed below.

Table 1. Carbonic anhydrase and olygomycin inhibition of ATPase activities from cells of different regions of M. domesfica larval midgut*

Mg*+-ATPase HCO;-ATPase

Carbonic anhydrase Control Control

(U/mg) (mU/mg) % Inhibition (mU/mg) % inhibition

Fore-midgut I .30 f 0.06 520 f 60 50* 10 70 f 10 0 Mid-midgut I .24 f 0.06 140*30 25 k 5 0 Hind-midgut 0.44 * 0.03 230 + 30 35 k 6 120+30 I*1

*Carbonic anhydrase was assayed in supematants, whereas ATPase activities were assayed in pellets obtained by centrifuging midgut cell homogenates at 6006 for 10 min at 4°C. Mg-ATPase were assayed in a medium containing: 50mM Tris-HCI pH 8.0, I mM MgSO, and I mM ATP. K+-ATPase and HCO,-ATPase was measured as the increase in the activity observed by adding respectively 20 mM KCI or 25 mM NaHCO to the assay medium of Mg 2+-ATPase. The detection limits of K+-ATPase (which was not found in any region) and for HCO;-ATPase were 10 mU/mg protein. Figures are means + SEM based on determinations carried out in three different preparations obtained from 50 larvae each.

468 WALTER R. TERRA et al.

A well-developed brush-border with a large num- ber of mitochondria present in the apical cytoplasm, together with plasma-membrane infoldings with asso- ciated mitochondria may provide the cells with the capacity for transporting several compounds (see Threadgold, 1976). The fact that the basal plasma- membrane infoldings constitute an extracellular com- partment, which has restricted access to the hae- molymph (due to the restricted openings into the underlying extracellular space), should permit the cell to concentrate solutes in that compartment to create an osmotic pressure gradient between compartment and lumen, which might assist the absorption of water (cf. Berridge, 1970). Otherwise, cells displaying basal plasma-membrane infoldings forming wide channels with many openings to the underlying space should be involved in water secretion (Berridge. 1970). Based on these concepts it was possible to propose, from ultrastructural analysis, the existence of fluid fluxes on the midgut of Rhynchosciara amer- icana (Ferreira et al., 1981) and Erinnyis ello (Santos et al., 1984). The proposed fluxes agreed with those suggested by physiological and biochemical obser- vations (Terra and Ferreira, 1981; Santos et al., 1983).

A restricted extracellular compartment may result from basal plasma-membrane infoldings, even if there is a large number of openings to the underlying space, provided that the infoldings form long and narrow channels. It is possible that this arrangement is more efficient in assisting water absorption than those based on wide and branched channels with few openings. The reasoning supporting this assertion is that long and narrow channels display a larger ratio of membrane-enclosed contents than wide and branched channels, thus making possible the exis- tence of a larger number of ion pumps per unit of volume. As a consequence, larger osmotic gradients may be created, resulting in larger volumes of water being absorbed per unit of time.

Interstitial cells, which occur in region I of the mid-midgut of M. domestica larvae, display charac- teristics of water-absorbing cells (Figs 1 and 2). It is tempting to speculate that the particles studding the cytoplasmic face of the basal infoldings of these cells (Fig. 2, inset) are ion pumps, which create osmotic gradients responsible for water absorption. This hy- pothesis is supported by the finding that similar structures, the portasomes, are involved in ion trans- location in several epithelia (Review: Harvey et al.,

1981). Otherwise, the only cells which ultrastructure suggests might secrete water are those present in region III of the hind-midgut (Figs 4 and 6), and perhaps also those in region II of the same region. Thus, based on ultrastructural observations, it is possible to propose in M. domestica larvae the exis- tence of a major flux of fluid from the hind-midgut into the mid-midgut. This proposal agrees with that based on direct experiments which were described above. Nevertheless, they do not permit one to infer the existence of a minor fluid flux, as proposed from dye experiments (Espinoza-Fuentes and Terra, 1987), from the fore-midgut into the mid-midgut. Other- wise, the amount of basal-membrane infoldings, and the small number of pores to the underlying space argue in favour of a role of the midgut caeca in absorption. Nevertheless, this has no support from the above-mentioned dye data.

In spite of the contrasting conclusions of Dimi- triadis and Kastritsis (1984) similar fluxes of fluids probably occur in Drosophila auraria larvae. Thus the interstitial cells of the anterior region of the mid- midgut of D. auraria larvae display, as in M. domes- tica, particles studding the cytoplasmic face of their basal infoldings (see Fig. 14 in Dimitriadis and Kastritsis, 1984). Furthermore, the cells in the poste- rior region of the hind-midgut of D. auraria present basal plasma-membrane infoldings forming wide channels (Dimitriadis and Kastritsis, 1984).

It is possible that only the cells from the midgut caeca, fore-midgut and chiefly from regions I and II of the hind-midgut of M. domestica larvae are in- volved in the secretion of soluble enzymes, since only they show what seem to be secretory processes (for a review on insect secretory processes, see Bignell et al., 1982): abundant rough endoplasmic reticulum and secretory-like vesicles which may be followed from the Golgi complex to the apex of the cells (Fig. 7). This agrees with the finding of amylase in the midgut caeca cells, of lysozyme and pepsin in the fore-midgut cells and of large amounts of amylase, maltase and trypsin in the hind-midgut cells (Espinoza-Fuentes and Terra, 1987). Otherwise, membrane-bound en- zymes (e.g. aminopeptidase) are found mainly in the anterior half of the hind-midgut (Espinoza-Fuentes and Terra, 1987). These data agree with the ultra- structural observations in Drosophila auraria (Dimi- triadis and Kastritsis, 1985).

The features discussed above have been incorpo- rated in a model of digestion in M. domestica midgut

Fig. 7. Diagrammatic representation of water fluxes and endo-ectoperitrophic circulation of digestive enzymes in the larval gut of M. domestica. Endogenous fluid (dotted arrows) is secreted in minor amounts into the fore-midgut and in major amounts into the posterior region of the hind-midgut by cells characterized by highly-developed basal plasma-membrane infoldings forming wide channels with many openings to the underlying space. The fluid moves towards the mid-midgut. At this site the fluid is absorbed by the interstitial cells which show basal plasma-membrane infoldings, arranged in long and narrow channels, with particles studding their cytoplasmic face. Polymer hydrolases (solid arrows) enter the endoperitrophic space in the fore-midgut and are directed towards the mid-midgut. Starch is partially digested in the fore-midgut and bacteria are killed in the mid-midgut through the combined action of low pH, lysozyme and pepsin. In the hind-midgut, polymer hydrolases (solid arrows) enter into the endoperitrophic space anteriorly, from which they are recovered stepwise as the polymeric molecules they hydrolyse become sufficiently small to accompany them through the peritrophic membrane. The enzymes and oligomeric molecules are then displaced towards the anterior region of the hind-midgut, where terminal digestion and absorption of food occur at the surface of the cells. ECS, ectoperitrophic space;

Ens, endoperitrophic space; PM, peritrophic membrane.

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469

470 WALTER R. TERRA et al.

1 FORE-HIDQUT zjc MID-MIDGUT HIND-MIDQUT

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Fig. 8. Diagrammatic representation of ion movements supposed to be responsible for the maintenance of the pH of the larval midgut contents of M. domestica. Carbonic anhydrase (CA) in midgut cells produces carbonic acid which dissociates into bicarbonate and a proton. In fore- and hind-midgut cells the proton is transported into the haemolymph through the basal membrane, whereas the bicarbonate is secreted into the midgut contents through the action of a ATP-driven pump. The bicarbonate neutralizes the acidic midgut contents. In the oxyntic cells of the mid-midgut, the bicarbonate is transported into the haemolymph, whereas the proton is actively translocated into the midgut lumen acidifying its contents.

Chloride ions probably follow the movement of protons.

(Fig. 7) [see also discussion in Espinoza-Fuentes and Terra, 1987).

The maintenance of midgut pH values

The pH changes remarkably along the midgut of M. domestica. The fore-midgut contents display a pH of 6.1, the mid-midgut contents a pH of 3.1 and the hind-midgut contents, a pH of 6.8 (Espinoza-Fuentes and Terra, 1987). The low pH observed in the mid-midgut of M. domestica, and of other cy- clorrhaphous flies, deserves special attention, since those insects are the only animals, other than verte- brates, to display such an acidic region in their guts (Vonk and Western, 1984). Only the oxyntic cells of region I of the mid-midgut seem to display sufficient mitochondria (besides the interstitial cells which might absorb water, see above) to do the osmotic work necessary to acidify the midgut contents, and only they display in their microvillar membranes particles which might be ion pumps (Fig. 3). Further- more, the oxyntic cells of the mid-midgut from M. domestica larvae are morphologically alike the oxyn- tic (also called parietal) cells from the mammalian stomach (compare Fig. 1 of this paper with Fig. 1 from Ito and Schofield, 1974). The oxyntic cells from the mammalian stomach are responsible for the secretion of protons, resulting from the dissociation of carbonic acid (originated through the action of carbonic anhydrase upon carbon dioxide), and of chloride ions, thus forming hydrochloric acid in the stomach lumen (review: Forte et al., 1980). It is

possible that the oxyntic cells from the M. domestica midgut also secrete hydrochloric acid, maintaining a low pH in the midgut contents. Further support for this belief comes from the finding that the carbonic anhydrase activity in M. domestica mid-midgut cells is very high (Table 1). Nevertheless, attempts to find a putative ATPase involved in proton translocation, which in mammalian stomachs corresponds to a potassium-activated ATPase not inhibited by oly- gomycin (review: Forte et al., 1980), were not success- ful (Table 1). Cup-shaped cells similar to M. domes- tica oxyntic cells (called cuprophilic cells by other authors) were found in the acidic mid-midguts from larvae of other cyclorrhaphous flies such as Droso- phila melanogaster (Filshie et al., 1971), Drosophila auraria (Dimitriadis and Kastritsis, 1984), but not in Lucilia cuprina (Waterhouse and Wright, 1960). Thus, proton pumps may not be necessarily related to cell shape, suggesting further studies on the sub- ject, mainly on Lucilia cuprina.

It has been pointed out earlier that the pH of the contents of the digestive tract rise suddenly from about 3.1 to 6.8 as the contents pass from the mid-midgut into the hind-midgut. A good guess as to the cause of this rise in pH is the secretion of bicarbonate ions by hind-midgut cells, in a way reminiscent to what occurs in the mammalian duo- denum. In mammals, stomach contents are alka- linized by bicarbonate ions secreted into the intestine through the action of an ATP-driven pump located on the plasma-membrane covering the enterocyte

Larval midgut of the housefly 471

microvilli (Humphreys and Chou, 1979; Gamer et al., (1987) Microvillar and secreted digestive enzymes from 1983). It is possible that the same occurs in the M. Muscu domestica larvae. Subcellular fractionation of mid-

domestica hind-midgut, since in this region a plasma- gut cells with electron microscopy monitoring. Insect

membrane-bound ATPase was found which was Biochem. 17, 819-827.

activated by bicarbonate ions, and which was not Ferreira C., Ribeiro A. F. and Terra W. R. (1981) Fine

inhibited by olygomycin (Table 1). structure of the larval midgut of the fly Rhynchosciuru

The pH of the food ingested by M. domestica americana and its physiological implications. J. Insect

larvae is about 5 (unpublished observations), whereas Physiol. 27, 559-570.

the pH of the fore-midgut contents is 6.1. It is Filshie B. K., Poulson D. F. and Waterhouse D. F. (1971)

possible that the observed rise in the pH of ingested Ultrastructure of the copper-accumulating region of the Drosophila larval midgut. Tissue Cell 3, 77-102.

food is accomplished as described above for the Forte J. G., Machen T. E. and Obrink K. J. (1980) buffering of hind-midgut contents. Mechanisms of gastric H+ and Cl- transport. A. Reu.

A model describing the secretion of ions proposed Physiol. 42, 11 I-126.

to be responsible for the maintenance of midgut pH Garner A., Peters T. J. and Wilkes J. (1983) Demonstration

values in M. domestica larval midguts is presented in of HCO,-activated Mg2+-dependent ATPase activity in

Fig. 8. rat duodenal microvillus membranes. J. Physiol. 342, 13P-14P.

Acknowlednements-Research in the Biochemistry De- Greenberg B. (1968) Micro-potentiometric pH deter-

partment was supported by grants from the Funda&o de minations of muscoid maggot digestive tracts. Ann. ent.

Amparo $ Pesquisa do Estado de SHo Paul0 (FAPESP), Sot. Am. 61, 365-368.

Conselho National de Desenvolvimento Cientifico e Tec- Harvey W. R., Cioffi M. and Wolfersberger M. G. (1981)

nol6gico (CNPq), and Financiadora de Estudos e Projetos Portasomes as coupling factors in active ion transport and

(FINEP); in the Biology Department, by CNPq thrbugh oxidative phosph&ylaiion. Am. Zool. 21, 775-791.

Plano Intearado de Genktica (PIG). We are much indebted Hobson R. P. (1931) Studies on the nutrition of blow-flv

to Mr V. c. Cruz, M. W. Caideira, Mrs I. M. Femandes, larvae. I. Str&ure and function of the alimentary tract.

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