With 8 figures
Printed in Great Britain
THE ACTION OF THE EXCRETORY APPARATUS
OF CALLIPHORA VOMITORIA IN HANDLING
INJECTED SUGAR SOLUTION
BY G. KNOWLES*
Department of Zoology, The University, Newcastle upon Tyne (Received 13 June 1975)
SUMMARY
Recent evidence suggests that the isolated Malpighian tubules of
Calli-phora possess mechanisms which restrict the loss of glucose and trehalose
from the insect. This report establishes that the intact, diuresing fly does not excrete glucose or trehalose when solutions of these sugars are injected. When solutions of non-metabolized sugars such as sorbose and xylose are injected into the fly, these sugars are rapidly excreted. High concentrations of sorbose and xylose are found in the urine, suggesting that rapid reabsorp-tion of fluid occurs in the excretory apparatus even during the diuresis which the injections provoke. However, injected sucrose is apparently not excreted in large amounts and it is possible that the Malpighian tubules when functioning in vivo are impermeable to disaccharides.
INTRODUCTION
The Malpighian tubules of insects were once considered freely and passively permeable to organic solutes, so that such molecules would be passively excreted, providing there was adequate fluid secretion and that they were not reabsorbed (Ramsay, 1958). The free permeability of the Malpighian tubules of some insects to organic solutes has recently been confirmed (Maddrell & Gardiner, 1974). Recovery of glycine which is thought to diffuse into the tubule fluid has been demonstrated in the recta of locust and cockroach (Balshin & Phillips, 1971; Wall & Oschman, 1970). However, evidence is accumulating that the Malpighian tubules of insects can pre-ferentially transport molecules whose presence could either be deleterious, e.g. dyes and acylamides (Maddrell et al. 1974), or beneficial, e.g. sugars (Knowles, 1975).
Recently, it has been suggested that the isolated Malpighian tubules of C. vomitoria reduce the excretion of trehalose and glucose (Knowles, 1975). It was proposed that the tubule hydrolyses trehalose to glucose derivatives and that the excretion of glucose is specifically restricted. This report describes the excretory pattern of a range of sugars after they have been injected into the haemolymph of the intact animal.
MATERIALS AND METHODS
All experiments were performed on male flies between 48 and 60 h after pupal emergence. They were fed on a mixture of sugar and dried milk, and water was given
ad libitum. Larvae were purchased from commercial suppliers.
Each fly, with its ventral side uppermost, was restrained on a slide covered with beeswax. A standard volume of Ringer (4-7 fil) was injected through the mesopleuron. The injection of this volume, about one third of the haemolymph volume, induced a diuresis in the fly. Gentle prodding of the abdomen caused the fly to release urine, which was collected at regular intervals. Haemolymph was taken from the site of injection, but was sampled less frequently than the urine and not taken more than three times from any one fly, thus preventing too great a loss of heamolymph. Sample volumes were calculated from the measured diameter of the droplets when immersed in paraffin oil. Sugars were universally labelled with 14C and purchased from the Radiochemical Centre, Amersham. Each batch of 50 /tCi of MC was dissolved in either 1-5 ml or 075 ml of Ringer. The higher specific activity was preferred for sugars which were considered metabolically active, namely glucose and trehalose. The concen-tration of sugars injected into each fly was 50 mM, this would be reduced by approxi-mately two-thirds by haemolymph volume. In some cases sugars were separated by thin layer chromatography (T.L.C). The procedure has been described elsewhere (Knowles, 1975).
RESULTS
(1) The rate of urine production
The rates of induced urine excretion resulting from fluid injection are shown in Fig. 1. It is apparent that diuresis is stimulated within 5 min. Excretion is fast at first, and then gradually reduced, reaching a low level after about an hour and a half.
(2) The excretion of sorbose and xylose
Excretory apparatus of Calliphora vomitoria
133
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20 60
Time (min)
[image:3.451.54.367.58.298.2]80 100
Fig. 1. The rate of urine issuing from the anus. Values are calculated by measuring the volume of each urine droplet and dividing by the time interval from the taking of the previous urine sample. Two series of experiments were performed, these are represented by different symbols. Error bars on all figures are ± o n e standard error of the mean. Figures adjacent to means give the total number of observations from which that meun was calculated.
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20 40 60
Time (min)
80 100
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3500-
3000-5
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20 40 60
Time (min)
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Fig. 3. The levels of radioactivity (cmp/jUl) in urine ( • — • ) and haemolymph ( O — O ) samples, taken from intact diuresing flies after the injection of ["CJxylose.
water reabsorption by either the ileum or rectum could elevate the final sorbose concentration in the urine. This third interpretation seems the most likely, since if xylose is injected similar results are again obtained (Fig. 3). Thus the non-metabolizable sugars are promptly removed from circulation, if this is coupled to high rates of urine excretion, then the combined effect would be to deplete the haemolymph of these sugars.
(3) The excretion of glucose
Excretory apparatus of Calliphora vomitoria
135
Trehalose Glucose
Solvent front
0-5 1-0 15 20 2-5 30 Distance along T.LC strip (in.)
[image:5.451.81.347.58.468.2]3-5 4-0
Fig. 4. The levels of radioactivity in chromatographed haemolymph samples (cpm//tl of sample spotted) taken 45 min after the injection of [14C]glucose.
(4) The excretion of trehalose and sucrose
60
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£> 5. •5: -S I 50 45 40L0-5 1-0 1-5 20 2-5 30 Distance along T.LG strip (in.)
[image:6.451.66.345.46.229.2]3-5 4-0
Fig. 5. The levels of radioactivity in chromatographed urine samples (cpm/sample spotted) taken 45 min after the injection of P*C]gIucose.
Table 1. The levels of radioactivity appearing in samples of haemolymph and
urine 45 min after the injection of either \}*C]sorbose or \}lC]ghicose
Urine Haemolymph Sorbose Glucose cpm//tl 6710 44° 8.E. 800 5° cpm//tl 1640 780 8.B. I3O 120
Table 2. The levels of radioactivity appearing in samples of haemolymph and
urine 45 mm after the injection of either ^*C\trehalose or ]}*C]sucrose
Trehalose Sucrose
cpm//*l
130
2 1 0
Unne S.B. 15 IS n 13 S f cpmlfA 2400 1300 Haemolymph S.E.
2 1 0
IOO n 9 7 Urine/ Haemolymph 0054 0-16
of the urine sample failed to distinguish any radioactivity peaks (Fig. 7), suggesting that general metabolism may have distributed the injected radioactivity among several species of molecules with a consequent lowering of specific activity for each substance: without preliminary fractionation there would be insufficient separation of several solutes by one dimensional chromatography.
It is noticeable from Table 2 and Fig. 8 that, after sucrose injection, the levels of radioactivity in the urine are well below those found in the haemolymph. Some pre-liminary experiments with inulin have yielded similar results (Knowles, 1974). This is in contrast to the experiments with sorbose and xylose where these solutes were concentrated in the urine. This could mean that the Malpighian tubule walls are much less permeable to disaccharides than monosaccharides.
(5) The appearance of amaranth in the excretory apparatus
Excretory apparatus of Calliphora vomitoria
137
12001-800
400
0-5 10 1-5 2-0 2-5 30 Distance along T.LC strip (in.)
[image:7.451.80.345.57.448.2]3-5 4-0
Fig. 6. The levels of radioactivity in the chromatographed haemolymph samples (cpm//il of sample spotted) taken 45 min after injection of P*C]trehalose.
with the radioactivity of the haemolymph from which that urine was secreted. There-fore, 100 nl of the dye amaranth were injected 5 min after diuresis had been stimulated by the injection of 4 7 /A of Ringer. Coloured urine gradually appeared after 3-4 min, and the colour intensity was fully developed by 5 min. Dissection of the excretory apparatus revealed that dye first appeared around the junction of the Malpighian tubule common collecting ducts with the gut. It is concluded that the lag period between initial secretion of tubule fluid and voidance of urine is around 5 min.
DISCUSSION
60
§ ? 55
50
45
0-5 1-0 1-5 2-0 2-5 30 Distance along T.LC strip (in.)
3-5 40
Fig. 7. The levels of radioactivity in chromatographed urine samples (cpm/sample spotted) taken 45 min after the injection of [14C]trehaloae.
2000 -i
3
I
.§ 1000
-.9 >, •5
20 40 60
Time (min)
100
Fig. 8. The levels of radioactivity (cmp//d) in the urine (•—•) and haemolymph (O—O) samples, taken from diuresing flies after the injection of [1<C]sucrose.
Excretory apparatus of Calliphora vomitoria
139Table 3. The estimated rates of Malpighian tubule fluid production and water
reabsorp-tion. Values calculated from the levels of radioactivity in the urine and haemolymph samples after the injection of [uC]sorbose (Fig. 2) and the diuresis profile of Fig. 1
Time interval
(min) a
0 - 4
5-io II-2O 21-30 31-40 4I-5O SI-60 6l-7O 7I-8O
Radioactivity of body fluids (cpm/nl)
Urine
b
0 - 2
o-8
1-4 i - 8
2 - 1
2 3 2 4 2 3 2 4 Haemolymph c o-8 0-8 0-67 0-65
0 6 5
0-65 0-65 0-65 0-65 Urine/ haemolymph ratio bjc^d
0 2 5 i - o
2 - 1
2-8 3 2 3 5 3 7 3-5 3 7 Volume of urine excreted in time interval (nl) e
1 0 0
2 2 0
4 6 0 5 6 0 4 0 0 3 0 0
2 0 0
190 150 Total 2580 Required volume of total tubule fluid produc-tion (nl) dxe=f —
2 2 0
97O
1600 1280 1005
7 4 0 6 6 0 55O
7025
Rate of reabsorption
(nl/min)
(J-e)ih = g
— —
1 0 2
104 88 7 0 54 57 4 0
The suggested rates of water reabsorption are also higher than the rates previously recorded from in situ, isolated rectal preparations of the blowfly. Phillips (1969) obtained values for rectal water reabsorption of around 20 nl/min/rectum. Perhaps other tissues of Calliphora, such as the lengthy ileum, provide additional water reabsorption. It is possible that the ileum absorbs large amounts of isomotic fluid, which would markedly reduce the tubule fluid volume, and that the rectum specifically regulates the osmotic pressure of the urine in accordance with the needs of the insect. Water reabsorption may appear contradictory to the immediate physiological needs of a diuresing insect in that such reabsorption would tend to extend the period of time before the fly has excreted the desired volume of fluid. However, during this period of prolonged diuresis the fly may circulate a far greater volume of haemolymph through the excretory apparatus than would occur if the tubule fluid was more directly excreted. The data from Table 3 show that the total volume of haemolymph secreted by the Malpighian tubules is probably at least 7-0 /il, well above the 3*0/41 of urine which is actually collected. This continuous recycling of haemolymph may facilitate solute excretion by increasing the volume of haemolymph filtered of unwanted substances.
tubules are functioning m vivo, they are less permeable to disaccharides than are isolated tubules. However, it is possible that the low sucrose clearance by the intact fly represents nothing more than recovery of the constituent monosaccharides of sucrose, following sucrose digestion in the ileum. Further evidence is required to resolve this argument, but if subsequent work shows that the in vivo tubule is signifi-cantly less permeable to molecules larger than monosaccharides, then the secretion-diffusion theory of insect excretion (Ramsay, 1958) will require amendment.
It is my pleasure to thank Professor J. Shaw for his encouragement and super-vision of this work. My thanks are also owing to the Science Research Council for their financial support.
REFERENCES
BALSHIN, M. & PHILLIPS, J. E. (1971). Active absorption of amino acids in the rectum of the desert locust. Nature, Lond., New Biol. 333, 53-5.
FRAENKEL, G. (1940). Utilization and digestion of carbohydrates by the adult blowfly. J. exp. Biol. 17, 18-29.
KNOWLES, G. (1974). The removal of carbohydrates and sulphate by the excretory system of the blow-fly Calliphora vomitoria. Ph.D. Thesis, Newcastle University.
KNOWLES, G. (1975). The reduced glucose permeability of the isolated Malpighian tubules of the blow-fly Calliphora vomitoria. J. exp. Biol. 63, 327-40.
MADDRELL, S. H. P. (1969). Secretion by the Malpighian tubules of Rhodnius. The movements of ions and water. J. exp. Biol. 51, 71-97.
MADDREIX, S. H. P. & GARDINER, B. O. C. (1974). The passive permeability of insect Malpighian tubules to organic solutes. J. exp. Biol. 60, 641-52.
MADDRELL, S. H. P., GARDINER, B. O. C , PILCHER, D. E. M. & REYNOLDS, S. E. (1974). Active
transport by insect Malpighian tubules of acidic dyes and acylamides. J. exp. Biol. 6i, 357-77. NORMANN, T. C. (1975). Neurosecretory cells in insect brain and production of hypoglycaemic hormone.
Nature, Lond. 354, 259-61.
PHILLIPS, J. E. (1969). Osmotic regulation and rectal absorption in Calliphora. Can.J. Zool. 47, 851-63. RAMSAY, J. A. (1958). Excretion by Malpighian tubules of the stick insect Dixipput morosus (Orthoptera,
Phasmidae): amino acids, sugars and urea. J. exp. Biol. as, 081—91.
WALL, B. J. & OSCHMAN, J. L. (1970). Water and solute uptake by the rectal pads of Periplaneta
americana. Am. J. Phytiol. 218, 1208-15.