Nonrelationship between plasma melatonin and background adaptation in the rainbow trout (Salmo gairdneri)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

Nonrelationship

DAVID

34,

459-467 (1978)

between Plasma Melatonin and Background Adaptation in the Rainbow Trout (&ho gairdneri)

W. OWENS,*

“Department

A. GERN,* CHARLES J. BOARDMAN?

WILLIAM AND THOMAS

of Zoology Colorado State

and Entomology University, Fort

and TDepartment Collins, Colorado

L. RALPH,*

of Statistics. 80523

Accepted November 3, 1977 Adult female rainbow trout (Salmo gairdneri) were maintained outdoors on black, white, and neutral colored backgrounds. Reflectometry readings were used to assess the color adaptation of the fish. Plasma samples were collected from the same individuals both before and after exposure to the backgrounds, as well as at both 1200 and 2400 hr. Using a sensitive radioimmunoassay, no significant differences in plasma melatonin levels could be detected for the fish kept on the three backgrounds. Since the fish did show significant color modification in response to 18-21.5 days on the backgrounds, it is concluded that melatonin may not participate in background adaptation in trout. A consistent nocturnal elevation in melatonin levels was noted on all backgrounds. The stress associated with taking multiple blood samples from the same fish may have caused a significant increase in post-treatment compared to pretreatment nocturnal melatonin titers.

A role for the indoleamide, melatonin, in the body color adaptation of fish has been suggested by several lines of research. Hafeez (1970) was able to obtain significant blanching in Salmo gairdneri both by intraperitoneal of melatonin injection (0.007 pglg) and by dissolving melatonin in the aquarium water (2 pg/ml). The enzyme, hydroxyindole-0-methyltransferase (HIOMT), which converts N-acetylserotonin to melatonin, has been shown to rise at night in S. gairdneri (Smith and Weber, 19’74, 1976a). This would correspond to the nocturnal blanching effect which Hafeez and Quay (1970) describe for trout maintained on black backgrounds in the laboratory. Blinded fish demonstrate nocturnal blanching; however, when they are also pinealectomized (the pineal is a probable source of melatonin) the effect disappears. Nocturnal lightening is also evident in the ammocoete larvae of a few species of lamprey (Young, 1935; Eddy and Strahan,

1968; Joss, 1973), where a close correlation between HIOMT activity levels and nocturnal blanching has recently been demonstrated (Joss, 1977). An even more dramatic indication of a possible role formelatonin in color adaptation is the circadian color change rhythm found in the pencil fish (Nannostomus heckfordi anomalus) which is highly and specifically sensitive to melatonin (Ruffin et al., 1969). Reed and Finnin (1973) have even utilized this species to develop a melatonin bioassay with a sensitivity down to 0.05 ng. As Young (1935) and Joss (1977) have pointed out, there is no obvious biological reason for larval lampreys to blanch at night. Similarly, with rainbow trout, it would seem to be selectively disadvantageous to blanch at night, perhaps leaving the animal more susceptible to predation. In fact, as the present study will show, trout do not blanch at night when maintained

459 0016~6480/78/0344-0459$01.00/0 Copyright @ 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

under a natural photic regime. On the other hand, Smith and Weber (1976b) have: suggested a more plausible role for mela@nin in the ability of fish to adapt to eariations in background coloration. Their very preliminary data suggest that HIOMT activity may be altered by maintaining trout on backgrounds of different colors. Bearing these reports in mind, the design of the present experiment was to place rainbow trout on backgrounds of three different reflectances to bee if ptasma melatonin levels would be altered as determined by a sensitive radioimmunoassay. In previous experiments we have noted considerable variability in trout melatonin levels (Gem rt ~1.. 1978. and unpublished data), thus samples were taken both before and after exposure to the backgrounds in order to utilize covariance statistical techniques. which can both reduce total variability and adjust for inadvertent sampling bias (Steel and Torrie, 1960). Since the postulated melatonin-background correlation might be evidenced either during the photophase or the scotophase. sampling was done at both times. METHODS The search

experiment Hatchery

was

conducted

(Colorado

at the

Division

Bellvue. Colorado, between April 1977. Thirty approximately Z-year-old

of 22

Bellvue

Re-

Wildlife)

and female

trout (Solnzcr ,+rdner-i. Richardson) of Montana. strain were used. They weighed

in

May 14. rainbow the Arlee. 87.5 + 24 g

t.r t SE). were fed pelleted chow daily (0.6% body wtiday). and were maintained in well water at approximately 12”. Prior to being placed in tanks with differ-

SAMPLING

The three te\t H’~K constructed \vay\

with

i.h;unhcr\ ~)i dii‘ferent lh;lzkground\ in i i 3-rn parallel concrctc I’IICC-

gravel

bottoms.

Black.

whifc.

ot-

c!c‘+~

polypropylene plartic sheet\ \+ct-c LIQXI IO trnr the treatment enclosure\. Black 01 white cruhhed gravel (2 cm) was spread on the bottom of the black or vIhitc r.icewaq. brown neutral

respectit

rly.

river grave! background

enclosure. :I brush

to

contrayting

to a depth

of

J-6

cm.

C~i-e~-

12 cm) &a\ \lmilarly used 111 the cle;t~ potyplopylzne

for

The tank4 were scruhhed periodic;jlly reduce algal growth and to maintain hackgrounds~

.4lthough

the iincd v+ith tile

quantification

of

the actual color\ of the backgrounds &mate was obtained by measut-ing from the bottom\ of the raceuay\

U;LS difficult. :in the reflectance at midda! on -I

day\

a Gos~n

during

the

experiment.

I_l,ing

I.una-

Pro light meteI&merged 111 ;I plastic hag and pointed at the bottom. rhe means c SF fol- the black. neutral, and white backgrouncl were iTY7 : 5%. 9022 + 37Y0, and 13X.775 i 7667 Blood sampling ,ind rrtlectance

!uY. r-e\prclivcl). measurement\

done on each li\h at 1301) and 2300 hr prior duction to the backgrounds and again at

were

to intro1200 and

Z400 hr after an I&day In order to reduce the

euposu~-c to the hackgrounds. \tress and hcmatacrit dilution

problem\

Hith

associated

multiple

zampllng.

we

waited X4 hr (3.5 cla)s~ between the rniddaq and midnight samples for both before ancf after \ampling prc,cedurc\ (Fig. I). M’c also divided each of the three treatment frst half

group? I IO troutigrotlp) in half. sampling the in day-night order (sequence No I I while

the second half was sampled in night-day ol-der quence No. 21. I’hiL was done both to eliminate to evaluate possible Reflectometry. chromatophol-e mediately after

:I\

\tatc netting

sampling ordcl:~n indru of

hiah, ot‘ the

each fi\h. was from the i-acewa)

IXand

rel::tive danc (within

imI.5

TIME

1 a a * 1 a a .w DAY SAMPLE+

0123456 I

rQ 2

,, 20212223242!5@ 3

FIG. I. Sampling protocol showing the sequence sampling technique. (No. 2) was taken, all fish were placed on their respective hackgrounds.

Immediately

4 after

the

IZOO-hr

sample

MELATONIN

AND

BACKGROUND

set). A portable reflectometer (Photovolt Corp., Model 610) was used to measure reflectance from three positions on the left side of the fish. Each position was midway between the lateral line and the middorsal line: (1) directly under the dorsal fin (rostral); (2) halfway between the dorsal and adipose fins (medial); and (3) directly under the adipose fin (caudal). The reflectometer head projects a disk of light 2 cm in diameter. At night the procedure was performed without the aid of additional illumination. Immediately after the reflectometry procedure, the fish was placed in a bucket containing I g of MS-222 anesthetic/ 40 liters of hatchery water. After l-2 min in t.he anesthetic, a blood sample was taken by cardia’c puncture and the fish was returned to the treatment raceway. A I in., 23-gauge needle on a 2.5~~ heparinized syringe was used to obtain the 2.0-ml sample. Two of the thirty fish died after the third of the four samples. Samples were kept on ice for l-2 hr until they were centrifuged for plasma collection. As a further control for handling effects, six additional fish were sampled in the same manner as the experimental groups. These fish, however, were not moved to a new background, but were maintained in the original concrete raceway with the larger group of 280 fish. The radioimmunoassay for melatonin was performed in triplicate on each sample using the assay developed by Rollag and Niswender (1976), as modified by Gern et rrl. (1978). ‘The statistical analyses of plasma melatonin levels and reflectance readings were performed on a CDC 6400 computer using either analysis of variance with repeated measures or analysis of covariance with repeated measures (Steel and Torrie, 1960). The two mortalities discussed above necessitated substituting treatment means for the respective missing measurements. This procedure was accompanied by an appropriate reduction in the degrees of freedom (df for all analyses. Because there was evidence of an effect of prior sampling on fish color (sequence effect), a separate analysis was also conducted using only the first sample from each fish after background exposure. This was done to eliminate any possible bias caused by repeated handling.

ADAPTATION

IN

461

TROUT

TABLE I SUMMARY OF FACTORIAL ANALYSIS OF VARIANCE WITH REPEATED MEASURES SHOWING THE RESULTS FOR THE POST-TREATMENT REFLECTOMETRY Analysis of variance Source of variation

d!

Mean square

F ratio

P

Treatment (T) Sequence (S) TxS Error [a]

2 I 2 24

1972.91 128.36 22.27 91.80

21.49 I .40 0.24

< 0.001 ns” ns

Diurnal (D) DxT DxS DxTxS Error [bl

I 2 I 2 24

41.09 25.27 1036.80 24.62 35.94

I.14 0.70 28.84 0.68

ns ns < 0.001 ns

Position (P) PxT PXS PxTxS PxD PxDxT PxDxS PxDxSxT Error [cl

2 4 2 4 2 4 2 4 90

747.61 21.26 53.27 8.94 124. I7 7.91 73.82 6.03 9.57

78.12 2.22 5.57 0.93 12.97 0.83 7.71 0.63

< 0.001 ns < 0.005 ns < 0.001 ns < 0.001 ns

” Not significant.

the black and neutral treatments (Fig. 2). This was assessed by the mean discrimination statistic known as the least significant difference (LSD) (Steel and Torrie, 1960). Similar results were obtained by the more conservative procedure of Tukey (HSD). The black and the neutral background adapted fish, however, were not significantly different by these tests. Thus, the background adaptation of the three treatments closely parallels the observed reflectance levels reported for the backRESULTS grounds under Methods. Background Adaptation The significant position (P) effect seen in Fundamental to the design of the present Table 1 and Fig. 2 indicates that the meaexperiment is the induction of a change in surement under the dorsal fin (rostral) was body color by altered backgrounds. Table 1 always darker than the other, more posteis a summary of the analysis of variance rior positions. There was no statistically table for the reflectometry data. The white significant diurnal (D) change in body color background treatment (T) induced signifi- observed in these fish which were maincantly lighter body color compared to both tained outdoors. However, there was a ten-

462 WHITE = .c, NEUTRAL = . BLACK = .

d 4 i f i i I

I 201

ROSTRAL

MEDIAL

CAmAL

POSITIONS FIG. 2. Color (percentage reflectance) for all fish after exposure to three baekgrounds (combined 1200and 2400-hr measurements). The means 2 0.5 LSD (least significant difference) are plotted. LSD = 3.40. Means with nonoverlapping 0.5 LSD are significantly different at the 0.05 level.

dency for black background fish to lighten slightly at night while the neutral and white background fish tended to darken. As Table 1 shows, the interactions between diurnal and sequence (D x S) and position and sequence (P x S), as well as the three-way interaction between position, diurnal, and sequence (P x D x S), are all significant. These interaction effects are due to a generalized and consistent darkening of all animals the second time they were sampled. Thus, sequence had a distinct effect on color. In order to determine if the sequence of sampling was influencing the significant treatment effect seen in Table 1. a separate analysis of variance was conducted using only the first measurements from each sequence, thus eliminating any possible sequence effect. In Table 2 it is shown that the treatment and position effects remain significant and were therefore not an artifact of the sequence of sampling. The position by diurnal (P x D) interaction also remains significant, inditing that the three positions on the fish do not behave similarly on a diurnal basis. The lack of any interaction effects involv-

Treatment (T) Diurnal (D) D x 7 Frror ]a]

7 / 2 24

792.6s

position (P) PXT P Y 1) I’ Y T x D Error [b]

3 4 2 3 4x

3 IO.88

36.06

9.68 34.X

i.12 3.97

6.97 X.62

O.XI

X.75

-

0.00s

I?. 10

0.13

ns’,

46.43 Yo.59

i).iI

!I\

0.001 ns .%.’0.05 ns

I’ Only the first measurement from each sequence was used. ” Not significant.

ing color treatments further indicates that the backgounds produced the desired consistent distinctions between the colors of the fish.

The melatonin levels in trout both before and after exposure to the three backgrounds are shown in Fig. 3. As the analysis

AFTER

Fro 3. Comparison of plasma melatoain levels both before and after exposure to 18-21,s days on three different backgrounds.

MELATONIN

AND

BACKGROUND

shown in Table 3 demonstrates, there were no significant differences in average posttreatment melatonin levels for the three treatments. The diurnal differences in melatonin levels were highly significant. However, there was no diurnal by treatment (D x T) interaction to suggest that the circadian dynamics of melatonin levels had been influenced by the background treatments. The significant treatment by sequence (T x !5) effect, as well as the diurnal by treatment by sequence (D x T x S) interaction, can be explained in part by some unusual variability encountered in the neutral background treatment group (Table 4). In the neutral group, the sequence No. 2 night level (first sample of pair) was high. On the othler hand, in both the white and black treatments, the sequence No. I night level (second sample of pair) was high. A highly significant correlation coefficient (r = 0.98) exists between the means for the pre- and postbackground melatonin sequences. This close correlation suggests that the significant treatment by sequence (T x S, Table 3) interaction is due to normal sampling bias and not to an effect of the neutral background. The covariance analyses

ADAPTATION

IN

TABLE MELATONIN

463

TROUT

LEVELS

4 FOR SEQUENCES

Melatonin Treatment group Neutral Day Sequence I Sequence 2 Combined Night Sequence I Sequence 2 Combined Black Day Sequence Sequence Combined Night Sequence Sequence Combined White Day Sequence Sequence Combined Night Sequence Sequence Combined

(pg/ml of plasma)

Prebackground

Postbackground

102 t 24” 139 ?z 52 120 ? 28

93 t 17 132 rt 45 II2 t- 24

262 f 26 411 2 130 336 + 68

307 t I8 551 2 135 429 2 76

I 2

136 k 26 I38 r 53 137 f 28

I64 t 41 II3 ?z 24 I38 & 24

I 2

437 k 77 357 k II0 397 k 65

637 i 99 408 rt 68 523 + 68

I 2

94 -c 23 855 I8 902 I4

875 I8 72 t I9 79r I2

I 2

419 t I50 248 rtr 40 334 c 79

541 k 85 332 2 34 436 5 56

” Mean 2 SE. TABLE SU~IMARY SHOWING

OF FACTORIAL THE RESULTS

3 ANALYSIS OF VARIANCE FOR THE MELATONIN

summarized in Tables 5 and 6 show that this significant treatment by sequence (T x S) interaction is attributable to the night and Analysis of variance not the day levels. We were still concerned Source Mean F that we might be masking an important of variation square ratio P df treatment effect by the use of the sequence averaging design (as in Table 3). Thus, Treatment (T) 2 30,25 I 1.13 nsb separate one-way analyses of covariance Sequence (S) I 20,167 0.75 ns T >: S 2 120,560 4.49 < 0.025 were conducted for both day and night Error [a] 26,844 24 levels separately, using only the first samDiurnal (D) I I ,867,723 141.77 < 0.001 ple from each sequence pair. Although this D x T 2 5,864 0.44 ns procedure reduced the number of animals D x S I 0.88 ns 11,648 per treatment to five, it allowed us to reD x T x S 2 63,815 4.84 < 0.025 move any possible effects of sequence. The Error [b] 22 13,183 F values of I.55 for the night measurements ” Unadjusted post-treatment levels. and 1.32 for the day measurements were b Not significant. both nonsignificant, indicating that melatoRADIOIMMUNOASSAY~

OWEN*

464 TABLE SUMMARY SHOWING

0F F;Ac~-ORIAL THE Rr-SULTS

5

ANAI ysI.7 of: C0vARIAN(.k. FOH THY- NIGHT MELL&TONI~ l>tstLS

-.

Analysis Source df’

---____

Treatment Sequence

(T) (S)

F

\quxc

ratio

P

8.637 12.391 80.626

0.48

ns”

0.68 4.44

ns c 0.025

-~

? I 2

TxS Error ” Not

of covariancc

Me21ll

of variation

23

I I

~

IX.ISS

significant.

nin levels had not been altered by the background treatments. As can be seen in Fig. 3, the night levels of melatonin for all treatments were elevated after the background exposures. An analysis of variance comparing pre- with postbackground levels for all individuals is significant at P < 0.05 (F = 4.66). The group of six individuals maintained in the original hatchery raceway and sampled in the same way as the experimentals also showed a similar increase in nocturnal levels of melatonin (before: 377 rt 84 pgiml; after: 552 t 79 pgiml). Therefore, the nocturnal rise in melatonin titers seen near the end of the experiment cannot be attributed to the background treatments. DISCUSSION After a thorough can find no support

analysis of the data, we for the hypothesis that TABLE

SUMMARY

OF FACTORIAL

SHOWING

THE

RESULTS

6

ANALYSIS FOR THE LEVELS Analysis

Source of variation Treatment Sequence TxS

df CT) (S)

23

Error I’ Not

2 I 2

significant

Mean square

2.575 1524 3236 2648

OF COVARIANCE DAY

MELATONIN

of covariance f ratio

P

0.97 0.58 1.22

ns” ns ns

ii

melatonin acts its a hormone in modifying chromatophores to facilitate background itdaptation in the rainbow trout. Smith and Weber (1976hi. on the other hand. strgge\t that HIOMT activity incr-cased during the day in steelhead trout (.\‘rrlnro ,tllii.cl/rc,r-i I maintained on ii white hackgrourrc!. I‘heir work, however. assumec, that HIOMT levels are linked directly to melatonin production, which may not be the case (Klein and Weller. 1973; Deguchi. 1973). Smith and Weber also used juvenile fish. while the fish used in this study were adult females. Although their comparisons, using fish ot different ages and from experiments conducted at different times of the year, are less than satisfactory. the possibility remains that a developmental change in background adaptation systems may occur in trout. Fain and Hadley (1966) have suggested a possible developmental change in the killifish Fundulus ~lrtctw~litrrs. In their study, the dispersion of melanin granules of adults was unresponsive to high doses of melatonin. In contrast, the melanophores of larvae are sensitive to melatonin (Nichols I-‘[ ui., 1966: Bagnara and Hadley, 1973). Hafeez (1970) states that the level of melatonin which he used to obtain the body blanching reaction in rainbow trout may have been pharmacological; since impaired body movements and loss of equilibrium were also seen. His 7-ngig dose would be 60- to lOO-fold higher than the total circuiating melatonin in a trout with unusually high night levels. a\ estimated from the titers obtained in the present experiment. Pencil fish (Ruffin et (il., 1969) and Xe~zoplrs (Bagnara, 1963; Quay and Bagnara, 1964; Quay, 1968) respond to doses as low as 0.1 ngiml, dissolved in the aquarium water. ‘These responses appear more nearly physiological. The pencil fish. however. does not simply blanch nocturnally; rather the granules of distinct melanophore populations actually disperse while others concentrate (Reed, 1968: Reed c’l nl., 1969). While the

MELATONIN

AND

BACKGROUND

melanophore response of the pencil fish seems intuitively to have a selective significance, the blanching of trout at night, which might follow the consistent nocturnal rise in melatonin levels which we have demonstrated, seems nonselective with regard to color adaptation. In fact, our data suggest that trout in a natural photic environment do not normally blanch at night, regardless of the background. Thus, we conclude that either the nocturnal levels of melatonin which we have demonstrated are below the minimum for chromatophore response or that other humoral and/or nervous controls compensate for the potential blanching effect of the high nocturnal melatonin levels. The repeated sampling protocol (four sarnples in 25 days) no doubt caused some stress to the fish. After the third sample, 2 of the 30 fish died. This stress may account for the slight darkening usually associated with the second of two samples taken only 84 hr apart. The darkening could be the result of either adrenocorticotrophin (ACTH) stimulation of melanization (Loud and Mishima, 1963) or direct melanophore expansion by catecholamines, as is known to occur in several species (Bagnara and Hadley, 1973). Melatonin levels were also elevated at night in repeatedly sampled trout, whether they were exposed to various backgrounds or retained in the original raceway. This increase may be the result of stress, as rats show elevated pineal melatonin levels when stressed (Lynch et al., 1977). Alternatively, the increased melatonin levels could be explained by a decrease in the rate of melatonin degradation, which, if melatonin production remained constant, would result in a longer half-life and higher plasma levels. Decreased enzymatic degradation may have been caused either by dilution of plasma proteins with extravascular fluid due to sampling-induced hypovolemia or by stress altering liver function. A further possibility which cannot be ruled out at this time is that the differences may

ADAPTATION

IN

TROUT

465

be related to seasonal changes (22 days) involving either solar or lunar cycles. There is considerable indirect evidence that a melanophore-stimulating hormone (MSH) from the pituitary is involved in background adaptation for some species of fish (Baker, 1963, 1965; Baker and Ball, 1970; Bagnara and Hadley, 1973). It is reasonable to expect, therefore, that our black background-adapted animals would have had higher levels of MSH. Sakai and coworkers (1976) have demonstrated a possible inhibitory effect of aMSH on the rat pineal. In addition, a selective accumulation of aMSH has also been shown in the rat pineal (DuPont et al., 1975). Assuming that melatonin levels reflect pineal activity in fish and considering that we can show no change in melatonin titers with background adaptation, our data suggest that, in trout, MSH may not modulate pineal function. Our experiment does not address the possibility of a very short-term or a very long-term adaptive change in melatonin with regard to background. We are also not able to comment on possible changes in melatonin turnover rate or the possibility that chromatophore response to melatonin may have been altered, rather than melatonin levels. Nevertheless, our data support the statement of Bagnara and Hadley (1973) that “There is no evidence . . . that any hormone other than MSH regulates vertebrate chromatic changes at the effector cell level in response to background adaptation.” ACKNOWLEDGMENTS This research was supported by NIff Grant No. NS-12257. We thank the Colorado Division of Wildlife, including Dr. Don Horak, Mr. Larry Harris, Mr. Tom Mandis, and Mr. Gene Rauch, for providing facilities and animals for this project. We are grateful to Mimi Owens and Alisa Ralph for sampling assistance, Jim zumBrunnen for statistical programming, and Dr. Bruce Firth and Scott Turner for commenting on the manuscript. Dr. Gordon Niswender of the Department of Physiology and Biophysics kindly provided melatonin antibody and RIA facilities.

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and color change in the lamprey, Lampetra. GUI. Cornp. Endocrinol. 21, I88- 195. Joss, J. M. P. (1977). Hydroxyindole-Omethyltransferase (HIOMT) activity and the uptake of “H-melatonin in the lamprey, Geofritr australis Gray. Gcn. Camp. Endocrinol. 31.

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Quay, W. B. (1970). The role of in the control of phototaxis and in rainbow trout (S&to Richardson). 2. VqqI. Physiol. 68,

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moube after injection of lz31 stimulating hormone. J. Endocrinol. Eddy, J. M. P., and Strahan, R. (1968). pineal complex of the lampreys,

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Y

goldfish

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drrl~.r

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.4. L

site of synthesis of the hormone in the trout 32, 397-398.

and the pituitary 48, 26-27.

DuPont. A., Puviani,

Fain,

E.

404.

B. I. (1965). The melanophore-stimulating pituitary. .I. Endouinol.

Baker.

(;uIz/~.

and COlOl Change. ‘. Englewood Cliffs. N.J. Effect of adaptation to black and

Prentice-Hall, Baker, B. I. (1963).

Baker,

light-

Gon.

J. R.. tuations (ASMT) steelhead

Bid.

Med.

Smith. J. R., tion of

Ctrn.

and

Marks,

on padrenergic pineal. Lfe .Cc+.

and Weber, L. J. (1974). Diurnal Aucin acetylserotonin methyltransferase activity in the pineal gland of the trout (S&n) grrirdneri). Prnc,. SW. Eup.

147, 441-443. and Weber, day-night

L.

indole-O-methyltransferase pineal gland of steelhead

nrri).

B.,

J. Loo/.

J. (1976a). The regulachanges in hydroxyactivity trout (S&no

54, 1530-1534.

in

the guird-

MELATONIN

AND

BACKGROUND

Smith, J. R., and Weber, L. J. (1976b). Alterations in diurnal pineal hydroxynindole-O-methyltransferase (HIOMT) activity in steelhead trout (Salmo gairdneri) associated with changes in environmental background color. Camp. Biochem. Physiol. 5X, 33-35.

ADAPTATION

IN

TROUT

467

Steel, R. G. D., and Torrie, J. H. (1960). “Principles and Procedures of Statistics.” McGraw-Hill, New York. Young, J. Z. (1935). The photoreceptors of lampreys. II. The functions of the pineal complex. J. Exp. Biol. 12, 254-270.