THE SHIFT IN VISUAL PIGMENT DOMINANCE IN THE RETINAE OF JUVENILE COHO SALMON (ONCORHYNCHUS KISUTCH): AN INDICATOR OF SMOLT STATUS

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1 J. exp. Biol. 195, (1994) Printed in Great Britain The Company of Biologists Limited THE SHIFT IN VISUAL PIGMENT DOMINANCE IN THE RETINAE OF JUVENILE COHO SALMON (ONCORHYNCHUS KISUTCH): AN INDICATOR OF SMOLT STATUS G. ALEXANDER, R. SWEETING AND B. MCKEOWN Department of Biological Sciences, Simon Fraser University, Burnaby BC, Canada V5A 1S6 Accepted 15 June 1994 Summary Smolting juvenile coho salmon were sampled to determine (1) whether a correlation between hypo-osmoregulatory ability and visual pigment composition existed and (2) whether the hormone 3,5,3 -tri-iodothyronine (T 3) was playing a role in the visual pigment conversion process. Plasma sodium levels of seawater-challenged fish (30 ) indicated that there was a 5 week period of optimal ability to excrete excess plasma sodium ions (hypo-osmoregulation) in the late spring/early summer that represented the window of opportunity for the entry or introduction to sea water of the salmon. Early in the smoltification process, the vitamin-a2-based visual pigment porphyropsin increased its dominance in the retinae, and radioimmunoassay of plasma indicated that T 3 levels were at a maximum prior to this increase in porphyropsin. As the parr smolt transformation continued, there was a steady decrease in the relative amounts of porphyropsin, indicating that the retinae were favouring the acquisition of rhodopsin. Rhodopsin dominance virtually coincided with the period of best hypo-osmoregulatory ability. Subsequently, the salmon showed a loss of hypo-osmoregulatory ability and concomitant increases in the amount of porphyropsin in the retina were observed. The relationship between the visual pigment shift and the smoltification process is discussed in terms of preparation for migration and thyroid hormone involvement, and the use of retinal visual pigment composition as an index of smolt status is proposed. Introduction The smoltification process transforms anadromous salmonids from stream-dwelling parr to seawater-adaptable smolts (Hoar, 1976; McKeown, 1984; Thorpe et al. 1984; McCormick and Saunders, 1987; Dickhoff et al. 1990) and is characterized by timely changes in metabolism, growth, osmoregulation (reviewed by Hoar, 1976; Wedermeyer et al. 1980; Barron, 1986; Hoar, 1988), behaviour and olfaction (Scholz et al. 1985; White et al. 1990). Hormones direct this metamorphic process (Barron, 1986; Dickhoff et al. 1990), with growth hormone (GH), cortisol (Richman and Zaugg, 1987), prolactin (PRL) and the thyroid hormones (TH) all being involved (Dickhoff et al. 1978). Thyroid hormones play perhaps the greatest role in this transformation; they have been shown to Key words: thyroid hormones, visual pigment, smoltification, Oncorhynchus kisutch.

2 186 G. ALEXANDER, R. SWEETING AND B. MCKEOWN affect body silvering, behaviour, osmoregulation, growth and learning ability (Higgs et al. 1982; Dickhoff and Sullivan, 1987; Morin et al. 1989). Salmonids have a paired-pigment visual system (Beatty, 1966; Allen et al. 1973, 1982; McFarland and Allen, 1977); that is, their photoreceptors may contain the visual pigments rhodopsin or porphyropsin. Although the total amount of visual pigment in the photoreceptors may remain relatively constant, the relative proportions of rhodopsin and porphyropsin may vary with season or developmental stage (Beatty, 1966; Allen and McFarland, 1973). Rhodopsin and porphyropsin have the same large transmembrane opsin protein; however, the prosthetic groups, or chromophores, found within these opsins distinguish the two visual pigments. Opsin proteins are found in the membranes of photoreceptor outer segments and the cis form of vitamin A1 aldehyde (retinal) or vitamin A2 aldehyde (3-dehydroretinal) is the chromophore found within the opsin, forming rhodopsin and porphyropsin, respectively. The second double bond seen in the carbon ring of 3-dehydroretinal is responsible for the max of porphyropsin being shifted to a longer wavelength than that of rhodopsin, effectively causing a similar shift in spectral sensitivity. Retinae with mixtures of rhodopsin and porphyropsin have intermediate spectral sensitivities. Juvenile salmonids in fresh water, a medium that favours longer wavelengths of light, possess porphyropsin-dominated retinae whereas juvenile salmonids in sea water, a medium favouring short wavelengths, have retinae dominated by rhodopsin (Bridges and Delisle, 1974). This type of shift in visual pigment dominance is accomplished by opsin apoproteins in the photoreceptors being renewed with retinal or 3-dehydroretinal to reflect the appropriate visual pigment. It is thought that this labile nature of the visual pigment composition permits the fish to change the spectral sensitivity of its visual system to match the light environment (Munz and McFarland, 1977; Bowmaker, 1990). This follows the sensitivity hypothesis; i.e. that the pigment composition is adjusted to effect the capture of the greatest number of quanta of light and to permit the organism to detect predator or prey better (Lythgoe, 1972; Muntz and Mouat, 1984). Several researchers have speculated that this change in visual pigment composition may be a feature of the smoltification process (Jacquest and Beatty, 1972; Dartnall, 1972; Evans and Fernald, 1990) and that visual pigment composition can be affected by the same hormones that direct smoltification (Cristy, 1974; Allen and Cristy, 1978). However, it has not been conclusively established whether these changes in visual pigment composition were part of the parr smolt transformation or were simply responses to the light environment and were only coincidental to the parr smolt transformation. The present study was undertaken to determine whether the change from a porphyropsin- to a rhodopsin-dominated visual system in coho salmon (Oncorhynchus kisutch) is one of the developmental processes of smoltification and may be used as an index of smoltification. Materials and methods Fish sampling protocol Over a period of 26 weeks (21 January to 14 July, 1992), yearling juvenile coho salmon

3 Visual pigments in juvenile salmon Daylight hours 16 Water temperature ( C) Water temperature Day length (h) Week Fig. 1. Water temperatures and day length in the outdoor raceways at Capilano Hatchery, Vancouver. The sampled coho salmon experienced the temperatures and day lengths shown throughout the course of the experiment. Day length data were provided by Environment Canada. [Oncorhynchus kisutch (Walbaum)] were sampled weekly from Capilano Hatchery at Vancouver, British Columbia, Canada. These fish were reared in outdoor raceways and during the sampling period experienced natural water temperatures and day length (Fig. 1). Twenty-four fish were randomly netted from the raceway and divided into two equal groups; one group was placed in a 100 l tank containing hatchery water, while the other group was placed into a 100 l tank containing 30 sea water, both at raceway temperature. These tanks were located in a lightproof cold-room and the fish were illuminated with a Kodak safelight (12 W) for 24 h. All subsequent manipulations of animals and retinal samples were performed under darkroom conditions. Fish (N=10) from each group were netted and killed by a sharp blow to the head. After mass and length measurements, blood samples were obtained by transection of the caudal peduncle and collected into ammonium-heparin-coated microhaematocrit tubes. These tubes were centrifuged (1800 g for 3 min) to obtain the plasma fraction and were immediately frozen and stored at 30 C. The plasma of the freshwater fish was used to determine circulating 3,5,3 -triiodothyronine (T 3 ) levels. The plasma of the seawaterchallenged fish was used to determine hypo-osmoregulatory ability (i.e. state of smoltification) by analysing plasma sodium concentration (Blackburn and Clarke, 1987; Franklin et al. 1992). Samples of visual pigments from the freshwater group were obtained by removing the eyes after blood collection. They were cleaned with distilled water and hemisected in a Petri dish containing a solution of ice-cold aluminium potassium sulphate (potassium alum) (4 % w/v in double-distilled water). The retinae and associated pigment epithelia of each fish were removed from the fundus with forceps and placed in a 15 ml opaque Nalgene bottle containing 10 ml of ice-cold potassium alum solution. This solution

4 188 G. ALEXANDER, R. SWEETING AND B. MCKEOWN eliminated contamination by blood proteins (Sillman et al. 1990). These bottles were then wrapped in aluminium foil, placed on dry ice and stored at 30 C. Preparation and analysis of visual pigments After thawing, the tissue samples of each fish were treated as follows. The retinal tissue was poured into a Petri dish containing ice-cold distilled water and the extraneous tissue was dissected away. The remaining tissue was poured into a 15 ml centrifuge tube, 10 ml of cold double-distilled water was added, and the tissue was centrifuged lightly (approximately 4000 g) for 5 min. The supernatant was discarded and the pellet resuspended and washed twice with 10 ml of ice-cold double-distilled water and a third time with 10 ml of ice-cold Tris maleate buffer at ph 7.3 (Sillman et al. 1990). The tissue was then placed in an ice-cold 15 ml rotary glass homogenizer and the visual pigments were extracted with 0.4 ml of 2 % digitonin prepared as described by Bridges (1977). After 8 10 strokes of the homogenizer, the homogenate was poured into 1.5 ml ultracentrifuge tubes (Beckman) and incubated at 23 C for 1 h. After centrifugation ( g) for 15 min at 4 C, the supernatant was removed with a Pasteur pipette and placed in a second 1.5 ml ultracentrifuge tube, buffered with saturated sodium borate (1/10 v/v) to bring the ph to approximately 8.6, and then stored in darkness at 20 C for 4 days to reduce the possibility of transient turbidity (Munz and Beatty, 1965). After thawing and vortexing, the extracts were transferred by Pasteur pipette to 1.5 ml microcuvettes for analysis. Spectophotometric analysis Prior to spectrophotometric analysis, each sample received 0.05 ml of freshly prepared 0.2 mol l 1 neutralized hydroxylamine sulphate (Dartnall, 1968). Hydroxylamine converts the photoisomerized chromophore into an oxime that has an absorbance peak of 387 nm for 3-dehydroretinal oxime (porphyropsin) and 365 nm for retinal oxime (rhodopsin). This not only reduces the interference of photoproducts with the visual pigment difference spectrum but also permits confirmation of visual pigment complement (Sillman et al. 1990). Retinal samples were analyzed using the bleaching method described by Munz and Beatty (1965), Beatty (1966, 1969) and Tsin (1979). Samples were placed into a temperature-controlled cell holder (23±0.3 C) in a computer-driven Spectronic 3000 array recording spectrophotometer (Milton Roy). The optical densities of the samples before bleaching were determined from 360 to 650 nm. The samples were then transferred to a bleaching apparatus, consisting of a cuvette holder and a 40 W quartz halogen lamp placed 40 cm above the cuvettes. The samples were exposed to fullspectrum light for 12 min, by which time the extracts had lost their characteristic pink colour. The cuvettes were then returned to the spectrophotometer and bleached spectra were measured. Referring to templates from Munz and Beatty (1965), the per cent porphyropsin was estimated by calculation from the normalized difference spectrum of each extract. These values were converted to molar per cent porphyropsin by using the molar extinction coefficients for rhodopsin (40 600) and porphyropsin (30 000) (Tsin and

5 Visual pigments in juvenile salmon 189 Beatty, 1977). The molar per cent porphyropsin represents the relative amount of porphyropsin as a percentage of total visual pigment content. For example, a retina with 30 molar per cent porphyropsin contains 30 % porphyropsin and 70 % rhodopsin. Plasma analysis Plasma T 3 of the freshwater fish was determined by using a T 3 radioimmunoassay (RIA) kit (Incstar Corp., Stillwater, MN). Duplicate 25 l plasma samples were placed in antibody-coated tubes and incubated with 125 I-labelled T 3 tracer. After incubation and aspiration, the tubes were counted on a gamma counter and the amount of T 3 in the plasma was determined by interpolation from a standard curve. The plasma sodium levels of seawater-challenged and control fish were determined by flame spectroscopy (Pye-Unicam, model SP 191), utilizing duplicate 5 l plasma samples diluted with 5 ml of double-distilled water. Statistical analyses Data shown in figures are means ± S.E.M. Analysis of variance (ANOVA) was used to compare means, and Bartlett s test was used for assessment of homogeneity of group variances. Asterisks in figures are assigned to means that are significantly different (P<0.05) from their immediate predecessor. Results Plasma sodium levels On the basis of the seawater challenges of the smoltifying coho salmon, temporal changes in their ability to maintain plasma sodium levels were observed to occur in four distinct phases (Fig. 2). Phase I was from the beginning (21 January) to the fourteenth week of the study (21 April). The mean plasma sodium values of the fish were initially 199±6 mmol l 1 which, over the next 13 weeks, increased slightly to 215±6 mmol l 1. During phase II, which occurred between weeks 15 and 18 (28 April to 19 May), there was a rapid and significant drop in plasma sodium levels to 175±4 mmol l 1. This last value was the lowest measured in this study. Phase III took place during the next 4 weeks (26 May to 9 June), when a plateau of continuing low plasma sodium values was seen (178±4 to 180±6 mmol l 1 ). Phase IV represents the final 4 weeks of the study (16 June to 14 July), when there was initially a significant and rapid increase of approximately 15 mmol l 1 in plasma sodium values followed by levels that were in the same range as in phase I (196±2 to 205±4 mmol l 1 ). Visual pigment composition The results of spectrophotometric analysis of retinal extracts are shown in Fig. 3. These data represent the mean molar per cent porphyropsin in the retinae of the freshwater fish that were sampled and are evaluated in the same four phases as established for the sodium results. During the first half of phase I, the mean molar per cent porphyropsin was %. These mean values rapidly increased to 78 % by week 8 and stayed above 60 % for the rest of phase I. During phase II, there was initially a significant drop to 45 %, and

6 190 G. ALEXANDER, R. SWEETING AND B. MCKEOWN [Plasma sodium] (mmol l 1 ) Phase I Week Fig. 2. Plasma sodium concentration of coho salmon transferred into 30 sea water for 24 h. Values are mean values ± S.E.M. (N=10). Phases (I IV) shown represent trends in hypoosmoregulatory ability of these salmon. For details see text. Values marked by an asterisk are significantly different (P<0.05) from the preceding point. the visual pigment composition then remained in the range % for the next 3 weeks. During the following week, which marks the beginning of phase III, there was a significant decline in mean per cent porphyropsin to 16 %, where it levelled off and remained for the rest of this phase and for the first week of phase IV. For the remainder of phase IV, the porphyropsin levels increased significantly to values between 22 and 33 %. Plasma T 3 titres The first 5 weeks of phase I saw rapid and significant declines in plasma T 3 levels from a high of 14.8±2.2 ng ml 1 to 3.2±0.1 ng ml 1 (Fig. 4). Low levels (2 4 ng ml 1 ) of plasma T 3 remained until the last week of phase I, when there was a significant increase to 7.2±1.0 ng ml 1. In phase II, the T 3 levels again dropped significantly; however, these levels were between 3.9 and 5.8 ng ml 1, approximately 2 3 ng ml 1 higher than the values of the previous phase. Phase III plasma T 3 titres, in terms of numerical value and scatter, were similar to those measured in phase II. Reduced plasma T 3 levels were measured in phase IV ( ng ml 1 ). II III IV Discussion In the first three phases in this study, the changes in the fishes ability to regulate plasma sodium levels can be related to the parr smolt transformation. During phase I, which encompassed the late winter to mid-spring seasons, the salmon showed poor ability to excrete excess sodium ions, an indication that the fish still had a physiology appropriate for fresh water. Phase II, which occurred during the late spring, documents a progressive improvement in hypo-osmoregulation; when the fish were exposed to 30

7 Visual pigments in juvenile salmon Molar per cent porphyropsin (%) Phase I Week Fig. 3. Molar per cent porphyropsin found in the retinae of smolting coho salmon. Values are mean ± S.E.M.(N=10). Values marked by an asterisk are significantly different (P<0.05) from the preceding point. Phases I IV are as in Fig. 2. II III IV sea water, they showed a greater ability to excrete excess sodium ions. This ability is important if the salmon are to be able to adapt to the marine environment, as premature chronic exposure to sea water can be lethal or metabolically costly. By the end of phase II, the coho salmon were able to maintain appropriate physiological levels of sodium when exposed to sea water. This hypo-osmoregulatory ability, which can be used as a index of smoltification (Blackburn and Clarke, 1987; Franklin et al. 1992), indicates that the fish in this study can be considered to have acquired smolt status in the last week of phase II. Continuing low sodium levels in phase III indicate that smolt status was maintained during this phase and that the fish were physiologically prepared for seawater entry during this period. The timing of release for downstream migration may be critical, as preparedness for the marine phase of the coho s anadromous life-history does not last indefinitely. This was shown by the reduction in hypo-osmoregulatory ability in phase IV. Loss of hypo-osmoregulatory ability indicates that the cohos were re-acquiring a freshwater physiology and, in essence, were preparing for another year in the freshwater environment. This reduction in hypo-osmoregulatory ability appeared to be rapid and may be characteristic of coho salmon undergoing the desmolting process. This process, also known as parr-reversion, occurs in coho salmon that are not exposed to sea water after they have smoltified (Baggerman, 1960; Zaugg and McLain, 1970). The results of these seawater challenges have provided us with a convenient measure of the progress of the parr smolt transformation of the fish of this study. As indicated, the change from parr to smolt can be described in four distinct phases: phase I, the pre-smolt stage; phase II, the rapid transition to smolt status; phase III, the maintenance of smolt status and, therefore the window of opportunity for seaward migration ; phase IV, a

8 192 G. ALEXANDER, R. SWEETING AND B. MCKEOWN [Plasma T3] (ng ml 1 ) Phase I II III IV Week Fig. 4. Plasma T 3 titres found in blood samples from smolting coho salmon. Values are mean ± S.E.M. (N=10). Values marked by an asterisk are significantly different (P<0.05) from the preceding point. Phases I IV are as in Fig. 2. reduction in smolt-like osmoregulatory ability. The distinct nature of these phases provides an investigator with the ability to assess the smolt status of salmonids from their hypo-osmoregulatory ability by subjecting them to seawater challenges. However, this kind of assessment, when used as a sole criterion and performed in isolated circumstances, could be misleading. For example, early in the smoltification process, there can be a lot of variation in the effects of seawater challenges as a result of handling stress or compromised health during the challenge procedure. This may result in higher plasma sodium values than appropriate for the actual stage of smoltification (Blackburn and Clarke, 1987). Estimating the smolt status of the fish in this study could be subject to the same pitfalls; however, they were sampled on a regular basis and the smolt status of the fish was estimated from measured trends over a number of weeks rather than single sampling episodes. Our sampling protocol provides confidence in the observation that the progress of the parr smolt transformation of coho salmon can be found in the four distinct phases previously described. The changes in the visual pigment composition of the fish were significantly correlated (P<0.005) with their ability to hypo-osmoregulate (Fig. 5). In the first 9 weeks of phase I, the fish exhibited an increase in the molar per cent porphyropsin in their retinae, followed by relatively constant levels of this visual pigment for the remainder of this phase. Interestingly, this initial increase in porphyropsin levels was not expected, as increases in light intensity and water temperature, the conditions the fish were experiencing (Fig. 1), have been shown to cause a decrease in relative amounts of

9 Visual pigments in juvenile salmon [Plasma sodium] (mmol l 1 ) Sodium Porphyropsin Molar per cent porphyropsin (%) 160 Phase I Week Fig. 5. Plots of mean plasma sodium concentration and molar per cent porphyropsin during the course of smoltification of the coho salmon (N=10). The trends in hypo-osmoregulatory ability and visual pigment composition are evident and correlation analysis of the mean values indicate a significant correlation (P<0.005) between plasma sodium concentration and molar per cent porphyropsin. For clarity, error bars are not shown. porphyropsin in the retinae of rainbow trout (Oncorhynchus mykiss) and kokanee salmon (Oncorhynchus nerka) (Beatty, 1966; Allen et al. 1973; Munz and McFarland, 1977; Tsin and Beatty, 1977; Tsin, 1979). It is possible that there was an endocrine influence directing this paradoxical visual pigment shift as a number of studies have shown that thyroid hormones can alter visual pigment composition in kokanee salmon (Beatty, 1969) and trout (Cristy, 1974). From week 10 to the end of phase I (week 14), while the fish were continuing the parr smolt transformation and were approaching smolt status, the porphyropsin trend reversed, and there was a steady decline in the mean molar per cent porphyropsin, indicating that the visual system was concomitantly increasing its molar per cent rhodopsin. During phase II, this increase in rhodopsin content continued and a dominance in rhodopsin (>50 % molar composition) was seen. This conversion process culminated in retinae containing over 80 % rhodopsin (18 % porphyropsin, 82 % rhodopsin) only 1 week after the fish acquired smolt status (phase III, week 19). The retinal domination by rhodopsin continued for 5 weeks. Although out of phase by 1 week, this domination of the retina by an A1-based chromophore suggests that the conversion to a rhodopsindominated retina may have been timed to coincide with the downstream migration period of these coho salmon. The coho salmon that are reared at the hatchery facility utilized in this study have historically migrated at this time and the results of downstream captures have indicated that cohorts to the coho salmon in this investigation entered the marine environment during this period (week 19). II III IV 0

10 194 G. ALEXANDER, R. SWEETING AND B. MCKEOWN Downstream migration ultimately results in the smolt entering the marine environment and it would not only be necessary for the fish to be prepared in hypo-osmoregulatory terms, it would also be an advantage if the visual system were also prepared. Compared with the freshwater light environment, the marine light environment favours the passage of shorter wavelengths of light, and retinae that have converted to rhodopsin will have spectral sensitivity curves that better match the marine light environment. Rhodopsin dominance would thus be of importance to the coho salmon in order to provide optimal visual acuity for the oceanic portion of their life-history. In concordance with the other preparatory changes seen in the salmonid while it is still in fresh water, such as the attainment of hypo-osmoregulatory ability, the visual pigment conversion process may also be considered to be a feature of the parr smolt transformation. The association between visual pigment composition and smolt status is strengthened by the observation that, when smolt status was being lost, corresponding changes in visual pigment composition were also seen. During phase IV, there was a small but significant decline in sodium regulation (Fig. 2), indicating a possible reversion to parrlike characteristics (Avella et al. 1990). Presumably, as the visual pigment shift from porphyropsin to rhodopsin appeared to be smoltification-associated, a return to a porphyropsin-dominated retina would also be expected in these parr-revertants. Indeed, as the coho salmon were losing their hypo-osmoregulatory abilities, the visual pigment composition began to follow the same trend: a return to levels seen in the pre-smolt stage. For those coho salmon that do not complete the parr smolt transformation, or are unable to migrate to the ocean, the overwintering period in fresh water would require a return to pre-smolt characteristics, such as a loss of hypo-osmoregulatory ability. This would also include a return to porphyropsin-dominated retinae so that visual acuity is maximized for the light environment found in fresh water. In this study, the changes in the visual system and in osmoregulatory ability were virtually coincident and suggest that the change in visual pigment composition may be as much a feature of the desmolting process as is the loss of sodium regulation. Thyroid hormones may have influenced the changes in visual pigment composition of the fish. Over the 8 weeks after the T 3 maximum in week 1 (Fig. 4), there was an increase in the molar per cent porphyropsin. Thyroid hormone studies by Beatty (1969) have demonstrated an 80 % increase in porphyropsin levels 3 4 weeks after T 4 (L-thyroxine) injections in kokanee salmon. The initial high levels of T 3, which were observed in this study, can thus be implicated in the rise of porphyropsin content of the retinae as these increases occurred in the 8 weeks following the T 3 maximum. Although the retinae may respond immediately to T 3 -directed production of porphyropsin, complete turnover of photoreceptor visual pigment content has been shown to take 6 weeks in frogs (Young, 1967), 3 6 weeks in the golden rudd (Scardinius erythrophthalmus L.) (Bridges and Yoshikami, 1970) and 4 7 weeks in rainbow trout (Tsin, 1979). As smoltification progressed, there was a steady shift to rhodopsin-dominance in the retinae, but the role of T 3 was not quite as evident. During the 10 week shift from a porphyropsin- to a rhodopsin-dominated retina (weeks 9 19), although there was a minor elevation in T 3 levels in week 14 (Fig. 4), and rhodopsin dominance was seen 4 weeks later, a cause-and-effect relationship cannot be inferred. The administration of T 4 has

11 Visual pigments in juvenile salmon 195 been shown to result in increases in porphyropsin levels in rainbow trout (Jacquest and Beatty, 1972; Cristy, 1974) and the same effect was seen in kokanee salmon with T 4 and T 3 injections (Beatty, 1969). One would expect higher plasma levels of T 3 to have the same result in coho salmon; however, it is possible that the minor elevations seen in week 14 were not sufficient to affect the visual pigment composition. Low levels of T 3, such as those seen in weeks 7 12, may permit the retina to respond more directly to other factors that may affect visual pigment composition, for example light (Allen, 1971), temperature (Allen and McFarland, 1973) or hormones such as prolactin (Cristy, 1974). It is possible that high plasma levels of T 3 are required for porphyropsin dominance, while loss of porphyropsin dominance may be associated with, or possibly depend on, lower levels of T 3. Beatty (1969) observed that, after injecting kokanee salmon with with a single dose of T 3, porphyropsin levels increased for the first 2 weeks, followed by a subsequent decline in porphyropsin content over the next 4 weeks. The effects of T 3 on changes in visual pigment composition of the coho salmon in this study did not show a strong cause-and-effect relationship. However, there was a strong association between visual pigment composition and hypo-osmoregulation. This kind of synchrony indicates that the measurement of visual pigment composition can be a useful tool in determining smolt status in coho salmon. As a steady decrease in molar per cent porphyropsin appeared to correlate with advancing smoltification (based on seawater challenges) and the lowest levels of porphyropsin were associated with smolt status, the lability of the coho salmon visual system permits the analysis of visual pigment composition to be used as an index of the smolt status. A protocol of regular sampling and analysis of retinal tissue would reveal the decrease in porphyropsin levels expected with the development of smolt status. When porphyropsin levels are at their minimum, the juvenile coho may be released for downstream migration or placed into sea pens. This index can be used alone or to augment other methods of determining smolt status, and could also be used to indicate when the salmon are losing or have lost their ability to survive and grow in the marine environment. We would like to thank Dr A. Kolok and Dr C. W. Hawryshyn for critically reading the manuscript. We also wish to express our gratitude to the Pacific Region Division of the Salmon Enhancement Branch of Fisheries and Oceans Canada and extend our thanks to the staff at Capilano Hatchery of Fisheries and Oceans Canada for their help in this study. This research was supported by a NSERC grant (no. 9434) to B.A.M. References ALLEN, D. M. (1971). Photic control of the proportions of two visual pigments in a fish. Vision Res. 11, ALLEN, D. M. AND CRISTY, M. (1978). Thiourea does not block visual pigment responses to prolactin in trout. Vision Res. 18, ALLEN, D. M., LOEW, E. R. AND MCFARLAND, W. N. (1982). Seasonal changes in the amount of visual pigment in the retinae of fish. Can. J. Zool. 60, ALLEN, D. M. AND MCFARLAND, W. N. (1973). The effect of temperature on rhodopsin porphyropsin ratios in a fish. Vision Res. 13,

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