STUDIES ON SALT AND WATER BALANCE IN CADDIS LARVAE (TRICHOPTERA)

Transcription

1 KJ. Exp. BioL (196a), 39, ith 7 text-figures nttd in Great Britain STUDIES ON SALT AND WATER BALANCE IN CADDIS LARVAE (TRICHOPTERA) III. DRINKING AND EXCRETION BY D. W. SUTCLIFFE Department of Zoology, University of Durham, King's College, Newcastle upon Tyne {Received 16 November 1961) I. INTRODUCTION In marine teleosts and some Crustacea the blood is maintained considerably hypoosmotic to the external medium, although at least some part of the external body surface is permeable to water. These animals are therefore subject to a continuous osmotic loss of water. In addition, water is lost during the production of urine isoosmotic or even hypo-osmotic to the blood. To compensate this steady loss of water, salt-free water must be regained from the external medium. For teleost fishes this osmotic problem was clearly formulated and solved in the classical investigation by Homer Smith (1930). In fresh water teleosts rarely drink, but in sea-water drinking is continuous and large quantities are imbibed. Water and monovalent ions are absorbed across the gut wall, and the excess salts are then excreted through the gills; a small quantity of divalent ions is excreted in the urine (Smith, 1930, 1932; Keys, 1931). Hence the kidneys have a minor role in maintaining salt balance, and contribute a deficit to water balance. This largely extrarenal control of salt and water balance appears to be common to a number of other salt-water animals, including Artemia satina which also regularly imbibes salt water (Croghan, 1958a, b, c), marine reptiles (Schmidt-Nielsen & Fange, 1958), some marine birds (Schmidt-Nielsen & Sladen, 1958) and probably at least some of the marine Crustacea which regulate hypoosmotically (Parry, i960; Robertson, i960; Shaw, i960). It appears, then, that in marine teleosts, and in Artemia, drinking is an essential part of the mechanism maintaining water balance. Drinking may be a direct response to water loss. This is suggested by the observation that marine elasmobranchs seldom drink (Smith, 1931). Here there is no water loss since the blood is slightly hyperosmotic to the external medium, and osmotic uptake is presumed to be sufficient to maintain a slow flow of hyper-osmotic urine (Smith, 1936). Keys (1933) has demonstrated the rapidly fatal effects of preventing the euryhaline teleost Anguilla from drinking sea water. Turning now to the salt-water insects, several are known to be strongly hypoosmotic to the external medium and the body wall is also slightly permeable to water and salts (Nemenz, 1960a, b; Sutcliffe, 1960a). Even in larvae of Aides detritus (Beadle, 1939) the body wall may not be completely impermeable to water. Again, as in the marine animals considered above, any osmotic loss must be replaced by

2 142 D. W. SUTCLIFFE osmotically free water gained from the medium. Relatively little is known about way in which salt-water insects have solved this problem. A. detritus larvae swallow salt water (Beadle, 1939) and, from measurements of gut-fluid osmotic pressure, it is clear that most of the salts are absorbed in the midgut (Ramsay, 1950). At least some of these salts are presumably excreted in the rectal fluid, as this is highly concentrated to become slightly hyper-osmotic to the external medium. This process is tentatively associated with a distinct region in the anterior part of the rectum (Ramsay, 1950). Nothing is known about the total water and salt fluxes in A. detritus larvae, but it is generally believed that both salt and water balance are maintained by elaboration of the concentrated rectal fluid, i.e. neither an active uptake of water through some special part of the external body surface nor an extrarenal excretion of salts is involved.* From the above considerations it appears that drinking may be an important part of the osmoregulatory mechanism of insects in salt water. Larvae of the freshwater insect Sialis lutaria drink when placed in salt water. Water and salts are absorbed across the gut wall and salts are excreted in the rectal fluid, although this does not become hypertonic to the blood (Shaw, 1955). Shaw deduced that drinking is controlled, so that the rate of salt intake remains at a fairly constant level. This behaviour contributes to the successful survival of Sialis larvae at quite high external salt concentrations. A caddis larva, Limnephilus affims, also survives at high external salt concentrations, up to at least 75 % sea water (Sutcliffe, 19606, 1961 a) whereas two freshwater species, L. stigma and AnaboHa nervosa, die rapidly in % sea water (Sutcliffe, 1961 b). Regulation of the concentration of body fluids in these caddis larvae has been reported (Sutcliffe, 1961 a, b). This paper investigates drinking behaviour and excretion in the same three species of caddis larvae, together with quantitative estimates of salt water intake by drinking and output in the rectum in Limnephilus affims larvae. II. METHODS The caddis larvae were kept individually in diluted sea water or Newcastle tap water as described previously (Sutcliffe, 1961 a, b). As before, the salt concentrations of seawater media are expressed in terms of equivalent concentrations of sodium chloride and, for convenience, sea-water media are referred to simply as solutions of NaCl (mm./l.). Larvae were acclimatized to experimental media for at least 5 days before the start of an experiment. In some experiments larvae were prevented from swallowing the external medium by fusing a small blob of 'Sira* wax over the mouthparts. Larvae not treated in this manner are referred to as normal larvae. Labelled sodium ( M Na) was employed in qualitative and quantitative investigations of drinking in L. affinis larvae (see IV6, c). Larvae previously acclimatized to the required external salt concentration were transferred singly into 10 ml. filtered seawater medium containing a very small quantity of M Na. During the next 80 hr. each larva was removed several times for radioactive assay of the whole larva. The procedure for each larva was as follows: (1) removed from experimental medium, washed in a jet of distilled water (to remove M Na from the body surface), dried with filter paper; (2) narcotized with CO 2 bubbled through a small quantity of non-radioactive experi- Croghan & Lockwood (1960) suggest that an extrarenal excretion of salts may occur in DrosophUa melanogaster larvbe reared in hypertonic media.

3 Salt and water balance in caddis larvae (Trichoptera). Ill 143 hnental medium; (3) placed in counting chamber (see below) filled with CO 2 -saturated medium used in (2); (4) radioactivity assayed; (5) replaced in radioactive experimental medium. Larvae recovered to normal activity within a few minutes after removal from the CO 2 -saturated medium. The counting chamber (Fig. 1) was constructed of celluloid, thickness 0-02 in. A rectangular piece of celluloid, a, 5-5 x 8-5 cm. was glued to the underside of a supporting piece of 0-25 in. thick Perspex, b. The Perspex had a central hole, leaving a celluloid 'window', W with a diameter equal to that of the end-window of a G.E.C. type GM^ Geiger counter. The chamber, 2-5 x 1-5 x 1-5 cm., was built onto the centre Lead / / brick Counting chamber Handle Fig. 1. A, The apparatus for measuring the radioactivity of L. affimt larvae. B, Plan view of the counting chamber, showing the position of a larva. C, Lid of counting chamber with hanging bars of celluloid to hold the larva firmly in a central position. of the 'window'. Two pairs of small struts, c, glued to the base of the chamber served to hold the caddis larva in a central position (Fig. 1B). A Perspex lid, d, had three hanging bars of celluloid each reaching to the base of the chamber and each with a central notch just large enough tofitover the body of a L. affinis larva (Fig. 1C). This arrangement held the ventral surface of the larva pressed gently but firmly against the celluloid 'window'. The chamber was filled with CO 2 -saturated non-radioactive medium. With the larva held in position the chamber was lowered onto the centre of the end-window of a counter mounted in a Perspex box (Fig. 1 A). Two pins, e, fixed in the lid of this box ensured that the chamber was always placed in exactly the same position over the centre of the counter. Counting time for each larva was usually 5 min., extended up to 20 min. for mouth-sealed larvae and near the end of an experiment, when the count rate fell off due to the rapid decay rate of M Na. Counting errors on individual larvae did not normally exceed ± 5 %. Appropriate corrections were made for background, decay rate (15-1 hr.) and dead-time periods when larvae were

4 144 D. W. SUTCLIFFE removed from radioactive media for counting. All counts are expressed as counts larva/5 mul - At the end of some experiments haemolymph samples were removed quantitatively, dried on planchets, and assayed for radioactivity in the usual way. By estimating the total volume of haemolymph in the larva the total radioactivity of the haemolymph was calculated, and this was compared with the radioactivity of the live larva previously obtained with the counting chamber. Counts on live larvae were roughly % of the calculated total haemolymph radioactivity. Although the counting 'efficiency' is low, the technique is considered to be satisfactory for demonstrating differences in a qualitative manner. The rate of rectal fluid production was estimated by collecting fluid in a fine glass pipette at intervals of about 3 hr. over a period of up to 12 hr. The technique was described previously (Sutcliffe, 1961a). Collections from a larva were continued during several consecutive days. The collected fluid was ejected from the pipette onto the arm of a 5 mg. torsion balance. The recorded weight is expressed in volumes by the approximation 1 mg. equivalent to 1 ftl. Attempts were made to collect rectal fluid by the method of Bone* & Koch (1942). The posterior abdominal segments of a larva were sealed into a celluloid cup containing liquid paraffin. The cup was floated on the experimental medium, with the larva suspended head-down in the medium. Bond & Koch collected fluid discharged from the rectum, which remained as discrete droplets in the paraffin. The method proved very unreliable with the caddis larvae used in the present investigation. Only a few specimens produced rectal fluid and this was nearly always contaminated with midgut fluid. Several modifications of the method were devised but these were also unreliable, and the method was rejected as unsuitable for quantitative estimates of rectal fluid production. Attempts to collect fluid by tying a fine glass cannula into the rectum were also unsuccessful. It appears that techniques involving a continued stress or pressure on the abdomen of caddis larvae are unsuitable. In some cases the result is a considerable discharge of fluid usually contaminated with midgut fluid. In others, discharge of rectal fluid may be completely stopped for several days. The technique finally employed in this investigation is also open to criticism as it involves frequent handling of the larvae, but at least it may be expected to provide comparative information concerning the gross features of rectal fluid production in larvae kept in a wide range of external salt concentrations. III. DRINKING IN THE FRESHWATER CADDIS LARVAE (a) Limnepkilus stigma Considerable fluctuations in body weight occurred when L. stigma larvae were transferred from tap water into 120 mm./l. NaCl. In most cases the body weight increased rapidly during the first 24 hr., gaining up to 50 % of the original weight, and this was followed by a loss which became particularly marked just prior to death. Some typical results are shown in Table 1. The rapid increase in weight of normal larvae must have been due to swallowing salt water, since larvae prevented from drinking showed no increase in weight (e.g. serial 89, Table 1). This was confirmed by adding small quantities of either Trypan blue or Amaranth (Azo-Rubin S) to the

5 Salt and water balance in caddis larvae {Trichopterd). Ill 145 Hiedium. After 24 hr. larvae were removed from the medium, killed rapidly in chloroform vapour, and dissected on a wax block. In most cases both foregut and midgut of normal larvae were greatly distended with fluid. In all cases the gut contained dye, often along its entire length, including the rectum. On the other hand no dye was found in the gut of larvae prevented from drinking. In these larvae the entire gut was usually flaccid and contained very little fluid. Table 1. Fluctuations in body weight of Limnephilus stigma larvae transferred from tap water into 120 mm./l. NaCl Serial 89* Initial body wt. (nig) I2-O Percentage change in body wt. at intervals of time (days) i i * Mouth sealed with wax to prevent drinking Dead Dead Dead Table 2. Fluctuations in body weight of Limnephilus stigma larvae transferred from tap water into 170 mm./l. NaCl Initial Percentage change in body wt. at intervals of time (days) body wt.,» s Serial (mg.) i i 2 2± 3 3! Dead 99 2i-o Dead Observation showed that the fluctuations in weight of larvae put into 120 mm./l. NaCl resulted from a rapid intake of considerable quantities of salt water followed by the regurgitation of this water through the mouth. Fortunately, the fluid in both foregut and midgut normally contains a dark brown pigment, and this was easily seen when discharged into the external medium. In almost all cases the medium was stained brown within 48 hr. after transferring larvae from tap water into 120 mm./l. NaCl, and the medium was often stained during the first 24 hr. This behaviour was even more marked when larvae were transferred from tap water into 170 mm./l. NcCl. Here, nearly all of the larvae regurgitated fluid from the gut during the first 24 hr., and they also rapidly lost weight (Table 2). From Table 2 it appears that the larvae did not initially drink the salt water but, in fact, drinking usually occurred within about 8 hr. after transferring larvae from tap water into 170 mm./l. NaCl. This was again demonstrated by adding dyes to the medium. Thus measurements of fluctuations in body weight, combined with observations on the regurgitation of gut fluid, indicate that at high external salt concentrations L. stigma larvae are very rapidly stimulated to drink large quantities of salt water, which is then regurgitated within a short time. In some cases the whole process must have occurred within a few hours, and it is possible that drinking and regurgitation may occur as an alternating sequence of events until the larvae die. 10 Exp. Biol. 39, 1

6 146 D. W. SUTCLIFFE (b) Anabotia nervosa Following direct transference from tap water into 120 mm./l. NaCl, fluctuations in body weight of A. nervosa larvae (Table 3) were not so marked as in L. stigma larvae. Moreover, although larvae were drinking salt water, only a few individuals regurgitated gut fluid during the first 2 days. It was also noticed that, in general, A. nervosa larvae survived for a slightly longer period than L. stigma larvae at this concentration. Nevertheless, regurgitation usually occurred within 4 days after transference into 120 mm./l. NaCl. Table 3. Fluctuations in body weight of Anabolia nervosa larvae transferred from tap water into 120 mm./l. NaCl trial 8 4» 85 # Initial body wt. (mg.) 810 6i-o Percentage change in body wt. at intervals of time (day i 1,J 2 2i 3i IO " " no 128 Mouth sealed with wax to prevent drinking. Table 4. Fluctuations in body weight of Anabolia nervosa larvae transferred from tap water into 220 mm./l. NaCl Serial Initial body wt. (nig.) o Percentage change in body wt. at intervals of time (days) \ no Dead Dead Dead Dead Dead At an external concentration of 220 mm./l. NaCl alternate drinking and regurgitation began immediately and continued to occur at frequent intervals until the larvae died, usually within about 3 days (Table 4). Judging by the rapid frequency of regurgitation in some larvae, the reaction to high concentrations of salt water was particularly violent. In many cases pieces of peritrophic membrane were found in the medium after 24 hr. in 220 mm./l. NaCl. The above observations will be dealt with further in the discussion. We are concerned here only with the fact that, to both L. stigma and A. nervosa larvae, external salt concentrations greater than about 60 mm./l. NaCl have a decided effect which becomes more pronounced as the external concentration is increased. This effect results in alternate drinking and regurgitation of large quantities of salt water, continuing until the death of the larvae.

7 Salt and water balance in caddis larvae (Trichoptera). Ill i<\rj IV. DRINKING IN THE EURYHALINE CADDIS LARVA, LIMNEPHILUS AFFINIS (a) Fluctuations in body weight Fluctuations in weight of a large number of individuals were followed during experiments in which larvae were kept in a wide range of external salt concentrations. The results indicated that, in nearly every case, only small fluctuations in body weight occurred, and that these fluctuations did not markedly increase at high external salt concentrations. A few examples are given below to illustrate these points. In one experiment three larvae were first acclimatized to tap water (group A). They were then transferred directly into 220 mm./l. NaCl and fluctuations in body weight were compared with those of six larvae previously acclimatized to an external concentration of 220 mm./l. NaCl (group B). The mean percentage change in body weight of these larvae over a period of 10 days is shown in Table 5. It is clear that the behaviour of larvae transferred directly from tap water did not differ from the behaviour of larvae already acclimatized to 220 mm./l. NaCl, although both group3 were drinking the medium. Drinking was demonstrated in other larvae kept in 220 mm./l. NaCl by the addition of either Trypan blue or Amaranth to the medium. Two days later these dyes were present in the foregut and midgut of dissected larvae. The results given in Table 5 suggest, therefore, that the amount of water swallowed was controlled, since the body weights remained fairly constant over a long period of time. Table 5. Fluctuations in body weight of Limnephilus affinis larvae in 220 mm./l. NaCl after previous acclimatization to tap water [group A) and to 220 mm./l. NaCl [group B) Serial Initial Percentage change in body wt. at intervals of time (days) or bodywt., *, group (mg.) O-o Dead ico 55 Dead A B This behaviour is strikingly different from that of L. stigma and A. nervosa larvae at similar external salt concentrations. Other differences were also marked. Thus, with very few exceptions, regurgitation of midgut fluid did not occur, at least in sufficient quantities to be visible in the medium. Altogether, several hundred larvae were kept singly in tubes containing media ranging in concentration from tap water to 410 mm./l. NaCl, and regurgitation was observed in less than 5 % of the larvae. In fact, regurgitation did not at first occur even when larvae were transferred directly from tap water into full-strength sea water, although the body weight of these larvae decreased by 30 % in 24 hr., presumably due to osmotic removal of water. Later, however, regurgitation did occur in these larvae, and the medium was strongly stained with the brown midgut fluid. In this case, and in others where regurgitation was observed, it was generally noted that the larvae died shortly afterwards. This is illustrated by serials 172 and 173 (Table 5). Serial 173 appeared to be quite normal on the fifth day of the experiment, but during the next 24 hr. there was a sudden loss in weight and the medium was strongly stained with brown pigment. Regurgitation was apparently accompanied by an osmotic loss of water, as the larva was very shrunken in

8 148 D. W. SUTCLIFFE appearance. The larva did not regain weight and was dead within 48 hr. Serial 17S also regurgitated gut fluid on the sixth day of the experiment and its subsequent behaviour was very similar to that of L. stigma and A. nervosa larvae at high external salt concentrations. In several instances it was also noted that pieces of peritrophic membrane were discharged into the medium. Thus the behaviour of L. affinis larvae in which regurgitation occurred was precisely the same as that of the freshwater species L. stigma and A. nervosa when in salt water. Clearly there is a very distinct difference in the normal behaviour of L. affinis larvae compared with larvae of the freshwater species. Salt water is swallowed, but is not regurgitated. Moreover, the body weight is maintained at a fairly constant level, and fluctuations did not exceed ±10% of the initial body weight even when larvae were placed into 410 mm./l. NaCl. This strongly suggests that the amount of water swallowed is actively controlled. Further evidence of controlled drinking was provided by the following experiment. Nine larvae acclimatized to tap water were treated as indicated in Table 6 and immediately put into 120 mm./l. NaCl. Fluctuations in body weight were followed over 7 days. The body weight of normal larvae (group 1) remained constant. In group 2 there was a small, gradual increase in weight due to osmotic uptake of water. In group 3 there was, presumably, a similar uptake of osmotic water which could not be excreted, since the rectum was ligatured. These larvae could, however, swallow the medium, but it is evident that they did not do so. This suggests that the normal behaviour of drinking in 120 mm./l. NaCl was held in abeyance, i.e. that drinking can be actively controlled by the larva. Table 6. Fluctuations in body weight of Limnephilus affinis larvae acclimatized to tap water, ligatured, and placed into 120 mm./l. NaCl Mean percentage change in body wt. at intervals of time (days) Group Treatment N I None II Mouth sealed and in abdominal ligature* III Abdominal ligature* 4 IOI 107 no 112 Ligatured between penultimate and terminal abdominal segments. (b) Qualitative investigations of controlled drinking Two techniques, both employing the same basic method, were used to estimate the rate of drinking in L. affinis larvae. The first technique, described in II, was devised to follow imbibition of salt water by a single larva over the course of about 3 days. This involved handling the larva at intervals and subjecting it to narcosis with CO 2. These experiments were followed by others in which larvae were not disturbed until their final removal from the medium. We are concerned here only with results obtained with the first technique. The method was to compare the total radioactivity of normal larvae with that of

9 Salt and water balance in caddis larvae (Trichoptera). Ill 149 farvae prevented from drinking the same radioactive medium. The latter provide an estimate of radioactivity due, in this case, to influx of M Na through the body wall. By comparison, excess radioactivity in normal larvae will be due to influx via the mouth, since there is no evidence to suggest that any significant influx occurs via the rectum. Now the gut wall of many insects is known to be highly permeable to ions, and it is assumed here that there is a very rapid exchange of M Na, across the gut wall, with ffl Na in the haemolymph, so that the rate of exchange across the gut wall is far greater than the rate of influx into the gut. In this case excess radioactivity in normal larvae will be a measure of the rate of drinking. If a larva swallows small quantities of radioactive medium at regular intervals, then the radioactivity of the larva should increase in a regular manner. This would be evidence of controlled drinking. On the other hand if drinking occurs at very irregular intervals, or if large quantities of water are suddenly imbibed, then this should be indicated by large irregular increases in radioactivity of the larva. -±5 3000, 2000, Jl Fig. a ^2000 i 1000 i? O-- t- O-" '^r- r oi --o--- l o O-- 1, o-p 1 1 -o -O -O- ^--O -o- -O to 70 Houn Fig Fig. 2. The increase in radioactivity of three L. affinis larvae at an external concentration of 120 mm./l. NaCl., serial 211-mouth sealed with war; O, serial 213-normal larva; 3, serial 215-normal larva. Serials 211 and 213 previously acclimatized for 6 days in 120 mm./l. NaCl, serial 215 previously acclimatized for 6 days in tap water. In Figs. 27 the small arrows indicate the radioactivity of the external medium, counts/jd./s min. Fig. 3. The increase in radioactivity of a L. affinis larva (serial 214) at an external concentration of 120 mm./l. NaCl. A, Previously acclimatized for 6 days in tap water. B, After 7 days in 120 mm./l. NaCl. C, After 14 days in 120 mm./l. NaCL In the first experiment serials 210 to 213 were acclimatized to an external concentration of 120 mm./l. NaCl and serials 214 and 215 were acclimatized to tap water. After 6 days acclimatization the mouth of serial 211 was sealed with wax and all six larvae were placed into 120 mm./l. NaCl containing a very small quantity of M Na. Some of the results are shown in Fig. 2. Serial 211, prevented from drinking the medium, showed a slow, regular increase in radioactivity. The increase in radioactivity of serials 210 and 212 was extremely similar to that of serial 213, shown in Fig. 2. In these three larvae the increase in radioactivity was gradual but at a higher rate than in serial 211, indicating that the normal larvae were swallowing small quantities of the medium at regular intervals. The experiment also indicates that the rate of drinking in the two larvae transferred

10 150 D. W. SUTCLIFFE directly from tap water into 120 mm./l. NaCl was no greater than that of the larvae already acclimatized to this salt concentration. The increase in radioactivity of serial 215 was identical with that of serials 212 and 213 (Fig. 2). The behaviour of serial 214 (previously acclimatized to tap water) was even more interesting since the larva apparently did not swallow any of the radioactive medium during the course of the experiment (Fig. 3 A). The increase in radioactivity of this larva can be attributed entirely to M Na influx through the body wall (cf. serial 211, Fig. 2). Once again this is strong evidence for suggesting that drinking is actively controlled. Further evidence was obtained by maintaining serial 214 at an external concentration of 120 mm./l. NaCl. Measurements of M Na influx were repeated again after 7 days and after 14 days in 120 mm./l. NaCl. The results are shown in Fig. 3 B, C. The increase in radioactivity during these two separate periods was practically identical and was now also very 11?* g g Z.S, i if 3000 Si 2000 < O~" ^- ) _ --' Hours Fig. 4. The increase in radioactivity of two L. affims larvae at an external concentration of 120 mm./l. NaCl. Both larvae previously acclimatized for 13 days in 120 mm./l NaCl. O, serial 313; ), serial 215. Compare with Fig. 2. similar to that of the other larvae in 120 mm./l. NaCl (Fig. 2). Finally, serials 213 and 215 were also maintained at an external concentration of 120 mm./l. NaCl and measurements of M Na influx were repeated during the following week. The results (Fig. 4) were very similar to the results obtained with these larvae during the previous week (Fig. 2). The consistent manner in which reproducible results were obtained on a single individual over a period of up to 3 weeks is remarkable. The results obtained on a number of individuals are also remarkably similar. There can be no doubt that this was due to a uniform kind of behaviour, viz. a strictly controlled rate of drinking maintained at the same rate over a long period of time. Observations on fluctuations in body weight ( IVa) suggested that drinking is actively controlled even at very high external salt concentrations. Further supporting evidence was obtained from the following experiments. Two groups of four larvae acclimatized to tap water and to an external concentration of 220 mm./l. NaCl respectively were transferred into radioactive 220 mm./l. NaCl. The increase in radioactivity of larvae in both groups was extremely similar; a typical example from each group is given in Fig. 5. Again it is clear that drinking was strictly controlled, and the

11 Salt and water balance in caddis larvae (Trichoptera). Ill 151 Behaviour of larvae transferred directly from tap water into 220 mm./l. NaCl is essentially the same as the behaviour of larvae already acclimatized to this external concentration. The larvae were maintained in 220 mm./l. NaCl and the experiment was repeated during the following week in order to demonstrate the effect of preventing a larva from drinking the radioactive medium. In the second week the mouth of serial 218 was sealed with wax, and the result is shown in Fig. 5. Similar results were also obtained on larvae acclimatized to an external concentration of 330 mm./l. NaCl. Two typical results for normal larvae are given in Fig. 6, together with the behaviour of two larvae prevented from drinking the medium. Finally, good evidence was obtained to show that the technique employed here provides a genuine picture of drinking behaviour in L. affinis larvae. In one of the i- ^ 11 SJ-S" h-«iht-i---ir o ""* w o-^->* ^100 v 9o * h - - -l«o I I! ic a fris 3000 T? _ Houn l f " ft ( _ - J ».. -#ft Hours Fig. s Fig. 6 * :»»* I i SO 60.Jioo J " ^iB 8 Fig. 5. The increase in radioactivity of two L. affinis larvae at an external concentration of 220 mm./l. NaCl. O, serial 218normal larva previously acclimatized for 6 days in 220 mm./l. NaCl;, serial 218mouth sealed with wax (see text); >, serial 221normal larva previously acclimatized for 6 days in tap water. Fig. 6. The increase in radioactivity of three L. affinis larvae at an external concentration of 330 mm./l. NaCl. Larvae previously acclimatized to this concentration for 6 days., serials 224, 225mouths sealed with wax; O, serial 227normal larva; ), serial 228normal larva. above experiments larvae acclimatized to an external concentration of 220 mm./l. NaCl were transferred into radioactive 220 mm./l. NaCl. Before transference to the radioactive medium the mouth of serial 216 was sealed with wax. The seal, however, was imperfect and the larva was still able to swallow the radioactive medium. Larvae treated in this manner usually responded by drinking abnormally large quantities of water. In this case serial 216 immediately drank about 10 fa. of the radioactive medium. Drinking was followed by regurgitation, the medium was strongly stained with brown fluid from the midgut, and the body weight returned to its initial level. The effect of this large intake on the total radioactivity of the larva is shown in Fig. 7. It is clear that the radioactivity of a larva reflects the intake (and loss) of radioactive salt water through the mouth. By comparing Fig. 7 with Figs. 2-6, it is also quite clear that the technique employed in these experiments is demonstrating that larvae in salt water normally drink only small quantities of the medium, and that drinking probably occurs at fairly regular intervals. The experiment with serial 216 was repeated during the following week, this time ensuring that the mouth was completely sealed. The results are also given in Fig

12 152 D. W. SUTCLIFFE (c) Quantitative estimates of controlled drinking Now that we have established that there is a regulated intake of small quantities of salt water we can attempt to describe it in quantitative terms. This can be done in the following manner. Salt water containing M Na is imbibed into the midgut, and it is assumed that even if no net movement of sodium occurs across the midgut wall there is a rapid exchange with ^Na in the haemolymph. Then in normal larvae the radioactivity of the haemolymph in excess of that accounted for by influx through the body wall will represent M Na gained from the medium by drinking. If the radioactivity of known amounts of medium and haemolymph is obtained a simple calculation will give an estimate of the quantity of medium imbibed. Let A m = the radioactivity of the Fig. 7. The increase in radioactivity of serial 216 at an external concentration of 220 mm./l. NaCl (see text). O, mouth not completely sealed;, mouth completely sealed with wax. medium, A n the radioactivity of haemolymph in normal larvae and A,, the radioactivity of haemolymph in larvae prevented from drinking, expressed as counts//j./ unit time, and let V = the total volume of haemolymph in a larva, in microlitres. Then the quantity (in microlitres) of radioactive medium imbibed is V. An V was not determined, but in Sialis the haemolymph'volume is equivalent to 56 % of the wet weight of the larva (Beadle & Shaw, 1950). It is assumed here that the haemolymph volume in L. affinis larvae is also equivalent to 56 % of the wet weight. It may be noted that even if the haemolymph volume is, in fact, equivalent to as much as 70 % of the wet weight, the resulting error in the estimation of V does not markedly affect the final result. Quantitative estimates of salt water intake were made with some of the larvae used in experiments described above ( IV b).). At the conclusion of these experiments haemolymph samples of known volume were removed and the radioactivity was assessed as described previously (Sutcliffe, 1961a). The relevant data obtained from these larvae is given in Table 7. 70

13 Salt and water balance in caddis larvae (Trichoptera). Ill 153 For larvae at an external concentration of 120 mm./l. NaCl the mean quantity of radioactive medium imbibed was 2-2 /xl. in 76-5 hr. or about 0-7 [A. per day. At an external concentration of 220 mm./l. NaCl the mean quantity imbibed was about o-6 [A. per day, and at 330 mm./l. NaCl serials 227 and 228 drank approximately 2-0 /A. per day. This suggests that drinking was considerably increased at the highest external salt concentration. However, it is important to be certain that drinking was not influenced by abnormal experimental conditions, such as the repeated handling and narcotization during the process of recording the radioactivity of live larvae. In an attempt to overcome these difficulties larvae were kept individually in semiisolated compartments of a Perspex box. Details of the technique are given in a Table 7. Data for quantitative estimations of salt-water intake by Limnephilus affinis larvae (see text) Medium (mm./l. NaCl) Serial Treatment* MS N N N MS N N N MS N N Body wt. (mg.) "5 4i' Means ' Means Means V </d.) At 35 35O Counts//u.L/5 min ] J 658 \ 715 I J J J 1250 An 2QOO I Time (hr.) 7S 78 \ 77 I f 72 J 7i 1 72 I f 68 \ 68 I MS = mouth-sealed larva: N = normal larva. previous paper (Sutcliffe, 1961 a). Briefly, larvae were first acclimatized to the required salt concentration, the medium was then gently poured away and fresh medium of the same concentration containing labelled sodium was run in with a syringe pipette. In this way larvae were not unduly disturbed until their final removal for measurements of the radioactivity of haemolymph samples. The results were presented in Figs. 3 and 4 of the previous paper. By applying the above calculation to counts obtained after 75 hr. in these experiments, where V = 20 /xl., it appears that larvae at an external sodium concentration of 100 mm./l. drank approximately 2-i (A. of the radioactive medium per day, and at an external sodium concentration of 300 mm./l. the larvae drank approximately 1-9 [A. per day. The latter estimate is in close agreement with that previously obtained for larvae in 330 mm./l. NaCl, but the estimate for larvae at an external concentration of 100 mm./l. sodium is three times greater than that obtained previously. In view of these differences an extensive investigation would be required in order to establish whether or not larvae drink significantly greater quantities of the medium when its salt concentration is increased. But we can conclude

14 154 D. W. SUTCLIFFE with some confidence that, over the entire range of external salt concentrations tolerated by L. affinis larvae, the daily intake of salt water is of the same order of magnitude, roughly 1-2 fil. or 3-7 % of the body weight per day. V. EXCRETION Whereas L. affinis larvae in salt water drink only small quantities of the medium, larvae of the freshwater species L. stigma and A. nervosa alternately drink and regurgitate large quantities of salt water. It is therefore of interest to examine the effect of this behaviour on the production of excretory fluid in the freshwater species, and to see whether the controlled intake of salt water by L. affinis larvae is balanced by an equivalent output of excretory fluid. Observations and results obtained with larvae of L. stigma were essentially the same as for A. nervosa larvae, and will not be considered further in this section. {a) Rectal fluid production in A. nervosa larvae Amounts varying from 0-2 /zl. to more than 1 /xl. were easily obtained at intervals of about 3 hr. from the rectum of larvae kept in tap water. A typical series of results obtained on 2 consecutive days is shown in Table 8. The mean amount of rectal fluid obtained from serials was 4-4 /A. per day, equivalent to 7 % of the mean body weight per day. This rate of excretion is very similar to the estimated rate of osmotic uptake, approximately equivalent to 7 % of the body weight per day (Sutcliffe, 1961 a). This close agreement between these estimates of water uptake and output in tap-water larvae indicates that the method employed for estimating the rate of excretion is a reasonable one. Day Table 8. The volume of rectal fluid collected at intervals during 2 consecutive days from Anabolia nervosa larvae kept in tap water erial Body wt. (mg) Vol. of rectal fluid W at intervals of time (hr.) o-6 o o-8 O-2 3 o-i Nil i-o 0-7 o-s O-Q 0-4 o-6 o-6 11 i-o NU 0-7 o Total volume rectal fluid in 11 hr i Calculated volume in 24 hr. (*d.) '4 In salt water the rate of excretion was considerably reduced. Thus at an external concentration of 120 mm./l. NaCl the daily output was reduced to less than one-third that of larvae in tap water (Table 9). At higher external salt concentrations the majority of larvae did not produce normal rectal fluid. From some larvae amounts of up to at least 5 pi. of fluid were collected at intervals of a few hours. This fluid was dark brown in colour and clearly derived from the midgut. In other larvae there appeared to be a continuous flow of water passing straight through the alimentary canal; the anal sphincter often remained permanently open, and in some cases part of the rectal wall was everted through the anus. This behaviour also occurred in larvae

15 Salt and water balance in caddis larvae {Trichoptera). Ill 155 ieft undisturbed at high external salt concentrations. Thus is was not entirely caused by frequent handling and the technique employed to obtain rectal fluid, although such treatment undoubtedly emphasized the behaviour. These observations indicate that high external salt concentrations produce strong disruptive effects on the alimentary canal. Table 9. The volume of rectal fluid collected pom Anabolia nervosa larvae transferred into 120 mm./l. NaCl, after 2 days (A) and 7 days (B) in the medium Serial A B Body wt. (mg.) 88-o o I2I - O Total volume rectal fluid collected in 12 hr. (A) and 9 hr. (B) o-i Calculated vol. in 24 hr. (/J-) i-8 o-6 23 i-3 o-8 Calculated vol as % body wt 2-O I-O Out of some twenty larvae one individual in salt water produced rectal fluid at a rate equal to that of tap-water larvae. This larva had been acclimatized to a salt concentration of 50 mm./l. NaCl for 3 weeks, and was then transferred directly into 170 mm./l. NaCl. The larva immediately began to drink the medium, but during the first 2 days it produced a regular flow of rectal fluid at a rate of output equivalent to 8-9 % of the body weight per day. It was hoped that this larva had successfully achieved a working balance between salt and water intake and output, but it died suddenly during the third day of the experiment. Two other larvae treated in the same way failed to produce any rectal fluid and both died within 3 days in 170 mm./l. NaCl. (b) Rectalfluidproduction in Limnephilus aflinis larvae The rate of excretion in L. afflnis larvae kept in tap water was very similar to that found in A. nervosa larvae. A typical series of results is given in Table 10. The mean quantity of rectal fluid produced by serial 240 during 3 days was equivalent to 2-6 /xl. per day or 7-5 % of the body weight per day. Similarly, serials produced 9-12% of the body weight per day. These estimates are similar to an estimated osmotic uptake of 7% of the body weight per day in tap water (Sutcliffe, 1961a). At an external concentration of 120 mm./l. NaCl the daily output of rectal fluid was reduced, and there was considerable individual variation in output. A typical series of results from larvae kept in 120 mm./l. NaCl for 6 days is given in Table 11. In this case serials 254 and 256 produced an amount equivalent to 4 % of the body weight per day, whereas in serials 255 and 257 the output was equivalent to only about 1 % of the body weight per day. Very similar results were obtained from larvae in 170 mm./l. NaCl. No satisfactory estimate of the rate of excretion could be obtained from larvae kept in 220 mm./l. NaCl. In one isolated instance, during 2 consecutive days, the output of a larva was equivalent to 7 % of the body weight per day. In practically every other case, however, the production of normal rectal fluid ceased; the fluid obtained was

16 156 D. W. SUTCLIFFE clearly derived from the midgut. This behaviour was undoubtedly due to disturbances (in water balance) caused by the technique employed in collecting the rectal fluid. It was nearly always possible to obtain normal rectal fluid from larvae which were only occasionally disturbed. A single collection was made at intervals of 2 or 3 days over a period of up to 2 weeks. At each collection the amount of rectal fluid usually varied between 0-2 and o-8 /A., although amounts of up to about 1-2 fa. were sometimes obtained from larvae at external concentrations varying between 220 and 410 mm./l. NaCl. Table 10. The volume of rectalfluidcollected at intervals during 4 days from Limnephilus affinis larvae kept in tap water* Vol. of rectal fluid (/J.) at intervals of time (hr.) during the ist day Calculated vol. in 24hr. Body y wt. Serial (mg.) o ist day 2nd day 4th day < o-i o i-o o-o o No collections made on the third day. Table 11. The volume of rectal fluid collected at intervals from Limnephilus affinis larvae kept in 120 mm./l. NaCl ierial Body wt. (mg.) Total volume rectal fluid collected in 12 hr. (id.) o-6o o-is o-8o o-is Calculated volume in 24 hr (/J-) 1-2 o-3 i-6 o-3 Although the rate of excretion at high salt concentrations cannot be accurately estimated, the following conclusions may be drawn. At external concentrations up to at least 410 mm./l. NaCl the larvae of L. affinis continue to produce normal excretory fluid in the rectum, and amounts of fluid equivalent to about 4 % of the body weight were obtained at a single collection. It is therefore quite possible that the output at high external salt concentrations is of the order of 4 % of the body weight per day. It is even possible that the daily output is greater than this, since in one larva at an external concentration of 220 mm./l. NaCl the output was equivalent to 7 % of the body weight per day. DISCUSSION Although entirely different methods were used to estimate water intake and output in L. affinis larvae the respective estimates are in surprisingly good agreement. Thus, bearing in mind the practical difficulties encountered in estimating the rate of rectal fluid production at high external salt concentrations, it is reasonable to conclude that over the entire range of salt concentrations tolerated by L. affinis larvae the salt water

17 Salt and water balance in caddis larvae (Trichoptera). Ill 157 swallowed is excreted in a roughly equivalent volume of rectal fluid.* Combining the observations presented in this paper and previously, it is suggested that salt and water regulation in L. affinis larvae is achieved in the following manner. In fresh water, osmotic uptake through the relatively permeable body wall is balanced by an equivalent output of rectal fluid strongly hypo-osmotic to the haemolymph. In salt water, osmotic uptake is reduced and stops when the external medium is iso-osmotic with the haemolymph. Any water used for the elimination of metabolic waste products must then be obtained by other means. Immersion in high salt concentrations also introduces a potential osmotic loss. In order to maintain a constant water content in the haemolymph and tissue cells, osmotically free water must be obtained from the medium. Salt water is swallowed at a strictly controlled rate, so that the absorbed salts can be eliminated in the rectal fluid as a concentrated (hyper-osmotic) solution of salts, a small quantity of osmotically free water being retained in the process. This free water will be available to offset dehydration, and at the same time a flow of (salt) water is maintained as a vehicle for the removal of waste products. As pointed out previously (Sutcliffe, 19616) the capacity of the excretory system to deal with excess salts gained by drinking is probably greatly enhanced in L. affinis larvae by a reduction in salt permeability of the body wall. This is important, since osmotically free water can only be gained from medium imbibed into the midgut (unless active uptake of water through the external cuticle is postulated). Hence, at any particular total osmotic pressure and volume of rectal fluid an increased capacity to excrete salts gained by drinking will increase the quantity of free water retained in the elaboration of rectal fluid hyper osmotic to the medium. The above regulatory mechanism may be common to a number of salt-water insects. Larvae of Aides detritus also drink salt water, and the major if not the only uptake of salts also occurs via the mouth and gut wall (Beadle, 1939). Beadle pointed out that in A. detritus larvae the regulatory mechanism must be concerned largely with compensating for or controlling the inward diffusion of salts, and he suggested that at least part of the mechanism is an active excretion of salts. This was confirmed by Ramsay's (1950) demonstration that the rectal fluid can be very slightly hyperosmotic to sea water. Beadle was unable to say whether or not drinking is controlled, but he did not pay particular attention to this point. Since the rectal fluid is apparently only slightly hyper-osmotic to sea water (as in L. affinis larvae) it is important that the intake of salts by drinking be strictly controlled so that the total uptake of salts, including any uptake through the cuticle, can be safely removed in the rectal fluid. It is suggested therefore that controlled drinking must also form an important part of the regulatory mechanism in A. detritus larvae. The same argument can be applied to larvae of Ephydra riparia. These maintain the haemolymph strongly hypoosmotic to very high external salt concentrations and elaborate rectal fluid considerably hyper-osmotic to the medium (Sutcliffe, 1960 a), which suggests a greater margin of safety in dealing with an excess salt load. However, E. riparia larvae have a salt tolerance range similar to larvae of E. cinerea, which also maintain a strongly hypo- If, in fact, the daily output corresponds to only one-half the daily intake of salt water at high external salt concentrations, extrarenal excretion of salts must be invoked to account for some of the excess salts gained both by drinking and by diffusion through the body wall. This conclusion is based on measurements of osmotic and salt concentration of haemolymph, rectal fluid and medium, and estimates of body wall permeability to water and salts (Sutcliffe, 1961 a).

18 158 D. W. SUTCLIFFE osmotic haemolymph. In E. cinerea permeability of the body wall both to water and salts is low (Nemenz, 1960 a, b), but it is not completely impermeable to water. Thus at very high external salt concentrations there will be a small but steady osmotic loss, and it seems probable that production of a highly hyper-osmotic rectal fluid in Ephydra larvae is necessary to retain sufficient osmotically free water to offset this osmotic loss. It appears then that the chief difference between larvae of salt-water Diptera and larvae of L. affinis lies in the relatively high permeability to water of the body wall in the caddis. In the latter, osmotic loss is reduced to a minimum by maintaining the haemolymph roughly iso-osmotic with the external medium (Sutcliffe, 1961a). In contrast, a reduction in permeability to water in the salt-water Diptera confers a considerable degree of osmotic independence, and regulation is homoiosmotic over a very wide range of external concentrations (Beadle, 1939; Beyer, 1939; Nemenz, 1960a; Sutcliffe, 1960 a). Unlike L. affinis, larvae of the freshwater species L. stigma and A. nervosa are unable to survive in salt water hyper-osmotic to the normal haemolymph level. Drinking does occur but is not controlled, and very large amounts of salt water are swallowed. Moreover, salt water has a decidedly adverse effect on the alimentary canal, manifested by massive regurgitation of fluid, eversion of the rectal wall and expulsion of the peritrophic membrane. This behaviour is accompanied by a rapid decrease in weight which must be due largely to exosmosis across the gut wall. In this respect the effect of salt water appears to be very similar to that observed by Wigglesworth (1933) in freshwater mosquito larvae placed in salt water. Here the gut epithelial cells separated from the basement membrane, and the gut was distended with fluid by exosmosis from the haemolymph. Wigglesworth also observed that the permeability of the gut wall was abnormally increased. The behaviour of Corethra (= Chaoborus) phimicornis larvae in salt water (Schaller, 1949) also closely resembles that of the freshwater caddis larvae. Thus drinking began immediately in hypertonic saline media, large quanties were swallowed, and strong pumping movements were initiated in the foregut. The pumping movements travelled posteriorly, forcing fluid into the rectal bladder which increased its rate of discharge. These observations by Schaller were confirmed by personal observations on C. phimicornis larvae (unpublished) which showed that drinking was immediate and violent in dilute sea-water media equivalent to 170 and 220 mm./l. NaCl. These external salt concentrations were rapidly fatal. As in the freshwater mosquito larvae (Wigglesworth, 1933) it wa3 found that the gut wall became abnormally permeable to dyes added to the medium. It is clear that successful adaptation to salt water must involve an increased tolerance of the gut wall to high concentrations of salt. This was fully recognized by Wigglesworth (1933) who demonstrated by means of elegantly simple observations that salt tolerance of the gut wall in A. aegypti larvae could be greatly increased by gradual acclimatization to high external salt concentrations. In this way larvae were successfully acclimatized to artificial sea water with a salt concentration roughly equivalent to a solution of 325 mm./l. NaCl. By gradually increasing the external salt concentration a small proportion of L. stigma and A. nervosa larvae were acclimatized to dilute sea water with a salt concentration equivalent to 220 mm./l. NaCl, although none survived more than 1 week at this concentration (Sutcliffe, 1961 b). In this connexion it would be interesting to investigate caddis larvae living in low salinity regions

19 Salt and water balance in caddis larvae (Trichoptera). HI 159 9f the Baltic Sea. Elsewhere these normally occur only in freshwater habitats, but in the Baltic Sea many species occur in a range of salt concentrations up to the equivalent of at least 100 mm./l. NaCl (Silfvenius, 1906; Nybom, i960). The behaviour of Sialis lutaria larvae in salt water is also interesting. Although this species is normally confined to freshwater habitats, drinking in salt water is controlled, and the gut wall tolerates a concentration of 256 mm./l. NaCl (Shaw, 1955). Shaw attributes the final death of Sialis larvae at this concentration as probably due to the rise in haemolymph osmotic pressure caused by salt penetration. In conclusion then, it appears that the successful adaptation of L. affinis to high concentrations of salt water has involved an increased salt tolerance of the gut wall. This feature, together with the strict regulation of salt-water intake by drinking, allows active control over the total salt and water content of haemolymph and tissue cells. The elaboration of a concentrated rectal fluid provides a fine adjustment to both the water and salt content of the larva. In the freshwater species L. stigma and A. nervosa, the absence of these adaptive features results in exosmosis across the gut wall, the haemolymph salt concentration rises, and production of rectal fluid cornea to a halt. The factors causing a cessation in rectal fluid production are uncertain. It could be due simply to a general lack of water following exosmosis, but other factors may also be involved. For example, Shaw (1955) has advanced the interesting suggestion that reduction in rectal fluid production in Sialis larvae is due to an increased sodium concentration in the haemolymph. In salt water, the haemolymph sodium level in freshwater caddis larvae increases rapidly whereas in L. affinis, in which rectal fluid production continues at high external salt concentrations, the haemolymph sodium is maintained at a low level (Sutcliffe, 1961a, b). SUMMARY 1. Two freshwater caddis larvae, L. stigma and A. nervosa, drink and regurgitate large quantities of salt water at frequent intervals. Drinking is not controlled, and larvae may drink an amount equivalent to 50 % of the body weight per day. The gut wall is adversely affected by salt water and exosmosis occurs across the gut wall. 2. L. affinis larvae drink only small quantities of salt water. Drinking is strictly controlled, and the intake is roughly equivalent to 3-7 % of the body weight per day over a wide range of external salt concentrations. The gut wall is not affected by high salt concentrations; regurgitation and exosmosis do not normally occur. 3. In the freshwater caddises the rate of rectal fluid production is approximately equivalent to 7 % of the body weight per day. Rectal fluid is not produced at high external salt concentrations. 4. L. affinis larvae continue to produce rectal fluid at very high external salt concentrations. The daily output is probably roughly equivalent to the daily intake of salt water by drinking. 5. The osmoregulatory mechanism in L. affinis larvae and other salt-water insects is discussed. It is suggested that controlled drinking forms an important part of this mechanism, together with the ability of the gut wall to withstand exposure to high salt concentrations and the ability to elaborate rectal fluid hyper-osmotic to the external medium.

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