Ion movements and water transport in the rectum of the locust Schistocerca gregaria

7. Insect Physiol., 1968, Vol. 14, pp. 269 to 275. Pergamon Press. Printed in Great Britain

ION MOVEMENTS AND WATER TRANSPORT IN THE RECTUM OF THE LOCUST SCHISTOCERCA GREGARIA R. H. STOBBART Department of Zoology, University of Newcastle upon Tyne, Newcastle upon Tyne 1 (Received

Abstract-The Schistocerca

3 August

1967)

fluxes of K and Na between the haemolymph and rectal wall of

gregaria have been studied (a) when the rectum is reabsorbing water

and (b) when it is not. Although the fluxes are high (about 0.5 and at least 1 PM/PM rectal ion per hr respectively) they do not differ significantly between the two categories. The results are discussed in relation to some current theories of water transport.

INTRODUCTION

WHILE it has been known for a long time that the recta of many insects can reabsorb water from the faeces back to the haemolymph (WIGGLESWORTH,1932), detailed information about the water and solute reabsorption has only recently become available (PHILLIPS, 1961, 1964a-c). In the locust Schistocerca gregaria Forskil active water transport is apparently performed by the rectal epithelium; water can be moved from the rectal lumen to the haemolymph against an osmotic gradient and independently of both the potential difference across and movement of ions through the rectal wall (PHILLIPS, 1964a). A recent electron microscope investigation of the rectum of the blowfly Calliphora has revealed in the papillae a complex system of intercellular spaces formed from infoldings of the cells’ lateral plasma membranes (BERRIDGE and GUPTA, 1967). As this rectum can also reabsorb water (PHILLIPS, 1961) it has been suggested (BERRIDGEand GUPTA, 1967) that in Calliphora this is achieved by the secretion of ions (probably K+ and Cl-) from the rectal lumen into the intercellular spaces; this secretion establishes an osmotic gradient across the rectal cells causing the diffusion of water into the intercellular spaces. The resultant increase in hydrostatic pressure within the spaces is then supposed to force the water (and ions) towards the haemolymph. The scheme is in fact the ‘double-membrane’ model advanced to explain water movement through other tissues (CURRAN, 1960; DUREJIN,1960). In view of the detailed information available on the rectum of Schbtocercu (PHILLIPS 1961, 1964a-c), examination of some of the implications of the double-membrane model when applied to the apparently unique water transport in this tissue is likely to be of value. 269

270

R. H. MATERIALS

STOBBART AND

METHODS

As in earlier work (PHILLIPS, 1961, 1964a-c) mature male Schistocerca gregaviu were used with the rectum tied off from the intestine. Such ligated recta can withdraw water from salt-free sugar solutions hyperosmotic to the haemolymph (PHILLIPS, 1964a), so that if local osmotic gradients dependent on concentrations of ions in certain regions of the rectal tissue are concerned in the reabsorption, such ions must come in the first instance from the haemolymph. If these concentrations are to be maintained in spite of the movement of water and ions away from the regions of concentration, one would expect appreciable exchanges of ions between rectal tissue and haemolymph to occur, and the rates of exchange to increase in some way with increase in the rate of reabsorption. Accordingly the rates of exchange of Na and K between the haemolymph and the rectal wall have been examined (at 20 to 22°C) in two categories of locust : ‘wet’ ones which were starved before use for 5 days at 20 to 22°C and 75 to 95 per cent r.h. with tap water to drink, and ‘dry’ ones which were treated similarly but at 40 to 50 per cent r.h. and with hyperosmotic saline to drink (PHILLIPS, 1964a). Dry locusts are known to show much the quickest rate of water reabsorption (which may be absent in wet ones), following the introduction of salt-free hyperosmotic sugar solution into the rectum (PHILLIPS, 1964a). The recta were ligated about 10 hr before use with Phillips’s technique. At the start of the experiment the locust was immobilized on a bed of plasticine and the rectum was rinsed with a large volume (about sucrose solution (Analar grade; Na and K contents, 300 ~1) of hyperosmotic* 0@48 and 0.028 mM/M respectively). It was then charged with about 50 ~1 of this solution, and the end of the abdomen sealed with beeswax-resin mixture. Known amounts of 22Na and 42K (as chlorides in 5 ~1 of water) were now injected into the haemolymph through the abdominal wall at the junction between segments 3 and 4, and the wound was sealed. The total amount of K injected caused not more than a 10 per cent increase in haemolymph K, while the amount of Na was negligible. After allowing 5 min to elapse for mixing, a 5 ~1 sample of haemolymph was taken from the neck and after the wound had been sealed was transferred to a planchette. About 30 min later a second was taken similarly, and then a third was taken for measurement of freezing point depression. A large amount of the rectal fluid was now moved to a ‘fluon’ slab by removing the wax seal and squeezing the end of the abdomen gently with forceps. A sample of the rectal fluid was then taken for measurement of freezing-point depression. Now the rectum was removed (the validity of the ligature being checked), freed as far as possible from other tissues, and dried on filter paper, during which step as much as possible of the remaining contents were squeezed out. Then it was rinsed very briefly in three changes of deionized water (total time taken about 2 set), dried again, weighed on a torsion balance, and then macerated onto a planchette. The following items were measured in the planchette samples : (1) radioactivity of 22Na and 42K; (2) radioactivity of 22Na (after decay of 42K) ; (3) N a and K contents (after ashing off the * Chosen

to allow water

reabsorption

by dry recta

only.

IONMOVEMENTS ANDWATER TRANSPORT IN RECTUM OFLOCUST

271

tissues at 450°C for 5 hr). These measurements allow (a) calculation of the specific activities of Na and K in the haemolymph and rectal tissue, and hence their fluxes between these tissues; (b) because of the small drop (only 4 per cent) in the haemolymph Na radioactivity during the experimental period (PHILLIPS, 1964b), calculation of haemolymph volume as given by the Na space; (c) calculation of specific activities of Na and K in the haemolymph using the haemolymph volume, the haemolymph Na and K concentrations, and the known amounts of 22Na and 42K injected into the haemolymph; the value of these indirect estimates will become apparent later. The measurements of radioactivity, corrected in the case of rectal samples for the increase in counting rate caused by the tissue (STOBBART,1967), were made with conventional counting equipment. Na and K were measured with the E. E. L. flame photometer, and the freezing-point depressions with the micro-cryoscopical technique of RAMSAYand BROWN(1955). RESULTS Table 1 shows some of the measurements made on the tissues. The dry treatment clearly reduces the Na space and increases the haemolymph Na and K concentrations and osmotic pressure, and probably increases the ionic concentration of the rectal tissue. The Na flux between the rectal tissue and the haemolymph is shown in Table 2, which indicates a rapid turnover of Na in the rectal tissue. The flux is absolutely about twice as great in dry recta but does not differ significantly between the two categories when expressed relatively as PM/PM rectal Na per hr. However, by the end of the experimental periods (0.61 to 0.73 hr) the specific activity of the Na in the rectal tissue is about 70 per cent of that in the haemolymph, so the exchange is nearing completion. It follows that the calculated fluxes will be only about 50 per cent of the true ones (assuming there to be only a single Na compartment in the rectal tissues). The situation with respect to K is more complex, for the exchange of K between haemolymph and tissues appears to behave as a two-compartment system. Five min after injection of the 42K the specific activity of the haemolymph K has dropped to about 20 per cent of the value expected if the 42K were distributed throughout the Na space. Thereafter the specific activity does not drop appreciably during the experimental periods. The rapid initial drop in specific activity probably represents an exchange with muscle and/or fat body K, but it has not yet been. investigated further. Its reality depends on the accuracy with which the Na space has been estimated. Comparison of the measured specific activity of Na in the haemolymph with that estimated indirectly as described above shows the estimated value to be 109.9 + 4.7 per cent (twenty observations) of the measured one. The Na space estimation is therefore sufficiently accurate to allow detection of the rapid exchange of K. If this rapid exchange is ignored and K fluxes into the rectal tissues are calculated on the basis of the haemolymph specific activity occurring after 5 min, the results shown in Table 3 are obtained. Here there is no significant difference between the two categories with respect to both absolute and relative flux values. The turnover of rectal K, though rapid, is slower than that of Na.

< 0.02

0.967 f 0.032 (5)

10.19 + 0.42 (10) -=I0.001

0.696 + 0.078 (4)

Osmotic pressure haemolymph (A”C)

6.51 + 0.53 (10)

Haemolymph K concentration (mM/l)

0.074

0.199 + 0.027 (5)

0.124 + 0.025 (5)

Na content of recta tMl0 mg rectal tissue

0.086

0,383 + 0.031 (5)

0.283 f 0.041 (5)

K content of recta PMPO mg rectal tissue

0,075

0.582 + 0.057 (5)

0407 f 0.064 (5)

Na+K content of recta tcM/lO mg rectal tissue

Here and elsewhere results are given as mean values k standard deviations, the figures in brackets referring to the numbers of observations.

< 0.01

< 0.001

138.9 f 8.3 (10)

357.0 + 23.5 (5)

(5)

76.5 + 5.0 (10)

(l-d)

493.8 + 25.6

Significance of difference between wet and dry (P)

=Y

Wet

Treatment

Haemolymph vol. as Na space

Haemolymph Na concentration (mM/l)

TABLE 1

ION MOVEMENTS AND WATER TRANSPORT IN RRCTUM OF LOCUST

273

TABLE 2

Treatment Wet Dry Significance of difference between wet and dry (P)

Na flux between rectal wall and haemolymph @M/10 mg rectal tissue per hr)

Na flux as PM/PM rectal Na per hr

0.116 + 0.009 (10) 0,239 + 0.034 (10)

1.01 f 0.070 (10) 1.15 +0*092 (10)

0.002

0.225

The procedure of ignoring the initial rapid exchange of haemolymph K is supported by the fact that at the end of the experimental periods the specific activity of the rectal tissue K is only about 30 per cent of the ‘5 min plus’ specific activity of haemolymph K, and so in this case the calculated flux values will not differ greatly from the true ones. TABLE 3

Treatment

K flux between rectal wall and haemolymph &M/l 0 mg rectal tissue per hr)

K flux as PM/PM rectal K per hr

0.129 * 0.019 (4) 0.184 ). 0.033 (5)

0.461 Z!Z 0.074 (4) 0.483 f. 0.075 (5)

0.19

O-8-0.9

Wet Dry Significance of difference between wet and dry (P)

TABLE 4

Wet

A’C of rectal fluid 1.513 3.683 * 2.188* 1.586 1.668

Change in A.“C of sucrose solution caused by recta

A.“C of rectal fluid

Change in A’C of sucrose solution caused by recta

-0.129 + 2.041 + 0.546 - 0.056 - 0.026

1.958 2.178 1.870 1.683 1.968

-to.316 +0..536 + 0.228 + 0.041 + 0.326

Freezing-point depressions (A’%) after 0*61-0*73 hr of the contents of ligated recta which had been charged with about 50 ~1 of sucrose solution of A.“C! 1.642. * Indicates that the contents were contaminated with faecal matter. 18

R. H. STOBBART

274

The reabsorption of water by the dry, but not the wet, recta is shown in Table 4. As sucrose cannot penetrate the cuticular lining of the rectum (PHILLIPS, 1961), and as under the conditions used ion movements to and from the rectal lumen are negligible (PHILLIPS, 1964a), the changes in osmotic pressure can be used as a rough indication of water movements to and from the lumen during the experimental periods. In the dry locusts water is reabsorbed in every case, the rates of removal being compatible with earlier results (PHILLIPS, 1964a). In three of the wet locusts water has entered the recta, again this is compatible with earlier results (PHILLIPS, 1964a), but in two there has apparently been a marked removal of water. This is certainly an artifact caused by an obvious contamination of the rectal fluid with faecal matter, as the osmotic gradients due to sucrose across these recta were much larger than those which ligated wet recta can develop (PHILLIPS, 1964a). In these two cases the rinsing procedure failed to clean out the recta thoroughly, and faecal matter was removed from them during their preparation for analysis, but the results are interesting in demonstrating the considerable osmotic pressures (essential for water excretion in wet locustsPHILLIPS, 1964c) which are obtainable with faecal matter. DISCUSSION The results reported here do not at first sight support the idea that water reabsorption is caused by a double-membrane mechanism driven by a recycling of ions, for there is no difference in the relative fluxes of Na and K (which could largely be due to exchange diffusion-STOBBART, 1967) between the wet and dry categories, and comparison of the Na and K concentrations in the rectal tissues and haemolymph suggests that the concentrations in the tissues are proportional to those in the haemolymph. To this extent the results support earlier work (PHILLIPS, 1964a) which suggests strongly that water itself is actively transported by the rectum. The fact that the ionic concentration in the rectal tissue water is lower than that in the haemolymph (PHILLIPS, 1964b) need not, however, conflict with a double-membrane mechanism as the regions of ionic concentration in the tissue may be small ; it is also possible that some of the recycled solutes may be organic in nature, for in Tenebrio it appears that organic solutes play some part in water reabsorption by the rectal complex (RAMSAY, 1964). Another possibility concerning the water reabsorption is perhaps worth consideration. In view of the rapid turnover of rectal Na and K, a double-membrane mechanism possibly operates continually in wet locusts, but the resulting water reabsorption is degraded by diffusion back into the rectum through the thin areas of rectal epithelium between the papillae; it may then be supposed that in dry locusts the back diffusion is reduced in some way, possibly through hormone action (VIETINGHOFF, 1966; WALL, 1966), to yield a net reabsorption of water from the rectal lumen. Acknowledgement-I

am grateful to Professor J. SHAWfor critical discussions.

ION MOVEMENTSAND

WATER TRANSPORT INRECTUM OFLOCUST

27.5

REFERENCES BERRIDGE M. J. and GUPTA B. L. (1967) Fine-structural changes in relation to ion and water transport in the rectal papillae of the blowfly, Calliphora. J. Cell Sci. 2, 89-112. CURRAN P. F. (1960) Na, Cl, and water transport by rat ileum in vitro. J. gen. Physiol. 43, 1137-1148. DURBIN R. P. (1960) Osmotic flow of water across permeable cellulose membranes. r. gen. Physiol. 44, 315-326. PHILLIPSJ. E. (1961) Rectal absorption of water and salts in the locust and blowfly. Ph.D. Thesis, University of Cambridge. PHILLIPSJ. E. (1964a) Rectal absorption in the desert locust Schistocercagregaria Forskll-I. Water. J. exp. Biol. 41, 15-38. PHILLIPS J. E. (1964b) Rectal absorption in the desert locust Schistocewa gregaria Forskil -11. Sodium, potassium and chloride. J. exp. Biol. 41, 39-67. PHILLIPS J. E. (1964~) Rectal absorption in the desert locust Schistocerca gregaria Forskil -111. The nature of the excretory process. J. exp. Biol. 41, 69-80. RAMSAY J. A. (1964) The rectal complex of the meal worm Tenebrio molitor L. (Coleoptera, Tenebrionidae). Phil. Trans. R. Sot. (B) 248, 279-314. RAMSAY J. A. and BROWN R. H. J. (1955) Simplified apparatus and procedure for freezingpoint determinations upon small volumes of fluid. J. scient. instrum. 32, 372-375. STOBBART R. H. (1967) The effect of some anions and cations upon the fluxes and net uptake of chloride in the larva of Aedes aegypti (L.), and the nature of the uptake mechanisms for sodium and chloride. J. exp. Biol. 47, 35-57. VIETINGHOFF U. (1966) Einfluss der Neurohormone C, und D, auf die ,4bsorptionleistung der Rectaldriisen der Stabheuschrecke (Carausius morosus Br.) Naturwissenschaften 53, 162-163. WALL B. J. (1966) Evidence for antidiuretic control of rectal water absorption in the cockroach Periplaneta americana L. J. Insect Physiol. 13, 565-578. WIGGLESWORTH V. B. (1932) On the function of the so-called ‘rectal glands’ of insects. Quart.J. micr. sci. 75, 131-150.