Fish Physiology and Biochemistry vol. 13 no. 2 pp 173-181 (1994) Kugler Publications, Amsterdam/New York

Osmoregulation in the stenohaline freshwater catfish, Heteropneustes fossilis (Bloch) in deionized water I. Parwez l , Fauzia A. Sherwanil and S.V. Goswami 2 Department of Zoology, lAligarh Muslim University, Aligarh-202 002, India and 2 University of Delhi, Delhi-110 007, India Accepted: February 19, 1994 Keywords: osmoregulation, catfish, Heteropneustesfossilis,deionized water, prolactin, renal function, hypoosmotic environment

Abstract Transfer of the stenohaline catfish, Heteropneustesfossilis from tap water (TW) to deionized water (DW) resulted in an increase in the glomerular filtration rate, urine volume and osmolar and free water clearance. In a closed system, where the DW was renewed only once a day, no change in the plasma osmolality was evident for up to 14 days. When DW was renewed four times a day for 25 days, a significant reduction in the plasma osmolality was observed within 24h. When the fish were transferred back to TW, plasma osmolality increased to normal freshwater level within 24h. These observations suggest the existence of highly efficient branchial mechanisms for active uptake of salts from an exceedingly dilute ambient medium. The fact that prolactin-secreting cells as well as corticotrophs in the pituitary of the fish in DW were highly stimulated suggests the involvement of the hormones in the adaptive responses of the catfish to DW.

Introduction The air-breathing catfish, Heteropneustesfossilis, is widely distributed in freshwater ponds and riverine backwaters of the Indian subcontinent. These water bodies are filled to capacity in the monsoon months due to influx of large volumes of rain water resulting in enormous dilution of almost all chemical constituents. With no influx during the rest of the year, the ponds are subject to water loss by evaporation and evapo-transpiration. Thus in these ponds, an indirect relationship exists between the water level and the salt concentration. Our earlier studies have shown that this catfish can survive in salinities up to 35°7%seawater (Parwez et al. 1979) by efficiently employing its renal mechanism (Goswami et al. 1983). However, no infor-

mation is available regarding the osmoregulatory mechanisms which enable the catfish to survive in the media of extremely low salt content. In the present study, an attempt has been made to study some parameters of osmoregulation following exposure of catfish to a salt-free environment.

Materials and methods Sexually mature male H. fossilis, weighing 30-50g, were obtained from the backwaters of the river Yamuna around Delhi and acclimated to the laboratory conditions (temperature 25 ± 1 C; photoperiod 12L:12D) for about 30 days in 20 1 aquaria containing stored tap water (TW: composition in mmoles/liter: Na+ , 0.87; K + , 0.1; Ca 2 + , 0.85;

Correspondence to: Dr. Iqbal Parwez, Department of Zoology, Aligarh Muslim University, Aligarh-202 002, India.

174 Mg 2 + , 0.37; Cl-, 0.37; SO42 - , 0.13). During this period they were fed ad libitum every day with Hindlever laboratory animal feed (Hindustan Lever Limited, Bombay, India) and the water was renewed daily by siphoning whilst replenishing simultaneously with fresh water adjusted to laboratory temperature. The fish were not fed during the experiments.

Experiment I Male catfish were allocated in batches of 6 to glass aquaria containing 20 1 of deionized water (DW, pH 6.3). Deionized water was obtained from a portable deionizer (Indion model CA 10U, Ion Exchange Ltd., Bombay, India) and tested every day for conductivity using a conductivity bridge (Elico, Model No. CM-82, Hyderabad, India). The fish maintained in tap water (TW) served as the control. Water in all the aquaria was renewed once a day. After 7, 14 and 21 days, blood and urine samples were obtained from experimental as well as control fish (each group sampled only once) and analyzed for osmolality. Urine flow rate (UFR) and osmolar and free-water clearance were measured according to techniques described earlier (Goswami et al. 1983). Briefly, plasma and urine osmolalities were measured in a 200 1 aliquot using an Osmette Osmometer (Model 2007, Precision Systems, Waltham, MA, U.S.A.). Urine was collected by 'retention catheter' method. One end of a 5-cm long polyethylene tubing (Ramson, India No. 46) was hermetically sealed within an impermeable rubber sachet which was provided with a 2.5-cm long thick-walled glass tubing at the other end for evacuation of air at the time of catheterization and drainage of urine at the termination of the experiment. The free end of polyethylene tubing was inserted into urogenital opening of the anesthetized fish and tied firmly to the muscular urogenital papilla. A loop of surgical suture thread was passed through the anal fin and around the rubber sachet to secure it alongside the tail of the fish. The rubber sachet was evacuated through the glass tubing whose tip was then sealed with sealing clay ('SealEase': Clay Adams, Parsippany, U.S.A.). The en-

tire process of catheterization could be completed within 3 min. The fish regained consciousness within 2-3 min of being returned to water. The total urine output during 14-16h was used to calculate the UFR which was expressed as ml/h/kg. Since the urogenital papilla is absent in females of this species, it was not possible to anchor the catheter for urine collection. Consequently, all experiments reported in the present investigation were conducted on the male catfish. Osmotic clearance (Cosm) and free water clearance (CH20) were calculated by the following formulae. Cosm = urine osmolality/ plasma osmolality x UFR and CH20 = UFR-Cosm. As an indirect estimate of electrolyte loss by the fish, 6 catfish (mean body weight 45 g) were placed in 20 1 of DW and the specific conductance of the water measured at various intervals over a 24h period and again after renewal of DW.

Experiment 2 This was similar to Experiment 1 except that water in all the aquaria was renewed four times every day throughout the duration of the experiment. Blood samples were drawn from the caudal vessels into heparinized glass syringes from batches of experimental and control fish every day for 25 days for estimation of plasma osmolality. Each group was bled only once. On day 25, one batch of fish in DW was transferred to TW, and blood samples obtained after 1, 4, 12, 24 and 48h for determination of plasma osmolality. The control fish in TW were transferred to another aquarium containing TW and processed in an identical manner. Another batch of fish in the above groups was used for estimation of glomerular filtration rate (GFR), UFR and fractional tubular reabsorption of water (o TH 20) by techniques described elsewhere (Goswami et al. 1983). Inulin clearance was employed as an index for GFR estimation. Urine collection as described before was initiated 2h after a single intraperitoneal administration of inulin (15 mg/0.3 ml). Pilot studies showed that this period was sufficient for uniform distribution of inulin in the extracellular space. Also, inulin administration did not cause any appreciable change in the

175 Table 1. Plasma and urine osmolality, urine flow rate and osmolar and free-water clearance of the catfish, Heteropneustesfossilisin tap water (TW) and following transfer to deionized water (DW) Days following transfer to DW Parameters

7

14

21

TW

DW

TW

DW

TW

DW

Plasma osmolality (mOsm/kg)

266 + 2

260 + 2

273 + 1

264 + 2

271 + 2

251 + 4**

Urine osmolality (mOsm/kg)

19 + 3

13 + 1

28 + 7

21 + 2

23 + 2

29

Urine flow rate (ml/h/kg)

8.8

0.6

13.4 + 0.7*

7.3 + 0.7

13.4 + 0.5**

8.1 + 1.0

13.2 + 1.3*

Osmolar clearance (ml/h/kg)

0.6

0.1

0.7 + 0.1

0.7 + 0.2

1.1 + 0.1

0.8

1.4 + 0.3

Free-water clearance (ml/h/kg)

8.1

0.5

12.7 + 0.7*

6.6 + 0.7

12.3 + 0.4**

7.3 + 0.9

All values represent mean *p < 0.005, **p < 0.001.

0.1

11.8

9

1.3*

SEM; number of fish for each time point ranged from 4-7; significance calculated by Student's t test;

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DEIONIZED WATER RENEWED

HOURS FOLLOWING TRANSFER TO DEIONIZED WATER

Fig. 1. The changes in the specific conductance of deionized water (DW) (20 1) at various intervals after introduction a group of six fish (mean body wt 45 g). The broken line depicts the change in specific conductance following transfer of the same group of fish to fresh DW.

176

280 E E 260 ,-

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DEIONIZEDWATER GROUP

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TRANSFERRED BACK TO TAP WATER

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8 10 12 14 16 18 20 FOLLOWING TRANSFER TO DEIONIZED WATER

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Fig. 2. The change in the plasma osmolality of H. fossilis, following transfer to deionized water and transfer back to tap water. Each time point represents the mean of 4-6 fish.

total urine output. Inulin concentration in plasma and urine was estimated by Schreiner's (1950) modification of Roe et al. (1949). Glomerular filtration rate was calculated using the following equation. GFR = Uin/Pin x UFR, where GFR was expressed as ml/h/kg and Uin and Pin were inulin concentrations (mg/ml) in urine and plasma respectively. At the end of the experiment (after 25 days in DW), fish were killed by decapitation. The pituitary along with the brain was fixed in Bouin's fixative, embedded in paraffin, sectioned at 5 ltm and stained with Herlant's tetrachrome stain (Herlant 1960). Prolactin-secreting cells and corticotrophs in the rostral pars distalis were identified on the basis of their cellular and tinctorial characteristics as described for H. fossilis pituitary by Baker et al. (1974). Diameters of 50 nuclei each of prolactin cells and corticotrophs of three fish from each group were measured with the help of calibrated eye-screw micrometer and used for calculation of nuclear areas (see Baker et al. 1974).

Results Experiment I No appreciable change was noticed in plasma osmolality up to 14 days of transfer of fish to DW (Table 1). However, UFR was about 50-80% higher (p