Salt and water balance in stonefly naiads, Pteronarcys californica newport

Camp. Biochem. Physiol., 1972, Vol. 41A, pp. 851 to 860. Pergamon Press. Printed in Great Britain

SALT AND WATER BALANCE IN STONEFLY NAIADS, PTERONARCYS CALIFORNICA NEWPORT CONRAD

COLBY”?

Department of Zoology, University of Montana, Missoula, Montana (Receiwed 14 April 1971) Final instar nymphs of Pteronarcys californica can produce a dilute Abstract-l. urine (17 mOsm/l.) and can most probably pick up inorganic ions actively from stream water. 2. They can fast for 28 days in stream water and still maintain control levels of weight, and of sodium, potassium and osmotic pressure of the hemolymph. 3. One half which were allowed to emerge as adults did so, the rest died. 4. These levels all drop in nymphs fasted in demineralized water, and no nymphs emerged as adults. INTRODUCTION

A LARGE variety of insect species from several different orders live all or part of their life cycles in fresh water. Because their body fluids are far more concentrated than the water in which they live, they are continually faced with water influx and salt loss. The manner in which they handle or tolerate hemolymph dilution varies from order to order (Shaw & Stabbart, 1963). Although osmoregulation has been studied in aquatic species of Hemiptera (Stadden, 1963), Trichoptera (Sutcliffe, 1961), Diptera (Wigglesworth, 1936; Koch, 1938; Ramsay, 1950; Stobbart, 1960) and Neuroptera (Beedle & Shaw, 1950; Shaw, 1955), osmoregulation in the exopterygote order, Plecoptera is little known. Plecopteran or stonefly nymphs are found mainly in well-aerated streams. When placed in water deficient in oxygen they become bloated with excess water (Knight & Gaufin, 1964), which suggests osmoregulating failure. Their presence and abundance in streams can thus be an indication or bio-assay of water quality. Furthermore, they provide a valuable source of food for trout (Pennak, 1953) and are thereby economically important. Changes in osmolality and of the concentrations of sodium and potassium in the hemolymph and urine were therefore measured in stonefly nymphs fasted in stream water, and in demineralized water. The species, Pteronarcys californica was chosen for study because it is large, locally abundant, easily collected and easily maintained in the laboratory. MATERIALS

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METHODS

Third-year naiads in their final instar were collected in late March from Rock Creek, a trout stream 20 miles southeast of Missoula, Montana. They were immediately transported * Present address: Department of Biology, Boise State College, Boise, Idaho 83707. t This study was completed in partial fulfilment of the M.A. degree in Zoology at the University of Montana. 851

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in thermally insulated containers containing Rock Creek (RC) water to the laboratory where they were maintained until experimentation in well-aerated RC water cooled to a constant 10°C. Hemolymph and urine were obtained from eight recently collected animals to serve as the control group. The remaining stonefly naiads were divided into five paired groups. One group of each pair would be maintained during the experiment in RC water and the other in demineralized (DM) water. Four of the five pairs were maintained in 1 1. Pyrex beakers which were three-fourths submerged in a large water-bath refrigerated at 10°C. Each of the eight groups of four pairs contained eight naiads respectively. After 18 days hemolymph was collected from one group maintained in Rock Creek water (RC naiads), and from its pair kept in demineralized water (DM naiads). At this time urine was also collected from another pair of RC and DM naiads. After 28 days of maintenance urine and hemolymph were likewise obtained from the remaining pairs of RC and DM naiads. Each member of the fifth pair of naiads contained six naiads. The twelve naiads were individually compartmentalized in plastic boxes to retain their identity. The boxes were also submerged in the water-bath. The water of all eleven groups was kept well aerated and was changed only daily to minimize the loss of ions leached from the naiads. All nymphs were fasted throughout the experiment. The twelve individuals in the fifth pair were weighed every 3 days. Before weighing, each naiad was allowed to crawl on blotting paper for 30 sec. It was then quickly weighed to the nearest mg and returned to its compartment within 1 min. No hemolymph or urine was collected from these insects. Hemolymph was gently aspirated into a capilIary tube which had one end inserted through a hole 0.5 mm in dia. drilled through the dorsal integument. The dorsal surface had been previously sprayed with a non-wetting agent containing Teflon. When at least 50 ~1 of hemolymph were collected as required for the subsequent analysis, one end of the capillary tube was carefully sealed by flame. The cellular matter was spun down in a microfuge and removed. The other end of the tube was sealed by flame, and the tube which now contained cell-free hemolymph was stored at - 4°C. Urine was collected by rectal cannulation. The polished end of a l-cm length of glass capillary tubing was barely inserted into the rectum and securely tied by a fine thread wrapped around the cerci. The other end was inserted into a section of polyethylene tubing (PE 90) which served to store the urine. Collodian was applied between the edge of the rectum and the cannula to seal the cannula to the rectum hermetically and securely. Dental acrylic tray cement was applied to the glass-polyethylene junction to create a water and air tight seal. Checks were made on several non-experimental naiads to establish that the connections were repeatedly sealed. To do this, the distal end of the polyethylene cannula was first sealed by heat. The urine excreted into the cannula compressed the air therein. When the distal end of the respective trial cannula was snipped off thereby returning the inside pressure to ambient, the fluid always moved rapidly up the cannula. It was assumed in the subsequent experimental collections that the system was air and water tight, and that the urine was neither contaminated nor lost to the ambient water. Consequently, the distal end was always left open. Once sufficient urine was collected (50 ~1) it was transferred to a glass capillary tube. One end of the tube was sealed by flame. If the urine contained solid fecal matter as it sometimes did from recently collected and recently fed animals the tube was centrifuged. The solid contents were removed, and after sealing the other end, the tube and the urine therein was stored at - 4°C. Osmolality of urine, hemolymph and RC water was determined by vapor pressure microsmometry. Potassium and sodium concentrations in the urine, hemolymph and RC water were determined by flame photometry. All means except those for body weights were statistically compared where appropriate by the t-test. The curves for body weights were compared by the x2 test.

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Because the nymphs were in their final instar it was of interest to determine whether they would emerge as adults or not. Consequently, sixteen animals were fasted in DM water and twelve nymphs in RC water. They were maintained relatively undisturbed at 10°C in 1 1. flasks until they either emerged or they died as naiads. The water was aerated and changed daily. RESULTS

1. Body weights Naiads kept in RC water essentially maintained their body weights (Fig. 1). Their body weights initially averaged l-22 g, and after 28 days of fasting their terminal body weights also averaged l-22 g. Three of the six nymphs gained some

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FIG. 1. Relationship of the changes of body weight with time expressed as the mean percentage of initial weight at each of the successive 3-day weighing periods. Open circles represent the means of nymphs fasted in Rock Creek water; closed circles, those for nymphs fasted in demineralized water.

weight, two lost some weight and one maintained its weight. Naiads in DM water significantly lost weight during the 28 days (P
maintained The initial about 5 per nine groups experiment.

2. Urinejlow No attempt was made to quantify the exact rates of urine flow. However, all of the control naiads required approximately 8 hr to produce 50 ~1 of urine (O-15 ml/ 24 hr) whereas animals at the eighteenth and twenty-eighth days required about 24 hr to produce that amount of urine (O-05 ml/24 hr).

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3. Sodium in the hemolymph and urine The mean concentration of sodium in the hemolymph of RC naiads dropped slightly but not significantly over the 28 days (Fig. 2). However, DM naiads could not maintain the concentration of hemolymph sodium. The mean dropped significantly after 18 days exposure to DM water and again after 28 days (Fig. 2). These two means were significantly lower than the respective means for RC naiads at 18 and 28 days (PC 0.001).

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FIG. 2. Relationship of the changes in sodium concentration in the hemolymph (left panel) and in the urine (right panel) with time. Open circles represent the means of:standard error (vertical lines) for sodium of nymphs fasted in Rock Creek water; closed circles, those for nymphs fasted in demineralized water. The numbers between interrupted connecting lines represent the P value for the statistical difference between the connected means. Uninterrupted lines between circles signify that the connected means do not differ significantly.

The concentration of sodium in the urine of RC nymphs collected at 18 days averaged somewhat higher than the control value (Fig. 2). The mean value of the 2%day collection returned to that of the initial control mean. Conversely, the urine sodium of DM naiads rose sharply and significantly in the 18-day collection and continued this rise appreciably in the last collection. The sodium levels in the terminal urine of DM naiads averaged significantly higher than the mean for both terminal RC naiads and for the controls (P-C 0.001).

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Potassium in the hemolymph and urine

The concentration of potassium in the urine of both groups of the pair collected at day 18 fell precipitously to levels which were less than 7 per cent of the control value (Fig. 3). The means for the K in the pair did not differ significantly. The mean K concentration in the urine of RC naiads fell slightly in the last collection. PTERONARCYS

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FIG. 3. Relationship with time of the mean potassium concentration in the hemolymph and urine of nymphs fasted in Rock Creek water (open circles), and of those fasted in demineralized water (closed circles). See legend for Fig. 2.

However, it rose significantly in the urine of DM naiads (Fig. 3) to reach a significantly higher value than that of the urine K collected from terminal RC naiads (P< 0.001). Both means, nevertheless, were significantly lower than the control value (P < 0.001).

The Na : K ratios were calculated for each animal and the values were averaged for each respective group rather than determining the ratio directly from the Na and K means for each group. This ratio for the hemolymph of RC naiads did not essentially change during the experiment (Fig. 4). However, the Na : K ratio dropped significantly for the hemolymph of DM nymphs during the first 18 days (Fig. 4), then rose appreciably to a mean value just below that for the controls. Individual values of terminal DM naiads varied considerably as indicated by the

CONRADCOLBY

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relatively large standard error of the mean. cantly from that for the controls.

ratio did not differ signifi-

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FIG. 4. Relationship of the changes with time of Na : K ratio of the hemolymph and urine of fasted nymphs. See legend for Fig. 2.

The Na : K ratio for control urine averaged only 0.12 k 0.02 (Fig. 4). The mean value for RC naiad urine at 18 days rose significantly above the control average, but this mean stabilized for 2%day RC naiads at a slightly lower value. The mean ratio for the urine of DM naiads at 18 days also reached a significantly higher value than that for the controls (Fig. 4). It then fell significantly at 28 days to a mean value of just over two. 6. Osmolality of hemolymph and urine The osmolality of the hemolymph of RC naiads did not change appreciably during the first 18 days (Fig. 5). The mean dropped appreciably but not significantly during the last 10 days. However, hemolymph osmolality of DM nymphs fell significantly throughout the experiment (Fig. 5) reaching a terminal mean the respective mean value of about 80 per cent of the control mean. Furthermore, hemolymph osmolality of DM naiads was significantly below that of RC naiad hemolymph at day 18 and day 28 respectively (PC O*OOl). The osmolality of the urine for both RC and DM naiads dropped significantly at the eighteenth day (Fig. 5). However, the osmolality of the urine of DM naiads did not fall as much and the mean value was significantly higher than that for the RC naiad urine (P< 0.001). The osmolality of the urine of terminal DM insects

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rose somewhat, whereas that of the urine of RC nymphs remained essentially the same (Fig. 5). The mean urine osmolality of RC animals at 28 days was significantly lower than that of terminal DM naiad urine (I’< 0.001).

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FIG. 5. Relationship of the changes with time of the osmolality of the hemolymph and urine of fasted nymphs. See legend for Fig. 2.

7. Emergence Clearly stonefly nymphs can tolerate fasting for 28 days without a general loss of health and vigor. The non-experimental animals in their final instar that were simply fasted until they either emerged or died as larvae showed that they can survive considerably longer than 28 days. Of the sixteen animals maintained in DM water, two died as nymphs after 33 days. The last naiad died after 45 days of fasting in DM water. None emerged. However, of the twelve nymphs fasted in RC water, the first died 30 days later and the sixth and last to die did so after a fast of 39 days. The remaining six emerged, molted and flew as adults. The first emerged after 37 days and the last after 51 days of fast. An examination of these emerged adults revealed the presence of numerous fat bodies and many seemingly developed eggs. 8. Composition of RC water Rock Creek water (eight samples) and mean concentrations of sodium respectively.

possessed a mean osmolality of 4 mOsm/l. and potassium of 1.1 and O-3 m-equiv/l.

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DISCUSSION

If stonefly nymphs are similar to other aquatic insects they are perpetually confronted with an influx of water and efflux of salts. Since RC water possessed both a relatively low osmolality and low concentrations of sodium and potassium, the respective high gradients across the permeable membranes in RC water would not differ appreciably from those in DM water. Furthermore, the influx of water would probably tend to increase body weight far more than the utilization of food stores would decrease it. Because the naiads fasted in either RC or DM water did not gain weight they apparently removed the influxed water via the extremely dilute urine. The rigidity of the exoskeleton could feasibly exert sufficient back pressure to restrict water entering across the osmotic gradient. However, this mechanism to limit volume was not required since swelling of naiads was never observed nor did any nymph that was weighed show an appreciable gain in weight. Furthermore, Knight & Gaufin (1964) reported that stonefly naiads become distended with excess fluid when oxygen in the medium becomes deficient. Apparently the lack of energy for active transport results in osmoregulatory failure. The large drop in urine flow of both groups at 18 days and the corresponding fall of potassium in that urine indicate that the nymphs had entered the postabsorptive state. Control naiads recently removed from the stream probably had imbibed water as well as algae and plant detritus high in potassium (Pennak, 1953), remaining in the gut. The control levels of urine potassium reflect removal of this potassium load. As the food is digested and assimilated, considerable amounts of urine would be required to excrete the ~monium ions produced, and to remove the excess potassium. At 18 days both groups had removed dietary potassium as shown by the drop in potassium and the rise in Na : K ratio of the urine. Likewise, the concomitant drop in urine osmolality at this time suggests that ammonium ions produced from the food had also been excreted. The osmolality and concentrations of sodium and potassium of the hemolymph of control Aeronarcys compares favorably with the respective values reported for the aquatic larvae of species from the exopterygote orders of Odonata, Ephemeroptera and Plecoptera (Sutcliffe, 1962). This investigator suggested that inorganic salts are largely ionized in the hemolymph of the Plecopteran, Perla bipwzctata and that free amino acids probably form part of the non-electrolyte fraction in the hemolymph. Even though amino acids may be present in Ptenmarcys hemolymph, the severe drop in hemolymph osmolality of DM naiads suggests that amino acids are not mobilized to increase the osmotic pressure of the hemolymph as inorganic ions are lost. Such a mechanism has been proposed for other aquatic insects (Shaw, 1955). The mean osmolality of 17 mOsm/l. measured for the urine of fasted RC larvae compares favorably with the osmolal equivalent of the mean of 12 mM/l. NaCl reported for the urine of mosquito larvae, Ai;des aegypti maintained in distilled water (Ramsay, 1950). Th e recta1 fluid of caddis larvae, Li~~ep~i~~s and ~n~~o~~ possessed osmotic pressures similar to those of the urine of control RC larvae (Sutcliffe, 1961). H owever, the urines of the neuropteran, Sialis (Shaw, 1955), and

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of the aquatic bug, Notonecta (Staddon, 1963), are many times more concentrated than the urine of fasted RC naiads. Fasting naiads in RC water maintained weight and the levels of sodium, potassium and osmolality of the hemolymph, whereas naiads kept in DM water failed to do so. Apparently, stonefly larvae can therefore actively extract these cations from RC water against a large gradient to offset those lost by leaching and in the urine. Conversely, fasting naiads in DM water were unable to replace these lost ions and as a consequence the levels of sodium, potassium and osmolality in the hemolymph slowly fell throughout the experimental period. The increase of these values in the urine suggests that DM naiads regulated volume both by removing influxed water via the urine and by reducing the osmotic gradient from medium to hemolymph. However, by doing so they lost valuable cations. Indeed, the 5 per cent loss of body weight of DM naiads reflects an overcompensation to regulate volume by removing inorganic ions. The data gathered and analyzed in this study support the conclusion that stonefly nymphs can actively transport ions inward against a gradient. It is yet unclear what structures are involved in this active transport. Nevertheless, this mechanism plus the ability to produce a dilute urine indicates that they are excellent osmoregulators. Not only can they withstand long periods of fasting but they could feasibly exist in fresh water possessing a lower ionic concentration than that of Rock Creek. Acknowledgements-Grateful acknowledgement is extended to Professor James R. Templeton, graduate committee chairman, who gave generous assistance, advice and encouragement, and to William Morrelles, who helped greatly in obtaining necessary equipment. REFERENCES BEADLEI,. C. & SHAWJ. (1950) The retention of salt and the regulation of the non-protein nitrogen fraction in the blood of the aquatic larva Sialis Maria. J. exp. Biol. 27, 96-109. KNIGHT A. & GAUFIN A. R. (1964) Relative importance of varying oxygen concentration, temperature, and water flow on the mechanical activity and survival of the Plecoptera nymph, Pteronarcys californica Newport. Proc. Utah Acad. Sci., Arts and Letters 41, Pt. 1, 14-28. KOCH H. J. (1938) The absorption of chloride ions by the anal papillae of dipteran larvae. J. exp. Biol. 15, 152-160. PENNAKR. W. (1953) Fresh-water Invertebrates of the United States. Ronald Press, New York. RAMSAYJ. A. (1950) Osmotic regulation in mosquito larvae. J. exp. Biol. 27,145-157. SHAWJ. (1955) Ionic regulation and water balance in the aquatic larva of Sialis Zutaria. J. exp. Biol. 32, 353-382. SHAWJ. & STOBBARTR. H. (1963) Osmotic and ionic regulation in insects. Advan. Insect Physiol. 1,315-399. STADDONB. W. (1963) Water balance in the aquatic bugs, Notonecta glauca 1,. and Notonecta marmorea. Fabr. (Hemiptera; Heteroptera). J. exp. Biol. 40, 563-571. STOBBARTR. H. (1960) Studies on the exchange and regulation of sodium in the larva of ACdes aegypti (L.)-II. The net transport and the fluxes associated with it. J. exp. Biol. 37, 594-608.

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SUTCLIFFED. W. (1961) Studies on salt and water balance in caddis larvae (Trichoptera)II. Osmotic and ionic regulation of body fluids in Linnephilus stigma Curtis and Anabolia nervosa. J. exp. Biol. 37, 521-530. SUTCLIFFED. W. (1962) The composition of haemolymph in aquatic insects. J. exp. Biol. 39, 325-343. WIGGLES~ORTHV. B. (1938) The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. exp. Biol. 15, 235-247. Key Word Index-Aquatic insects; osmoregulation; water balance; stonefly; Pteronarcys californica.

sodium and potassium balance;