Magei et al.: Recovery of visual function in Hippoglossus stenolepis after exposure to bright light 
569 
fish, namely the percentage of maximal response at 
each tested light intensity. Finally, the data from each 
individual ERG curve was fitted by using a second-or¬ 
der polynomial equation with SYSTAT software, vers. 
13 (Systat Software, Inc., San Jose, CA) or Microsoft 
Office 2013 (Microsoft Corp., Redmond, WA), because 
the ERG response curves generally were of a sigmoid 
shape. To provide a summary measure of visual im¬ 
pairment, we calculated log-scale illumination required 
to produce a 50% p-max response from each fish. In 
the left and right eye, and exposure recovery experi¬ 
ments, ERG responses presented as voltages and p- 
max responses were examined with repeated measures 
analysis of variance (ANOVA) (Sokal and Rohlf, 1969). 
For examination of the light level required to produce 
a 50% p-max response, we compared treatment groups, 
using one-way ANOVA (Sokal and Rohlf, 1969). Where 
appropriate, we employed a Tukey’s honestly signifi¬ 
cant difference (HSD) test (Sokal and Rohlf, 1969) to 
examine differences in treatment means. Tests were 
considered significant at the P<0.05 level. 
Behavioral evaluation of fish in relation to visual function 
Individual 3-year-old Pacific halibut (21-27 cm TL) 
were anesthetized with MS-222 as described above, but 
in this case both eyes were subjected to a 15-min ex¬ 
posure to simulated sunlight before behavioral experi¬ 
ments. After light exposure, pairs of fish were moved 
into 1.9-m diameter x 80-cm deep circular tanks to re¬ 
cover. The tanks were located within a light-controlled 
laboratory and supplied with constantly flowing sea¬ 
water at ~9°C. 
Experiments were conducted with 8-10 pairs of fish 
at six light intensities simulating environmental con¬ 
ditions typical for Pacific halibut (-90-900 m): IxlO -3 , 
IxlO -4 , IxlO -5 , and IxlO -6 , IxlO -7 pmol-m -2 -s -1 , and 
complete darkness (<0.01xl0 -7 ). Light levels were mea¬ 
sured on the bottom of the experimental tank with a 
IL17Q0 Research Radiometer equipped with a photo- 
synthetically active radiation-filtered waterproof sen¬ 
sor. To reduce shadows, all lighting was attached to an 
overhead ring suspended 1.8 m above the tank bottom 
and approximately 0.7 m outside the tank circumfer¬ 
ence. Four cone lamps with green LED (~555-nm) clus¬ 
ters were mounted on the ring. The LED clusters were 
linked to a rheostat that was used to vary light inten¬ 
sity. The lights were placed directed perpendicular to 
the tanks to avoid glare and hot spots. 
We recorded fish movements with an overhead video 
camera (Ikegami Electronics, Inc., Mahwah, NJ) and 
under infrared illumination. Infrared illumination 
ranged from 760-880 nm, which is a range undetect¬ 
able by Pacific halibut (John, 1964; Higgs and Fuiman, 
1996; Brill et al., 2008). Infrared lights were placed 
below the bottom of the tank and provided a silhouette 
of the fish; these lights were left on for all experimen¬ 
tal trials, regardless of the light treatment being used. 
Each experimental tank had a clear Plexiglas tube 
placed in the middle that held a white fishing jig that 
was attached to the ceiling with a counter-weighted 
line and to the bottom of the tank with an elastic band. 
The bottom 20 cm of the Plexiglas tubes were covered 
with black tape, such that the jig would not be visible 
to the fish when not in use. 
Fish were allowed to recover for at least 48 h after 
exposure to simulated sunlight before use in further 
trials. Each pair of fish was tested at all 6 levels of 
illumination: 2 illumination levels on each of the first 
2 days, and a single illumination level on the last day. 
The illumination level was set with the rheostat and 
fish were allowed to acclimate for 2 h before the trial 
began, 2 h were allowed between trials, and the order 
of testing with respect to illumination level was ran¬ 
domized. A trial at each illumination level consisted 
of two 5-min periods before and after presentation of 
the visual stimulus (white jig). After the first 5-min 
period, the jig was moved up and down rapidly (within 
the Plexiglas tube) for 60 s and then allowed to sink 
back below the masked bottom of the Plexiglas tubes, 
where it was out of sight. Each minute was split into 
10-s intervals and scored as to whether the pair of fish 
reacted to the visual stimuli. A reaction was considered 
positive if the fish either 1) moved one body length, 2) 
made oral contact with the column while attempting 
to bite at the jig, or 3) re-oriented itself such that the 
long axis of the fish was pointing toward the jig (-10°). 
Scoring behavior of fish 
Scores were recorded as either 0 (no reaction by ei¬ 
ther fish), 1 (reaction by one fish), or 2 (reaction by 
both fish). For each 1-min trial, the 10-s scores were 
summed to arrive at an activity index. We compared 
activity indexes of fish exposed to simulated sunlight 
and control fish over time at each light level by us¬ 
ing repeated measures ANOVA (n=6-9). Where ANO¬ 
VA results indicated significant differences, a Tukey’s 
HSD was used to determine differences between group 
means. During the scoring process and in preliminary 
analysis it became apparent there was no difference 
between the lowest light levels (IxlO -5 , IxlO -6 , and 
IxlO -7 pmol-m -2 -s -1 and complete darkness). Hence, 
we decided to show only the highest 4 light intensities 
(IxlO -3 , IxlO -4 , IxlO -5 , and IxlO -6 pmol-m -2 -s -1 ). 
Results 
Electroretinography experiment 
At the same light intensities, voltages measured on the 
corneal surface of the right eyes of control fish were 
significantly higher than those measured on the cor¬ 
neal surface of left eyes. This finding was manifest by 
a significant interaction between eye (left vs. right) and 
light intensity in our ANOVA (F [16 32 ]=4.18, P<0.0001). 
The difference in the responses of right and left eyes 
increased with increasing light intensities (Fig. 1). 
When voltage data for each fish were converted to p- 
