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Smoltification and seawater adaptation in Atlantic salmon (Salmo salar): Plasma prolactin, growth hormone, and thyroid hormones
G E N E R A L A N D COMPARATIVE E N D O C R I N O L O G Y
74, 355-364 (1989)
Smoltification and Seawater Adaptation in Atlantic Salmon (Salmo salar): Plasma Prolactin, Growth Hormone, and Thyroid Hormones PATRICK P R U N E T , * GILLES B O E U F , t JONATHAN P. BOLTON,* AND GRAHAM YOUNG:~
*Laboratoire de Physiologic des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cddex, France; ?IFREMER, Centre de Brest, BP 70, 29263 Plouzan~, France; and SDepartment of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720 Accepted October 18, 1988 To obtain more information on the role of prolactin and growth hormone during the parr-smolt transformation of Atlantic salmon, a population of fish in fresh water was sampled from January to June during two consecutive years. Gill Na+,K+-ATPase activity increased steadily during smoltification and a plasma thyroxine peak was observed 2-3 weeks before the gill Na+,K+-ATPase peak. On the basis of these two parameters, smoltification was considered complete in our populations in April 1985 and May 1986. Two peaks in plasma growth hormone levels occurred in 1986, one in mid-April and the second in mid-May. In both cases, these peaks coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. Moreover, the second peak which lasted for 1 month coincided with maximal gill Na + ,K +-ATPase activity. A decrease in plasma prolactin levels was observed during smoltification of Atlantic salmon in 2 consecutive years. During this period of decreasing and low plasma prolactin levels, gill Na +,K+-ATPase activity increased to its highest values. Atlantic salmon smolts were also directly transferred into seawater. After 2 days or more in seawater, plasma prolactin levels were not significantly different from those on Day 0, whereas in fresh water they showed large fluctuations. All these data indicate that growth hormone may play an important role in the development of hypoosmoregulatory activity. Increased hypoosmoregulatory ability also appears to be associated with low prolactin levels. 9 1989AcademicPress, Inc.
The parr-smolt transformation (smoltification) in salmonids involves complex physiological changes and biochemical events. One of the most characteristic features of the smolt is its enhanced seawater adaptability (see Hoar, 1976; Folmar and Dickhoff, 1980; Wedemeyer et al., 1980). Although thyroid hormones have been implicated in the control of smoltification (see Folmar and Dickhoff, 1981; Grau et al., 1985; Barron, 1986), evidence for a precise role of thyroxine and triiodothyronine in the acquisition of seawater adaptability is not yet forthcoming (Boeuf, 1987). Increasing evidence suggests that cortisol, another important hormone in teleost osmoregulation, causes physiological changes associated with seawater adaptation (Specker and
Schreck, 1982; Barton et al., 1985; Virtanen and Soivio, 1985; Young, 1986; Richman and Zaugg, 1987; Bjfrnsson et al., 1987). H o w e v e r , in Atlantic salmon, plasma cortisol does not appear to be directly related to the increase in gill Na § ,K+-ATPase activity observed during smoltification (Langhorne and Simpson, 1986). Although the importance of pituitary hormones in teleost osmoregulation is well documented, few studies have focused on this aspect during smoltification. Several studies indicate that prolactin (PRL) is not necessary for freshwater survival of hypophysectomized salmonids (Komourdjian and Idler, 1977; BjOrnsson and Hanson, 1983; Nishioka et al., 1987). However, 355 0016-6480/89 $1.50 Copyright 9 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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m e a s u r e m e n t o f p l a s m a P R L l e v e l s in s a l m o n i d s t r a n s f e r r e d f r o m f r e s h w a t e r to seawater or the converse support a possible role of PRL as a freshwater-adapting horm o n e ( H i r a n o e t al., 1985; P r u n e t e t al., 1985; P r u n e t a n d B o e u f , 1985). G r o w t h h o r m o n e ( G H ) h a s b e e n i m p l i c a t e d as a s e a w a t e r - a d a p t i n g h o r m o n e in s a l m o n i d s ( K o m o u r d j i a n e t al., 1976; C l a r k e a n d N a g a h a m a , 1977; M i w a a n d Inui, 1985; B o l t o n e t al., 1987) a n d a r i s e in p l a s m a G H l e v e l s during coho salmon smoltification has been r e p o r t e d ( S w e e t i n g e t al., 1985). I n a c o m p a n i o n p a p e r ( Y o u n g e t al., 1989), p l a s m a P R L a n d G H l e v e l s d u r i n g smoltification and seawater adaptation have been reported for coho salmon. The p r e s e n t p a p e r r e p o r t s o n c h a n g e s in p l a s m a PRL and GH levels during Atlantic salmon s m o l t i f i c a t i o n in r e l a t i o n to t h y r o i d h o r m o n e l e v e l s a n d gill N a § 2 4 7 activity. The results are compared with those from studies on the coho salmon (Young et al., 1989; B j O r n s s o n e t al., 1989) a n d a r e d i s c u s s e d in t e r m s o f t h e p o s s i b l e r o l e o f t h e s e h o r m o n e s in t h e d e v e l o p m e n t o f h y poosmoregulatory ability during smoltification.
MATERIALS AND METHODS Animals. Atlantic salmon eggs imported from Norway in 1984 and 1985 were reared in Le Conquet hatchery (Brittany). Yearlings were isolated in 4-m2 Ewos tanks in November preceding the sampling period and were fed on manufactured dry pellets by automatic feeders (ration, 2% body wt/day). The photoperiod was natural and the water temperature during the experimental period (January to June) increased from 5 to 15~ in 1985 and from 3 to 14~ in 1986. In comparison to 1985, in January-February 1986, fish had to face a lower temperature which delayed the natural temperature increase usually observed in spring. Sampling. As our population of Atlantic salmon show a characteristic bimodal distribution of body length and weight, fish were sampled in consecutive years from February to May (1985) and from February to July (1986). Blood samples were obtained from the posterior aorta with a heparinized syringe always at the same time of the day (9--11 AM). Fish were stunned by cranial concussion without anesthesia. Plasma was
collected after centrifugation of blood and kept frozen at - 2 0 ~ until analysis. Gill filaments were taken from the same fish, rinsed in 0.25 M sucrose buffered at pH 7.4 and immediately frozen in liquid nitrogen. Gill Na+,K+-ATPase were measured according to the methods described by Lasserre et al. (1978). During the first year of sampling, 100 fish were transferred from the hatchery to the experimental farm at Brest late in April and were divided between two Ewos tanks. On April 22, the seawater supply of one tank was turned on and the freshwater supply was turned off. In this manner seawater replaced fresh water within 2 hr. Neither group was fed for 3 days before transfer. The salinity of the seawater stayed constant (35 -+ 1%o)during the experiment. Both groups of fish were sampled just before transfer and 1, 2, 4, and 7 days after transfer. Plasma osmolarity was measured using a Roebling freezing point osmometer. Hormone radioimmunoassays. Plasma PRL levels were measured using the method of Prunet et al. (1985). GH levels were determined according to the method of Bolton et al. (1986). The cross-reactivity between these two radioimmunoassays has been estimated at less than 0.3% (Hirano et al., 1985; Bolton et al., 1986). Triiodothyronine and thyroxine levels were measured using the methods of McKenzie et al. (1978) as described in Boeuf and Prunet (1985). Statistical analysis. One-way analysis of variance and the Newman-Keuls multiple range test were used to assess the significance of seasonal changes in plasma hormone levels and gill Na+,K+-ATPase activity. In the transfer experiment, differences at each sampling date between fish in fresh water and in seawater were assessed using Student's t test.
RESULTS Gill N a + , K + - A T P a s e L e v e l s Significant increases (P < 0.01; A N O V A ) in gill N a + , K + - A T P a s e a c t i v i t y o c c u r r e d in b o t h y e a r s o f s t u d y . I n 1985, Na§ activity increased from F e b r u a r y to A p r i l a n d r e a c h e d a p l a t e a u in m i d - A p r i l w h i c h l a s t e d until m i d - M a y (Fig. 1). A s i m i l a r p a t t e r n o f N a + , K § c h a n g e s w a s o b s e r v e d in 1986, a l t h o u g h t h e c u r v e w a s s h i f t e d to t h e r i g h t , w i t h t h e Na§247 increase delayed for a b o u t 1 m o n t h (Fig. 2). Plasma Thyroid Hormone
Levels
F r o m F e b r u a r y to M a r c h 1985 (Fig. 3), plasma thyroxine levels remained low.
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HORMONES AND ATLANTIC SALMON SMOLTIFICATION
~
/i_ ~176176 thyroxine-
triiodothyronine ---
lO
Feb 9
r
1
r
jan feb mar apr may june 1965
FIG. I. Gill Na+,K+-ATPase activity during the parr-smolt transformation of Atlantic salmon reared in fresh water in 1985. Each point represents a mean and SEM for 10 fish.
However, a sharp rise was observed on March 25 (P < 0.01 compared to values from January and February). Levels stayed significantly higher (P < 0.01) until April 4 but then dropped to initial values and did not show further significant changes. Plasma triiodothyronine stayed low in February and March. However, a slow but steady increase was observed in April and later in the month levels reached a significantly higher value compared to FebruaryMarch (P < 0.05). In 1986 (Fig. 4) the general patterns of plasma triiodothyronine
March
April
May
FIG. 3. Change in plasma thyroxine (-) and triiodothyronine (- - -) levels during the parr-smolt transformation of Atlantic salmon reared in fresh water in 1985. Each point represents a mean and SEM for 8-10 fish.
and thyroxine were similar to those observed in 1985. However, this year was characterized by a delay in the thyroxine peak (April I5 to 21, P < 0.0t compared to values from February and March). After May 25, plasma thyroxine levels increased again and stayed significantly higher until July (P < 0.01 compared to values from April 29 to May 15). Changes in triiodothyronine levels in 1986 were of greater ampli-
0
thyroxine(ng/ml ) triiodothyronine ( ng/ml ) . . . . .
-
-
feb
mar
apr
may
june
T
T
feb
mar
T
apt
T
may
[
june
1986
FIG. 2. Gill Na§ activity during the parr-smolt transformation of Atlantic salmon in 1986. Each point represents a mean and SEM for 10 fish.
FIG. 4. Change in plasma thyroxine (-) and triiodothyronine (- - -) levels during the parr-smolt transformation of Atlantic salmon in fresh water in 1986, Each point represents a mean and SEM for 8-10 fish.
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tude than in 1985. A first peak (April 15, P < 0.01 compared to values from preceding dates) was observed just preceding a thyroxine peak (April 24) and later in the season two other triiodothyronine peaks occurred (May 26 and June 24, P < 0.01 for both peaks compared to values from April 29 to May 15). feb
Prolactin Levels In 1985 (Fig. 5), plasma PRL levels decreased significantly (P < 0.01) from an initial value of 7-9 ng/ml in February to 2-3 ng/ml in March. After this initial decrease, PRL levels stayed around 2-4 ng/ml in March-April and no significant difference was observed during this period. In 1986 (Fig. 6), the pattern was similar to 1985. Until early March, levels were high (8-12 ng/ml) and a significant decrease (P < 0.01) was observed on March 14 followed by a long period of consistently low PRL levels (no significant difference from March 15 until July 11). In 1985, smolts were transferred to seawater on April 22. At this time, fish showed high seawater adaptability as indicated by plasma osmotic pressure which stabilized at its seawater level within 24 hr (Fig. 7). An increase in the plasma PRL level, although not significant, was observed 24 hr after transfer to seawater and was followed by a decrease (Fig. 7). However, on Days 2, 4, and 7, PRL levels in the seawater group were not significantly dif-
FIG. during salmon resents
mar
may
june
6. Change in plasma prolactin (PRL) levels the parr-smolt transformation of Atlantic reared in fresh water in 1986. Each point repa mean and SEM for 8-10 fish.
ferent from the initial level on Day 0. In the freshwater control group, PRL levels fluctuated during the experiment, and freshwater levels were only significantly higher than seawater levels on Days 2 and 4.
Growth Hormone Levels (Fig. 8) Plasma GH levels were only measured in 1986. After an initial decrease (February l l, P < 0.01 compared to values from March), no further significant change was observed until April 8 with GH levels re-
A
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FIG. during salmon resents
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April
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5. Change in plasma prolactin (PRL) levels the parr-smolt transformation of Atlantic reared in fresh water in 1985. Each point repa mean and SEM for 8-10 fish.
.
:
,'.
2
4
6
8 days
FIG. 7. Change in plasma osmotic pressure (A) and prolactin (PRL) levels (B) in Atlantic salmon smolt transferred from fresh water to seawater. The solid line corresponds to the control group kept in fresh water and the dotted line to the group transferred to seawater. Each point represents a mean and SEM for five fish. Asterisks denote significant difference (P < 0.01) in levels between freshwater and seawater fish on the same day.
HORMONES AND ATLANTIC SALMON SMOLTIFICATION
30
20
T
feb
f"
I
mar
apr
r may
june
FIG. 8. Change in plasma growth hormone (GH) levels during parr-smolt transformation of Atlantic salmon reared in fresh water in 1986. Each point represents a mean and SEM for 8-10 fish.
maining low (10-13 ng/ml). A peak in levels occurred on April 15 (P < 0.01 compared to values in March and early April) followed by a sharp decline (P < 0.01). In May, a significant increase (P < 0.01) was observed on May 6; later, GH levels rose steadily to achieve maximum levels of 38 ng/ml on May 26. In June, GH levels stayed significantly (P < 0.01) higher than levels in February and March until early July when the initiation of a decrease occurred. DISCUSSION
The purpose of this study was to obtain information on the possible involvement of PRL and GH in the endocrine control of smoltification in Atlantic salmon by measuring plasma levels of these two hormones. To characterize the stage of the parr-smolt transformation, plasma thyroid hormone levels and gill Na+,K+-ATPase activity were followed. Both parameters are possible indicators of smolt stage (Folmar and Dickhoff, 1981; Langdon and Thorpe, 1984; Boeuf and Prunet, 1985). In 1985 and 1986, these parameters changed during smoltification as already described in Atlantic salmon (Saunders and Henderson, I978; Lindahl et al., 1983; Youngson
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and Simpson, 1984; Boeuf and Prunet, 1985; Virtanen and Soivio, 1985; Langhorne and Simpson, 1986). Thus, gill Na + ,K+-ATPase activity increased steadily from February to April and this increase paralleled the increase in seawater adapta b i l i t y w h i c h was m a x i m u m w h e n Na + ,K+-ATPase reached a plateau (Boeuf et al., 1985; Langdon and Thorpe, 1985). The most prominent change in thyroid hormone levels was the appearance of a thyroxine peak which occurred during the Na+,K+-ATPase increase, generally 2-3 weeks before peak enzyme activity was achieved. Although this thyroxine peak occ u r r e d during a period of high gill Na +,K +-ATPase activity, Boeuf (1987) did not find a coincidence of this peak with maximum seawater adaptability in Atlantic salmon. Monitoring of these parameters indicated that on the basis of seawater adaptability, smoltification was complete in our population in April 1985 and May 1986. The delay in 1986 compared with 1985 may be due to the lower temperatures during the presmoltification period in 1986. Temperature is an important modulator of salmon smoltification (see Saunders and Henderson, 1970; Wedemeyer et al., 1980; Clarke and Shelbourn, 1985). An important fnding in the present study was the increase in plasma GH levels during smoltification of Atlantic salmon, in agreement with the increase reported in plasma GH during coho salmon smoltification (Sweeting et al., 1985; Young et al., 1989). Histological studies of coho pituitaries also indicate increased secretory activity of GH cells during smoltifcation (Clarke and Nagahama, 1978; Nishioka et al., 1982). In our study, two GH peaks are evident, the first one occurring in mid-April and the second occurring in mid-May. In both cases, these GH changes coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. These data substantiate several reports indicating a close reIationship between GH and thyroid hormones in fish. The coinci-
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dence between the triiodothyronine peak and the GH increase agrees with the classical picture in mammals of the stimulation by triiodothyronine of GH synthesis and release (Reichlin, 1974; Martial et al., 1977). However, this effect was not observed in tilapia using incubated pituitaries (Nishioka et al., 1985). Salmon GH has also been shown to enhance peripheral conversion of thyroxine to triiodothyronine in eels (De Luze and Leloup, 1984). However, it is not clear from the present study if such a relationship exists in Atlantic salmon as high GH levels were not always coupled with high triiodothyronine levels. Clearly, more investigations are needed to clarify the relationship between GH and thyroid hormones during smoltification. The second GH increase, which lasted for about I m o n t h , c o i n c i d e d with m a x i m a l gill Na+,K+-ATPase activity. Moreover, the GH decline in late June/early July occurred at t h e s a m e t i m e as a d e c r e a s e in Na+,K+-ATPase activity. These results are in agreement with those of previous studies indicating a stimulatory effect of GH on this enzyme's activity in presmolt coho salmon and in hypophysectomized yearling coho salmon (Richman and Zaugg, 1987; Bj6rnsson et al., 1987). However, other workers did not observe an effect of GH treatment on Na + ,K +-ATPase activity (Miwa and Inui, 1985; Richman et al., 1987). When data from all sampling dates in this study were pooled (n = 148), a significant correlation (r = 0.3, P < 0.01) was obtained b e t w e e n GH levels and gill Na+,K§ activity, supporting the importance of a GH peak for seawater adaptation of Atlantic salmon smolts. Recently, Nanc6 et al. (1989), using an isolated perifused head preparation, have shown that Atlantic salmon smolts are able to establish a negative Na § flux at the level of the gill filament immediately after seawater transfer. Moreover, using the same perifusion technique, we have observed a similar phenomenon in rainbow trout pretreated with GH in fresh water just before
seawater transfer (Prunet et al., 1989). In general, these data support the evidence from the present study for a possible role of GH in salmon smoltification. A decrease in plasma PRL levels was observed during smoltification in 2 consecutive years. The d e c r e a s e o c c u r r e d in March; thereafter PRL levels remained low even during the desmoltification period (June-July). During this period of decreasing a n d l o w p l a s m a P R L l e v e l s , Na+,K§ activity increased and reached its maximal level. In coho salmon, the PRL peak in late March/early April is followed by an abrupt decrease and PRL remained at a basal level for about 1 month before increasing again (Young et al., 1989). Interestingly, this decrease in PRL level c o i n c i d e d w i t h an i n c r e a s e o f Na§ activity (Bj6rnsson et al., 1989). Several studies have already shown an inhibitory effect of PRL on this gill enzyme's activity in other fish species (Pickford et al., 1970; Kamiya, 1972; Gallis et al., 1976; Foskett et al., 1982). Hence, it is conceivable that during smoltification and the preadaptive development of seawater osmoregulatory mechanisms, the decrease in PRL levels might permit gill Na + ,K +-ATPase activity to increase. This hypothesis is reinforced by recent data indicating that decreasing plasma PRL levels are characteristic of smolts, as parr from the same population sampled during the same period did not show this phenomenon (Prunet and Boeuf, 1989). The transfer experiment on Atlantic salmon smolts partially supports this hypothesis. In the freshwater group sampled between April 22 and 27, fluctuations in PRL levels were observed. Because these animals were transferred from the hatchery to the experimental farm only several days before the beginning of the experiment and were maintained under different rearing conditions, these fluctuations may reflect stress: recently, chronic stress has been shown to increase PRL levels in coho salmon and in rainbow trout (Avella et al.,
H O R M O N E S A N D A T L A N T I C SALMON S M O L T I F I C A T I O N
1989; Prunet and Gonnet, unpublished data). In the present study, plasma PRL increased 24 hr after transfer to seawater and thereafter decreased to basal levels. After 7 days in seawater, no significant differences were observed between the seawater group and the freshwater control group. These data are in agreement with other similar transfer experiments using Atlantic salmon smolts (Prunet and Boeuf, 1985). It is also interesting to note that after 2 days or more in seawater, plasma PRL levels in seawater-adapting fish were not significantly different to those measured on Day 0 in freshwater smolts. This is clearly different from what has been observed in a nonsmoltifying salmonid, the rainbow trout, which shows a sharp decline in plasma PRL levels after seawater transfer (Prunet et al., 1985). The transfer of coho salmon smolts to seawater induced a pattern of PRL change similar to that observed in rainbow trout (Young et al., 1989), although no significant decrease in plasma PRL levels was observed after transfer of chum salmon to s e a w a t e r (Hasegawa et al., 1987). The lack of a further decrease in plasma PRL levels after transfer of Atlantic salmon smolts to seawater suggests that the decrease in PRL levels in fresh water during smoltification is part of the mechanism bringing about changes preparatory for seawater entry. Although any comparison of data obtained in Atlantic salmon and coho salmon must be qualified by acknowledging that differences in rearing conditions may have contributed to some of the differences observed between this and the companion study on coho salmon (Bj6rnsson et al., 1989; Young et al., 1989), there are certain areas where the physiology of the Atlantic smolt seems to diverge from that of the coho smolt. Atlantic salmon smolts appear to undergo a greater development of gill Na+,K+-ATPase activity in fresh water, compared to coho smolts, relative to the level seen in seawater-adapted animals (e.g., L a n g h o r n e and Simpson, 1986; Bj6rnsson et al., 1988). Although some of
361
the reported differences in enzyme activity between the two species may be due to differences in rearing conditions, we have observed similar differences between coho (Boeuf et al., 1978) and Atlantic salmon (Boeuf et al., 1989) reared under identical conditions, although in different years. Additionally, while cortisol enhances gill Na§ activity in coho salmon (Richman and Zaugg, 1987; Bj6rnsson et al., 1987) and changes in plasma cortisol after seawater transfer are consistent with a role of cortisol in hypoosmoregulation (Redding et al., 1984; Young et al., 1989), work on Atlantic salmon has so far failed to implicate cortisol in seawater adaptation (Langdon et al., 1984; Nichols and Weisbart, 1985; Langhorne and Simpson, 1986). From these data, Langhorne and Simpson (1986) have argued that Atlantic salmon smolts are more highly preadapted for seawater life than coho smolts where entry into seawater is accompanied by increases in circulating cortisol and, frequently, relatively large increases in Na§ activity. Whether these differences between the two species are related to the more pronounced decrease seen in the present study in plasma PRL levels (both in duration and value) and the reduction in plasma PRL levels in coho but not Atlantic salmon upon transfer to seawater, rather than an artifact of different rearing conditions, needs further study. In summary, similar changes in thyroid hormones and GH occur during the period of enhanced seawater adaptability of Atlantic and coho salmon. These data support other evidence for a role of GH in hypoosmoregulation in the two species. Although we have observed a different pattern of plasma PRL between the two species during the parr-smolt transformation, and have suggested that this may be related to differences in the d e v e l o p m e n t of gill Na§ activity, relative to seawater values, it is striking that PRL levels declined early in development and that all other hormones measured subsequently in-
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creased. The possibility that PRL influences gill N a + , K + - A T P a s e activity in salmon deserves further investigation. ACKNOWLEDGMENTS We thank A. Leroux and M. Ollitrault for their excellent technical assistance. We gratefully acknowledge Professors T. Hirano and H. Kawauchi and Dr. T. Ogasawara for the provision of salmon growth hormone and antisera. We also express our gratitude to Professor H. A. Bern, Dr. B. Th. Bj6rnsson, and Dr. S. D. McCormick for critical reading of this manuscript. This work was supported in part by IFREMER under Grant 3957b, in part by a NATO grant to P. Prunet, and in part by NOAA, National Sea Grant College Program, Department of Commerce, under Grant NA85AA-D-SG140, projects R/F-101 and R/F-I17 to H. A. Bern, through the California Sea Grant College Program, and in part by the California State Resources Agency. The U.S. government is authorized to reproduce and distribute for governmental purposes.
REFERENCES Avella, M., Schreck, C. B., and Prunet, P. (1989). Influence of stress on plasma prolactin levels in coho salmon, Oncorhynchus kisutch. Submitted for publication. Barron, M. G. (1986). Endocrine control of smoltitication in anadromous salmonids. J. Endocrinol. 108, 313-319. Barton, B. A., Schreck, C. B., Ewing, R. D., Hemingsen, A. R., and Patin6, R. (1985). Change in plasma cortisol during stress and smoltification in coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 59, 468-471. Bj6rnsson, B. Th., and Hansson, T. (1983). Effect of hypophysectomy on the plasma ionic and osmotic balance in rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 49, 240-247. Bjfrnsson, B. T., Yamauchi, K., Nishioka, R. S., Deftos, L. J., and Bern, H. A. (1987). Effect of hypophysectomy and subsequent hormonal replacement therapy on hormonal and osmoregulatory status of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 68, 421-430. Bjfrnsson, B. T., Young, G., Lin, R. J., Deftos, L. J., and Bern, H. A. (1989). Smoltification and seawater adaptation of coho salmon (Oncorynchus kisutch): Plasma calcium regulation, osmoregulation, and calcitonin. Gen. Comp. Endocrinol. 74, 346--354. Boeuf, G. (1987). " C o n t r i b u t i o n ~ l'I~tude de l'Adaptation /i l'Eau de Mer chez les Poissons Salmonid6s--D~termination de Crit~res de Smoltification par Mesure de l'Activit6 (Na § ,K §
ATPasique des Microsomes de la Branchie et des Hormones Thyroi'diennes Plasmatiques," p. 370. Th6se Doct. d'Etat, Universit6 de Bretague Occidentale, Brest, France. Boeuf, G., Lasserre, P., and Harache, Y. (1978). Osmotic adaptation of Oncorhynchus kisutch Walbaum. II. Plasma osmotic and ionic variations and gill Na+,K + ATPase activity of yearling coho salmon transferred to sea water. Aquaculture 15, 35-52. Boeuf, G., Le Bail, P. Y., and Prunet, P. (1989). Growth hormone and thyroid hormones during and after transfers to seawater in the Atlantic salmon, Salmo salar L. Aquaculture, in press. Boeuf, G., and Prunet, P. (1985). Measurement of gill Na+,K+-ATPase activity and plasma thyroid hormones during smoltification in Atlantic salmon (Salmo salar L.). Aquaculture 45, 111-119. Bolton, J. P., Takahashi, A., Kawauchi, H., Kubota, J., and Hirano, T. (1986). Development and validation of a salmon growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 62, 230-238. Bolton, J. P., Collie, N. L., Kawauchi, H., and Hirano, T. (1987). Osmoregulatory actions of growth hormone in rainbow trout (Salmo gairdneri). J. Endocrinol. 112, 63-68. Clarke, W. C., and Nagahama, Y. (1977). Effect of premature transfer to sea water on growth and morphology of the pituitary, thyroid, pancreas and interrenal in juvenile coho salmon, Oncorhynchus kisutch. Canad. J. Zool. 55, 1620-1630. Clarke, W. C., and Shelbourn, J. E. (1985). Growth and development of seawater adaptability by juvenile fall chinook salmon, Oncorhynchus tshawytscha, in relation to temperature. Aquaculture 45, 21-31. De Luze, A., and Leloup, J. (1984). Fish growth hormone enhances peripheral conversion of thyroxine to triidothyronine in the eel Anguilla anguilla L. Gen. Comp. Endocrinol. 56, 308-312. Folmar, L. C., and Dickhoff, W. W. (1980). The parrsmolt transformation (smoltification) and seawater adaptation in salmonids. A review of selected literature. Aquaculture 21, 1-37. Folmar, L. C., and Dickhoff, W. W. (1981). Evaluation of some physiological parameters as predictive indices of smoltification. Aquaculture 23, 309-324. Foskett, J. K., Machen, T. E., and Bern, H. A. (1982). Chloride secretion and conductance of teleost opercular membrane: Effect of prolactin. Amer. J. Physiol. 242, R380-R389. Gallis, J. L., Lasserre, P., and Belloc, F. (1979). Freshwater adaptation in the euryhaline teleost, Chelon labrosus. L Effects of adaptation, prolactin, cortisol, and actinomycin D on plasma os-
HORMONES AND ATLANTIC SALMON SMOLTIFICATION motic balance and (Na§247 in gill and kidney. Gen. Comp. Endocrinol. 38, 1-10. Grau, E. G., Nishioka, R. S., Specker, J. L., and Bern, H. A. (1985). Endocrine involvement in the smoltification of salmon, with special reference to the role of thyroid gland. In "Current Trends in Comparative Endocrinology" (B. Lofts and W. N. Holmes, Eds.). Hong Kong Univ. Press, Hong Kong. Hasegawa, S., Hirano, T., Ogasawara, T., Iwata, M., Akiyama, T., and Arai, S. (1987). Osmoregulatory ability of chum salmon, Oncorhynchus keta, reared in fresh water for prolonged periods. Fish Physiol. Bichern. 4, 101-110. Hirano, T., Prunet, P., Kawauchi, H., Tokahashi, A., Kubota, J., Nishioka, R. S., Bern, H. A., Takada, K., and Ishii, S. (1985). Development and validation of a salmon prolactin radioimmunoassay. Gen. Comp. Endocrinol. 59, 266-276. Hoar, W. S. (1976). Smolt transformation: Evolution, behaviour and physiology. J. Fish. Res. Board. Canad. 33, 1234-1252. Johnston, C. E., and Saunders, R. L. (1981). Parrsmolt transformation of yearling Atlantic salmon (Salrno salar) at several rearing temperatures. Canad. J. Fish. Aquat. Sci. 38, 1189-1198. Kamiya, M. (1972). Hormonal effect on Na§ § ATPase activity in the gill of Japanese eel, Anguilla anguilla, with special reference to seawater adaptation. Endocrinol. Japon. 19, 483--493. Komourdjian, M. P., and Idler, D. R. (1977). Hypophysectomy of rainbow trout Salmo gairdneri and its effect on plasmatic sodium regulation. Gen. Cornp. Endocrinol. 32, 536-542. Komourdjian, M. P., Saunders, R. L., and Fenwick, J. C. (1976). The effect of porcine somatotropin on growth and survival in seawater of Atlantic salmon (Salmo salar) parr. Canad. J. Zool. 54, 531-535. Langdon, J. S., and Thorpe, J. E. (1984). Response of the gill Na+,K§ activity, SDH activity and chloride cells to saltwater adaptation in Atlantic salmon Salmo salar L. parr and smolt. J. Fish. Biol. 24, 323-331. Langdon, J. S., and Thorpe, J. E. (1985). The ontogeny of smoltification: Developmental patterns of gill Na + ,K+-ATPase, SDH, and chloride cells in juvenile Atlantic salmon Salmo salar L. Aquaculture 45, 83-95. Langhorne, P., and Simpson, T. H. (1986). The interrelationship of cortisol, gill Na § ,K § and homeostasis during the parr-smolt transformation of Atlantic salmon (Salmo salar L.). Gen. Comp. Endocrinol. 61, 203-213. Lasserre, P., Boeuf, G., and Harache, Y. (1978). Osmotic adaptation of Onchorhynchus kisutch Wal-
363
baum. I. Seasonal variations of gill Na+,K § ATPase activity in coho salmon, 0 +-age and yearling, reared in fresh water. Aquaculture 14, 365382. Lindhal, K., Lundqvist, H., and Rydevik, M. (1983). Plasma thyroxine levels and thyroid gland histology in Baltic salmon (salmo salar L.) during smoltification. Canad. J. Zool. 61, 1954-1958. Martial, J. A., Baxter, J. D., Goodman, H. M., and Seaburg, P. H. (1977). Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones. Proc. Natl. Acad. Sci. USA 74, 1816-1820. McKenzie, D., Licht, P., and Papkoff, H. (1978). Thyrotropin from amphibian (Rana catesbeiana) pituitaries and evidence for heterothyrotropic activity of bullfrog luteinizing hormone in reptiles. Gen. Cornp. Endocrinol. 36, 566--574. Miwa, S., and Inui, Y. (1985). Effects of L-thyroxine and ovine growth hormone on smoltification of amago salmon (Oncorhynchus rhodurus). Gen. Comp. Endocrinol, 58, 436--442. Nanc6, J. M., Sola, F., Bornancin, M., Boeuf, G., and Dutil, J. D. (1989). Studies of transepithelial Na § exchange in Salmo salar srnolt and postsmolt transferred to seawater. Aquaculture, in press. Nichols, D. J., and Weisbart, M. (1985). Cortisol dynamics during seawater adaptation of Atlantic salmon Salmo salar. Arner. J. Physiol. 248, R651R659. Nishioka, R. S., Bern, H. A., Lal, K. V., Nagahama, Y., and Grau, E. G. (1982). Changes in the endocrine organs of coho salmon during normal and abnormal smoltification--An electron microscope study. Aquaculture 28, 21-38. Nishioka, R. S., Grau, E. G., and Bern, H. A. (1985). In vitro release of growth hormone from the pituitary gland of Tilapia, Oreochrornis mossambicus. Gen. Comp. Endocrinol. 60, 90--94. Nishioka, R. S., Richman, N. H., Young, G., Prunet, P., and Bern, H. A. (1987). Hypophysectomy of coho salmon (Oncorhynchus kisutch) and survival in fresh water and seawater. Aquaculture 65, 343352. Pickford, G. E., Griffith, R. W., Torreti, J., Hendler, E., and Epstein, F. H. (1970). Branchial reduction and renal stimulation of Na § § by prolactin in hypophysectomized killifish in fresh water. Nature (London) 228, 378-379. Prunet, P., and Boeuf, G. (1985). Plasma prolactin level during transfer of rainbow trout (Salmo gairdneri) and Atlantic salmon (Salmo salar) from fresh water to sea water. Aquaculture 45, 167176. Prunet, P., and Boeuf, G. (1989). Plasma prolactin levels during smolting in Atlantic salmon, Salmo salar. Aquaculture, in press.
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Prunet, P., Boeuf, G., and Houdebine, L. M. (1985). Plasma prolactin levels in rainbow trout during the adaptation to different salinities. J. Exp. Zool. 235, 187-196. Prunet, P., Nancr, J. M., Sola, F., Gostan, N., and Bornancin, M. (1989). The effect of ovine growth hormone on Na § and water gill exchange in rainbow trout directly transferred into seawater. Submitted for publication. Reichlin, S. (1974). Regulation of somatotrophic hormone secretion. In "Handbook of Physiology, Sect 7, Endocrinology" (E. Knobil and W. H. Sawyer, Eds.), Vol. 4, pp. 405--447. Williams and Wilkins, Baltimore. Richman, N. H., III, Nishioka, R. S., Young, G., and Bern, H. A. (1987). Effects of cortisol and growth hormone replacement on osmoregulation in hypophysectomized coho salmon (Oncorhynchus kisutch). Gen. Cornp. Endocrinol. 67, 194--201. Richman, N. H., III, and Zaugg, W. W. (1987). Effects of cortisol and growth hormone on osmoregulation in pre- and desmolthqed coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 65, 189-198. Redding, J. M., Schreck, C. B., Birks, E. K., and Ewing, R. D. (1984). Cortisol and its effects on plasma thyroid hormone and electrolyte concentrations in fresh water and during seawater adaptation in yearling coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 56, 146-155. Saunders, R. L., and Henderson, E. B. (1970). Influence of photoperiod on smolt development and growth of Atlantic salmon (Salmo salar). J. Fish. Res. Board Canad. 27, 1295-1311. Saunders, R. L., and Henderson, E. B. (1978).
Changes in gill ATPase activity and smolt status of Atlantic salmon (Salmo salar). J. Fish. Res. Board Canad. 35, 1542-1546. Specker, J. L., and Schreck, C. B. (1982). Changes in plasma corticosteroids during smoltification of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 46, 53-58. Sweeting, R. M., Wagner, G. F., and McKeown, B. A. (1985). Change in plasma glucose, amino acid nitrogen and growth hormone during smoltification and seawater adaptation in coho salmon, Oncorhynchus kisutch. Aquaculture 45, 185-197. Virtanen, E., and Soivio, A. (1985). The pattern of T3, T4, cortisol and Na+,K+-ATPase during smoltification of hatchery-reared Salmo salar and comparison with wild smolts. Aquaculture 45, 97-109. Wedemeyer, G. A., Saunders, R. L., and Clarke, W. C. (1980). Environmental factors affecting smoltification and early marine survival of anadromous salmonids. Mar. Fish. Rev. 42, 1-14. Young, G. (1986). Cortisol secretion in vitro by the interrenal of coho salmon (Oncorhynchus kisutch) during smoltification: Relationship with plasma thyroxine and plasma cortisol. Gen. Comp. Endocrinol. 63, 191-200. Young, G., Bj6rnsson, B. T., Prunet, P., Lin, R. J., and Bern, H. A. (1989). Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch): Plasma prolactin, growth hormone, thyroid hormones, and cortisol. Gen. Comp. Endocrinol. 74, 346-354. Youngson, A. F., and Simpson, T. H. (1984). Changes in serum thyroxine levels during smolting in captive and wild Atlantic salmon, Salmo salar L. J. Fish. Biol. 24, 2%39.
74, 355-364 (1989)
Smoltification and Seawater Adaptation in Atlantic Salmon (Salmo salar): Plasma Prolactin, Growth Hormone, and Thyroid Hormones PATRICK P R U N E T , * GILLES B O E U F , t JONATHAN P. BOLTON,* AND GRAHAM YOUNG:~
*Laboratoire de Physiologic des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cddex, France; ?IFREMER, Centre de Brest, BP 70, 29263 Plouzan~, France; and SDepartment of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720 Accepted October 18, 1988 To obtain more information on the role of prolactin and growth hormone during the parr-smolt transformation of Atlantic salmon, a population of fish in fresh water was sampled from January to June during two consecutive years. Gill Na+,K+-ATPase activity increased steadily during smoltification and a plasma thyroxine peak was observed 2-3 weeks before the gill Na+,K+-ATPase peak. On the basis of these two parameters, smoltification was considered complete in our populations in April 1985 and May 1986. Two peaks in plasma growth hormone levels occurred in 1986, one in mid-April and the second in mid-May. In both cases, these peaks coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. Moreover, the second peak which lasted for 1 month coincided with maximal gill Na + ,K +-ATPase activity. A decrease in plasma prolactin levels was observed during smoltification of Atlantic salmon in 2 consecutive years. During this period of decreasing and low plasma prolactin levels, gill Na +,K+-ATPase activity increased to its highest values. Atlantic salmon smolts were also directly transferred into seawater. After 2 days or more in seawater, plasma prolactin levels were not significantly different from those on Day 0, whereas in fresh water they showed large fluctuations. All these data indicate that growth hormone may play an important role in the development of hypoosmoregulatory activity. Increased hypoosmoregulatory ability also appears to be associated with low prolactin levels. 9 1989AcademicPress, Inc.
The parr-smolt transformation (smoltification) in salmonids involves complex physiological changes and biochemical events. One of the most characteristic features of the smolt is its enhanced seawater adaptability (see Hoar, 1976; Folmar and Dickhoff, 1980; Wedemeyer et al., 1980). Although thyroid hormones have been implicated in the control of smoltification (see Folmar and Dickhoff, 1981; Grau et al., 1985; Barron, 1986), evidence for a precise role of thyroxine and triiodothyronine in the acquisition of seawater adaptability is not yet forthcoming (Boeuf, 1987). Increasing evidence suggests that cortisol, another important hormone in teleost osmoregulation, causes physiological changes associated with seawater adaptation (Specker and
Schreck, 1982; Barton et al., 1985; Virtanen and Soivio, 1985; Young, 1986; Richman and Zaugg, 1987; Bjfrnsson et al., 1987). H o w e v e r , in Atlantic salmon, plasma cortisol does not appear to be directly related to the increase in gill Na § ,K+-ATPase activity observed during smoltification (Langhorne and Simpson, 1986). Although the importance of pituitary hormones in teleost osmoregulation is well documented, few studies have focused on this aspect during smoltification. Several studies indicate that prolactin (PRL) is not necessary for freshwater survival of hypophysectomized salmonids (Komourdjian and Idler, 1977; BjOrnsson and Hanson, 1983; Nishioka et al., 1987). However, 355 0016-6480/89 $1.50 Copyright 9 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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m e a s u r e m e n t o f p l a s m a P R L l e v e l s in s a l m o n i d s t r a n s f e r r e d f r o m f r e s h w a t e r to seawater or the converse support a possible role of PRL as a freshwater-adapting horm o n e ( H i r a n o e t al., 1985; P r u n e t e t al., 1985; P r u n e t a n d B o e u f , 1985). G r o w t h h o r m o n e ( G H ) h a s b e e n i m p l i c a t e d as a s e a w a t e r - a d a p t i n g h o r m o n e in s a l m o n i d s ( K o m o u r d j i a n e t al., 1976; C l a r k e a n d N a g a h a m a , 1977; M i w a a n d Inui, 1985; B o l t o n e t al., 1987) a n d a r i s e in p l a s m a G H l e v e l s during coho salmon smoltification has been r e p o r t e d ( S w e e t i n g e t al., 1985). I n a c o m p a n i o n p a p e r ( Y o u n g e t al., 1989), p l a s m a P R L a n d G H l e v e l s d u r i n g smoltification and seawater adaptation have been reported for coho salmon. The p r e s e n t p a p e r r e p o r t s o n c h a n g e s in p l a s m a PRL and GH levels during Atlantic salmon s m o l t i f i c a t i o n in r e l a t i o n to t h y r o i d h o r m o n e l e v e l s a n d gill N a § 2 4 7 activity. The results are compared with those from studies on the coho salmon (Young et al., 1989; B j O r n s s o n e t al., 1989) a n d a r e d i s c u s s e d in t e r m s o f t h e p o s s i b l e r o l e o f t h e s e h o r m o n e s in t h e d e v e l o p m e n t o f h y poosmoregulatory ability during smoltification.
MATERIALS AND METHODS Animals. Atlantic salmon eggs imported from Norway in 1984 and 1985 were reared in Le Conquet hatchery (Brittany). Yearlings were isolated in 4-m2 Ewos tanks in November preceding the sampling period and were fed on manufactured dry pellets by automatic feeders (ration, 2% body wt/day). The photoperiod was natural and the water temperature during the experimental period (January to June) increased from 5 to 15~ in 1985 and from 3 to 14~ in 1986. In comparison to 1985, in January-February 1986, fish had to face a lower temperature which delayed the natural temperature increase usually observed in spring. Sampling. As our population of Atlantic salmon show a characteristic bimodal distribution of body length and weight, fish were sampled in consecutive years from February to May (1985) and from February to July (1986). Blood samples were obtained from the posterior aorta with a heparinized syringe always at the same time of the day (9--11 AM). Fish were stunned by cranial concussion without anesthesia. Plasma was
collected after centrifugation of blood and kept frozen at - 2 0 ~ until analysis. Gill filaments were taken from the same fish, rinsed in 0.25 M sucrose buffered at pH 7.4 and immediately frozen in liquid nitrogen. Gill Na+,K+-ATPase were measured according to the methods described by Lasserre et al. (1978). During the first year of sampling, 100 fish were transferred from the hatchery to the experimental farm at Brest late in April and were divided between two Ewos tanks. On April 22, the seawater supply of one tank was turned on and the freshwater supply was turned off. In this manner seawater replaced fresh water within 2 hr. Neither group was fed for 3 days before transfer. The salinity of the seawater stayed constant (35 -+ 1%o)during the experiment. Both groups of fish were sampled just before transfer and 1, 2, 4, and 7 days after transfer. Plasma osmolarity was measured using a Roebling freezing point osmometer. Hormone radioimmunoassays. Plasma PRL levels were measured using the method of Prunet et al. (1985). GH levels were determined according to the method of Bolton et al. (1986). The cross-reactivity between these two radioimmunoassays has been estimated at less than 0.3% (Hirano et al., 1985; Bolton et al., 1986). Triiodothyronine and thyroxine levels were measured using the methods of McKenzie et al. (1978) as described in Boeuf and Prunet (1985). Statistical analysis. One-way analysis of variance and the Newman-Keuls multiple range test were used to assess the significance of seasonal changes in plasma hormone levels and gill Na+,K+-ATPase activity. In the transfer experiment, differences at each sampling date between fish in fresh water and in seawater were assessed using Student's t test.
RESULTS Gill N a + , K + - A T P a s e L e v e l s Significant increases (P < 0.01; A N O V A ) in gill N a + , K + - A T P a s e a c t i v i t y o c c u r r e d in b o t h y e a r s o f s t u d y . I n 1985, Na§ activity increased from F e b r u a r y to A p r i l a n d r e a c h e d a p l a t e a u in m i d - A p r i l w h i c h l a s t e d until m i d - M a y (Fig. 1). A s i m i l a r p a t t e r n o f N a + , K § c h a n g e s w a s o b s e r v e d in 1986, a l t h o u g h t h e c u r v e w a s s h i f t e d to t h e r i g h t , w i t h t h e Na§247 increase delayed for a b o u t 1 m o n t h (Fig. 2). Plasma Thyroid Hormone
Levels
F r o m F e b r u a r y to M a r c h 1985 (Fig. 3), plasma thyroxine levels remained low.
357
HORMONES AND ATLANTIC SALMON SMOLTIFICATION
~
/i_ ~176176 thyroxine-
triiodothyronine ---
lO
Feb 9
r
1
r
jan feb mar apr may june 1965
FIG. I. Gill Na+,K+-ATPase activity during the parr-smolt transformation of Atlantic salmon reared in fresh water in 1985. Each point represents a mean and SEM for 10 fish.
However, a sharp rise was observed on March 25 (P < 0.01 compared to values from January and February). Levels stayed significantly higher (P < 0.01) until April 4 but then dropped to initial values and did not show further significant changes. Plasma triiodothyronine stayed low in February and March. However, a slow but steady increase was observed in April and later in the month levels reached a significantly higher value compared to FebruaryMarch (P < 0.05). In 1986 (Fig. 4) the general patterns of plasma triiodothyronine
March
April
May
FIG. 3. Change in plasma thyroxine (-) and triiodothyronine (- - -) levels during the parr-smolt transformation of Atlantic salmon reared in fresh water in 1985. Each point represents a mean and SEM for 8-10 fish.
and thyroxine were similar to those observed in 1985. However, this year was characterized by a delay in the thyroxine peak (April I5 to 21, P < 0.0t compared to values from February and March). After May 25, plasma thyroxine levels increased again and stayed significantly higher until July (P < 0.01 compared to values from April 29 to May 15). Changes in triiodothyronine levels in 1986 were of greater ampli-
0
thyroxine(ng/ml ) triiodothyronine ( ng/ml ) . . . . .
-
-
feb
mar
apr
may
june
T
T
feb
mar
T
apt
T
may
[
june
1986
FIG. 2. Gill Na§ activity during the parr-smolt transformation of Atlantic salmon in 1986. Each point represents a mean and SEM for 10 fish.
FIG. 4. Change in plasma thyroxine (-) and triiodothyronine (- - -) levels during the parr-smolt transformation of Atlantic salmon in fresh water in 1986, Each point represents a mean and SEM for 8-10 fish.
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PRUNET ET AL.
tude than in 1985. A first peak (April 15, P < 0.01 compared to values from preceding dates) was observed just preceding a thyroxine peak (April 24) and later in the season two other triiodothyronine peaks occurred (May 26 and June 24, P < 0.01 for both peaks compared to values from April 29 to May 15). feb
Prolactin Levels In 1985 (Fig. 5), plasma PRL levels decreased significantly (P < 0.01) from an initial value of 7-9 ng/ml in February to 2-3 ng/ml in March. After this initial decrease, PRL levels stayed around 2-4 ng/ml in March-April and no significant difference was observed during this period. In 1986 (Fig. 6), the pattern was similar to 1985. Until early March, levels were high (8-12 ng/ml) and a significant decrease (P < 0.01) was observed on March 14 followed by a long period of consistently low PRL levels (no significant difference from March 15 until July 11). In 1985, smolts were transferred to seawater on April 22. At this time, fish showed high seawater adaptability as indicated by plasma osmotic pressure which stabilized at its seawater level within 24 hr (Fig. 7). An increase in the plasma PRL level, although not significant, was observed 24 hr after transfer to seawater and was followed by a decrease (Fig. 7). However, on Days 2, 4, and 7, PRL levels in the seawater group were not significantly dif-
FIG. during salmon resents
mar
may
june
6. Change in plasma prolactin (PRL) levels the parr-smolt transformation of Atlantic reared in fresh water in 1986. Each point repa mean and SEM for 8-10 fish.
ferent from the initial level on Day 0. In the freshwater control group, PRL levels fluctuated during the experiment, and freshwater levels were only significantly higher than seawater levels on Days 2 and 4.
Growth Hormone Levels (Fig. 8) Plasma GH levels were only measured in 1986. After an initial decrease (February l l, P < 0.01 compared to values from March), no further significant change was observed until April 8 with GH levels re-
A
FW-FW--
FW-SW---
3,oJ
,
.
T4 peak § j Na - K+ATPase
/
/ pe?k
~oi, 0
1.7....-------...~
Feb
FIG. during salmon resents
apr
Morch
April
May
5. Change in plasma prolactin (PRL) levels the parr-smolt transformation of Atlantic reared in fresh water in 1985. Each point repa mean and SEM for 8-10 fish.
.
:
,'.
2
4
6
8 days
FIG. 7. Change in plasma osmotic pressure (A) and prolactin (PRL) levels (B) in Atlantic salmon smolt transferred from fresh water to seawater. The solid line corresponds to the control group kept in fresh water and the dotted line to the group transferred to seawater. Each point represents a mean and SEM for five fish. Asterisks denote significant difference (P < 0.01) in levels between freshwater and seawater fish on the same day.
HORMONES AND ATLANTIC SALMON SMOLTIFICATION
30
20
T
feb
f"
I
mar
apr
r may
june
FIG. 8. Change in plasma growth hormone (GH) levels during parr-smolt transformation of Atlantic salmon reared in fresh water in 1986. Each point represents a mean and SEM for 8-10 fish.
maining low (10-13 ng/ml). A peak in levels occurred on April 15 (P < 0.01 compared to values in March and early April) followed by a sharp decline (P < 0.01). In May, a significant increase (P < 0.01) was observed on May 6; later, GH levels rose steadily to achieve maximum levels of 38 ng/ml on May 26. In June, GH levels stayed significantly (P < 0.01) higher than levels in February and March until early July when the initiation of a decrease occurred. DISCUSSION
The purpose of this study was to obtain information on the possible involvement of PRL and GH in the endocrine control of smoltification in Atlantic salmon by measuring plasma levels of these two hormones. To characterize the stage of the parr-smolt transformation, plasma thyroid hormone levels and gill Na+,K+-ATPase activity were followed. Both parameters are possible indicators of smolt stage (Folmar and Dickhoff, 1981; Langdon and Thorpe, 1984; Boeuf and Prunet, 1985). In 1985 and 1986, these parameters changed during smoltification as already described in Atlantic salmon (Saunders and Henderson, I978; Lindahl et al., 1983; Youngson
359
and Simpson, 1984; Boeuf and Prunet, 1985; Virtanen and Soivio, 1985; Langhorne and Simpson, 1986). Thus, gill Na + ,K+-ATPase activity increased steadily from February to April and this increase paralleled the increase in seawater adapta b i l i t y w h i c h was m a x i m u m w h e n Na + ,K+-ATPase reached a plateau (Boeuf et al., 1985; Langdon and Thorpe, 1985). The most prominent change in thyroid hormone levels was the appearance of a thyroxine peak which occurred during the Na+,K+-ATPase increase, generally 2-3 weeks before peak enzyme activity was achieved. Although this thyroxine peak occ u r r e d during a period of high gill Na +,K +-ATPase activity, Boeuf (1987) did not find a coincidence of this peak with maximum seawater adaptability in Atlantic salmon. Monitoring of these parameters indicated that on the basis of seawater adaptability, smoltification was complete in our population in April 1985 and May 1986. The delay in 1986 compared with 1985 may be due to the lower temperatures during the presmoltification period in 1986. Temperature is an important modulator of salmon smoltification (see Saunders and Henderson, 1970; Wedemeyer et al., 1980; Clarke and Shelbourn, 1985). An important fnding in the present study was the increase in plasma GH levels during smoltification of Atlantic salmon, in agreement with the increase reported in plasma GH during coho salmon smoltification (Sweeting et al., 1985; Young et al., 1989). Histological studies of coho pituitaries also indicate increased secretory activity of GH cells during smoltifcation (Clarke and Nagahama, 1978; Nishioka et al., 1982). In our study, two GH peaks are evident, the first one occurring in mid-April and the second occurring in mid-May. In both cases, these GH changes coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. These data substantiate several reports indicating a close reIationship between GH and thyroid hormones in fish. The coinci-
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dence between the triiodothyronine peak and the GH increase agrees with the classical picture in mammals of the stimulation by triiodothyronine of GH synthesis and release (Reichlin, 1974; Martial et al., 1977). However, this effect was not observed in tilapia using incubated pituitaries (Nishioka et al., 1985). Salmon GH has also been shown to enhance peripheral conversion of thyroxine to triiodothyronine in eels (De Luze and Leloup, 1984). However, it is not clear from the present study if such a relationship exists in Atlantic salmon as high GH levels were not always coupled with high triiodothyronine levels. Clearly, more investigations are needed to clarify the relationship between GH and thyroid hormones during smoltification. The second GH increase, which lasted for about I m o n t h , c o i n c i d e d with m a x i m a l gill Na+,K+-ATPase activity. Moreover, the GH decline in late June/early July occurred at t h e s a m e t i m e as a d e c r e a s e in Na+,K+-ATPase activity. These results are in agreement with those of previous studies indicating a stimulatory effect of GH on this enzyme's activity in presmolt coho salmon and in hypophysectomized yearling coho salmon (Richman and Zaugg, 1987; Bj6rnsson et al., 1987). However, other workers did not observe an effect of GH treatment on Na + ,K +-ATPase activity (Miwa and Inui, 1985; Richman et al., 1987). When data from all sampling dates in this study were pooled (n = 148), a significant correlation (r = 0.3, P < 0.01) was obtained b e t w e e n GH levels and gill Na+,K§ activity, supporting the importance of a GH peak for seawater adaptation of Atlantic salmon smolts. Recently, Nanc6 et al. (1989), using an isolated perifused head preparation, have shown that Atlantic salmon smolts are able to establish a negative Na § flux at the level of the gill filament immediately after seawater transfer. Moreover, using the same perifusion technique, we have observed a similar phenomenon in rainbow trout pretreated with GH in fresh water just before
seawater transfer (Prunet et al., 1989). In general, these data support the evidence from the present study for a possible role of GH in salmon smoltification. A decrease in plasma PRL levels was observed during smoltification in 2 consecutive years. The d e c r e a s e o c c u r r e d in March; thereafter PRL levels remained low even during the desmoltification period (June-July). During this period of decreasing a n d l o w p l a s m a P R L l e v e l s , Na+,K§ activity increased and reached its maximal level. In coho salmon, the PRL peak in late March/early April is followed by an abrupt decrease and PRL remained at a basal level for about 1 month before increasing again (Young et al., 1989). Interestingly, this decrease in PRL level c o i n c i d e d w i t h an i n c r e a s e o f Na§ activity (Bj6rnsson et al., 1989). Several studies have already shown an inhibitory effect of PRL on this gill enzyme's activity in other fish species (Pickford et al., 1970; Kamiya, 1972; Gallis et al., 1976; Foskett et al., 1982). Hence, it is conceivable that during smoltification and the preadaptive development of seawater osmoregulatory mechanisms, the decrease in PRL levels might permit gill Na + ,K +-ATPase activity to increase. This hypothesis is reinforced by recent data indicating that decreasing plasma PRL levels are characteristic of smolts, as parr from the same population sampled during the same period did not show this phenomenon (Prunet and Boeuf, 1989). The transfer experiment on Atlantic salmon smolts partially supports this hypothesis. In the freshwater group sampled between April 22 and 27, fluctuations in PRL levels were observed. Because these animals were transferred from the hatchery to the experimental farm only several days before the beginning of the experiment and were maintained under different rearing conditions, these fluctuations may reflect stress: recently, chronic stress has been shown to increase PRL levels in coho salmon and in rainbow trout (Avella et al.,
H O R M O N E S A N D A T L A N T I C SALMON S M O L T I F I C A T I O N
1989; Prunet and Gonnet, unpublished data). In the present study, plasma PRL increased 24 hr after transfer to seawater and thereafter decreased to basal levels. After 7 days in seawater, no significant differences were observed between the seawater group and the freshwater control group. These data are in agreement with other similar transfer experiments using Atlantic salmon smolts (Prunet and Boeuf, 1985). It is also interesting to note that after 2 days or more in seawater, plasma PRL levels in seawater-adapting fish were not significantly different to those measured on Day 0 in freshwater smolts. This is clearly different from what has been observed in a nonsmoltifying salmonid, the rainbow trout, which shows a sharp decline in plasma PRL levels after seawater transfer (Prunet et al., 1985). The transfer of coho salmon smolts to seawater induced a pattern of PRL change similar to that observed in rainbow trout (Young et al., 1989), although no significant decrease in plasma PRL levels was observed after transfer of chum salmon to s e a w a t e r (Hasegawa et al., 1987). The lack of a further decrease in plasma PRL levels after transfer of Atlantic salmon smolts to seawater suggests that the decrease in PRL levels in fresh water during smoltification is part of the mechanism bringing about changes preparatory for seawater entry. Although any comparison of data obtained in Atlantic salmon and coho salmon must be qualified by acknowledging that differences in rearing conditions may have contributed to some of the differences observed between this and the companion study on coho salmon (Bj6rnsson et al., 1989; Young et al., 1989), there are certain areas where the physiology of the Atlantic smolt seems to diverge from that of the coho smolt. Atlantic salmon smolts appear to undergo a greater development of gill Na+,K+-ATPase activity in fresh water, compared to coho smolts, relative to the level seen in seawater-adapted animals (e.g., L a n g h o r n e and Simpson, 1986; Bj6rnsson et al., 1988). Although some of
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the reported differences in enzyme activity between the two species may be due to differences in rearing conditions, we have observed similar differences between coho (Boeuf et al., 1978) and Atlantic salmon (Boeuf et al., 1989) reared under identical conditions, although in different years. Additionally, while cortisol enhances gill Na§ activity in coho salmon (Richman and Zaugg, 1987; Bj6rnsson et al., 1987) and changes in plasma cortisol after seawater transfer are consistent with a role of cortisol in hypoosmoregulation (Redding et al., 1984; Young et al., 1989), work on Atlantic salmon has so far failed to implicate cortisol in seawater adaptation (Langdon et al., 1984; Nichols and Weisbart, 1985; Langhorne and Simpson, 1986). From these data, Langhorne and Simpson (1986) have argued that Atlantic salmon smolts are more highly preadapted for seawater life than coho smolts where entry into seawater is accompanied by increases in circulating cortisol and, frequently, relatively large increases in Na§ activity. Whether these differences between the two species are related to the more pronounced decrease seen in the present study in plasma PRL levels (both in duration and value) and the reduction in plasma PRL levels in coho but not Atlantic salmon upon transfer to seawater, rather than an artifact of different rearing conditions, needs further study. In summary, similar changes in thyroid hormones and GH occur during the period of enhanced seawater adaptability of Atlantic and coho salmon. These data support other evidence for a role of GH in hypoosmoregulation in the two species. Although we have observed a different pattern of plasma PRL between the two species during the parr-smolt transformation, and have suggested that this may be related to differences in the d e v e l o p m e n t of gill Na§ activity, relative to seawater values, it is striking that PRL levels declined early in development and that all other hormones measured subsequently in-
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creased. The possibility that PRL influences gill N a + , K + - A T P a s e activity in salmon deserves further investigation. ACKNOWLEDGMENTS We thank A. Leroux and M. Ollitrault for their excellent technical assistance. We gratefully acknowledge Professors T. Hirano and H. Kawauchi and Dr. T. Ogasawara for the provision of salmon growth hormone and antisera. We also express our gratitude to Professor H. A. Bern, Dr. B. Th. Bj6rnsson, and Dr. S. D. McCormick for critical reading of this manuscript. This work was supported in part by IFREMER under Grant 3957b, in part by a NATO grant to P. Prunet, and in part by NOAA, National Sea Grant College Program, Department of Commerce, under Grant NA85AA-D-SG140, projects R/F-101 and R/F-I17 to H. A. Bern, through the California Sea Grant College Program, and in part by the California State Resources Agency. The U.S. government is authorized to reproduce and distribute for governmental purposes.
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