The MalpighianC Tubules Of Drosophila Melanogaster: A Novel Phenotype For Studies Of Fluid Secretion And Its Control

To extend studies of ion transport beyond that revealed by the techniques of classical physiology, it is necessary to adopt a molecular genetic approach, in which the relevant genes are identified, characterised and mutated. However, the generalised molecular genetic dissection of the major genes responsible for a stimulus–secretion pathway in any vertebrate epithelium remains a daunting task, as the genetic tools available are relatively unsophisticated. Encouraged by previous physiological work on the larger, but less well genetically mapped, Drosophila hydei (Bertram et al. 1991; Wessing and Eichelberg, 1978; Wessing et al. 1987), we have investigated the feasibility of adapting the established tubule secretion assay to the study of tubules of larval and adult Drosophila melanogaster. The results (i) demonstrate that these Malpighian tubules, to our knowledge the smallest yet studied, are amenable to physiological study; (ii) describe a novel modification of the tubule assay system that allows virtually the entire tubule length to be employed in secretion studies; and (iii) show that the epithelial transport mechanisms identified in D. melanogaster tubules are comparable with those in tubules of other insect species. We propose, therefore, a complete physiological and molecular dissection of this tissue, which may not be possible in any other transporting epithelium.

The Oregon R strain of D. melanogaster were kept on standard fly medium in tubes at 23°C and in ambient humidity (Ashburner, 1989). Mature third-instar larvae were taken from their food: they were not sexed before use. Adults of around 1 week post-emergence were preferred; although no consistent age effects were observed, the tubules of younger adults appeared to be less easily damaged by dissection. Although no obvious sex difference in tubular secretion was observed, only female adults were selected, partly in order to control for undetected differences and partly because their tubules are larger than those from the smaller males.

Larvae and adults were dissected under standard D. melanogaster saline (see below) by gripping neighbouring abdominal tergites laterally with two pairs of fine watchmakers’ forceps and tearing the body wall open (Fig. 1). The two halves of the body were then drawn apart, uncoiling the alimentary canal. The anterior tubules would then unravel and part from their anterior attachments (Fig. 1). Further dissection was required to free the posterior pair of tubules. Each pair of tubules was then cut free at the junction with the common ureter that connects them to the alimentary canal (Fig. 1).

Standard Drosophila saline (Ashburner, 1989) was used for dissection. For the secretion assays, a range of experimental salines was tested, based on mixtures of Grace’s or Schneider’s insect culture media (Gibco BRL) and D. melanogaster saline. Tubule function appeared to be relatively insensitive to the choice of saline, but secretion was faster and maintained for longer in a 1:1 mixture of standard saline with Schneider’s medium. The concentrations of major ion (mmol l⁻¹) in the standard saline were: Na⁺, 132; K⁺, 20; Cl⁻, 158; Mg²⁺, 8.5; Ca²⁺, 2; HCO₃⁻, 10.2.; H₂PO₄⁻, 4.3. The mixture combined the advantages of a saline containing bicarbonate (Thomas, 1989) with those of a tissue-culture medium. Pharmacological reagents were obtained from Sigma (Poole, Dorset, UK), Research Biochemicals Incorporated (Natick, MA, USA) or Calbiochem-Novabiochem (Nottingham, UK).

The assay system was adapted from that classically employed for tubules (Maddrell, 1991). Drops of bathing medium, each of 6 μl, were placed in depressions in a base of paraffin wax under mineral oil (to prevent evaporation) in a Petri dish, and a pair of tubules – still linked by their attachment to the remaining part of the common ureter – was placed in each drop. One of the pair of tubules was pulled out of the drop and wrapped around a thin steel pin, to which it adhered by surface tension (Fig. 1). The use of one tubule of each pair as an anchor for the other allowed virtually the entire length of a tubule to be bathed in the saline drop. Additionally, it allowed the application of classical techniques even to these tubules, the smallest yet studied. The secreted fluid emerged from the aperture at the cut end of the common ureter. Droplets of secreted fluid were removed at intervals with a fine glass rod. Measurement of the diameter of the spherical droplet with an ocular micrometer allowed calculation of the volume of fluid secreted, and measurement of the time interval in which the drop was secreted allowed the secretion rate to be calculated.

Data were analysed using Excel 4.0 (Microsoft) on an Apple Macintosh computer. All data are reported as mean ± S.E.M., with the number of tubules shown in the figure legends. Where error bars are not visible, they are smaller than the symbol used. Wherever possible, the same ordinate scaling has been used throughout, to allow direct comparisons. Where appropriate, the significance of differences between treatments was assessed using Student’s t-test (two tailed), taking P=0.05 as the critical level.

Both larval and adult tubules can be used for secretion assays; however, adult tubules proved more robust and so were used for the definitive studies. The results of a typical experiment with adult tubules are shown in Fig. 2. Unstimulated tubules secreted fluid steadily for over 5 h. The average rate of fluid secretion during the first hour after isolation was 0.74±0.03 nl min⁻¹ (N=217). In other experiments, tubules have continued to secrete for up to 15 h, unusual even in insect transporting systems. Additionally, the small size of tubules allows up to 20 tubules to be assayed simultaneously in a single dish, permitting good internal controls for extraneous variables.

No difference in secretion rate was observed between anterior or posterior tubules in either larvae or adults and, accordingly, both were used in subsequent experiments. Three morphologically distinct regions are distinguishable (at both light and electron microscopic levels) in the anterior tubules and two in the posterior tubules of Drosophila (Wessing and Eichelberg, 1978). The possibility that fluid secretion might be restricted to tubule subregions was studied in a series of experiments, in which varying lengths of tubule were allowed to remain in the drop. The most distal (white) region of the anterior tubules failed to secrete measurable volumes of fluid over several hours; the main central segment of both anterior and posterior tubules secreted fluid; in both, the lower tubule and collecting duct showed weak reabsorptive activity. Provided that the entire main segment was included in the bathing drop, variation in the precise overall length of tubule included did not therefore introduce serious variability in the measured readings.

It is generally accepted that acceleration of fluid secretion in tubules is accomplished – at least in part – through cyclic AMP (Coast et al. 1991; Maddrell, 1971). Accordingly, it is not surprising that cyclic AMP and forskolin (an activator of adenylate cyclase) are potent agonists for secretion in D. melanogaster (Fig. 3A,B). However, cyclic-AMP-stimulated and forskolin-stimulated rates fall well short of those observed in some other studies, suggesting that cyclic AMP is only one of the stimulatory second messengers acting in this tissue. This was confirmed by the observation that a much more potent stimulation of fluid secretion could be elicited by extracts of the fused thoracic ganglia (Fig. 3C). Typically, such extracts could elicit secretion rates of 3–4 nl min⁻¹; the peak rate measured in one tubule exceeded 6 nl min⁻¹. Using a micrometer graticule, we determined the outside diameter of pumping tubules to be 35 μm and the luminal diameter to be 17 μm. Given that the active length of each tubule was 2 mm, it is clear that each tubule cell in the main segment must pump its own volume of fluid in less than 15 s. We are not aware of other epithelia, even the tubules of Rhodnius prolixus (Maddrell, 1991), which can match this rate.

Bafilomycin is a potent and selective inhibitor of V-ATPases (Bowman et al. 1988). Fluid secretion, in both stimulated and unstimulated tubules, was rapidly abolished by bafilomycin at 5×10⁻⁵mol l⁻¹ (Fig. 4A).

Fluid secretion was reduced by 20 μmol l⁻¹ amiloride to values below basal, although even at 10⁻⁴ mol l⁻¹ secretion was not completely abolished (Fig. 4B). This implies that amiloride-sensitive exchanger(s) play a major – though not obligatory – part in fluid secretion. Amiloride is a relatively non-specific inhibitor of Na⁺-selective transport processes, and the possibility that amiloride is acting on a basolateral Na⁺ channel (for example) cannot be excluded. However, ion-selective electrode data (M. J. O’Donnell, personal communication) suggest that the fluid secreted by D. melanogaster tubules, like that secreted by the larger D. hydei (Bertram et al. 1991), is K⁺-rich, with minimal Na⁺ content.

Ouabain, an inhibitor of Na⁺/K⁺-ATPase, had no effect even at 10⁻³ mol l⁻¹ either on basal (Fig. 4C) or stimulated (not shown) secretion rates. Relative insensitivity to ouabain is a characteristic of many insect tubules: in fact, fluid secretion by resting Rhodnius prolixus tubules is stimulated by ouabain (Maddrell and Overton, 1988). This is interesting, because the D. melanogaster tubule basolateral membrane has been shown to contain particularly high levels of Na⁺/K⁺-ATPase a.-subunit by antibody staining (Lebovitz et al. 1989). This implies either that (i) while a Na⁺/K⁺-ATPase is present, it does not contribute significantly to the transport process overall, presumably because of the dominant role played by the apical V-ATPase, as in Rhodnius prolixus (Maddrell and Overton, 1988) or that (ii) as in some other species of insect (Holzinger et al. 1992), the Na⁺/K⁺-ATPase is highly insensitive to ouabain.

Tubules were insensitive to bumetanide, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransport, at 4×10⁻⁴ mol l⁻¹, a concentration 40 times higher than that required to inhibit fluid secretion by 80% in Rhodnius prolixus tubules (O’Donnell and Maddrell, 1984). In most insects, basal ion entry is thought to be via separate channels for Na⁺, K⁺ and Cl⁻, whereas in Rhodnius prolixus (a bloodsucker), it is via a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransport. The lack of bumetanide sensitivity suggests that D. melanogaster tubules, like those of most insects, fall into the former group.

This pharmacological sensitivity of fluid secretion allows us to confirm the general model developed for other insect epithelia. Ion translocation is energised by an apical, bafilomycin-sensitive V-ATPase, which drives net K⁺ secretion via an alkali metal cation/proton exchanger (Wieczorek et al. 1991).

Tubules excrete toxins and metabolites, particularly (in some species) certain plant alkaloids or small organic acid metabolites, by a combination of filtration and selective active transport. The latter process will also transport certain organic dyes (Maddrell et al. 1974). We have confirmed that such a transport occurs in D. melanogaster; both Phenol Red and Amaranth were concentrated by the tubules in the secreted fluid (not shown).

In summary, it seems clear that the tubules of D. melanogaster possess all the richness of secretory machinery found in the epithelia of other insects, and that these processes are similar to those observed in the epithelia of other animals. Our impression is that the Malpighian tubule is by far the most robust and accessible epithelium for experimental study in this species. We have now undertaken a complete physiological and molecular genetic characterisation of the stimulus/secretion coupling pathway of D. melanogaster tubules.

The range of genetic manipulations available to aid a parallel molecular dissection of ion transport in this tissue is impressive. The D. melanogaster genome is already mapped to very high resolution, and many thousands of genes have been identified by classical or reverse genetic techniques (Lindsley and Zimm, 1992). Accordingly, sequence information or mutants may already be available for many elements of the tubule stimulus/secretion pathway. Intracellular signalling pathways have also been intensively studied from a neurobiological perspective, and several relevant genetic loci have been cloned or mutagenised, often in the context of studies on learning and memory (Tully, 1991). Genes can be selectively targetted for inactivation by transposon-mediated ‘site-selected’ mutagenesis (Kaiser and Goodwin, 1990). Ectopic and conditional expression of constructs encoding mutant genes, peptide inhibitors or antisense RNA can be mediated by a range of tissue-specific or heat-shock promoters (Rubin, 1988).

In principle, a similar level of physiological knowledge is obtainable for a wide range of animal epithelia. However, the scope for further elucidation of function by genetic intervention is unique to Drosophila. In this context, the results presented here, which establish that there is an epithelium in this classical genetic model which is amenable to physiological study, take on a particular significance. Accordingly, we think that it will become an important model system in the future.

This work was supported by MRC grant G9120579CB, by a Nuffield Foundation Research Fellowship to J.A.T.D. and by a grant from Gonville and Caius College, Cambridge, to S.H.P.M.. We are grateful to Dr S.-A. Davies for helpful discussion.