ORIGINAL PAPER

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 eect of an induced salmonid parr-to-
smolt metamorphosis (smoltication) on the optical
quality of the ocular lens was studied. In two separate
experiments, rainbow trout (Oncorhynchus mykiss) parr
were fed thyroxine in their diet to induce the metamor-
phosis. Lenses were excised at regular samplings during
the treatment period and optically scanned using a
custom scanning laser monitor. Radioimmunoassay was
used to measure serum titers of thyroxine and 3,5,3-
triiodo-L-thyronine. It was found that lens optical
quality was consistently negatively correlated with
3,5,3-triiodo-L-thyronine levels, but not with thyroxine
levels. To test if thyroid hormones are directly respon-
sible for the change in optical quality, rainbow trout
lenses were cultured for 72 h in a medium containing
3,5,3-triiodo-L-thyronine, but no eect was observed.
The signicance of these ndings in the contexts of the
shes visual capabilities and smolting physiology is
discussed.
Keywords Thyroxine Thyroid hormone Rainbow
trout Ocular lens Metamorphosis
Abbreviations BVD: Back vertex distance RIA:
Radioimmunoassay SLM: Scanning laser monitor
Introduction
In several salmonid species can be found individuals and
distinct populations whose life cycles take them from
fresh water, where they hatch, to seawater in which they
feed and grow. They then return to fresh water where
they spawn or simply wait for the next opportunity to
feed at sea. The shifts in environment that accompany
the migrations are signicant, and the shes must be pre-
adapted to the new environment before exposure or they
are unlikely to survive. They must contend with the
changes in water osmolarities, predator and prey dis-
tributions, and sensory environment. The fresh water to
seawater migration and the accompanying process of
pre-adaptation, known as smoltication, consists of
behavioral (Hoar 1988; Iwata 1995), sensory (Beatty
1966; Morin et al. 1989), morphological (Boeuf 1993)
and physiological (Hoar 1988, and Boeuf 1993) changes.
There is evidence of a circannual rhythm to smoltica-
tion (Eriksson & Lundqvist 1982), which is entrained by
seasonal variations in temperature and photoperiod.
Several hormones such as the thyroid hormones,
growth hormone, and cortisol are involved in eecting
and mediating the transformation. But though they act
in concert to eect the complete, fully adaptive meta-
morphosis, certain specic changes (or at least the ini-
tiations of these changes) seem to often be associated
with a single hormone. For example, the thyroid hor-
mone thyroxine (T
4
) has been shown to stimulate sil-
vering of the body, cause behavioral changes (Hutchison
and Iwata 1998), and cause photopigment ratio changes
(Beatty 1972) and the loss of ultraviolet-sensitive cones
in the ventral retina of the rainbow trout, Oncorhynchus
mykiss (Browman and Hawryshyn 1992, 1994; Allison
et al. 2003).
T
4
and the thyroid hormone 3,5,3-triiodo-L-thyro-
nine (T
3
) are iodinated amino acids. They and related
iodinated compounds are found almost ubiquitously
throughout the animal kingdom as eectors and medi-
K. L. H. van Doorn (&) J. G. Sivak
School of Optometry, University of Waterloo, 200 University
Avenue W., Waterloo, ON, N2L 3G1, Canada
E-mail: klhvandoorn@delagar.com
Tel.: +613-674-1752
Fax: +519-725-0784
K. L. H. van Doorn J. G. Sivak M. M. Vijayan
Department of Biology, University of Waterloo, 200 University
Avenue W., Waterloo, ON, N2L 3G1, Canada
Present address: K. L. H. van Doorn
2220 County Rd 10 (RR 1), St-Eugene, ON, K0B 1P0, Canada
J Comp Physiol A (2005) 191: 649657
DOI 10.1007/s00359-005-0615-y
ators of growth and metabolism. They are also present
in some plants and protists, in some cases due to
endogenous production and in others because of exog-
enous uptake (Eales 1997). Among the higher verte-
brates, the thyroid gland is the organ responsible for
thyroid hormone production, mostly by secreting T
4
,
although in some species it secretes proportionally
smaller quantities of T
3
as well. After its release into the
body uids, T
4
action is regulated via a process of
deiodination to T
3
in peripheral tissues (Ramsden 1977;
Plate et al. 2002). T
3
is physiologically the more active
form, being bound with a higher anity by cellular
receptors (Oppenheimer 1983).
Thyroxine and/or T
3
are known to be involved in
several cases of vertebrate metamorphosis, such as the
amphibian tadpole to adult metamorphosis (Dodd and
Dodd 1976; Galton 1983), and the metamorphic matu-
rations of several species of sh. The larva-to-juvenile
attening of pleuronectid atshes (Sklower 1930;
Hoar 1951; Miwa et al. 1987, 1988), the maturation of
eel leptocephali (Sklower 1930), and salmonid smolti-
cation (Hoar 1939) all involve T
4
or T
3
to some degree.
In the case of salmonid smoltication specically, there
is a surge in T
4
production soon after the onset of the
process. This can bring T
4
levels up to 2090 ng/ml from
520 ng/ml depending on age, species, population, and
year (Hoar 1988; Dickho et al. 1982; Boeuf 1993).
Salmonids are highly visual sh. They depend heavily
upon vision for navigation (Groves et al. 1968; Parkyn
et al. 2003), mate localization (Duker 1982), and prey
localization and capture (Hoar 1958; Kato 1991;
Browman et al. 1993). It is now well established that
exposure to saltwater can induce cataractogenesis in
salmonids (Iwata et al. 1987), a problem that continues
to vex the aquaculture industry (Ersdal et al. 2001; Breck
and Sveier 2001; Menzies et al. 2002). The likelihood of
developing cataracts depends largely upon the meta-
morphic stage of the sh. The exact mechanism by which
saltwater aects the lens is not known, although Bjerka s
et al. (2003) suggest that defective osmoregulation in
smolts, such as might be caused by improper smolti-
cation, is responsible for cataract formation through
incompetence at maintaining an optimal lens hydration
state.
Because smoltication involves regulation of osmo-
regulatory mechanisms, it is conceivable that the process
itself could aect the ocular lens. Mechanisms that di-
rectly or indirectly aect the osmolarity of the aqueous
humor could alter the environment of the lens beyond
that with which the lens is able to cope via its own
osmoregulatory mechanisms, such as the Na/K pump. It
is also possible that adjustment of the lens own osmo-
regulatory mechanisms could take place during smol-
tication, similar to the increase in gill NaK-ATPase
activity that is known to occur (Hoar 1988; Boeuf 1993).
The study described here aimed to characterize optical
quality changes of the lens that occur during the sal-
monid parr-to-smolt metamorphosis, specically that
which can be accounted for by a T
4
surge.
Research on salmonid metamorphosis physiology has
traditionally been accomplished in one of the following
three ways: (1) by sampling wild populations, (2) by
inducing 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 Hawryshyn
1992; 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 the
ease with which confounding factors can be controlled in
a laboratory.
The experiments presented here specically involved
the induction of metamorphic changes by exposing
rainbow trout to T
4
. Although rainbow trout are re-
cently landlocked and non-anadromous, meaning that
they do not migrate in the wild, they nevertheless retain
the capacity to undergo smoltication-like metamor-
phosis when exposed to the hormones involved (Eales
1979). Because T
4
-treated salmonids do not undergo all
the metamorphic changes necessary to pre-adapt them
to saltwater, Eales (1979) referred to them as pseudo-
smolts. Nevertheless, they remain a useful model for
studying metamorphic physiology, for specic changes
can be observed in isolation from the many other
physiological factors that characterize true smoltica-
tion.
Materials and methods
Experiment 1: assessment of lens optical quality
during T
4
-induced metamorphosis of rainbow trout
Two similar experiments, experiment 1A and experiment
1B, are presented here. There are two major dierences
between them. The rst dierence is in the experimental
paradigms: experiment 1A compares treated and control
groups in which the animals were at the same develop-
mental stage while experiment 1B is a before-and-after
comparison of a treated group with conspecics from
prior to the initiation of the treatment. The second dif-
ference is in the treatment itself: during experiment 1A,
the dose of T
4
given to the treated animals was in-
creased, whereas a single high dose was given to the
animals in experiment 1B.
Experimental set-up
One hundred non-anadromous rainbow trout parr were
obtained from the Humber Springs Trout Club, a local
hatchery in Orangeville, ON, Canada. They were housed
in two groups of 50 in 415 l tanks in open circulation
ow-through systems at a consistent temperature of
12C and on a 12:12 h light:dark cycle. The sh were
650
allowed to acclimate for 3 weeks prior to beginning the
experiments. Their status as parr was ascertained by
visual inspection, notably by their conspicuous and well-
dened parr markings. The presence of these markings
was used to monitor individuals developmental stages
throughout the experiments.
The duration of the experiment 1A was 54 days. At
the beginning of the experiment, sh weighed on average
30.34.7 g, and by the end, they had grown to
54.711.3 g. Control sh were sampled for the rst
19 days, after which time the remaining control sh were
used as treated sh in experiment 1B, the duration of
which was 26 days. In this experiment, sh weighed
43.38.1 g at the beginning, and by the end, they had
grown to 47.78.0 g. All experimental procedures were
in accordance with the animal utilization guidelines of
the University of Waterloo, the Canadian Council of
Animal Care, and the Ontario Animals for Research
Act.
The sh feed served as the vehicle for T
4
, as was
described by Tagawa and Iwata (1991). The process of
lacing the feed began by dissolving T
4
in 1 ml 1N NaOH
solution and 500 ml absolute ethanol. The feed was then
bathed in this solution overnight in a fume hood,
allowing the liquids to evaporate and leaving the T
4
bound to the feed. Two concentrations were prepared in
this way: 0.25 mg/g (T
4
:feed), and 0.4 mg/g (T
4
:feed).
Feed given to the control sh was treated in the same
way, but without the addition of T
4
.
Fish were fed daily to satiation. In experiment 1A,
sh were fed with food containing 0.25 mg/g (T
4
:feed)
for the rst 15 days, followed by 0.4 mg/g (T
4
:feed) for
the remaining 27 days. Fish were then returned to an
untreated diet and sampled over 12 days. Four to six
sh were sampled at each sampling point in this
experiment. In experiment 1B, sh were fed throughout
with food containing 0.4 mg/g (T
4
:feed). In this
experiment, three sh were sampled at each sampling
time. Fig. 1 shows an experiment timeline that includes
T
4
dosages and sampling times. Sampling began by
anaesthetizing the animals in a 30-mg/l clove oil
solution, after which they were weighed and blood was
drawn from the caudal vessel. The blood was kept on
ice for up to an hour before being centrifuged for
10 min at 6,000 rpm. The plasma was kept at 80C
until radioimmunoassay (RIA) analyses could be
performed. After drawing blood, each animal was
decapitated and its head was placed on ice until lens
dissections were conducted. Heads were kept on ice for
up to 3 h.
Lenses were dissected out of the eye and placed in
customized lens culture cells containing H
10
medium
supplemented 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 specically for lens culture and
optical scanning. Complete H
10
medium contains in
tissue culture grade water, NaCl (135.5 mM), KCl
(5.0 mM), CaCl
2
(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 sh
ocular humors. During dissection, care was taken never
to touch the lenses themselves but with surgical sponges
and custom made glass scoops, thus avoiding inic-
tion of surface abrasions.
Assessing lens optical quality
Lenses were scanned using a custom designed scanning
laser monitor (SLM) based on the Scantox in vitro lens
assay system as described in Weerheim and Sivak (1992).
The SLM is a computer automated laser scanning device
that can provide a general measure of a lens optical
quality 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 point
light source at innity.
To briey explain the functioning of the SLM, len-
ses in their culture cells are placed into a light tight
chamber. 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 digital
camera photographs the 20 refracted beams, the images
of which are then collated and analyzed by custom
software that outputs the BVD statistics of the lens.
Each lens is scanned along two axes at 90 to each
other, and the BVD SE results of these scans are
averaged. 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 (T
4
:food) for the rst
15 days, after which the dose was increased to 0.4 mg/g (T
4
:food).
Treatment of this group ended on day 42. Animals in the control
group were given vehicle-treated food for 19 days to ensure that the
vehicle solution had no eect. The remaining sh in this group
became the subjects of experiment 1B in which the animals were
given a single dose of 0.4 mg/g (T
4
:food) for 26 days. Circles
indicate sampling points, and numbers beneath the circles indicate
the day at which the sampling occurred
651
T
4
and T
3
radioimmunoassays (RIA)
Regular testing of the sh serum T
4
titer was necessary
to ensure adequate dosage of T
4
. Serum T
3
titers were
also measured to ensure that T
4
to T
3
conversion was
taking place appropriately and was not impeded by
stress or other factors. Serum samples from each sh
were analyzed using MediCorp T
4
and T
3
RIA kits
(MediCorp, Montre al, QC, Canada; catalog numbers:
06B-254011 and 06B-254215, respectively).
Experiment 2: lenses cultured with thyroid hormone T
3
The results obtained in experiment 1 suggest a role for
T
3
in eecting lens optical quality changes (see the Re-
sults section). Experiment 2 was conducted to determine
if the eect observed is directly and exclusively attrib-
utable to T
3
.
Ten lenses were dissected from seven rainbow trout
parr obtained from the Humber Springs Trout Club in
Orangeville, 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 sh,
if used in the experiment, were always placed in separate
groups. The lenses were cultured at 12C in H
10
medium
supplemented with 10% dialyzed FBS. They were al-
lowed to acclimate to the environment for 1 week prior
to beginning treatment. This also allowed any abrasions
or other mechanical damage incurred during dissection
to become obvious, thus permitting the removal of
damaged lenses prior to beginning the experiment.
The culture medium of the treated lenses was then
supplemented with 50 ng/ml of T
3
and lenses were
cultured for another 72 h. During this 72 h period,
lenses were scanned after 0, 4, 24, 48, and 72 h of
treatment using an SLM. Culture media were changed
every 48 h.
Statistical analyses
All statistical analyses were performed using the Systat
10.0 statistical software. Comparisons between treated
and control groups were conducted via analyses of
variance (ANOVA). Pearson correlation coecients
were used to determine correlations between BVD SE
and serum hormone titers, and Bonferroni probabilities
were calculated based on these values. Probability values
equal to or less than 0.05 were considered statistically
signicant.
Results
Experiment 1: assessment of lens optical quality during
T
4
-induced metamorphosis of rainbow trout
No dierence in lens optical quality was observed be-
tween samples of the control group (P=0.360), leading
to the conclusion that there was no eect of time over
which the sham-treatment took place. To increase the
power of all subsequent treated-versus-control statistical
tests, data from all samples of the control group were
pooled, and all samples from treated groups were each
compared against this pool. In doing this, it was possible
to reduce the total number of animals required in these
experiments.
Experiment 1A
The two-step dosage regime (sh fed for 15 days with
0.25 mg/g (T
4
:feed) and for the following 27 days with
0.4 mg/g (T
4
:feed)) resulted in a progressive increase in
overall T
4
concentrations to 60.7214.99 ng/ml during
the experiment (Fig. 3). T
3
levels progressively increased
as well, reaching a peak of 8.880.19 ng/ml on day 40
(Fig. 4). It can be inferred that conversion of T
4
to T
3
was occurring normally. Silvering of the body and the
loss of parr marks were becoming evident by day 12, and
by day 33, all sh were well silvered. This conrmed that
metamorphosis was taking place.
A signicant reduction in overall lens optical quality
was evident 24 h after treatment began (P<0.000) and
this drop was sustained throughout the 6 weeks of
treatment (at each sampling time, P 0.042). On the 5
th
day post-treatment (day 47), lens optical quality of the
treated sh appeared to have recovered (P=0.398), but
a sample taken on the 12th day post-treatment again
showed a signicant dierence in comparison to the
control group (P=0.033) (Fig. 5).
Fig. 2 Diagrammatic illustration of the SLM scanning mechanism.
Twenty parallel beams are passed through the lens, using a
computer-controlled laterally-mobile mirror to position the beam
at each increment. This simulates a point light source located at
innity. A digital camera captures images of the refracted beams.
This allows the general optical quality of the lens to be measured by
quantifying its capacity to form a sharp image of a point source
located at innity. In practice, beams are passed along two axes at
90, each passing through the optical center, and results are
averaged
652
BVD SE and serum T
4
titers in the treated group
were found to be positively correlated (Pearsons
R=0.563, p 0.000). Even when data from the control
group were omitted from the calculation, BVD SE and
serum T
4
titers were positively correlated (Pearsons
R=0.474, P=0.002). BVD SE and serum T
3
titers were
also positively correlated (Pearsons R=0.394,
P=0.005) but only when data from the control group
were included. With control data omitted, Pearsons
R=0.313 and P=0.071. Because serum samples were
tested rst for T
4
, too little remained in some cases to
also test for T
3
, thus reducing the sample size available
for T
3
titers. This may explain the non-signicant
probability.
Experiment 1B
This treatment method, in which sh were fed
throughout with 0.4 mg/g (T
4
: feed), initially raised
serum T
4
titers more rapidly than observed with the two-
step dosage regime (Fig. 3). T
3
levels progressively de-
creased after day 6 (Fig. 4), suggesting that the rate of
conversion of T
4
to T
3
was decreasing or that the
clearing rate of T
3
was increasing.
A signicant reduction in lens optical quality was
observed 24 h after the rst treatment (P < 0.000),
and again this was maintained throughout the treat-
ment until the 19
th
day, by which time recovery ap-
peared to have taken place (P=0.004. However, as
seen in Fig. 5, this is due to the BVD SE having fallen
below that of the control group). The sample taken on
day 26 showed no dierence from that of the control
group (P=0.124).
BVD SE and serum T
4
titers in the treated group
were not found to be correlated, regardless of whether
data from the control groups were included or omitted
(Pearsons R=0.262, P=0.112; and Pearsons R=0.264,
P=0.343). BVD SE and serum T
3
titers were positively
correlated (Pearsons R=0.483, P=0.008) but only
when data from the control group were included. With
control data omitted, Pearsons R=0.413 and P=0.142.
As in experiment 1A, serum samples were tested rst for
T
4
, and so little remained in some cases to also test for
T
3
. This reduced the sample size available for T
3
titers,
compounding the already lesser sample size in this
experiment, which could explain the non-signicant
probability, in spite of the R value.
Fig. 3 Serum T
4
titers. Little change in serum T
4
titers was
observed in the control group over the course of the vehicle
treatment. A gradual increase in T
4
titers was seen in experiment
1A, in which there was a dosage increase on day 15. After ceasing
treatment on day 42, hormone titers returned to baseline levels. In
experiment 1B, there appeared to be relatively constant high levels
of T
4
, despite some uctuations, which could be attributed to small
sample size variability (n=3)
Fig. 4 Serum T
3
titers. T
3
levels remained relatively constant in the
control group, whereas it progressively increased in the experiment
1A treated group, before diminishing upon removal of the
treatment on day 42. The sample taken on day 54 showed a very
high unexplained variability. T
3
titers of the treated animals in
experiment 1B were signicantly elevated at the rst sampling
point, but began a progressive decrease by day 12. The reason for
this decrease is unclear, but it would likely be due to either a
decrease in the rate of T
4
deiodination, an increase in the rate of T
3
clearing from the serum, or both
Fig. 5 BVD SE of the 3 groups (1A, 1B and Control). No
statistically signicant dierences were observed between samples
of the control group, suggesting no eect due to the vehicle-treated
feed they were given. As a result, samples of this group were pooled
and all analyses of the treated were done against this pool.
Asterisks indicate statistically signicant dierences from the
control pool. At all sampling points in experiment 1A, samples
were signicantly dierent from the control pool (P 0.042) until
day 47 (P=0.398), 5 days after the treatment ended. BVD SE again
increased by day 54, resulting in a signicant dierence from the
control pool (P=0.033). At all sampling points in experiment 1B
up to and including those taken on day 12, statistical analyses were
signicant (P 0.000). By day 19, complete recovery had occurred
despite the ongoing treatment. Statistical signicance is indicated
on day 19 due to the BVD SE of the treated group having fallen
below that of the control pool
653
Experiment 2: lenses cultured with thyroid hormone
No dierence in lens optical quality was detected be-
tween those lenses treated with T
3
and the controls (re-
peated measures ANOVA P=0.555). See Fig. 6 for a
graphical summary of the results.
Discussion
This study shows conclusively that a reduction of lens
optical quality can occur as a result of a T
4
surge, such
as that which occurs during smoltication. In both
experiments 1A and 1B, the decrease of lens optical
quality was apparent 24 h after the rst dose of T
4
. The
exact mechanism by which the change is eected is not
yet known.
Bjerka s et al. (1996) found a correlation between ra-
pid growth and cataract formation in farmed Atlantic
salmon (Salmo salar) in freshwater, although they could
not exclude potential compounding eects due to
smoltication. By feeding all groups according to the
same 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 dierences be-
tween groups in this experiment.
Upon exposure to saltwater, the aqueous humor
osmolality of coho salmon increases more rapidly than
that of the plasma (Iwata et al. 1987). Since aqueous
osmolarity is typically lower than plasma osmolarity in
teleosts (Nicol 1989), this suggests that ion inux from
the seawater or water outux from the aqueous through
a permeable cornea (Edelhauser 1968) occurs upon
saltwater 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 in
this state of osmolar ux, the composition of the aque-
ous may be altered as well as that of the lens. Lens
hydration state could be altered enough during this
period to aect its optical quality.
A recent medical study (Age-Related Eye Disease
Study Research Group, 2001) showed, with borderline
signicance, a link between thyroid hormone treatment
in humans and the presence of moderate cortical opac-
ities. Due to the borderline signicance, however, the
authors suggest further research before drawing any
conclusions. The administration of thyroid hormones to
adult frogs has been shown to increase lens epithelial
mitotic activity (Weinsieder et al. 1972), although direct
exposure to T
3
showed no eect on cell proliferation in
cultured lenses. The potential link between mitotic
activity and cataract development (i.e. lens optical
quality reduction) upon thyroid hormone exposure is
intriguing, and may be worth pursuing in the context of
smoltication.
Methionine deciency in the diet of rainbow trout has
been shown to cause cataracts (Cowey et al. 1992). It has
also been shown to induce an elevation in plasma T
3
in
chickens (Carew et al. 2003). Bearing in mind the phy-
logenetic separation between these species, it is inter-
esting to consider the possibility that the cataracts
observed by Cowey et al. might have been attributable
to an increase of T
3
titers in the sh. If so, their ndings
would parallel those described in this study, although
the severities of the observed lenticular changes were
dierent in both studies.
Like Weinsieder et al. (1972), the work presented here
ruled out a direct and exclusive eect attributable to T
3
,
because culturing lenses in media containing T
3
showed
no eect on lens optical quality in experiment 2. It might
be argued that the lenses were cultured for too short a
time (72 h) for an optical eect to be observed, but be-
cause an eect was observed after 24 h in experiments
1A and 1B, we would assume that if it were due to T
3
exclusively, it would have been manifested by the 24
th
hour of culture. This of course does not rule out an
optical eect due to a long-term exposure to T
3
, or an
optically unrelated physiological eect. However, if such
phenomena were to occur, it still could not explain the
results observed as a result of in vivo exposure to T
4
.
There is a curious dierence between the results ob-
tained from experiments 1A and 1B: in the latter, in
which sh were given 0.4 mg/g (T
4
:feed) from the start,
lens optical quality completely recovered during the
treatment period around day 19. The reason for this is
not known, but there were three conspicuous dierences
between these experiments, one or more of which may be
responsible.
Firstly, the sh in experiment 1B were somewhat
larger at the beginning of the experiment than those in
experiment 1A (mean weights: 43 g vs 29.8 g), although
they 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 H
10
medium with
10% FBS with (treated) and without (control) the addition of
50 ng/ml T
3
. No dierence was observed between the two groups
(P=0.555). Thus there is no exclusive and direct optical eect
attributable to T
3
654
Secondly, serum T
3
titers in experiment 1B dimin-
ished after reaching a peak on day 6. At no time did such
a decreasing trend occur in experiment 1A during the
period of treatment. This suggests a drop in the rate of
conversion of T
4
to T
3
or an increased rate of T
3
clearing
from the serum (Plate et al. 2002). It is possible that a
stressful factor may have aected the sh in experiment
1B, such as the overall lower stocking densities that they
experienced (18 sh of 43 g vs 50 sh of 29.8 g, both in
415 l tanks).
Thirdly, experiment 1B was begun 30 days later than
experiment 1A. Farbridge & Leatherland (1991) de-
scribed an endogenous biweekly pattern in rainbow
trout food consumption and hormone levels that was
correlated with the phase of the moon. This phenom-
enon was ruled out as the cause of the strange response
in experiment 1B, since both experiments were begun at
approximately the same point in the lunar cycle. But
because experiment 1B was begun 30 days later in the
autumn season, the potential inuence of the endoge-
nous circannual rhythm present in smolting salmonids,
which may potentiate or inhibit the conversion of T
4
to
T
3
depending on its current state, should be considered.
The correlational analyses showed a positive corre-
lation between lens optical quality and serum T
4
titers in
experiment 1A but not in experiment 1B. That no cor-
relation was evident in experiment 1B is understandable
considering that lens optical quality recovered during
the treatment period, while T
4
levels were still elevated.
BVD SE and T
3
were positively correlated in both
experiments, when data from the control group were
included in the analyses. Bearing in mind that T
3
is
physiologically more active than T
4
, it is not surprising
that a positive correlation with T
3
was found in experi-
ment 1B, but no correlation with T
4
.
It is perplexing that the apparent optical recovery in
experiment 1A on day 47 was short-lived. The sample
taken on day 54 again showed a signicant dierence
with the control group. As can be seen in Fig. 4, there is
great variability in the T
3
titers observed in this sample.
Unfortunately, we are at a loss to explain the T
3
vari-
ability, especially in light of the low T
4
titers as shown in
Fig. 3. This may again be an artifact of the somewhat
small sample sizes, which would emphasize the contri-
butions of exceptional individual sh.
One nal comment should be made about the results
obtained as they relate to the mechanism by which lens
optical quality was measured. Throughout the experi-
ment, lenses were scanned along a 22.5 mm axis
depending on the size of the aperture of the scanning cell
in which the lenses were kept, which was in turn
dependent on the diameter of the lens. Lenses less than
2.5 mm in diameter were mounted on a 2-mm aperture,
and those equal to or greater than 2.5 mm in diameter
were mounted on a 2.5 mm aperture. The 20 beams
passed through the lenses were always equally spaced
such that they scanned across the whole aperture. As the
sh grew throughout the experiments, their lenses also
grew, but regardless of their size, only the central
2.5 mm of the diameter was scanned. As a result, optical
quality data from the periphery of the lenses will be
absent toward the end of the experiment. Because new
growth occurs at the periphery of the lens, this region
may be most disturbed by whatever is aecting the
optical quality of the lens. Since data on this region of
the lens is lacking in the later samples, it is possible that
lens optical quality from these lenses was being under-
estimated. While this would not change the conclusions
drawn from the results, it may be relevant when con-
sidering the functional impact on the shes vision.
Functional impact on visual capabilities
The functional impact on the shes vision remains un-
known. That the BVD SE measurements were done in
vitro 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 to
have a resolving power up to ten times that of their
retinal photoreceptor density (Fernald 1988), some de-
bate exists as to the signicance of this (Sivak 1990). A
lens with such a high resolving power may benet min-
imum viewable acuity and hyperacuity (types of visual
acuity that oer resolutions smaller than photoreceptor
separation), and it could also improve image contrast
(Snyder et al. 1986) though at the expense of aliasing
artifacts. Should it be the case in late parr stage O.
mykiss and other salmonids that the resolution of the
retina is the limiting factor in their visual acuity, a
reduction in lens optical quality might have little to no
functional impact on vision. We would be left wondering
if evolution might thus have favored an over-engineered
lens in these animals to compensate for the deleterious
eects observed during smoltication and saltwater
exposure.
In their study of osmotic cataracts in wild seagoing
salmon, Bjerka s et al. (2003) noted that the presence of
cataracts did not aect the condition factor (weight/
length
3
) of the sh, indicating that they were still able to
nd food. Under certain conditions, it would thus seem
that a severe reduction in visual capacity is not delete-
rious to the animals in that particular population, al-
though stocking density and food availability are
important factors to consider, as well as the consistence
of the diet.
In summary, the research presented here showed that
exposure of rainbow trout to T
4
levels similar to those
which are observed during the natural smoltication of
O. mykiss (steelhead trout specically) can reduce the
optical quality of their lenses. This is, to our knowledge,
the rst time that the lenses of these sh were shown to
be aected by the physiological metamorphosis itself
(induced or natural). The investigation of lens quality in
wild salmonid populations at dierent stages of the life
cycle is a compelling area for future research, to deter-
mine if lens eects are a natural occurrence. If so, one
655
might begin investigating environmental and genetic
factors relevant to the onset of and potential recovery
from optical distress. It will also be important to eluci-
date the physiological mechanisms responsible for such
a change in lens optical quality.
Acknowledgements We kindly thank Vladimir Bantseev, Nancy
Gibson, Robin Jones, Kelley Moran and Martin Ryan of the
University of Waterloo for their practical insights and technical
support during the course of this research. Special thanks are of-
fered to Drs. Munehico Iwata and Geo Eales for methodological
assistance. We would nally like to thank two anonymous
reviewers for raising points which allowed us to improve the
manuscript. This research was funded by the Natural Sciences and
Engineering Research Council of Canada. These experiments
comply with the Principles of animal care, publication no. 8623,
revised 1985 of the National Institute of Health, and also with the
current laws of Canada.
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