Osmoregulatory changes accompanying smoltification in coho salmon

Aquaculture, 28 (1982) 67-74 Elsevier Scientific Publishing Company,

OSMOREGULATORY IN COHO SALMON

CHRISTOPHER and HOWARD

67 Amsterdam

CHANGES

A. LORETZ*, A. BERN**

NATHAN

- Printed

in The Netherlands

ACCOMPANYING

L. COLLIE,

SMOLTIFICATION

NURNEY

Department of Zoology and Cancer Research Laboratory, Berkeley, CA 94720 (U.S.A.)

H. RICHMAN

University of California,

*Present address: Department of Biological Sciences, State University Buffalo, Buffalo, NY 14260 (U.S.A.) **To whom requests for reprints should be addressed. (Accepted

15 January

III

of New York at

1982)

ABSTRACT Lore& C.A., Collie, N.L., Richman III, N.H. and Bern, H.A., 1982. Osmoregulatory changes accompanying smoltification in coho salmon. Aquaculture, 28: 67-74. During the spring smoltification of the coho salmon (Oncorhynchus kisutch), alterations in osmoregulatory function occur in the urinary bladder, intestine and opercular membrane. Na and Cl reabsorption by the urinary bladder of seawater-adapted fish, initially at freshwater levels, is abolished during the period of smoltification. Intestinal fluid absorption of freshwater-adapted fish increases to seawater levels. The abundance of mitochondria-rich “chloride cells” in freshwater-adapted fish opercular membranes increases. All of these changes occur some weeks after the plasma thyroxine surge and coincide with increased brancial Na,K-ATPase activity. These data suggest that the appropriate time for seawater entry or placement of hatchery-reared coho salmon may be several weeks after the new moon-related thyroxine peak.

INTRODUCTION

Two important aspects of the life history of many salmonids, including the coho salmon (Oncorhynchus hisutch), are the seaward migration followed by a period of growth in the sea and the anadromous spawning migration to freshwater home lakes and streams; both migrations are highly regular seasonal events. In several salmonid species, salinity tolerance (survival) and preference for saline waters generally increase with age and size and occur maximally at periods of seaward migration; salinity tolerance and saline preference decline somewhat in fish held in fresh water after the migratory period ends (see reviews by Hoar, 1976; Folmar and Dickhoff, 1980). Changes in salinity tolerance, of course, represent changes in the integrated function of a number of osmoregulatory organs, including the gills, skin, kidney, urinary bladder and gut. Whereas the adaptive changes in function

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of these organs may not be expected to differ radically from those of other euryhaline teleosts (cf. Prosser, 1973; Gordon, 1977), the timing and development of these changes in relation to smoltification are of substantial interest. Better understanding of the timing of these processes can have important and direct application to salmon fisheries through the determination of optimum release time of hatchery-reared fish. We have studied the development of ion and water transport processes in several osmoregulatory organs of yearling coho salmon obtained from the Mad River Hatchery during study years 1979 (Mad River stock) and 1980 (Trinity stock) and from the Irongate Hatchery during study year 1981 (Irongate stock). We summarize briefly herein findings on the urinary bladder, intestine and opercular membrane. URINARY

BLADDER

The urinary bladder is an important osmoregulatory organ in many teleosts (Hirano et al., 1973). In fresh water, salt losses are reduced by active reabsorption of Na and Cl from kidney urine while a relatively water-impermeable bladder reduces water reabsorption. In seawater, generally, bladder water permeability is increased to promote water reabsorption and, thereby, reduce urinary water losses. The coho salmon urinary bladder, from freshwater-adapted (FW) or seawater-adapted (SW) fish, is a tubular structure, 5 --lo mm in length and l-2 mm in diameter, formed as a fusion of the mesonephric ducts and lined by a simple columnar epithelium (average cell height, 20 pm). Compared with the bladder volumes of a number of other teleost species (Hirano et al., 1973), the calculated bladder volume at 5-30 ~1 for fish used in this study (25-75 g) is among the smallest. Despite the small size and probably small contribution to osmoregulation of the bladder, in view of its embryonic origin, ion and water transport functions of the bladder may reflect renal tubular functions. Urinary bladders were mounted in Ussing-type chambers, bathed in salmon Ringer solution, and studied using electrophysiological and radiotracer techniques previously described (Loretz and Bern, 1980). Table I summarizes the electrophysiological characteristics of urinary bladders from FW and SW fish. TABLE I Electrophysiological

characteristics

of urinary bladder from 0. kisutck

Source

n

TEP (mV)

R(n :cm’)

SCC (rA/cm’)

FW fish SW fish

15 19

+0.2 + 0.1 -0.1 f 0.1

1150 f 202 420 i 94*

+0.7 P 0.2 +0.2 + 0.5

FW, freshwater-adapted; SW, seawater-adapted. Data presented as mean + SEM. *P < 0.01 (Student t-test) compared with FW fish urinary bladder.

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There were no differences in the transepithelial potential (TEP), resistance (R) or short-circuit current (SCC) at different times for FW fish and for SW fish during the period of study (March-July 1980) and the data, therefore, for all FW and for all SW groups, respectively, have been pooled. As with the bladder of another salmonid, the rainbow trout Sulmo irideus in fresh water (Lahlou and Fossat, 1971), the coho salmon urinary bladder exhibited near-zero TEP and SCC, indicating the absence of any electrogenic ion transport. R across FW coho salmon urinary bladder was significantly greater than across SW urinary bladder; R across bladders of salmon from either medium was substantially higher than across trout bladder (278 Q cm* ; Fossat and Lahlou, 1979). The net transports of Na and Cl (Jf$ and ci,), calculated as the difference between unidirectional 22Na and 36C1fluxes, respectively, were measured in vitro using standard radiotracer techniques (Loretz and Bern, 1980) and are presented in Fig. 1. Jzzt and tit by FW coho urinary bladder compare well with those by bladders from FW rainbow trouts S. guirdneri and S. irideus (Lahlou and Fossat, 1971; Hirano et al., 1973; Fossat et al., 1974; Fossat and Lahlou, 1979). The similar rates of Na and Cl transport and the lack of TEP and SCC across the coho salmon urinary bladder might suggest a neutral, coupled NaCl cotransport like that described for the S. irideus bladder (Fossat and Lahlou, 1979). In May, Jze: and <‘,t by SW coho urinary bladders were at FW levels whereas in July, J::i and czt were decreased to values not significantly different from zero. Whereas environmental salinity does not effect Na and Cl ab-

Fig. 1. Net absorption (Jnet) of Na and Cl by FW (upper panel) and SW (middle panel) yearling coho salmon urinary bladders during 1980. SW fish were adapted to seawater (34Oj,,, ) for 2 weeks prior to study. Data are presented as mean + SEM. n = 4 for all J net except J&; on 23-24 July where n = 2. Survival after 2 weeks of the three groups transferred to seawater is presented in the lower panel. Jnet values for small numbers of animals during the 1981 season are similar to +%se presented here.

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sorption in S. gairdneri bladders (Hirano et al., 1973), Na absorption by the S. irideus bladder is reduced by nearly 50% with seawater adaptation (Fossat et al., 1974). Thus, in two of three salmonid species studied to date, reductions in electrolyte reabsorption occur with seawater adaptation. Renal changes accompanying seawater adaptation of S. gairdneri include 90% reductions in glomerular filtration rate and urine flow rate (Henderson et al., 1978). Together, these data suggest that renal function in SW salmonids may be reduced to principally excretion of divalent ions, especially Mg (Hickman and Trump, 1969). Whereas Na and Cl reabsorption by salmon urinary bladder (to reduce urinary loss) may be essential for maintenance of hydromineral balance in fresh water, seawater survival is not impaired, and is actually improved, in those SW salmon exhibiting cessation of Na and Cl transport (Fig. 1). Urinary losses of water could be detrimental in the dehydrating marine environment; curtailment of renal function would reduce such losses. In non-salmonid species which do not reduce renal function there may be a greater dependence on intestinal fluid absorption and branchial electrolyte secretion to maintain positive water balance and allow continued renal function. It has been proposed that a surge in plasma thyroxine may trigger seaward migration by salmon (Grau et al., 1981) and increase seawater survival (Folmar and Dickhoff, 1981). It is interesting that the reduction in bladder ion absorption and increased seawater survival occur at least 2 months after the thyroxine surge (18 March; although the thyroxine peak for the year of this study (1980) was not pronounced). Thyroid hormone appears not to be a direct trigger of alterations in urinary bladder water reabsorption, either, since in vivo treatment by triiodothyronine or thyrotropin of starry flounder urinary bladder (Johnson et al., 1972) and thyroxine treatment of S. gairdneri urinary bladder (C.A. Loretz and T. Hirano, unpublished data, 1981), were without effect. INTESTINE

The euryhaline teleost intestine serves two major functions: nutrition and osmoregulation. The latter changes markedly as a function of environmental salinity (Skadhauge, 1969; Hirano et al., 1976). The FW teleost minimizes osmotic water gain from the hypoosmotic environment by possession of a low body-surface water permeability and by drinking very little and maintaining a low rate of intestinal water absorption. The SW teleost encounters a dehydrating hyperosmotic environment and exhibits much higher rates of both drinking and intestinal fluid absorption (Smith, 1930; Gordon, 1977). Our studies show that intestinal osmoregulatory function assumes the seawater pattern prior to migration and thus preadapts the fish for life in the sea. Histologically, the coho salmon intestine is similar to that of other salmonids. A prominent annulo-spiral septum separates the anterior from the

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posterior intestine (Collie, 1981). This latter segment was used in an in vitro sac preparation to measure net fluid absorption gravimetrically as described in detail elsewhere (Collie, 1981; Collie and Bern, 1982). In FW fish in vivo, intestinal fluid absorption is probably less than that indicated by in vitro measurements presented below since the gut lumen of FW fish contains a thick gelatinous material and very little fluid (Shehadeh and Gordon, 1969; N.L. Collie, this study). In contrast, the posterior intestine of SW fish was fully distended with fluid. Analysis of this fluid showed it to be essentially isoosmotic to plasma. These data, together with the findings that 10m4 M ouabain completely inhibits in vitro fluid absorption (Collie and Bern, 1982), supports the notion that ingested seawater equilibrates osmotically with the plasma and solute-linked water absorption occurs from an isoosmotic luminal fluid (House and Green, 1965; Shehadeh and Gordon, 1969; Skadhauge, 1969; Hirano and Mayer-Gostan, 1976). Two years of seasonal studies during the period of smoltification have revealed that the low rate of intestinal fluid absorption in FW parr increases prior to seawater entry (Fig. 2). Absorption rates in FW fish remain elevated for several months before returning to low levels in the fall. The plasma thyroxine peak measured in these fish by Grau et al. (1981) precedes the elevated plateau in fluid absorption by as many as 4 weeks. The thyroxine surge and elevated intestinal fluid absorption may be temporally linked since the latter increased in both years at similar times following the thyroxine peak (cf. Collie and Bern, 1982).

OTJ

FMAMJJ

AS0 Months

Fig. 2. Seasonal variation in net water fIux (Jv) across posterior intestine of FW yearhng coho salmon. The 1980 season data (closed circles) are plotted on the Julian calendar; the 1979 curve (open circles) is aligned according to the lunar calendar so that the lunar phases and, hence, the thyroxine peaks (indicated by arrow) of both years coincide. Hori zontal broken line and shaded region represent the mean + SEM (n = 10) of Jv measured in long-term (> 3 months) SW yearling coho salmon.

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However, several lines of evidence suggest that thyroid hormone (thyroxine or triiodothyronine) is not directly responsible for stimulating intestinal fluid transport. First, fluid absorption rates remain elevated long after the plasma thyroxine concentration has decreased. Second, short-term (1-4 days) injections into coho of bovine thyrotropin at doses which increase plasma thyroxine levels fail to alter significantly net fluid transport. Finally, in vitro addition of thyroxine (200 ng/ml) to the intestinal sac incubation media does not alter fluid absorption. OPERCULAR

MEMBRANE

The branchial epithelium, in part because of its great surface area and the presence therein of the ion-transporting “chloride cells”, is an important ion-regulatory organ. The flat, chloride cell-rich opercular membrane preparation of the killifish Fundulus heteroclitus has been developed as a model for branchial chloride cell function (Karnaky et al., 1977). The transport properties of a similar preparation of the coho salmon opercular membrane can be studied in vitro. The distribution of the mitochondria-rich chloride cells can be visualized and quantified with the aid of a,fluorescent mitochondrial probe, dimethylaminostyrylethylpyridiniumiodine (DASPEI; Bereiter-Hahn, 1976). The opercular membranes of coho salmon were examined throughout smoltification using a DASPEI-staining technique. Opercular membranes were sampled every 2 weeks from February through May 1981. Initially, very few scattered DASPEI-positive cells were observed. This pattern was maintained, with few exceptions, from February through April. In late May, however, the number of DASPEI-positive cells increased approximately three-fold and were evenly distributed throughout the tissue. The coho salmon opercular membrane is currently being examined using electrophysio, logical and radiotracer techniques to determine its potential contribution as an osmoregulatory organ. The results of these studies, although still only suggestive, raise the possibility that chloride cell proliferation may be associated with smoltification. CONCLUSIONS

It is clear that developmental changes in urinary bladder and intestine function of the coho salmon occur during smoltification. Branchial Na,KATPase activity in these coho salmon increases coincident with the changes we have described (Zaugg and McLain, 1972; and data from animals used in these studies). Whereas passage through the thyroxine surge has been related to seawater survival (Folmar and Dickhoff, 1981), the thyroxine surge does not appear to be the proximate stimulus for the changes we discuss. Cortisol is recognized as a seawater-adapting hormone in teleost (cf. Bern, 1975), and it is interesting that an increase in circulating cortisol occurs in

73

coho salmon several weeks after the thyroxine surge (Specker, 1982). Nevertheless, the factors directly regulating changes in osmoregulatory function remain to be elucidated. In the organs studied herein, it appears that osmoregulatory “maturation” for seawater life occurs some weeks after the thyroxine surge. This strongly suggests that the time appropriate for entry or placement of hatchery-reared coho into seawater may be several weeks after the new moon-related thyroxine peak (Grau et al., 1982). ACKNOWLEDGEMENTS

Supported in part by NOAA, National Sea Grant College Program, Department of Commerce, under Grant NOAA 04-8-MOl-189, Project R/F-45 through the California Sea Grant College Program. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. Also supported by NSF grant PCM 78-10348 and by NIH fellowship AM-05918 to C.A.L. We also thank the California Department of Fish and Game for their kind cooperation. REFERENCES Bereiter-Hahn, J., 1976. DimethylaminostyryImethyIpyridiniumiodine (DASPMI) as a fluorescent probe for mitochondria in situ. Biochim. Biophys. Acta, 423: l-14. Bern, H.A., 1975. Prolactin and osmoregulation. Am. Zool., 15: 947-948. Collie, N.L., 1981. Changes in intestinal fluid transport associated with smoltification and seawater adaptation in coho salmon (Oncorhynchus hisutch). M.A. Dissertation, University of California, Berkeley, CA, 33 pp. Collie, N.L. and Bern, H.A., 1982. Changes in intestinal fluid transport associated with smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch). J. Fish. Biol. (in press). Folmar, L.C. and Dickhoff, W.W., 1980. The Parr---smolt transformation (smoltification) and seawater adaptation in salmonids. A review of selected literature. Aquaculture, 21: I-37. Folmar, L.C. and Dickhoff, W.W., 1981. Evaluation of some physiological parameters as predictive indices of smoltification. Aquaculture, 23 : 309-324. Fossat, B. and Lahlou, B., 1979. The mechanism of coupled transport of sodium and chloride in isolated urinary bladder of the trout. J. Physiol., 294: 211-222. Fossat, B., Lahlou, B. and Bornancin, M., 1974. Involvement of a Na-K-ATPase in sodium transport by fish urinary bladder. Experientia, 30: 376-377. Gordon, M.S., 1977. Animal Physiology: Principles and Adaptations. MacMillan, New York, NY, 3rd edn, 699 pp. Grau, E.G., Dickhoff, W.W., Nishioka, R.S., Bern, H.A. and Folmar, L.C. 1981. Lunar phasing of the thyroxine surge preparatory to seawater migration of salmonid fish. Science, 211: 607-609. Grau, E.G., Specker, J.L., Nishioka, R.S. and Bern, H.A., 1982. Factors determining the occurrence of the surge in thyroid activity in salmon during smoltification. Aquaculture, 28: 49-57. Henderson, I.W., Brown, J.A., Oliver, J.A. and Haywood, G.P., 1978. Hormones and single nephron function in fishes. In: P.J. Gaillard and H.H. Boer (Editors), Comparative Endocrinology. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 217-222

74 Hickman Jr., C.P. and Trump, B.F., 1969. The kidney. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. 1. Academic Press, New York, NY, pp. 91-239. Hirano, T. and Mayer-Gostan, N., 1976. Eel esophagus as an osmoregulatory organ. Proc. Nat. Acad. Sci., U.S.A., 73: 1348-1352. Hirano, T., Johnson, D.W., Bern, H.A. and Utida, S., 1973. Studies on water and ion movements in the isolated urinary bladder of selected freshwater, marine and euryhaline teleosts. Comp. Biochem. Physiol., 45A: 529-540. Hirano, T., Morisawa, M., Ando, M. and Utida, S., 1976. Adaptive changes in ion and water transport mechanism in the eel intestine. In: J.W.L. Robinson (Editor), Intestinal Ion Transport. Medical and Technical Publ., Lancaster, pp. 301-317. Hoar, W.S., 1976. Smolt transformation: evolution, behavior, and physiology. J. Fish. Res. Board Can., 33: 1233-1252. House, C.R. and Green, K., 1965. Ion and water transport in isolated intestine of the marine teleost, Cottus scorpius. J. Exp. Biol., 42: 177-189. Johnson, D.W., Hirano, T., Bern, H.A. and Conte, F.P., 1972. Hormonal control of water and sodium movements in the urinary bladder of the starry flounder, Platichthys stelZ&us. Gen. Comp. Endocrinol., 19: 115-128. Karnaky Jr., K.J., Degnan, K.J. and Zadunaisky, J.A., 1977. Chloride transport across isolated opercular epithelium of killifish: a membrane rich in chloride cells. Science, 195: 203-205. Lahlou, B. and Fossat, B., 1971. Mecanisme du transport de l’eau et du se1 i travers le vessie urinaire d’un Poisson teleosteen en eau deuce, la truite arc-en-ciel. C.R. Acad. Sci., 273: 2108-2110. Loretz, CA. and Bern, H.A., 1980. Ion transport by the urinary bladder of the gobiid teleost, Gillichthys mirabilis. Am. J. Physiol., 239: R415-R423. Prosser, C.L., 1973. Comparative Animal Physiology. Saunders, Philadelphia, PA, 3rd edn, 966 pp. Shehadeh, Z.H. and Gordon, M.S., 1969. The role of the intestine in salinity adaptation of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol., 30: 397-418. Skadhauge, E., 1969. The mechanism of salt and water absorption in the intestine of the eel (Anguilla anguilla) adapted to waters of various salinities. J. Physiol., 204: 135-158 Smith, H.W., 1930. The absorption and excretion of water and salts by marine teleosts. Am. J. Physiol., 93: 480-505. Specker, J.L., 1982. Interrenal function and smoltification. Aquaculture, 28: 59-66. Zaugg, W.S. and McLain, L.R., 1972. Changes in gill adensine-triphosphatase activity associated with Parr-smolt transformation in steelhead trout, coho, and spring chinook salmon. J. Fish. Res. Board Can., 29: 161-171.