Journal of Cell Science 101, 349-361 (1992) 349 Printed in Great Britain © The Company of Biologists Limited 1992

Transport of the cationic fluorochrome 123 in an insect's Malpighian tubule: indications of a reabsorptive function of the secondary cell type

W. MEULEMANS and A. De LOOF

Zoological Institute of the University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium

Summary

The pathway of was examined after the lumen was found using Lucifer Yellow CH, injection into Sarcophaga flies and after in vitro labeling fluorescent dextrans and fluorescent albumin. Prior of the Malpighian tubules. After in vitro labeling the incubation with the ionophores valinomycin, nigericin, primary cells only retained this potential-sensitive CCCP (all 1 pg/ml), dinitrophenol (1 mM) and NaN3 for a short period while all secondary cells accumulated (10~2 M) inhibited the selective accumulation of rhoda- the dye from the tubule lumen. In vivo the secondary mine 123 to a large extent while monensin (1-5 /ig/ml) cells also accumulated rhodamine 123 from the lumen, showed little inhibitory effect. Furthermore, only cat- but the primary cells in the distal parts of all four tubules ionic and no anionic or neutral were accumulated retained the dye for prolonged periods. This was most by the secondary cells. In the fleshfly Calliphora and the pronounced in the distal part of the anterior Malpighian fruitfly Drosophila, the dye rhodamine 123 also selec- tubules, where rhodamine 123 was eventually precipi- tively accumulated in the secondary cells, as well in vitro tated on the luminal concretions. Rhodamine 123 as in vivo. initially accumulated in the secondary cell mitochondria and eventually in intensely fluorescing vesicles, probably Key words: Malpighian tubules, dye transport, lysosomes. No evidence for endocytotic processes from reabsorption, secondary cells, rhodamine 123.

Introduction has been described (Lison, 1937; Maddrell et al., 1974; Bresler et al., 1990). The excretion of basic dyes like The Malpighian tubules are the so-called 'kidney neutral red and methylene blue (Lison, 1938; Maddrell, tubules' of the insects. Extensive research has been 1977) in some insect Malpighian tubules has been linked devoted to the study of the mechanisms underlying the to the capability of these tubules to actively excrete primary urine secretion by Malpighian tubules (Mad- toxic alkaloids like nicotine, atropine and morphine, drell, 1978a; Bradley, 1985; Maddrell and Overton, which are also organic bases (Maddrell, 1977). 1990). It has been shown that along with the large In this paper we present evidence that in vitro and in amount of fluid passing through or in between the cells, vivo the secondary (S) cells of larval and adult many small molecules also enter the primary urine Malpighian tubules of some Diptera (Brachycera) are (Maddrell and Gardiner, 1974; Bradley, 1985). Small accumulating membrane-permeant cationic dyes but essential molecules like amino acids or sugars leaking not anionic or neutral dyes from the luminal fluid. into the lumen would then be reabsorbed along their Furthermore, in the distal part of the anterior Mal- passage through the long Malpighian tubules or in parts pighian tubules rhodamine 123 was precipitated on the of the gut such as the rectum. Until now, only an active luminal concretions. reabsorption of glucose from the primary urine has been described in the Malpighian tubules of some Besides the Malpighian tubules of the fleshfly insects (Knowles, 1975; Rafaeli-Bernstein and Mordue, Sarcophaga bullata, some comparative investigations 1979). Direct uptake of any molecule by a particular cell were also conducted with the Malpighian tubules of the type in the Malpighian tubules has not been described. fruitfly Drosophila melanogaster and a related fleshfly, Coloured organic molecules have often been used to Calliphora erythrocephala. characterize the excretory systems of insects. Acidic organic molecules like , indigocarmine and Materials and methods amaranth are actively cleared from the haemolymph by Animals the Malpighian tubules, but no accumulation in the cells Sarcophaga bullata (Parker) was reared as described by 350 W. Meulemans and A. De Loof

Huybrechts and De Loof (1977). In our experiments, male observed under the microscope. Larvae were and female flies of day 3 were used, unless mentioned also injected with 100 /ig/ml. otherwise. Drosophila melanogaster was reared as described by Callaerts et al. (1988). The wild type of Calliphora In vitro application of drugs and antibiotics erythrocephala was caught in Leuven and reared as described The drugs and antibiotics were tested at different concen- for Sarcophaga. trations for 5-120 minutes on Malpighian tubules in vitro. The following provided the best minimum incubation time and Supravital staining of the Malpighian tubules lowest concentration for a clearly detectable effect: valinomy- In vitro staining of the tubules cin (Janssen Chimica, Beerse, Belgium), 30 min in 1 ^g/ml; nigericin (Sigma), 30 min in 1 jug/ml; monensin (Serva), up to The Malpighian tubules were carefully dissected in Ringer's solution (adapted from Chan and Gehring (1971), in mM: 120 120 min in 5 ^g/ml; CCCP (Serva), 30 min in 1 ^M; 2,4-DNP (Serva), 30 min in 1 mM; sodium azide (Merck), 30 min in NaCl, 10 KC1, 15 MgSO4, 5 CaCl2, 10 Tricine, 20 glucose, 50 2 sucrose, pH 6.8). In this solution the tubules could actively 10~ M. Whenever appropriate, the same concentration of secrete fluid for at least 24 hours without oxygen supply, as the primary solvent acetone or was included in the was measured with the in vitro bio-assay according to Ramsay Ringer's solution in the control conditions (max. 0.5%). Only (1954). Some control experiments were conducted in a tubules with viable cells were used in the experiments. glucose-free bicarbonate buffer (Sarcophaga Ringer's (in mM): 121.5 NaCl, 10 KC1, 1 NaH PO , 10 NaHCO , 0.7 2 4 3 Results MgCl2, 2.2 CaCl2, pH 6.8, aerated with 95% Oj/5% CO2), according to Verachtert and De Loof (1988). After staining and rinsing in Ringer's solution, the tubules General morphology and terminology were transferred to a hollow glass slide in a few drops of The fleshfly Sarcophaga bullata (like most other Ringer's fluid, and covered with a coverglass. In this covered Diptera) contains 4 Malpighian tubules (Fig. 1), 2 slide the tubule cells remained viable for many hours (cell oriented anteriorly (anterior Malpighian tubules or viability tested by Trypan blue exclusion; Ashburner, 1989). AMT) and 2 oriented posteriorly (posterior Malpighian For immediate photography of these whole-mounts, a flat tubules or PMT) in the abdomen. Each AMT contains a glass slide with two glass bridges was used. morphologically distinct distal part (dAMT), while the The fluorescently stained tubules were observed under a Leitz Laborlux S fluorescence microscope, using the rhoda- mine and fluorescein excitation filters. Rhodamine 123, Lucifer Yellow CH, sulphorhodamine B, FTTC-labeled dex- trans of 4.4 and 35 kDa and a TRTTC-labeled dextran of 10 kDa were purchased from Sigma. Bovine serum albumin (Serva) was conjugated with FITC (Sigma) as described by Haaijman (1983). Eosins Y and B and were purchased from Merck; Pyronin Y(G) was purchased from Serva; merocyanin 540 from Fluka; and fluorescein (sodium) from UCB. Fluorescence was recorded using Kodak T MAX 400 ASA film.

In vitro injection of fluorochromes into the lumen The lumen of the proximal half of a freshly dissected tubule was perfused with a short pulse of dye solution by means of small glass capillaries with an outer tip diameter of 25-40 /zm. The dye containing Ringer's solution was injected into the lumen until the coloured fluid was seen to leave the other open tubule end. After 1 to 5 minutes of incubation the lumen was rinsed with Ringer's solution and the tubule was mounted in fresh Ringer's fluid in a hollow slide, and covered with a coverglass.

In vitro transport of fluorochromes in the lumen For these experiments, whole Malpighian tubules were set up between a fluorochrome solution and a dye-free Ringer's solution in two separate bathing droplets under paraffin oil (see Maddrell and Overton, 1990).

Intravital staining of the Malpighian tubules In these experiments 2 )A of rhodamine 123 (100 ^g/ml) or other dyes in Ringer's fluid (0.1-1 mg/ml) were injected into Fig. 1. General morphology and orientation of Malpighian the thorax of adult Sarcophaga bullata by means of small glass tubules in Sarcophaga bullata (after Wessing and capillaries with a tip diameter of about 100 /an. In each Eichelberg, 1969). Notice the swollen distal part (d) of the experiment a control group, injected with Ringer's solution anterior Malpighian tubule (A) and the morphologically alone, was kept under identical conditions. After the proper homogeneous posterior Malpighian tubule (P), which incubation time, the tubules of experimental and control flies resembles the proximal part (p) of the anterior tubule (M, were removed in Ringer's fluid, and were immediately midgut, H, hindgut). Dye transport in insect Malpighian tubules 351

PMT is morphologically homogeneous and similar to labeled P cells had lost most of the fluorochrome (Fig. the proximal part of the AMT (pAMT) (see Fig. 1). 2B). Even the P cells in the most distal part of the PMT Each tubule consists of two cell types, the primary (P) lost their fluorescence after two hours in vitro. Separate and the secondary (S) cell type. Although in dipteran mitochondria were clearly recognized as tubular ves- Malpighian tubules most of the S cells have a stellate icles throughout the entire S cell. This pattern re- cell shape, we prefer naming them secondary instead of sembled that described for vertebrate cultured cells stellate cells, since similar cell types in other insect (Johnson et al., 1980). At first this selective staining of groups are not stellate (Bradley, 1985). In comparison the S cells was thought to be an artefact caused by the to the P cells the smaller S cells in Diptera have shorter prolonged incubation of the tubule in the covered microvilli, which contain little or no mitochondria, and hollow slide. However, when the same tubule was again they have a smaller nucleus and a transparent cyto- stained in a fluorochrome-containing Ringer's solution plasm containing little or no granular deposits. the P cells were labeled as intensely as after the first Alkassis and Schoeller-Raccaud (1984) named the staining. So their mitochondria did not lose the ability cells in the distal part of the AMT of larval Calliphora to accumulate Rh 123. The dye must have leaked from erythrocephala differently. We suggest that these cells the P cells to the lumen. are also named S and P cells, since as well as some When such a tubule with brightly labeled S cells was differences the cells in distal and proximal tubule parts left in the Ringer's solution for another hour or longer, have some apparent characteristics in common: a small large round vesicles were formed (Fig. 2D). Concomi- nucleus and a transparent cytoplasm in all S cells tantly the typical mitochondrial pattern disappeared. compared to a granular cytoplasm containing a large Possibly these are enlarged, poisoned mitochondria or nucleus in all P cells. Furthermore, a very similar F- lysosomal vesicles that finally accumulated the dye actin cytoskeleton was found in the S and P cells in both molecules. parts of the AMT (Meulemans and De Loof, 1990a,b). We will however always indicate whether the cells are Anterior tubules. Immediately after in vitro labeling all found in the distal or proximal part of the AMT. P cells in the AMT were brightly labeled, even those in the distal part of the AMT. As in the PMT, the S cells Supravital and intravital labeling of the Malpighian only displayed minor fluorescence. When a tubule was tubules with rhodamine 123 and other fluorochromes left in the Ringer's solution for two hours, even the S Rhodamine 123 (Rh 123) is a very fluorescent rhoda- cells in the distal part of the AMT selectively accumu- mine derivative that is known to be non-toxic to lated the dye (Fig. 2C). After prolonged incubation in vertebrate cells after a short application of 10 ^g/ml Ringer's solution, the S cells also displayed bright (Johnson et al., 1980, 1981; Chen, 1989). This fluoro- round vesicles in both distal and proximal parts of the chrome is known to be attracted by the high potential AMT. The results of the in vitro staining of both AMT gradient over the mitochondrial membranes in living and PMT are summarised in Table 1. cells. Experiments with the ion-selective ionophore nigericin (K+/H+ exchanger) showed that it is the In vitro injection into the Malpighian tubule lumen potential difference across the mitochondrial mem- Posterior tubules. To test whether the S cells had indeed branes rather than the pH gradient that attracts the dye accumulated the dye from the luminal cavity, proximal molecules (Johnson et al., 1981). parts of posterior Malpighian tubules were luminally injected with a 10 /Ug/ml solution of Rh 123 in Ringer's Supravital labeling of the Malpighian tubules fluid. After incubating for 1 or 2 minutes, it was obvious In vitro labeling that only the S cells had accumulated the fluorochrome; Posterior tubules. In a posterior Malpighian tubule the P cells were not labeled (Fig. 2E). It was even stained in vitro in 5 jig/ml Rh 123 for 10 minutes and unnecessary to rinse the lumen, since little dye washed with the Ringer's solution for 5 minutes, the remained in the luminal cavity. If the same tubule was mitochondria were well labeled. All P cells displayed an then labeled at its haemolymph side, the P cells were intense labeling while the S cells appeared largely brightly stained. They did not lose their ability to take unstained (Fig. 2A). Because of the granular cytoplasm up the Rh 123 but their apical membrane seemed to be and the extensive labeling of the P cells, separate quite impermeable to this cationic fluorochrome. If Rh mitochondria could not be detected. But the high 123 (10 ^g/ml) was injected into the lumen of the most amount of mitochondria in the P cell microvilli was distal part of the PMT, no selective accumulation in the clearly recognized as an intense apical zone upon these S cells was noticed. The P cells accumulated most of the cells. This was most obvious when looking at the P cell dye while the S cells only displayed minor labeling. So brush border through the transparent S cells. Also, due only the more proximal S cells accumulated the dye to the intensive staining of the underlying P cells the from the lumen in these posterior tubules. The apical mitochondria in the S cells, which were poorly labeled, membranes of the P cells in the distal part of the PMT were hard to detect. seem to be permeable to this cationic dye, while the If such a living Rh 123-stained tubule was left in the proximally located P cells were largely impermeable. Ringer's solution for about two hours, an entirely different staining pattern was found. Only the S cells Anterior tubules. Injection of the dye solution into the were labeled very intensely while the previously well lumen of the proximal part of the AMT resulted in the 352 W. Meulemans and A. De Loof

Fig. 2. Malpighian tubules were stained with Rh 123 in vitro. (A) Immediately after staining for 10 min in 5 |Ug/ml; the secondary cells (S cells) remain largely unstained; (B and C) proximal (B) and distal (C) part of AMT, 120 min in Ringer's fluid after staining; all S cells accumulated the dye while all P cells lost it; (D) S cell, 180 min in Ringer's solution after staining; the mitochondnal pattern has disappeared and round vesicles are found; (E) Rh 123 (10 jig/ml) was injected into the lumen of the proximal part of a tubule; only the S cells accumulated the dye from the lumen; (F-I) the following fluorochromes were injected into the proximal part of a tubule: 10 /ig/ml (F), 20 j/g/ml merocyanin 540 (G), 20 jig/ml fluorescein (H) and 20 ng/m\ pyronine Y (I). Only the latter (cationic) dye is accumulated by the S cells. The anionic dyes (in G and H) are found in high concentrations between the P cell microvilli (arrowheads). Bars, 10 jwn, A, B, D, F-I; 20 ion, C and E. Dye transport in insect Malpighian tubules 353

Table 1. Relative fluorescence intensities after in vitro labeling of adult Malpighian tubules Time after labeling

10 min 120 min 180 min Prox. Dist. Prox. Dist. Prox. Dist. PMT S cell *•* *•* Pcell + AMT S cell T r I 4+ • •* **• Pcell

The tubules were stained for 10 min in 5 /

Fig. 3. Injection of Rh 123 into intact flies: (A) 30 min after injection; (B) 24 hours after injection; proximal part of the PMT showing selective accumulation in the S cell mitochondria; (C) same fly 24 hours after injection; more distal part of the PMT showing P cells that retain dye longer while the S cells (S) appear largely unlabeled; (D) 30 hours after injection, showing intensely fluorescing round vesicles in the S cells; (E) distal part of the AMT 30 hours after injection; the lumen contains brightly labeled concretions while the cells appear unstained; (F) granules taken from the lumen of the distal AMT 30 hours after injection, showing a rim of Rh 123 around most granules; (G) 90 hours after injection; Rh 123 is found in the centre of most granules. Bars, 5 /m\, F and G; 10 ^m, B-D; 20 /.im, A and E. Dye transport in insect Malpighian tubules 355

Table 2. Relative labeling intensities of the different cell types after Rh 123 injection Time after injection

10 min 60 min 40h Prox. Dist. Prox. Dist. Prox. Dist. PMT Scell ••** ***• P cell + + AMT Scell r + + + r *•+• Pcell

2 jA of Rh 123 at 100 /ig/ml was injected into adults. See Table 1 legend for abbreviations. "**S cells are diffusely labeled but contain some very fluorescently labeled vesicles. These vesicles are smaller but intenser as those found 180 min after in vitro labeling. periods post injection, even the mitochondria in some S Table 3. Relative labeling intensities after injection of cells in the distal AMT were labeled more intensely 2 [d rhodamine 123 (100 jJgJml) into third-instar than neighbouring P cells. At only about 40 hours after feeding larvae injection into the adult, the dye had left the primary Time after labeling cells of all tubules. At about the moment the S cells started losing their 10 min 120 min mitochondrial pattern, the so-called concretions Prox. Dist. Prox. Dist. (Brown, 1982; Sohal et al., 1976) in the lumen of the PMT S cell distal part of the AMT were fluorescently labeled (Fig. Pcell 3E). When comparing the granular deposits in the distal AMT S cell part of the AMT lumen with those of control flies, it was Pcell obvious that the concretions in the dye-injected flies No long-term experiments were conducted. contained the dye rhodamine 123 in a rim around their periphery (Fig. 3F). After about 90 hours post injection, the concretions were still labeled, but now the fluorescence was found in the central part of the Selective accumulation of other dyes? concretions (Fig. 3G). These were preformed granules In these experiments only the proximal part of adult upon which non-fluorescent material had been de- posterior Malpighian tubules was used. In experiments posited in the lumen. The latter granules were clearly in which 10 mg/ml Lucifer Yellow CH in Ringer's larger than those in earlier stages. These granules have solution was injected into the lumen of adult tubules no also been seen to descend along the lumen of the evidence was found of endocytotic processes occurring proximal part of the AMT. in the S cell type. Lucifer Yellow CH is a highly After about 40 hours post injection, the S cells in the fluorescent naphthalimide dye that normally does not AMT also displayed a rather diffuse staining of the penetrate membranes of living cells (Stewart, 1978) and whole cell instead of the mitochondrial pattern. As in can be employed as a marker for endocytosis (Swanson, the posterior adult tubules, most S cells contained some 1989). After injection of FITC- or TRITC-labeled dextrans with relative molecular masses of 4.4, 10 and very fluorescent vesicles in their cytoplasm (Fig. 3D). 3 All the different processes in intact flies could not be 35X10 (10 mg/ml) or FITC-labeled bovine serum accurately timed, since the injected flies showed great albumin (10 mg/ml) in the lumen of the proximal part of variation in their speed of dye clearance. Some flies a Malpighian tubule no uptake of fluorescence was cleared the dye from all their cells in 40 hours, while in noticed after 60 minutes of incubation. No endocytosis some other flies brightly labeled P cells could still be was detected after incubation in the Ringer's solution or found at this time. All Rh 123-injected flies survived as in the complex Schneider's medium (Serva). well as the controls injected with Ringer's solution. Injection of other fluorochromes indicated that only After injection of Rh 123 into third-instar feeding positively charged molecules were accumulating in the larvae, the S cells of the proximal part of the AMT were proximal S cells. The cationic dyes rhodamine 6G and brightly labeled after about two hours. In contrast to pyronin Y (Fig. 21) were also accumulating in the S cells the adult, neither the P cells nor the S cells in the distal after injection into the tubule lumen (20 /xg/ml) and part of the AMT accumulated any dye: two hours after after injection into adults (1 mg/ml). The acid (anionic) injection both S and P cells appeared unstained. So in dyes eosin Y and B, sodium fluorescein (Fig. 2H), larvae, most P cells had lost the dye after some time sulphorhodamine B and merocyanin 540 (Fig. 2G) were while all S cells (except those in the distal part of the not accumulating in the S cells after injection into the AMT) had accumulated the dye in their mitochondria. tubule lumen (10-20 ^g/ml) or into adults (1 mg/ml). No deposition of dye was found in concretions in the Also, the neutral dye rhodamine B did not selectively lumen of the distal part of larval AMT. The results of accumulate in the S cells after injection into the tubule the intravital staining with Rh 123 are summarised in lumen (10 jig/ml) or into adults (100 /xg/ml; Fig. 2F). Table 2 for adults and in Table 3 for larvae. This neutral and the acid dyes entered the P cells after 356 W. Meulemans and A. De Loof

injection into the lumen, while the S cells were poorly the S cells to a great extent (Fig. 4C). At the low labeled. concentrations (1-10 ng/ml) used on cell cultures, it was No difference in response to the various dyes was ineffective on the Malpighian tubules. noticed between the sexes. Treatment with 10 fJM CCCP for 5 minutes prior to injection of Rh 123 in the lumen inhibited the uptake of Comparison with other Diptera (Brachycera) Rh 123 in the S cells (Fig. 4D). After 15-60 minutes in 1 The Malpighian tubules of adult Drosophila melanogas- /zM CCCP some diffuse labeling was still found in the S ter and adult Calliphora erythrocephala also showed an cells, but no mitochondria accumulated the dye. So accumulation of Rh 123 in their S cells, after in vitro and when CCCP was applied prior to injection of Rh 123 in vivo labeling. Remarkably, the S cells in the distal into the lumen little uptake was seen, but when CCCP part of the AMT also, which are often described as a was applied after staining a whole tubule the S cells different cell type, accumulated Rh 123 from the accumulated much more dye, although not in their Malpighian tubule lumen. mitochondria. In the latter condition most fluorescence leaks from the P cells, so the S cells are surrounded by a In vitro administration of drugs and antibiotics high concentration of Rh 123. 2 In an effort to understand the process of selective After treatment with 1 mM 2,4-DNP or 10~ M NaN3 accumulation of Rh 123 and other cationic dyes by the for 20 minutes before injection of Rh 123 into the tubule secondary cells, some ion-selective ionophores (Press- lumen, no accumulation of Rh 123 was found in the S man, 1976) and drugs that interfere with the metab- cell mitochondria (Fig. 4E), although some diffuse olism were applied to posterior Malpighian tubules intracellular accumulation could often be found. before or after staining with Rh 123. Drug administration after luminal dye-injection Drug administration after in vitro labeling If tubules were incubated for 15 minutes in nigericin (1 When tubules previously stained at their haemolymph ;Ug/ml, Fig. 4F) or monensin (1 jig/ml) after injection of side with Rh 123 in vitro were treated with the Rh 123 into the lumen, the S cell mitochondria were protonophore 2,4-dinitrophenol (2,4-DNP, 1 mM) or very brightly labeled, more then in control tubules with the inhibitor of electron transport sodium azide incubated in Ringer's solution (Fig. 4G). This differ- (10~2 M), for 30 minutes, the S cells also accumulated ence in mitochondrial membrane potential was most the dye after about two hours of incubation in fresh obvious after injection of lower concentrations of Ringer's solution, but less than in the control. The rhodamine 123 (0.5-1 jig/ml) into the tubule lumen. staining was diffuse, no mitochondria were labeled. The This increase in concentration of the dye can be P cell mitochondria lost the dye more rapidly in these ascribed to the expected increase in mitochondrial tubules, while the S cells still accumulated some dye potential, as described for nigericin (Johnson et al., diffusely throughout their cytoplasm. Administration of 1981). the protonophore carbonyl cyanide 3-chlorophenyl Treatment with valinomycin (1 ^g/ml), CCCP (1 hydrazone (CCCP) to tubules previously stained with 3 2 /iM), 2,4-DNP (10~ M) and NaN3 (10~ M) after Rh 123 showed that after 60 minutes in 10 [M CCCP injection of Rh 123 into the lumen caused the dye to much dye was accumulated by the S cells, although no leak from the S cell mitochondria. In the case of CCCP, mitochondria were stained. The fluorescence was much of the fluorescence was retained in large round spread diffusely throughout the cell, and was also vesicles in the S cells. All the latter treatments are concentrated in some round vesicles in the S cell known to abolish the mitochondrial membrane poten- cytoplasm. Although the mitochondrial membrane tial (Johnson et al., 1981; Harold, 1986). Together with potential, and thus after a while also the cell membrane the stimulation of dye uptake by nigericin, it may be potential, is inhibited in such cells, the dye still concluded that the S cell mitochondria were the accumulated in the S cells to a large extent (although organelles accumulating Rh 123. not in the mitochondria). Influence of other parameters Drug administration prior to luminal dye-injection Injection of Rh 123 into tubules bathed for 60 minutes Treatment of tubules with several ionophores prior to in Ringer's solution with NaCl, or KC1 replaced by injection of Rh 123 in the tubule lumen gave interesting equimolar quantities of choline chloride (Serva), had results: little loss of uptake was seen in S cells treated no effect on the accumulation of Rh 123. with monensin (1-5 /zg/ml for upto 120 minutes, Fig. Incubating adult tubules in Sarcophaga Ringer's fluid 4B). Treatment with the ionophore nigericin (1 ^g/ml (Verachtert and De Loof, 1988) for two hours after in for 15-60 minutes, Fig. 4A) greatly reduced the amount vitro labeling or dissection of tubules in this Ringer's of Rh 123 accumulating in the S cells. Although they + + + solution from dye-injected flies did not result in any can both transport Na and K against H , nigericin is different results. So whenever a possible change in essentially a K+/H+ ionophore, while monensin is + + internal pH was caused by the bicarbonate-free essentially a Na /H ionophore (Pressman, 1976). Ringer's that we used (Thomas, 1989), this did not Incubation for 15-60 minutes in 1 /ig/ml of the K - result in an inhibition of dye uptake by the S cells. The S selective ionophore valinomycin prior to luminal dye- cell mitochondria were also brightly labeled in this injection also inhibited the uptake of fluorochrome by Ringer's fluid while the P cells were mostly unlabeled. Dye transport in insect Malpighian tubules 357

Fig. 4. Injection of Rh 123 into the Malpighian tubule lumen after addition of drug or antibiotic: little or no dye is accumulated by the S cells (S) after addition of nigericin (A, 1 /ig/ml for 20 min), valinomycin (C, 1 /ig/ml for 60 min), CCCP (D, 10 fM for 30 min) and 2,4-DNP (E, 1 raM for 60 min), while the S cell mitochondria remain well labeled after 120 min in 1 /ig/ml monensin (B). Incubation in nigericin (1 /ig/ml for 15 min) after injection of Rh 123 (1 /ig/ml) into the tubule lumen shows brightly labeled S cells (F), while another tubule of the same fly, incubated in Ringer's fluid, shows S cells that accumulated much less dye (G). Bars, 10 fan A-E; 20 fan, F and G. 358 W. Meulemans and A. De hoof

Incubating a tubule for two hours in Ringer's solution potential is increased. The very specific (electrogenic) at 4°C prior to injection of cold Rh 123 into the lumen potassium ionophore valinomycin also inhibits the dye did not decrease the accumulation of Rh 123 into the S uptake, but this ionophore also dissipates the electro- cell mitochondria. If a tubule stained in Rh 123 at its chemical gradient across the mitochondrial membranes haemolymph side was left for three hours in Ringer's (Johnson et al., 1981). solution at 4°C, the P cells were still brightly labeled No inhibition of accumulation of Rh 123 by the S cells while the S cells had not accumulated any dye. So the was found with ouabain (10~4 M). Atzbacher et al. low temperature inhibited the release of the dye (1974) described inhibitory effects of ouabain on the molecules from the P cell mitochondria. The S cells can excretion of acidic dyes in Drosophila, and ouabain- still accumulate the dye from the lumen under this sensitive ATPases have been characterized in some condition, but since little dye was leaking from the P insect Malpighian tubules (Bradley, 1985). The uptake cells they appear largely unstained. of Rh 123 by the S cells was also not inhibited by the Injection of a pulse of the non-ionic detergent Triton absence of extracellular Na+ or K+. Bresler et al. (1985, X-100 (0.1% in Ringer's solution) into the tubule lumen 1990) described Na+-dependent fluorescein transport in prior to injection of Rh 123 (10 /ig/ml) decreased the the Malpighian tubules of several insects. Rafaeli- dye accumulation in the S cell mitochondria, but the Bernstein and Mordue (1979) found no inhibitory effect proximal P cells also were diffusely labeled. So of very low sodium concentrations on glucose reabsorp- permeabilisation of the S and P cell apical membranes tion in Locusta Malpighian tubules, although according decreases the mitochondrial staining, but the P cells to these authors this transport should be sodium- become permeable to the dye molecules. dependent. Incubating a tubule for up to two hours in Ringer's The process of accumulation by the S cells is quite solution containing the cardiac glycoside ouabain (10~4 fast. This might be an indication of facilitated diffusion M, Serva) prior to injection of Rh 123 into the lumen of Rh 123 through the apical S cell membrane. Possibly had no detectable effect on the accumulation of the dye the S cells contain carrier proteins in their apical in the S cells. membrane that facilitate passage into the S cells. Rather non-specific carriers have been described for the transport of organic acids and bases in the vertebrate Discussion kidney tubules (Ullrich, 1979) and for organic acids in insect Malpighian tubules (Bresler et al., 1985, 1990). Our experiments show that secondary cells can accumu- This transport is believed to be carrier-mediated, since late some small organic molecules with positive charge it was completely inhibited by competitive inhibitors. (at physiological pH) from the luminal fluid. The Strictly speaking, the transport of organic bases in the primary cell apical membrane in the more proximal mammalian proximal kidney tubules is not active, since parts of the tubules seems to be largely impermeable to it represents facilitated diffusion that is largely depen- these cationic dyes. As seen in the experiments with dent on the cell membrane potential and thus on CCCP, 2,4-DNP and NaN3, the large fluid flow through cellular metabolism (Ullrich, 1979). the P cells towards the lumen is not responsible for the The mechanism for selective accumulation by the S impermeability of the P cell apical membrane. These cells remains unknown, but the impermeability of the drugs completely abolish fluid transport by the Mal- apical P cell membrane might play an important role in pighian tubules (Berridge, 1966). Furthermore, after + this process. If the apical P cell membrane is largely incubation in Ringer's solution without K no uptake of impermeable, then Rh 123 can only diffuse into the S luminal dye by the P cells was noticed. Fluid transport cell. In the distal parts of the posterior tubules, little through the P cells should be strongly inhibited in the + accumulation by the S cells is noted, while the P cells absence of K (Berridge, 1968). The experiments with take up the Rh 123 in these parts. When after some time nigericin and some inhibitors of the mitochondrial the P cells start loosing the dye in this part, the S cells membrane potential indicate that Rh 123 was indeed accumulate the dye from the lumen. The mitochondrial accumulating in the S cell mitochondria. membrane potential seems to be the major driving force Experiments using the K+/H+ ionophore nigericin for this accumulation. Since Rh 123 only labels the S prior to injection of Rh 123 into the lumen showed a cells well when presented at their apical membrane, strong inhibition of dye accumulation by the S cells, some process at this membrane must facilitate the entry although their mitochondrial membrane potential into the cell, or the basal cell membrane must be quite should be increased by this treatment (Johnson et al., impermeable to the dye. The specific inhibition of 1981). In cultured vertebrate cells, no inhibition of dye uptake after incubation in nigericin also indicates some uptake by nigericin was found (Johnson et al., 1981). + + process at the apical membrane that might facilitate Monensin (a Na /H ionophore) on the contrary does entry into the S cell. The lack of information concerning not inhibit the uptake of Rh 123. These two ionophores + + ion transport phenomena in the secondary cell type cause a non-electrogenic dissipation of Na or K makes it difficult to explain the accumulation by the S gradients, with a concomitant increase or decrease in + cells. cellular pH. So altering intracellular K concentration Also of interest is the apparent regionalisation of the and/or pH with nigericin seems to inhibit the uptake of morphologically uniform PMT, at least in relation to rhodamine 123, although the mitochondrial membrane the release and uptake of Rh 123. Maddrell (1978b) also Dye transport in insect Malpighian tubules 359 described physiological differences in the morphologi- No evidence was found for endocytosis of the cally homogeneous lower Malpighian tubule of Rhod- fluorescently labeled dextrans, fluorescently labeled nius prolixus. bovine serum albumin or Lucifer Yellow CH from the Johnson et al. (1981) indicated that cells with Malpighian tubule lumen. This of course does not differing mitochondrial membrane potentials can be exclude any endocytosis of other molecules, but it distinguished by means of the degree of their uptake of strongly suggests that there is no bulk pinocytosis of Rh 123. This probably does not apply to the primary luminal contents. The speed with which Rh 123 cells, since after application of the dye to their basal cell accumulates in the S cells also indicates a non- side the P cells are labeled intensely. We suggest, endocytotic process. Vigorous endocytosis of luminally rather, that there would be a difference in membrane injected dextrans has been described in mammalian permeability for this cationic dye of apical versus basal kidney tubules (Schwartz and Al-Awqati, 1990). P cell membranes, and of proximal versus distal regions Many potassium-transporting insect tissues like the of the tubules. Bernal et al. (1982) described an midgut and the Malpighian tubules contain (apical) accumulation of rhodamine 123 in all the cell types that membrane-bound particles (Gupta and Berridge, 1966; they examined, but the cell types differed in their ability Berridge and Oschman, 1969). These 'portasomes' to retain the dye. Carcinoma cells retained the dye for were first described as apical K+- transporting pumps two to five days, while normal epithelial cells lost the (Harvey et al., 1983). Finally, the potassium transport dye within one to 16 hours. They suggested that this in lepidopteran goblet cells was found to depend upon difference in retention of rhodamine 123 was caused by apical proton ATPases (Wieczorek et al., 1989, 1991). the sensitivity of carcinoma cells to the dye, while non- These vacuolar-type proton ATPases were found to carcinoma cells would possess mechanisms for inacti- provide the energy for potassium transport out of the vating rhodamine 123 or for excluding it from vulner- cell, by means of K+/hH+ antiport. Although in most able cell compartments. Similarly, most P cells seem to animal cells Na+ gradients are used as an energy source possess mechanisms for losing the dye rather quickly, for organic ion transport, several proton-gradient- while the S cells lose the dye very slowly. The S cells based organic ion transport systems have been found in eventually accumulate the dye in small, round vesicles. animal cells. Ganapathy and Leibach (1991) described These vesicles might be of lysosomal origin, and may proton-coupled solute transporter in different ver- destroy the dye molecules or any mitochondria poi- tebrate cell types, among which is an organic cation soned by Rh 123. Lysosomal vesicles containing transporter. This transport system would be driven by a mitochondria have been described (De Duve and proton gradient, using both the chemical and electrical Wattiaux, 1966). Although after a short application this components of this proton gradient as an energy source. dye is non-toxic, prolonged administration of the dye In the brush border of renal proximal tubule cells, has been found to disrupt mitochondrial functions in Na+/H+ exchange would provide the energy for the some vertebrate cultured cells (Bernal et al., 1982). transport of the cation N-methylnicotinamide (NMN) It is also very interesting that Rh 123 is precipitated by creating a H+ gradient (Ross and Holohan, 1983). on the luminal granules in the distal part of the AMT. Vacuolar-type proton ATPases generate the electro- Much confusion still exists in the literature about the chemical gradient that permits the accumulation of origin and function of these extracellular concretions neutral or positively charged biogenic amines into (Sohal et al., 1976; Brown, 1982). After injection of Rh acidic vesicles (Mellman et al., 1986). Possibly the 123 in adults the dye was seen to precipitate on the organic cation Rh 123 could use similar routes for luminal granules, and the granules were seen to grow in entering the S cells, by a possible combination with H+- the tubule lumen by deposition of material upon ATPase. However, it would seem unlikely that the preformed granules. Wigglesworth (1965) had already positively charged (and permeant) dye molecule would indicated that these luminal granules could be formed need such a system to enter the (negatively charged) S by the precipitation of excreted materials within the cell's cytoplasm. More probably, any Rh 123 entering lumen. This precipitation of Rh 123 seems to be limited the P cells could be returned to the lumen by means of to the distal part of the AMT, although similar such a system. This system could eliminate cationic concretions can be found in the distal part of the PMT. molecules via the urine under normal circumstances. Brown (1982) described several types of such metal- However, no evidence of the short-circuiting of such a containing granules in invertebrate tissues. Periodic mechanism by means of proton ionophores was found precipitation seems to be responsible for the concentric in this study. nature of the granules in some invertebrates. Whether Recently, Bertram et al. (1991) described the inhi- the extracellular granules originate from intracellular bition of in vitro fluid secretion in Drosophila Mal- ones or whether they are formed entirely extracellularly pighian tubules by bafilomycin A± and other inhibitors remains unclear in many insects, but they may be of vacuolar-type ATPases. Leyssens et al. (1991) found important in detoxification of metallic (cationic!) ions. evidence for an electrogenic proton ATPase at the In our experiments the exogeneous molecule Rh 123 apical membrane of Formica Malpighian tubules. The was precipitated on granules in a specialised part of the same group described an alkalinisation of intracellular Malpighian tubule lumen and then carried along the pH upon stimulation of KCl and fluid secretion (Zhang tubule lumen towards the gut (where it was probably et al., 1991). So evidence is accumulating to suggest that excreted). the Malpighian tubules of insects secrete primary urine 360 W. Meulemans and A. De Loof by means of proton ATPase-dependent cation (K+) Meigen (Diptera: Calliphoridae). Int. J. Insect Morph. Embryol. transport. Recently, Klein et al. (1991) have even 13, 215-231. Ashburner, M. (1989). Drosophila: a Laboratory Manual. Cold localised the vacuolar proton ATPase in the apical Spring Harbor Laboratory Press, New York. membranes of midgut and Malpighian tubules of Atzbacher, U., Hevert, F., Weber-Von Grothuss, E. and Wessing, A. Manduca sexta moths. According to the authors, these (1974). The influence of ouabain on the elimination of injected and Malpighian tubules only comprise one type of cell. Such orally applied dyes in Drosophila hydei. J. Insect Physiol. 20, 1989- plasma membrane proton ATPases were also detected 1997. + Bernal, S. D., Lampidls, T. J., Summerhayes, I. C. and Chen, L. B. in the K -transporting insect sensilla by means of (1982). Rhodamine-123 selectively reduces clonal growth of monoclonal antibodies (Klein and Zimmermann, carcinoma cells in vitro. Science 218, 1117-1119. 1991). Further studies will have to be focussed on the Berridge, M. J. (1966). Metabolic pathways of isolated Malpighian presence and possible involvement of vacuolar-type tubules of the blowfly functioning in an artificial medium. J. Insect ATPases in the reabsorption and/or exclusion of organic Physiol. 12, 1523-1538. Berridge, M. J. (1968). Urine formation by the Malpighian tubules of cations. Calliphora. I. Cations. J. Exp. Biol. 48, 159-174. We have already indicated the suitability of the Berridge, M. J. and Oschman, J. L. (1969). A structural basis for fluid fluorochrome rhodamine 123 as a selective supravital secretion by Malpighian tubules. Tissue and Cell 1, 247-272. label for the secondary cell type in the Malpighian Bertram, G., SchleithofT, L., Zimmerman, P. and Wessing, A. (1991). Bafilomycin A, is a potent inhibitor of urine formation by tubules of Sarcophaga bullata (Meulemans and De Malpighian tubules of Drosophila hydei: is a vacuolar-type ATPase Loof, 1991). This also seems to hold for the intravital involved in ion and fluid secretion? J. Insect Physiol. 37, 201-209. labeling of the other Diptera (Brachycera) examined. Bradley, T. J. (1985). The excretory system: structure and Such selective vital labeling may facilitate future physiology. In Comprehensive Insect Physiology, Biochemistry and research on these relatively unknown secondary cells. Pharmacology (ed. Kerkut, G.A. and Gilbert, L.I.), vol. 4, pp. 421-465. Pergamon Press, Oxford. This dye also clearly shows the polarity of the different Bresler, V. M., Belayeva, E. A. and Mozhayeva, M. G. (1990). A Malpighian tubule cells. In the P, as well as in the S comparative study on the system of active transport of organic acids cells, differences in intracellular fluorescence intensity in Malpighian tubules of insects. J. Insect Physiol. 36, 259-270. are found after application of the dye to the apical or Bresler, V. M., Mozhayeva, M. G. and Belyaeva, E. A. (1985). A basolateral cell membranes. As well as facilitated comparative study on the system of active transport of organic acids transport processes, the possible influence of extracellu- in Malpighian tubules of the tropical cockroach, Blaberus giganteus. Comp. Biochem. Physiol. 80A, 393-397. lar (charged) molecules of the cell's glycocalyx on the Brown, B. E. (1982). The form and function of metal-containing differential permeability of membranes should not be 'granules' in invertebrate tissues. Biol. Rev. 57, 621-667. overlooked (Gupta, 1989). Callaerts, P., Huybrechts, R. and De Loof, A. (1988). Distribution of a methionine enkephalin-like substance in gonads of Drosophila Berridge and Oschman (1969) suggested that the S melanogaster. Int. J. Invert. Reprod. Dev. 13, 147-156. cells in Calliphora could reabsorb sodium from the Chan, L. and Gehring, W. (1971). Determination of blastoderm cells lumen (hypothetically), while Taylor (1971) described in Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 68, 2217- similar cells in Carausius and other insects as mucocytes 2221. because of their positive reaction for acid mucopolysac- Chen, L. B. (1989). Fluorescent labeling of mitochondria. In Methods in Cell Biology (ed. Y.-L. Wang and D.L. Taylor), vol. 29, charides. In many insect Malpighian tubules cells with Fluorescence Microscopy of Living Cells in Culture, part A, similar morphology to the secondary cells of Diptera Fluorescent Analogs, Labeling Cells and Basic Microscopy, pp.103- are found (Martoja and Ballan-Dufrancais, 1984). 123. Academic Press Inc, New York. Whether they also exhibit similar characteristics re- De Duve, C. and Wattiaux, R. (1966). Functions of lysosomes. Annu. mains to be investigated. Principal and intercalated cell Rev. Physiol. 28, 435^92. Ganapathv, V. and Lelbach, F. H. (1991). Proton-coupled solute types have also been distinguished by means of a transport in the animal cell plasma membrane. Curr. Opin. Cell mitochondria-selective (carbocyanine) dye in the mam- Biol. 3, 695-701. malian kidney tubules (Schwartz and Al-Awqati, 1990). Gupta, B. L. (1989). The relationship of mucoid substances and ion This differential staining seemed to be based upon and water transport, with new data on intestinal goblet cells and a model for gastric secretion. In Mucus and Related Topics, Symp. differences in numbers of mitochondria rather then on Soc. Exp. Biol., vol. 43, pp. 81-110 (ed. Chantler, E. and Ratcliffe, differences in membrane permeability or dye retention. + N.A.), Company of Biologists Limited, Cambridge. Cells with opposite polarities of H secretion have been Gupta, B. L. and Berridge, M. J. (1966). A coat of repeating subunits described in the kidney collecting duct (Schwartz et al., on the cytoplasmic surface of the plasma membrane in the rectal 1985). papillae of the blowfly, Calliphora erythrocephala (Meig.), studied in situ by electron microscopy. J. Cell Biol 29, 376-382. Haaijman, J. J. (1983). Labelling of proteins with fluorescent dyes: The authors thank Prof. M. Quaghebeur for the use of the quantitative aspects of immunofluorescence microscopy. In fluorescence microscope. We also thank P. Callaerts for Immunohistochemistry (ed. Cuello, A. C), pp. 47-85. John Wiley helpful discussions, Prof. H. Wieczorek for providing recent and Sons, New York. information on insect vacuolar-type ATPases and Mrs. J. Harold, F. M. (1986). The Vital Force: a Study of Bioenergetics (ed. Puttemans for drawing. W.M. thanks the I.W.O.N.L. of F.M. Harold), chap. 7: Mitochondria and Oxidative Belgium for financial support. Phosphorylation, pp.197-250. W.H. Freeman and Company, New York. Harvey, W. R., Cioffl, M. and Wolfersberger, M. G. (1983). Chemiosmotic potassium ion pump of insect epithelia. Amer J. References Physiol. 244, R163-R175. Huybrechts, R. and de Loof, A. (1977). Induction of vitellogenin Alkassls, W. and SchoeUer-Raccaud, J. (1984). infrastructure of the synthesis in male Sarcophaga bullata by ecdysterone. J. Insect Malpighian tubules of blow fly larva, Calliphora erythrocephala Physiol. 23, 1359-1362. Dye transport in insect Malpighian tubules 361

Johnson, L. V., Walsh, M. L., Bockus, B. J. and Chen, L. B. (1981). Pressman, B. C. (1976). Biological applications of ionophores. Annu. Monitoring of relative mitochondrial membrane potential in living Rev. Biochem. 45, 500-531. cells by fluorescence microscopy. J. Cell Biol. 88, 526-535. Rafaell-Bernstein, A. and Mordue, W. (1979). The effects of Johnson, L. V., Walsh, M. L. and Chen, L. B. (1980). Localization of phlorizin, phloretin, and ouabain on the reabsorption of glucose by mitochondria in living cells with rhodamine 123. Proc. Nat. Acad. the Malpighian tubules of Locusta migratoria migratoroidcs. J. Sci. USA 77, 990-994. Insect Physiol. 25, 241-247. Klein, U., Loffelmann, and Wieczorek, H. (1991). The midgut as a Ramsay, J. A. (1954). Active transport of water by the Malpighian model system for insect K+-transporting epithelia: tubules of the stick insect, Dixippus morosus (Orthoptera, immunocytochemical localization of a vacuolar-type H+ pump. /. Phasmidae). J. Exp. Biol. 31, 104-113. Exp. Biol. 161, 61-75. Ross, C. R. and Holohan, P. D. (1983). Transport of organic anions Klein, U. and Zimmerman, B. (1991). The vacuolar-type ATPase and cations in isolated renal plasma membranes. Annu. Rev. from insect plasma membrane: immunocytochemical localization in Pharmacol. Toxicol. 23, 65-85. insect sensilla. Cell and Tissue Res. 266, 265-273. Schwartz, G. J. and Al-Awqati, Q. (1990). Identification and study of Knowles, G. (1975). The reduced glucose permeability of the isolated specific cell types in isolated nephron segments using fluorescent Malpighian tubules of the blowfly, Calliphora vomitoria. J. Exp. dyes. Meth. Enzymol. 191, 253-265. Biol. 62, 327-340. Schwartz, G. J., Barasch, J. and Al-Awqati, Q. (1985). Plasticity of Leyssens, A., Zhang, S. L., Weltens, R., Van Kerkhove, E. and Steels, functional epithelial polarity. Nature 318, 168-371. P. (1991). Evidence for the presence of an electrogenic cation pump Sohal, R. S., Peters, P. D. and Hall, T. A. (1976). Fine structure and in the isolated Malpighian tubule cells of Formica. Arch. Int. X-ray microanalysis of mineralized concretions in the Malpighian Physiol. Biochim. Biophys. 99, P10. tubules of the housefly, Musca domestica. Tissue and Cell 3, 447- Llson, L. (1937). Etudes histophysiologiques sur le tube de Malpighi 458. des insectes. I. Elimination des colorants acides par le tube de Stewart, W. W. (1978). Functional connections between cells as Malpighi chez les Orthopteres. Arch. Biol. (Paris) 48, 321-360. revealed by dye-coupling with a highly fluorescent naphtalimide Llson, L. (1938). Etudes histophysiologiques sur le tube de Malpighi tracer. Cell 14, 741-759. des insectes. II. L'elimination des colorants basiques chez les Swanson, J. (1989). Fluorescent labeling of endocytotic Orthopteres. Z. Zellforsch. Mikrosk. Anat. 28, 179-209. compartments. In Methods In Cell Biology, vol.29 (ed. Wang, Y.- Maddrell, S. H. P. (1977). Insect Malpighian tubules. In Transport of L. and Taylor, D. L.): Fluorescence Microscopy of Living Cells in Ions and Water in Animals (ed. Gupta, B.L., Moreton, R.B., Culture, part A: Fluorescent Analogs, Labeling Cells and Basic Oschman, J.L. and Wall, B.J.), pp.541-569, Academic Press, New Microscopy: pp.137-151, Academic Press Inc, New York. York. Taylor, H. H. (1971). The fine structure of the type 2 cells in the Maddrell, S. H. P. (1978a). Transport across insect exdetory Malpighian tubules of the stick insect, Carausius morosus. Z. epithelia. In Transport Across Multi-membrane Systems (ed. Zellforsch. Mikrosk. Anat. 122, 411-424. Giebisch, G.), pp.240-271. Springer Verlag, Heidelberg. Thomas, R. C. (1989). Cell growth factors: Bicarbonate and pH| Maddrell, S. H. P. (1978b). Physiological discontinuity in an response. Nature 337, 601. epithelium with an apparently uniform structure. J. Exp. Biol. 75, Ullrich, K. J. (1979). Renal transport of organic solutes. In Transport 133-145. Organs (ed. Giebisch, G.), pp. 413-448. Springer Verlag, Maddrell, S. H. P. and Gardiner, B. O. C. (1974). The passive Heidelberg. permeability of insect Malpighian tubules to organic solutes. J. Verachtert, B. and De Loof, A. (1988). Experimental reversal of the Exp. Biol. 60, 641-652. electric field around vitellogenic follicles of Sarcophaga bullata. Maddrell, S. H. P., Gardiner, B. O. C, Pilcher, D. E. and Reynolds, Comp. Biochem. Physiol. 90A, 253-256. S. E. (1974). Active transport by insect Malpighian tubules of acidic Wessing, A. and Eichelberg, D. (1969). Elektronenoptische dyes and of acylamides. /. Exp. Biol. 61, 357-377. Untersuchungen an den Nierentubuh (Malpighische gefa&e) von MaddreU, S. H. P. and Overton, J. A. (1990). Methods for the study Drosophila melanogaster. I. Regionale Gliederung der Tubuli. Z. of fluid and solute transport and their control in insect Malpighian Zellforsch. Mikrosk. Anat. 101, 285-322. Wieczorek, H., Putzenlechner, M., Zelske, W. and Klein, U. (1991). tubules. Meth. Enzymol. 192, 617-632. + + Martoja, R. and Ballan-Dufrancals, C. (1984). The ultrastructure of A vacuolar-type proton pump energizes K /H -antiport in an the digestive and excTetory organs. In Insect Ultrastructure (ed. animal plasma membrane. /. Biol. Chem. 266, 15,340-15,347. King, R. C. and Akai, H.), vol. 2, pp. 199-268. Plenum Press, New Wieczorek, H., Weerth, S., Schlndlbeck, M. and Klein, U. (1989). A York. vacuolar-type proton pump in a vesicle fraction enriched with Mellman, I., Fuchs, R. and Helenlus, A. (1986). Acidification of potassium transporting plasma membranes from tobacco endocytic and exocytic pathways. Annu. Rev. Biochem. 55, 663- hornworm midgut. J. Biol. Chem. 264, 11,134-11,148. 700. Wigglesworth, V. B. (1965). Excretion. In The Principles of Insect Meulemans, W. and De Loof, A. (1990a). Cytoskeletal F-actin Physiology, 6th edn (ed. V.B. Wigglesworth), chap. XII, pp. 496- patterns in the Malpighian tubules of insects. Tissue and Cell 22, 529. Methuen and Co, London. 283-290. Zhang, S. L., Van Kerkhove, E. and Steels, P. (1991). The Meulemans, W. and De Loof, A. (1990b). The dynamics of thick actin intracellular pH in Malpighian tubule cells of Formica is alkaline: bundles localised at the basal membrane of whole-mounted insect effect of stimulation of secretion by increasing basolateral excretory epithelia during metamorphosis. Eur. J. Cell Biol. 53, potassium concentration. Arch. Int. Physiol. Biochim. Biophvs. 99, suppl. 31, 23. P17. Meulemans, W. and De Loof, A. (1991). Methods for the selective vital staining of stellate cells in the Malpighian tubules of the fleshfly Sarcophaga bullata. Gen. Comp. Endocrinol. 82(2), 250, (Received 15 July 1991 - Accepted, in revised form, abstract 111. 25 October 1991)