Van Doorn Et Al 2005

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O R IGINA L PA PE R

Kevin L. H. van Doorn J. G. Sivak M. M. Vijayan

Optical quality changes of the ocular lens during induced parr-to-smolt

metamorphosis in Rainbow Trout (Oncorhynchus mykiss)

Ocular lens optical quality during induced salmonid metamorphosis

Received: 12 August 2004 / Revised: 24 January 2005 / Accepted: 29 January 2005 / Published online: 11 May 2005 Springer-Verlag 2005

Abstract The effect of an induced salmonid parr-to-smolt metamorphosis (smoltification) on the opticalquality of the ocular lens was studied. In two separate

experiments, rainbow trout (Oncorhynchus mykiss) parrwere fed thyroxine in their diet to induce the metamor-phosis. Lenses were excised at regular samplings duringthe treatment period and optically scanned using acustom scanning laser monitor. Radioimmunoassay wasused to measure serum titers of thyroxine and 3,5,3-triiodo-L-thyronine. It was found that lens opticalquality was consistently negatively correlated with3,5,3-triiodo-L-thyronine levels, but not with thyroxinelevels. To test if thyroid hormones are directly respon-sible for the change in optical quality, rainbow troutlenses were cultured for 72 h in a medium containing3,5,3-triiodo-L-thyronine, but no effect was observed.

The significance of these findings in the contexts of thefishes visual capabilities and smolting physiology isdiscussed.

Keywords Thyroxine Thyroid hormone Rainbowtrout Ocular lens Metamorphosis

Abbreviations BVD: Back vertex distance RIA:Radioimmunoassay SLM: Scanning laser monitor

Introduction

In several salmonid species can be found individuals anddistinct populations whose life cycles take them fromfresh water, where they hatch, to seawater in which theyfeed and grow. They then return to fresh water wherethey spawn or simply wait for the next opportunity tofeed at sea. The shifts in environment that accompanythe migrations are significant, and the fishes must be pre-adapted to the new environment before exposure or theyare unlikely to survive. They must contend with thechanges in water osmolarities, predator and prey dis-tributions, and sensory environment. The fresh water toseawater migration and the accompanying process ofpre-adaptation, known as smoltification, consists ofbehavioral (Hoar 1988; Iwata 1995), sensory (Beatty1966; Morin et al. 1989), morphological (Boeuf 1993)and physiological (Hoar1988, and Boeuf1993) changes.There is evidence of a circannual rhythm to smoltifica-tion (Eriksson & Lundqvist1982), which is entrained byseasonal variations in temperature and photoperiod.

Several hormones such as the thyroid hormones,growth hormone, and cortisol are involved in effectingand mediating the transformation. But though they actin concert to effect the complete, fully adaptive meta-morphosis, certain specific changes (or at least the ini-tiations of these changes) seem to often be associatedwith a single hormone. For example, the thyroid hor-mone thyroxine (T4) has been shown to stimulate sil-vering of the body, cause behavioral changes (Hutchisonand Iwata1998), and cause photopigment ratio changes(Beatty 1972) and the loss of ultraviolet-sensitive conesin the ventral retina of the rainbow trout, Oncorhynchusmykiss (Browman and Hawryshyn 1992, 1994; Allisonet al.2003).

T4 and the thyroid hormone 3,5,3-triiodo-L-thyro-nine (T3) are iodinated amino acids. They and relatediodinated compounds are found almost ubiquitouslythroughout the animal kingdom as effectors and medi-

K. L. H. van Doorn (&)

J. G. SivakSchool of Optometry, University of Waterloo, 200 UniversityAvenue W., Waterloo, ON, N2L 3G1, CanadaE-mail: [email protected].: +613-674-1752Fax: +519-725-0784

K. L. H. van Doorn J. G. Sivak M. M. VijayanDepartment of Biology, University of Waterloo, 200 UniversityAvenue W., Waterloo, ON, N2L 3G1, Canada

Present address: K. L. H. van Doorn2220 County Rd 10 (RR 1), St-Eugene, ON, K0B 1P0, Canada

J Comp Physiol A (2005) 191: 649657DOI 10.1007/s00359-005-0615-y


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ators of growth and metabolism. They are also presentin some plants and protists, in some cases due toendogenous production and in others because of exog-enous uptake (Eales 1997). Among the higher verte-brates, the thyroid gland is the organ responsible forthyroid hormone production, mostly by secreting T4,although in some species it secretes proportionallysmaller quantities of T3as well. After its release into thebody fluids, T4 action is regulated via a process ofdeiodination to T3 in peripheral tissues (Ramsden1977;Plate et al. 2002). T3 is physiologically the more activeform, being bound with a higher affinity by cellularreceptors (Oppenheimer1983).

Thyroxine and/or T3 are known to be involved inseveral cases of vertebrate metamorphosis, such as theamphibian tadpole to adult metamorphosis (Dodd andDodd 1976; Galton 1983), and the metamorphic matu-rations of several species of fish. The larva-to-juvenileflattening of pleuronectid flatfishes (Sklower 1930;Hoar 1951; Miwa et al. 1987, 1988), the maturation ofeel leptocephali (Sklower 1930), and salmonid smoltifi-cation (Hoar1939) all involve T4 or T3 to some degree.

In the case of salmonid smoltification specifically, thereis a surge in T4 production soon after the onset of theprocess. This can bring T4levels up to 2090 ng/ml from520 ng/ml depending on age, species, population, andyear (Hoar 1988; Dickhoff et al. 1982; Boeuf1993).

Salmonids are highly visual fish. They depend heavilyupon vision for navigation (Groves et al. 1968; Parkynet al. 2003), mate localization (Duker 1982), and preylocalization and capture (Hoar 1958; Kato 1991;Browman et al. 1993). It is now well established thatexposure to saltwater can induce cataractogenesis insalmonids (Iwata et al.1987), a problem that continuesto vex the aquaculture industry (Ersdal et al. 2001; Breck

and Sveier2001; Menzies et al. 2002). The likelihood ofdeveloping cataracts depends largely upon the meta-morphic stage of the fish. The exact mechanism by whichsaltwater affects the lens is not known, although Bjerkaset al. (2003) suggest that defective osmoregulation insmolts, such as might be caused by improper smoltifi-cation, is responsible for cataract formation throughincompetence at maintaining an optimal lens hydrationstate.

Because smoltification involves regulation of osmo-regulatory mechanisms, it is conceivable that the processitself could affect the ocular lens. Mechanisms that di-rectly or indirectly affect the osmolarity of the aqueous

humor could alter the environment of the lens beyondthat with which the lens is able to cope via its ownosmoregulatory mechanisms, such as the Na/K pump. Itis also possible that adjustment of the lens own osmo-regulatory mechanisms could take place during smol-tification, similar to the increase in gill NaK-ATPaseactivity that is known to occur (Hoar1988; Boeuf1993).The study described here aimed to characterize opticalquality changes of the lens that occur during the sal-monid parr-to-smolt metamorphosis, specifically thatwhich can be accounted for by a T4 surge.

Research on salmonid metamorphosis physiology hastraditionally been accomplished in one of the followingthree ways: (1) by sampling wild populations, (2) byinducing metamorphosis through photoperiod control(Clarke et al. 1978, 1981, 1989), (3) by inducing meta-morphosis by exposure to a hormone (Allen 1977;Tagawa and Iwata 1991; Browman and Hawryshyn1992; Finnson and Eales 1999), or (4) by exposure di-rectly to saltwater (Duston 1994). The research pre-sented in this study took the third route, because of theease with which confounding factors can be controlled ina laboratory.

The experiments presented here specifically involvedthe induction of metamorphic changes by exposingrainbow trout to T4. Although rainbow trout are re-cently landlocked and non-anadromous, meaning thatthey do not migrate in the wild, they nevertheless retainthe capacity to undergo smoltification-like metamor-phosis when exposed to the hormones involved (Eales1979). Because T4-treated salmonids do not undergo allthe metamorphic changes necessary to pre-adapt themto saltwater, Eales (1979) referred to them as pseudo-

smolts. Nevertheless, they remain a useful model forstudying metamorphic physiology, for specific changescan be observed in isolation from the many otherphysiological factors that characterize true smoltifica-tion.

Materials and methods

Experiment 1: assessment of lens optical qualityduring T4-induced metamorphosis of rainbow trout

Two similar experiments, experiment 1A and experiment1B, are presented here. There are two major differencesbetween them. The first difference is in the experimentalparadigms: experiment 1A compares treated and controlgroups in which the animals were at the same develop-mental stage while experiment 1B is a before-and-aftercomparison of a treated group with conspecifics fromprior to the initiation of the treatment. The second dif-ference is in the treatment itself: during experiment 1A,the dose of T4 given to the treated animals was in-creased, whereas a single high dose was given to theanimals in experiment 1B.

Experimental set-up

One hundred non-anadromous rainbow trout parr wereobtained from the Humber Springs Trout Club, a localhatchery in Orangeville, ON, Canada. They were housedin two groups of 50 in 415 l tanks in open circulationflow-through systems at a consistent temperature of12C and on a 12:12 h light:dark cycle. The fish were

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allowed to acclimate for 3 weeks prior to beginning theexperiments. Their status as parr was ascertained byvisual inspection, notably by their conspicuous and well-defined parr markings. The presence of these markingswas used to monitor individuals developmental stagesthroughout the experiments.

The duration of the experiment 1A was 54 days. Atthe beginning of the experiment, fish weighed on average30.34.7 g, and by the end, they had grown to54.711.3 g. Control fish were sampled for the first19 days, after which time the remaining control fish wereused as treated fish in experiment 1B, the duration ofwhich was 26 days. In this experiment, fish weighed43.38.1 g at the beginning, and by the end, they hadgrown to 47.78.0 g. All experimental procedures werein accordance with the animal utilization guidelines ofthe University of Waterloo, the Canadian Council ofAnimal Care, and the Ontario Animals for ResearchAct.

The fish feed served as the vehicle for T4, as wasdescribed by Tagawa and Iwata (1991). The process oflacing the feed began by dissolving T4in 1 ml 1N NaOH

solution and 500 ml absolute ethanol. The feed was thenbathed in this solution overnight in a fume hood,allowing the liquids to evaporate and leaving the T4bound to the feed. Two concentrations were prepared inthis way: 0.25 mg/g (T4:feed), and 0.4 mg/g (T4:feed).Feed given to the control fish was treated in the sameway, but without the addition of T4.

Fish were fed daily to satiation. In experiment 1A,fish were fed with food containing 0.25 mg/g (T4:feed)for the first 15 days, followed by 0.4 mg/g (T4:feed) for

the remaining 27 days. Fish were then returned to anuntreated diet and sampled over 12 days. Four to sixfish were sampled at each sampling point in thisexperiment. In experiment 1B, fish were fed throughoutwith food containing 0.4 mg/g (T4:feed). In thisexperiment, three fish were sampled at each samplingtime. Fig.1 shows an experiment timeline that includesT4 dosages and sampling times. Sampling began byanaesthetizing the animals in a 30-mg/l clove oilsolution, after which they were weighed and blood wasdrawn from the caudal vessel. The blood was kept onice for up to an hour before being centrifuged for10 min at 6,000 rpm. The plasma was kept at 80Cuntil radioimmunoassay (RIA) analyses could beperformed. After drawing blood, each animal wasdecapitated and its head was placed on ice until lensdissections were conducted. Heads were kept on ice forup to 3 h.

Lenses were dissected out of the eye and placed incustomized lens culture cells containing H10 mediumsupplemented with 5% dialyzed fetal bovine serum(FBS) (Hikida and Iwata 1987; Dorfman-Hecht et al.

1994) supplied by GIBCO, Grand Island, NY, USA.These cells are designed specifically for lens culture andoptical scanning. Complete H10 medium contains intissue culture grade water, NaCl (135.5 mM), KCl(5.0 mM), CaCl2 (2.0 mM), glucose (5.0 mM), HEPES(10.0 mM), penicillin:streptomycyin (100 U/ml :100 lg/ml), and enough 0.1 N NaOH to bring the pH to 7.4.This medium is designed to mimic the osmolarity of fishocular humors. During dissection, care was taken neverto touch the lenses themselves but with surgical spongesand custom made glass scoops, thus avoiding inflic-tion of surface abrasions.

Assessing lens optical quality

Lenses were scanned using a custom designed scanninglaser monitor (SLM) based on the Scantox in vitro lensassay system as described in Weerheim and Sivak (1992).The SLM is a computer automated laser scanning devicethat can provide a general measure of a lens opticalquality via measurements of its back vertex distance(BVD) variability (standard error, SE), essentially mea-suring the lens capacity to form a sharp image of a pointlight source at infinity.

To briefly explain the functioning of the SLM, len-

ses in their culture cells are placed into a light tightchamber. A red diode laser (k = 630 nm) located be-neath the cell emits 20 equally spaced parallel colli-mated beams along the diameter of the lens. A digitalcamera photographs the 20 refracted beams, the imagesof which are then collated and analyzed by customsoftware that outputs the BVD statistics of the lens.Each lens is scanned along two axes at 90 to eachother, and the BVD SE results of these scans areaveraged. Fig.2 diagrammatically illustrates the scan-ning mechanism.

Fig. 1 Timeline of experimental treatments and samplings. Ani-mals in experiment 1A were given 0.25 mg/g (T4:food) for the first15 days, after which the dose was increased to 0.4 mg/g (T4:food).Treatment of this group ended on day 42. Animals in the controlgroup were given vehicle-treated food for 19 days to ensure that thevehicle solution had no effect. The remaining fish in this groupbecame the subjects of experiment 1B in which the animals weregiven a single dose of 0.4 mg/g (T4:food) for 26 days. Circlesindicate sampling points, and numbers beneath the circles indicatethe day at which the sampling occurred

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T4 and T3 radioimmunoassays (RIA)

Regular testing of the fish serum T4 titer was necessaryto ensure adequate dosage of T4. Serum T3 titers werealso measured to ensure that T4 to T3 conversion wastaking place appropriately and was not impeded bystress or other factors. Serum samples from each fishwere analyzed using MediCorp T4 and T3 RIA kits(MediCorp, Montreal, QC, Canada; catalog numbers:06B-254011 and 06B-254215, respectively).

Experiment 2: lenses cultured with thyroid hormone T3

The results obtained in experiment 1 suggest a role forT3 in effecting lens optical quality changes (see the Re-sults section). Experiment 2 was conducted to determineif the effect observed is directly and exclusively attrib-utable to T3.

Ten lenses were dissected from seven rainbow troutparr obtained from the Humber Springs Trout Club inOrangeville, ON, Canada. Lenses were partitioned into

two groups, a control group (n=4) and a treated group(n=6), while ensuring that both lenses from a given fish,if used in the experiment, were always placed in separategroups. The lenses were cultured at 12C in H10mediumsupplemented with 10% dialyzed FBS. They were al-lowed to acclimate to the environment for 1 week priorto beginning treatment. This also allowed any abrasionsor other mechanical damage incurred during dissectionto become obvious, thus permitting the removal ofdamaged lenses prior to beginning the experiment.

The culture medium of the treated lenses was thensupplemented with 50 ng/ml of T3 and lenses were

cultured for another 72 h. During this 72 h period,lenses were scanned after 0, 4, 24, 48, and 72 h oftreatment using an SLM. Culture media were changedevery 48 h.

Statistical analyses

All statistical analyses were performed using the Systat10.0 statistical software. Comparisons between treatedand control groups were conducted via analyses ofvariance (ANOVA). Pearson correlation coefficientswere used to determine correlations between BVD SEand serum hormone titers, and Bonferroni probabilitieswere calculated based on these values. Probability valuesequal to or less than 0.05 were considered statisticallysignificant.

Results

Experiment 1: assessment of lens optical quality duringT4-induced metamorphosis of rainbow trout

No difference in lens optical quality was observed be-tween samples of the control group (P=0.360), leadingto the conclusion that there was no effect of time overwhich the sham-treatment took place. To increase thepower of all subsequent treated-versus-control statisticaltests, data from all samples of the control group werepooled, and all samples from treated groups were eachcompared against this pool. In doing this, it was possibleto reduce the total number of animals required in theseexperiments.

Experiment 1A

The two-step dosage regime (fish fed for 15 days with0.25 mg/g (T4:feed) and for the following 27 days with0.4 mg/g (T4:feed)) resulted in a progressive increase inoverall T4 concentrations to 60.7214.99 ng/ml duringthe experiment (Fig. 3). T3levels progressively increasedas well, reaching a peak of 8.880.19 ng/ml on day 40(Fig. 4). It can be inferred that conversion of T4 to T3was occurring normally. Silvering of the body and theloss of parr marks were becoming evident by day 12, and

by day 33, all fish were well silvered. This confirmed thatmetamorphosis was taking place.

A significant reduction in overall lens optical qualitywas evident 24 h after treatment began (P


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BVD SE and serum T4 titers in the treated group

were found to be positively correlated (PearsonsR=0.563, p 0.000). Even when data from the controlgroup were omitted from the calculation, BVD SE andserum T4 titers were positively correlated (PearsonsR=0.474, P=0.002). BVD SE and serum T3 titers werealso positively correlated (Pearsons R=0.394,P=0.005) but only when data from the control groupwere included. With control data omitted, PearsonsR=0.313 and P=0.071. Because serum samples weretested first for T4, too little remained in some cases toalso test for T3, thus reducing the sample size availablefor T3 titers. This may explain the non-significantprobability.

Experiment 1B

This treatment method, in which fish were fedthroughout with 0.4 mg/g (T4: feed), initially raisedserum T4titers more rapidly than observed with the two-step dosage regime (Fig.3). T3 levels progressively de-creased after day 6 (Fig. 4), suggesting that the rate ofconversion of T4 to T3 was decreasing or that the

clearing rate of T3 was increasing.A significant reduction in lens optical quality was

observed 24 h after the first treatment (P < 0.000),and again this was maintained throughout the treat-ment until the 19th day, by which time recovery ap-peared to have taken place (P=0.004. However, asseen in Fig.5, this is due to the BVD SE having fallenbelow that of the control group). The sample taken onday 26 showed no difference from that of the controlgroup (P=0.124).

BVD SE and serum T4 titers in the treated groupwere not found to be correlated, regardless of whetherdata from the control groups were included or omitted

(Pearsons R=0.262,P=0.112; and PearsonsR=0.264,P=0.343). BVD SE and serum T3 titers were positivelycorrelated (Pearsons R=0.483, P=0.008) but onlywhen data from the control group were included. Withcontrol data omitted, Pearsons R=0.413 andP=0.142.As in experiment 1A, serum samples were tested first forT4, and so little remained in some cases to also test forT3. This reduced the sample size available for T3 titers,compounding the already lesser sample size in thisexperiment, which could explain the non-significantprobability, in spite of the R value.

Fig. 3 Serum T4 titers. Little change in serum T4 titers wasobserved in the control group over the course of the vehicletreatment. A gradual increase in T4 titers was seen in experiment1A, in which there was a dosage increase on day 15. After ceasingtreatment on day 42, hormone titers returned to baseline levels. Inexperiment 1B, there appeared to be relatively constant high levelsof T4, despite some fluctuations, which could be attributed to smallsample size variability (n=3)

Fig. 4 Serum T3titers. T3 levels remained relatively constant in thecontrol group, whereas it progressively increased in the experiment1A treated group, before diminishing upon removal of thetreatment on day 42. The sample taken on day 54 showed a veryhigh unexplained variability. T3 titers of the treated animals inexperiment 1B were significantly elevated at the first samplingpoint, but began a progressive decrease by day 12. The reason forthis decrease is unclear, but it would likely be due to either adecrease in the rate of T4deiodination, an increase in the rate of T3clearing from the serum, or both

Fig. 5 BVD SE of the 3 groups (1A, 1B and Control). Nostatistically significant differences were observed between samplesof the control group, suggesting no effect due to the vehicle-treatedfeed they were given. As a result, samples of this group were pooledand all analyses of the treated were done against this pool.Asterisks indicate statistically significant differences from thecontrol pool. At all sampling points in experiment 1A, sampleswere significantly different from the control pool (P 0.042) untilday 47 (P=0.398), 5 days after the treatment ended. BVD SE againincreased by day 54, resulting in a significant difference from thecontrol pool (P=0.033). At all sampling points in experiment 1Bup to and including those taken on day 12, statistical analyses were

significant (P 0.000). By day 19, complete recovery had occurreddespite the ongoing treatment. Statistical significance is indicatedon day 19 due to the BVD SE of the treated group having fallenbelow that of the control pool

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Experiment 2: lenses cultured with thyroid hormone

No difference in lens optical quality was detected be-tween those lenses treated with T3 and the controls (re-peated measures ANOVA P=0.555). See Fig.6 for agraphical summary of the results.

Discussion

This study shows conclusively that a reduction of lens

optical quality can occur as a result of a T4 surge, suchas that which occurs during smoltification. In bothexperiments 1A and 1B, the decrease of lens opticalquality was apparent 24 h after the first dose of T4. Theexact mechanism by which the change is effected is notyet known.

Bjerkas et al. (1996) found a correlation between ra-pid growth and cataract formation in farmed Atlanticsalmon (Salmo salar) in freshwater, although they couldnot exclude potential compounding effects due tosmoltification. By feeding all groups according to thesame regime (daily to satiation) with the same base feed,we have attempted to rule out rapid growth and nutri-

tional factors as the causes of observed differences be-tween groups in this experiment.

Upon exposure to saltwater, the aqueous humorosmolality of coho salmon increases more rapidly thanthat of the plasma (Iwata et al. 1987). Since aqueousosmolarity is typically lower than plasma osmolarity inteleosts (Nicol 1989), this suggests that ion influx fromthe seawater or water outflux from the aqueous througha permeable cornea (Edelhauser 1968) occurs uponsaltwater exposure. To pre-adapt to this encounter,regulation of ion pumps in the cornea and/or the lenses

as well as alterations in the aqueous production mech-anisms could occur during the metamorphosis, and inthis state of osmolar flux, the composition of the aque-ous may be altered as well as that of the lens. Lenshydration state could be altered enough during thisperiod to affect its optical quality.

A recent medical study (Age-Related Eye DiseaseStudy Research Group, 2001) showed, with borderlinesignificance, a link between thyroid hormone treatmentin humans and the presence of moderate cortical opac-ities. Due to the borderline significance, however, theauthors suggest further research before drawing anyconclusions. The administration of thyroid hormones toadult frogs has been shown to increase lens epithelialmitotic activity (Weinsieder et al. 1972), although directexposure to T3 showed no effect on cell proliferation incultured lenses. The potential link between mitoticactivity and cataract development (i.e. lens opticalquality reduction) upon thyroid hormone exposure isintriguing, and may be worth pursuing in the context ofsmoltification.

Methionine deficiency in the diet of rainbow trout has

been shown to cause cataracts (Cowey et al.1992). It hasalso been shown to induce an elevation in plasma T3 inchickens (Carew et al. 2003). Bearing in mind the phy-logenetic separation between these species, it is inter-esting to consider the possibility that the cataractsobserved by Cowey et al. might have been attributableto an increase of T3titers in the fish. If so, their findingswould parallel those described in this study, althoughthe severities of the observed lenticular changes weredifferent in both studies.

Like Weinsieder et al. (1972), the work presented hereruled out a direct and exclusive effect attributable to T3,because culturing lenses in media containing T3 showed

no effect on lens optical quality in experiment 2. It mightbe argued that the lenses were cultured for too short atime (72 h) for an optical effect to be observed, but be-cause an effect was observed after 24 h in experiments1A and 1B, we would assume that if it were due to T3exclusively, it would have been manifested by the 24th

hour of culture. This of course does not rule out anoptical effect due to a long-term exposure to T3, or anoptically unrelated physiological effect. However, if suchphenomena were to occur, it still could not explain theresults observed as a result of in vivo exposure to T4.

There is a curious difference between the results ob-tained from experiments 1A and 1B: in the latter, in

which fish were given 0.4 mg/g (T4:feed) from the start,lens optical quality completely recovered during thetreatment period around day 19. The reason for this isnot known, but there were three conspicuous differencesbetween these experiments, one or more of which may beresponsible.

Firstly, the fish in experiment 1B were somewhatlarger at the beginning of the experiment than those inexperiment 1A (mean weights: 43 g vs 29.8 g), althoughthey were still at the parr stage as judged by their con-spicuous lateral parr markings.

Fig. 6 BVD SE of lenses cultured for 72 h in H10 medium with10% FBS with (treated) and without (control) the addition of50 ng/ml T3. No difference was observed between the two groups(P=0.555). Thus there is no exclusive and direct optical effectattributable to T3

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Secondly, serum T3 titers in experiment 1B dimin-ished after reaching a peak on day 6. At no time did sucha decreasing trend occur in experiment 1A during theperiod of treatment. This suggests a drop in the rate ofconversion of T4to T3or an increased rate of T3clearingfrom the serum (Plate et al. 2002). It is possible that astressful factor may have affected the fish in experiment1B, such as the overall lower stocking densities that theyexperienced (18 fish of 43 g vs 50 fish of 29.8 g, both in415 l tanks).

Thirdly, experiment 1B was begun 30 days later thanexperiment 1A. Farbridge & Leatherland (1991) de-scribed an endogenous biweekly pattern in rainbowtrout food consumption and hormone levels that wascorrelated with the phase of the moon. This phenom-enon was ruled out as the cause of the strange responsein experiment 1B, since both experiments were begun atapproximately the same point in the lunar cycle. Butbecause experiment 1B was begun 30 days later in theautumn season, the potential influence of the endoge-nous circannual rhythm present in smolting salmonids,which may potentiate or inhibit the conversion of T4 to

T3depending on its current state, should be considered.The correlational analyses showed a positive corre-

lation between lens optical quality and serum T4titers inexperiment 1A but not in experiment 1B. That no cor-relation was evident in experiment 1B is understandableconsidering that lens optical quality recovered duringthe treatment period, while T4 levels were still elevated.BVD SE and T3 were positively correlated in bothexperiments, when data from the control group wereincluded in the analyses. Bearing in mind that T3 isphysiologically more active than T4, it is not surprisingthat a positive correlation with T3 was found in experi-ment 1B, but no correlation with T4.

It is perplexing that the apparent optical recovery inexperiment 1A on day 47 was short-lived. The sampletaken on day 54 again showed a significant differencewith the control group. As can be seen in Fig. 4, there isgreat variability in the T3titers observed in this sample.Unfortunately, we are at a loss to explain the T3 vari-ability, especially in light of the low T4titers as shown inFig.3. This may again be an artifact of the somewhatsmall sample sizes, which would emphasize the contri-butions of exceptional individual fish.

One final comment should be made about the resultsobtained as they relate to the mechanism by which lensoptical quality was measured. Throughout the experi-

ment, lenses were scanned along a 22.5 mm axisdepending on the size of the aperture of the scanning cellin which the lenses were kept, which was in turndependent on the diameter of the lens. Lenses less than2.5 mm in diameter were mounted on a 2-mm aperture,and those equal to or greater than 2.5 mm in diameterwere mounted on a 2.5 mm aperture. The 20 beamspassed through the lenses were always equally spacedsuch that they scanned across the whole aperture. As thefish grew throughout the experiments, their lenses alsogrew, but regardless of their size, only the central

2.5 mm of the diameter was scanned. As a result, opticalquality data from the periphery of the lenses will beabsent toward the end of the experiment. Because newgrowth occurs at the periphery of the lens, this regionmay be most disturbed by whatever is affecting theoptical quality of the lens. Since data on this region ofthe lens is lacking in the later samples, it is possible thatlens optical quality from these lenses was being under-estimated. While this would not change the conclusionsdrawn from the results, it may be relevant when con-sidering the functional impact on the fishes vision.

Functional impact on visual capabilities

The functional impact on the fishes vision remains un-known. That the BVD SE measurements were done invitro makes inferential speculation on the in vivo abso-lute resolving capabilities of these lenses somewhat ten-uous.

Although the lenses of adult teleosts are considered tohave a resolving power up to ten times that of their

retinal photoreceptor density (Fernald 1988), some de-bate exists as to the significance of this (Sivak 1990). Alens with such a high resolving power may benefit min-imum viewable acuity and hyperacuity (types of visualacuity that offer resolutions smaller than photoreceptorseparation), and it could also improve image contrast(Snyder et al. 1986) though at the expense of aliasingartifacts. Should it be the case in late parr stage O.mykiss and other salmonids that the resolution of theretina is the limiting factor in their visual acuity, areduction in lens optical quality might have little to nofunctional impact on vision. We would be left wonderingif evolution might thus have favored an over-engineered

lens in these animals to compensate for the deleteriouseffects observed during smoltification and saltwaterexposure.

In their study of osmotic cataracts in wild seagoingsalmon, Bjerkas et al. (2003) noted that the presence ofcataracts did not affect the condition factor (weight/length3) of the fish, indicating that they were still able tofind food. Under certain conditions, it would thus seemthat a severe reduction in visual capacity is not delete-rious to the animals in that particular population, al-though stocking density and food availability areimportant factors to consider, as well as the consistenceof the diet.

In summary, the research presented here showed thatexposure of rainbow trout to T4 levels similar to thosewhich are observed during the natural smoltification ofO. mykiss (steelhead trout specifically) can reduce theoptical quality of their lenses. This is, to our knowledge,the first time that the lenses of these fish were shown tobe affected by the physiological metamorphosis itself(induced or natural). The investigation of lens quality inwild salmonid populations at different stages of the lifecycle is a compelling area for future research, to deter-mine if lens effects are a natural occurrence. If so, one

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