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Thermal Tolerance of Diploid Versus Triploid Rainbow Trout and Brook Trout
Assessed by Time to Chronic Lethal Maximum

Article  in  Environmental Biology of Fishes · February 2006

DOI: 10.1007/s10641-006-0008-2

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Environmental Biology of Fishes (2006) 75:183–193 Ó Springer 2006
DOI 10.1007/s10641-006-0008-2

Thermal tolerance of diploid versus triploid rainbow trout and brook trout
assessed by time to chronic lethal maximum

Peter F. Galbreatha,c, Nathan D. Adamsa, Lee. W. Sherrill IIIa & Thomas H. Martinb
a
Mountain Aquaculture Research Center, Western Carolina University, Cullowhee 28723, NC, USA (e-mail:
galp@critfc.org)
b
Department of Biology, Western Carolina University, Cullowhee 28723, NC, USA
c
Columbia River Inter-Tribal Fish Commission, 729 NE Oregon St., Suite 200, Portland 97232, OR, USA

Received 3 January 2005 Accepted 6 January 2006

Key words: ploidy, triploidy, critical thermal maximum, brook charr

Synopsis

An effect of ploidy on thermal tolerance in juvenile trout was assessed in a series of tests comparing time to
chronic lethal maximum (CLMax). Diploid and triploid fish were produced from a common spawn for
three different groups each of brook trout Salvelinus fontinalis and of rainbow trout Oncorhynchus mykiss.
One or two CLMax tests were performed per group, on between 15 and 50 individuals per ploidy within
groups. The tests involved exposure of fish to a progressive 2°C day)1 water temperature increase and
recording of the time at which each individual fish reached loss of equilibrium (LE). The time to LE data
were rank transformed and analyzed as a randomized complete block design. Although relative perfor-
mance varied among trials, the analysis indicated overall differences due to ploidy were small and non-
significant among both brook trout and rainbow trout. Size proved to be significantly correlated with time
to LE in the brook trout trials, but not in the rainbow trout trials. Two of the six groups included a large
proportion of fish which had received a heat shock following fertilization, but were not successfully tri-
ploidized. In both cases, thermal tolerance of the heat-shocked diploids was similar to that of the non-heat
shocked control diploids, indicating no persistent effect of the heat shock on thermal tolerance.

Introduction tion in flesh quality, reduced dress-out percentage,

Induction of triploidy is an effective means to
produce functionally sterile trout and salmon. Use
of sterile triploids presents certain advantages in
both commercial culture and fisheries management
of salmonids. In culture of rainbow trout On-
corhynchus mykiss as a food fish, which is per-
formed primarily with monosex female stocks,
gonadal development in triploid females is arrested
and the fish do not acquire commercially undesir-
able secondary sexual characteristics – darkening
of skin pigmentation, increased aggression, reduc-
etc. Intentional stocking of triploids into natural
water bodies, as well as escapement of triploid fish
from commercial facilities, poses no direct threat to
the genetic integrity of natural stocks, as the arti-
ficially produced triploids cannot successfully
interbreed with wild fish (see reviews by Utter et al.
1983, Ihssen et al. 1990, Thorgaard 1992, Tave
1993, Lutz 2001). Despite these advantages, pro-
duction of triploid salmonids has not been widely
accepted, due in part to the perception that trip-
loids do not perform as well as normal diploids
under conditions of stress. This perception is based
184
in large part on anecdotal evidence from certain
experiences of poor performance of triploids in
commercial culture (Jungalwalla 1991, Sutterlin &
Collier 1991, Johnstone 1996, McGeachy et al.
1996, Stuart 1996, Benfey 1999, Altimiras et al.
2002). However, it contrasts with results from
controlled studies of behavior and physiology,
including some that looked specifically at response
to various stressors, which generally indicate
comparable performance of triploid and diploid
salmonids (see review by Benfey 1999, and sub-
sequent reports by Benfey & Biron 2000, Dillon
et al. 2000, Sadler et al. 2000a, b, Gillet et al. 2001,
Altimiras et al. 2002, Mercier et al. 2002, Hynd-
man et al. 2003a, Johnson et al. 2004). Nonethe-
less, there have been a few studies where decreased
survival and growth of triploid relative to diploid
salmonids were reported. In some of these cases,
decreased tolerance of triploids to stress associated
with high temperature was suggested as the cause
for their poorer performance (Quillet et al. 1987,
Quillet & Gagnon 1990, Myers & Hershberger
1991, Blanc et al. 1992, Simon et al. 1993, Ojolick
et al. 1995, Bonnet et al. 1999, Cotter et al. 2002).
Thermal tolerance of fish is often reported as
critical thermal maximum (CTMax). A CTMax test
involves exposure of a group of experimental fish to
conditions of progressively increasing water tem-
perature, generally in the range of 1°C h)1 to
1°C min)1, and calculation of the average temper-
ature at which they reach a behavioral or physio-
logical endpoint such as loss of equilibrium (LE, the
inability to maintain an upright position within the
water column), opercular spasms, or cessation of
opercular ventilation (see reviews by Brett 1956,
Hutchison 1976, Becker & Genoway 1979, Paladi-
no et al. 1980, Kilgour & McCauley 1986). In the
only published report which directly investigated
an effect of ploidy in a thermal tolerance test,
Benfey et al. (1997) compared CTMax measures of
diploid (2n) and triploid (3n) brook trout Salveli-
nus fontinalis. Their tests involved exposure of fish
both as underyearlings (average weight = 25 g)
and as yearlings (average weight = 668 g) at
heating rates of either 2°C h)1 or 15°C h)1. Results
consistently indicated no significant difference in
CTMax attributable to ploidy.
However, the acute nature of a CTMax test
contrasts with the more chronic nature of ther-
mal stress as experienced by fish in natural or
commercial settings, or in protracted growth trials.
For this reason, various researchers have ques-
tioned the relevance of CTMax measures as indi-
cators of thermal tolerance, and proposed tests of
longer duration using slower rates of temperature
change (Lutterschmidt & Hutchison 1997, Beitin-
ger et al. 2000, Selong et al. 2001). Beitinger et al.
(2000) referred to such tests as measuring chronic
lethal maximum (CLMax). To assess the influence
of heating rate on the relative sensitivity of lethal
maximum tests, Galbreath et al. (2004) measured
relative thermal tolerance of juvenile brook trout,
rainbow trout and brown trout in trials at four
different heating rates: 24, 8, 4 and 2°C day)1. They
used LE as the thermal tolerance end point. This
behavior, unlike onset of opercular spasms or ces-
sation of opercular ventilation, could be readily
observed from above the water’s surface by a re-
searcher standing alongside the experimental tank.
Additionally, time to LE, as opposed to tempera-
ture at LE, was calculated as the measure of ther-
mal resistance. Time to LE provides a more direct
measure of the additive effects of thermal stress
(Kilgour & McCauley 1986), and potentially
greater sensitivity to small differences in tolerance
between individuals than does temperature at LE.
Results indicated that as heating rate decreased,
differences in thermal maximum between species
were increasingly evident. Galbreath et al. (2004)
therefore adopted this modified CLMax protocol,
using a 2°C day)1 heating rate and measurement of
time to LE, for assessing relative thermal tolerance
of groups or individual fish within an experiment.
It is possible that the lack of an effect of ploidy
on thermal tolerance observed by Benfey et al.
(1997) could be attributable to insufficient sensi-
tivity of their CTMax protocol. The present study
reexamined whether ploidy has an effect on ther-
mal tolerance in salmonids. A series of CLMax
tests, using the modified protocol described by
Galbreath et al. (2004), were conducted on juve-
nile diploid and triploid brook trout and rainbow
trout.
Materials and methods
Diploid and triploid rainbow trout (Rbt) and
brook trout (Bkt) were produced during the fall
spawning seasons of 2000, 2001 and 2002. Eggs

were obtained from broodfish held by the Moun-
tain Aquaculture Research Center (MARC; Wes-
tern Carolina University, Cullowhee, North
Carolina) at the Lonesome Valley Aquaculture
Research Station (Cashiers, North Carolina), or
they were obtained from a nearby state or federal
fish hatchery. In each spawning, a sample of eggs
was collected from several females and fertilized
with sperm from several males using a factorial
mating design – the eggs were pooled, mixed then
sublotted, and each sublot was fertilized with the
milt from one male. The fertilized eggs were then
repooled, mixed and divided in half. At 10 min
post-fertilization, one half of the eggs was placed
in a water bath at 28°C for a period of 10 min.
This thermal shock protocol is effective in induc-
ing triploidy in the eggs of trout, by causing
retention of the set of chromosomes destined for
extrusion as the second polar body (Chourrout
1980, Galbreath & Samples 2000). The eggs were
then transferred to separate trays within a vertical
tray incubator in the MARC on-campus fish
rearing facilities. Following hatch, the fry were
transferred to separate tanks within a common
indoor recirculated-water system for initial rear-
ing. At 4 – 6 months of age, triploid fish were
adipose fin-clipped, to distinguish them from dip-
loids, and the fish were transported to the Lone-
some Valley Aquaculture Research Station, for
continued rearing in side-by-side troughs supplied
with flow-through stream water, or they were
pooled into the same rearing trough.
Between 6 and 12 months post-initiation of
feeding, the diploid and triploid fish from each
group were transferred back to the on-campus
MARC facilities and placed in separate 560 l cir-
cular fiberglass tanks within a common recirculat-
ed-water rearing system (3400 l total water
volume) supplied with 3 l min)1 well water. Within
the week, each fish was blood-sampled and its
ploidy confirmed by flow cytometric analysis
(Galbreath & Samples 2000). Occasional diploids
found among the triploid groups, due to incom-
plete triploidization by the heat shock, were dis-
carded. For two groups (Rbt (3a and Bkt (1),
however, 20 – 30% of the putatively triploid fish
were determined to be diploid. (The reason for this
is unknown, though we suspect that the heat
shock protocol for triploidization was not fully
respected.) In these cases, the heat-shocked
185
diploids were tattooed on the ventral side with
Alcian blue applied with a Panjetä needleless
injector (Wright Health Group Ltd., West Dundee,
Scotland; Herbinger et al. 1990, Thedinga &
Johnson 1995), and retained in the experiment.
Data collected from these individuals permitted
comparison with that of the non-heat shocked
control diploids, to test for a persistent effect of the
heat shock on thermal tolerance, separate from
that of the change in ploidy.
The experimental fish were held for a minimum
of three weeks to promote acclimatization to
conditions within the recirculated-water rearing
system prior to thermal tolerance testing (Hutchi-
son & Maness 1979, Lutterschmidt & Hutchison
1997). Water temperature during this period gen-
erally varied less than ±1°C, and averaged be-
tween 12 and 17°C depending on the experiment.
During this acclimation period the fish were fed
daily to near satiation with a commercial trout diet
until 1 or 2 days prior to the thermal tolerance
test, at which time feeding ceased.
For each of the six groups of fish, a CLMax test
was conducted using a 2°C day)1 heating rate,
generally following procedures described by Gal-
breath et al. (2004). In three cases (Rbt #1, Rbt #3
and Bkt #2), sufficient fish were available to repeat
the test with a second group of fish. In each trial, a
randomly chosen sample of fish of both ploidies
were pooled in a 560 l experimental tank within
the same rearing system. Water flow in this tank
was then switched from recirculation to flow-
through at 1 – 2 l min)1, initially with water from
the recirculation system, and later with 18 – 20°C
well water. Water temperature in the experimental
tank was increased using an 1800 W immersion
heater controlled by a programmable digital
thermostat (Process Technology, Mentor, Ohio).
Water circulation within the tank was maintained
with a 0.5 A submersible pump. Throughout the
7 – 8 day period of each trial, temperature in the
experimental tank was recorded once per minute
with a HOBO Water Temp Proä temperature data
logger (Onset Computer Corporation, Bourne,
Massachusetts). Supplemental aeration main-
tained dissolved oxygen concentration at 80 – 90%
saturation throughout each experiment, as mea-
sured periodically with a YSI Model 50ä dissolved
oxygen meter (Yellowsprings Instrument Com-
pany, Yellowsprings, Ohio).
186
When temperature increased above 25°C, the
fish were observed frequently for the erratic
swimming and gasping behaviors typically ob-
served in fish prior to reaching LE (Paladino
et al. 1980, Elliot 1981, Lutterschmidt &
Hutchison 1997). After the first fish reached LE,
observation was constant until the end of the
experiment. When a fish initially lost equilib-
rium, it rolled to its side or upside down,
revealing it lighter colored underside and per-
mitting observation by the researcher standing
beside the tank and looking down into the wa-
ter. If the fish completed a full rotation of the
tank in this manner, it was prodded with a dip
net. If it did not right itself and swim off, it was
considered as having definitively arrived at LE
and was removed from the experimental tank.
The time was recorded and the fish was identi-
fied to ploidy group by presence or absence of
an adipose fin (and tattoo in two trials), and it
was measured for length and weight. Condition
factor (K ) was calculated according to the for-
mula: K = weight (g)  100/length (cm)3 (Moyle
& Cech 1996), and time to LE was calculated as
the number of hours between initiation of the
experiment and the time the fish reached LE.
Additional information specific to the experi-
mental fish in each trial is provided below:
Rbt #1 – Fish were produced on September 11,
2001, from 30 female and 15 male Erwin  Arlee
strain rainbow trout at the Erwin National Fish
Hatchery, Erwin, Tennessee. Two CLMax tests
were conducted on the diploid and triploid
progeny – the first (Rbt (1a) commenced on
October 13, 2002 with an initial water tempera-
ture of 13.9°C, and the second (Rbt #1b) on
October 27, 2002 with an initial water tempera-
ture of 13.1°C.
Rbt #2 – Fish in this trial were the pooled
progeny from two different spawns of broodstock
held at the Lonesome Valley Aquaculture Re-
search Station; this stock originated from fish
obtained from the Pisgah Forest State Fish
Hatchery, Brevard, North Carolina. The first
spawning was conducted on October 2, 2001 and
the second on October 16, 2001. Less than 5 fish
per sex were used within each day’s spawning, al-
though the exact number was not recorded. The
CLMax test began on December 11, 2002, with an
initial water temperature of 14.2°C.
Rbt #3 – Fish were produced on November 5,
2002, from the spawning of approximately 20 fish
per sex of Arlee strain rainbow trout at the Erwin
National Fish Hatchery. Two CLMax trials were
performed, the first (Rbt #3a) beginning on Jan-
uary 8, 2004 with an initial water temperature of
11.6°C, and the second (Rbt #3b) on January 18,
2004 with an initial water temperature of 11.4°C.
The first trial (Rbt #3a) also contained a group of
heat-shocked but non-triploidized diploids.
Bkt #1 – Fish were produced from the spawn of
15 females and 3 males on October 24, 2000. The
broodstock were held at the Lonesome Valley
Aquaculture Research Station; they were the
progeny of fish obtained as juveniles from the
Pisgah Forest State Fish Hatchery. The CLMax
trial commenced on July 15, 2001, with an initial
water temperature of 16.5°C.
Bkt #2 – Fish were produced from 10 female
and 6 male MARC broodstock on November 1,
2000. Two CLMax trials were performed, the first
(Bkt #2a) on July 31, 2001 with an initial water
temperature of 14.5°C, and the second (Bkt #2b)
on September 25, 2001 with an initial water tem-
perature of 12.9°C.
Bkt #3 – Fish in this trial were the pooled
progeny of two different spawns of MARC
broodstock, the first conducted on October 16,
2001 and the second on October 30, 2001. Less
than 5 fish per sex were used within each spawn,
although the exact number was not recorded. The
CLMax test began on December 2, 2002 with an
initial water temperature of 12.5°C.
Although temperature at LE data was also
recorded, measurement of time to LE offers
greater sensitivity to differences between fish
within a trial (Galbreath et al. 2004) and time to
LE was therefore used as the dependent variable.
Due to slight variations in heating rate among
trials, as well as inevitable small differences in size
and genetic make-up of experimental subjects
among trials, the experiment was analyzed as a
randomized complete block design with trials treated
as blocks. Through preliminary analysis, we found
that residuals tended to be significantly non-
normal (Shapiro–Wilk’s test, PROC UNIVARI-
ATE, SAS/STAT Release 8.02, SAS Inc., Cary,
North Carolina). Therefore, the data were rank-
transformed as suggested by Iman et al. (1984),
and analyses were then conducted using PROC
 187

MIXED (SAS/STAT Release 8.02, SAS Inc.,
Cary, North Carolina). Similar analyses on ranks
have been shown to be equivalent to Friedman’s
nonparametric tests for complete block designs
(Conover & Iman 1981). Ploidy effects were trea-
ted as fixed, while trial and trial  ploidy effects
were treated as random effects and their covari-
ance components tested using the COVTEST op-
tion of PROC MIXED. Preliminary examination
of the data suggested the possibility of correlations
between time to LE and size, so total length (TL)
and condition factor were included in the statisti-
cal model as covariates.
Additionally, separate statistical analyses were
performed for trials Rbt 3a and Bkt, each of which
included three groups of fish: heat shocked trip-
loids (3n+), non-heat shocked control diploids
(2n)), and heat shocked diploid fish (2n+). These
analyses tested for an effect of the heat shock
treatment used to induce triploidy, independent of
the resultant ploidy. Analyses were completed as
described above, with the exception that there were
no trial nor trial  ploidy effects included in the
model. Pair-wise differences between ploidy groups
were tested using the Tukey–Kramer adjustment
option of the LSMEANS statement (PROC GLM,
SAS/STAT Release 8.02, SAS Inc., Cary, North
Carolina).
Results
Length and condition factor were generally similar
between ploidies within a trial, but differed
somewhat among trials (Table 1). The effects of
fish size (total length and condition factor) were
found to be significantly positively correlated with
ranked time to LE for brook trout, but not for
rainbow trout (Table 2).
While the random effects of trial and the trial 
ploidy interaction contributed large covariance
components, they were not significant for either
species (Tables 3 and 4). In the rainbow trout
studied, there was no evidence of an effect of

Table 1. Data (mean ± standard deviation) for fish size (length and weight) and condition factor, temperature at LE, time to LE, and
rank (sequential order of the time at which fish arrived at LE) for a series of CLMax tests between diploid (2n) and triploid (3n)
rainbow trout (Rbt) and brook trout (Bkt).

Species Ploidy No. Fish Length Weight (g) Condition Temp. at Time to LE Rank
Trial # (mm) factor (K) LE (°C) (hours)

Rbt
1a 2n 23 131.0 ± 19.8 25.1 ± 12.6 1.04 ± 0.05 28.14 ± 0.18 179.4 ± 2.0 26.3 ± 14.3
3n 23 139.3 ± 17.6 30.2 ± 10.6 1.07 ± 0.15 28.07 ± 0.17 178.8 ± 2.1 20.7 ± 12.2
1b 2n 23 133.7 ± 17.3 24.2 ± 8.7 0.97 ± 0.08 27.93 ± 0.19 176.6 ± 1.7 26.3 ± 12.8
3n 20 134.4 ± 21.1 25.7 ± 13.8 0.98 ± 0.09 27.85 ± 0.17 175.4 ± 1.6 17.0 ± 10.4
2 2n 40 142.4 ± 18.0 30.7 ± 12.0 1.01 ± 0.09 28.00 ± 0.23 166.8 ± 2.0 28.3 ± 16.6
3n 15 146.7 ± 19.7 33.3 ± 14.0 1.00 ± 0.08 28.00 ± 0.16 166.7 ± 1.4 27.3 ± 14.9
3a 2n) 36 199.2 ± 21.3 89.9 ± 32.4 1.10 ± 0 09 27.73 ± 0.19 195.7 ± 2.3 40.7 ± 28.5
2n+ 30 205.9 ± 20.6 91.9 ± 28.3 1.02 ± 0.08 27.76 ± 0.12 196.2 ± 1.6 47.6 ± 22.7
3n 30 180.6 ± 26.7 64.9 ± 25.0 1.04 ± 0.07 27.87 ± 0.22 197.2 ± 2.4 58.8 ± 29.3
3b 2n 33 202.6 ± 26.5 81.6 ± 35.4 0.93 ± 0.07 28.19 ± 0.14 202.8 ± 1.4 27.6 ± 18.4
3n 30 175.0 ± 28.1 53.8 ± 22.6 0.94 ± 0.06 28.25 ± 0.14 203.5 ± 1.4 36.9 ± 17.3
Bkt
1 2n) 27 111.4 ± 14.8 15.2 ± 6.3 1.04 ± 0.08 28.03 ± 0.24 138.7 ± 2.3 67.2 ± 36.7
2n+ 44 116.3 ± 14.8 17.6 ± 6.6 1.06 ± 0.06 28.06 ± 0.20 139.2 ± 1.8 72.0 ± 31.1
3n 45 109.7 ± 14.1 13.6 ± 4.8 0.97 ± 0.07 27.88 ± 0.18 137.4 ± 2.1 40.1 ± 25.6
2a 2n 39 118.1 ± 14.7 16.0 ± 6.1 0.92 ± 0.06 27.96 ± 0.12 156.7 ± 2.1 60.1 ± 21.0
3n 50 115.7 ± 19.4 14.3 ± 6.7 0.85 ± 0.06 27.75 ± 0.31 153.3 ± 5.0 33.3 ± 23.2
2b 2n 43 169.4 ± 17.6 51.6 ± 18.5 1.01 ± 0.10 27.56 ± 0.14 159.1 ± 1.2 52.0 ± 25.4
3n 43 160.9 ± 21.8 39.7 ± 14.8 0.90 ± 0.06 27.46 ± 0.16 158.1 ± 1.4 35.1 ± 21.7
3 2n 41 134.1 ± 13.9 26.5 ± 8.6 1.06 ± 0.07 27.64 ± 0.22 173.9 ± 2.4 38.2 ± 25.2
3n 48 162.2 ± 20.1 43.7 ± 14.8 0.99 ± 0.09 27.73 ± 0.13 175.1 ± 1.4 50.8 ± 25.2

In Trials Rbt #3a and Bkt #1, 2n- indicates control diploid fish (did not receive a thermal shock following fertilization) and 2n+
indicates diploid fish which did receive a thermal shock following fertilization but were not triploidized.
188

Table 2. Covariant coefficient estimates and test summaries for a series of CLMax tests between diploid and triploid rainbow trout
(Rbt) and brook trout (Bkt).

Species Covariant Coefficient SE df t p

Rbt Total length )0.087 0.050 262 )1.75 0.081

Condition factor )15.399 13.172 264 )1.17 0.243
Bkt Total length 0.757 0.081 308 9.30 <0.001
Condition factor 38.455 13.588 328 1.96 0.051

Table 3. Summary of analyses of a series of CLMax tests between diploid and triploid rainbow trout.

Source Numerator df Denominator df F p

Total length 1 262 3.06 0.081

Condition 1 264 1.37 0.243
Ploidy 1 3.92 0.00 0.951
Covariance SE z p
Trial 7233.1 5133.4 1.41 0.079
Trial X ploidy 29.5 30.6 0.96 0.243
Residual 331.4 29.0 11.42 <0.001

Individual trials and trial by ploidy interactions were treated as random effects while ploidy was treated as a fixed effect. Total length
and condition were included as covariates. The Satterthwaite approximation was used to insure appropriate degrees of freedom for all
tests.

Table 4. Summary of analyses for a series of CLMax tests between diploid and triploid brook trout.

Source Numerator df Denominator df F p

Total length 1 308 86.42 <0.001

Condition 1 328 3.85 0.051
Ploidy 1 3.44 6.55 0.073
Covariance SE z p
Trial 8399.3 6895.1 1.22 0.112
Trial X Ploidy 61.4 63.6 0.97 0.167
Residual 682.5 53.5 12.77 <0.001

Individual trials and trial by ploidy interactions were treated as random effects while ploidy was treated as a fixed effect. Total length
and condition were included as covariates. The Satterthwaite approximation was used to insure appropriate degrees of freedom for all
tests.

ploidy on time to LE (p=0.951, Table 3, Fig-
ure 1), and the median temperature at LE was
similar (Figure 1). In the brook trout, the differ-
ence in rank between ploidy groups was very close
to the chosen alpha level of 0.05 (p=0.073, Ta-
ble 4, Figure 2), although the median temperature
at LE was similar (Figure 2).
Results of ANOVA for trials Rbt #3a and Bkt
#1 indicate no significant differences in time to LE
between diploid fish which had or had not received
as newly fertilized eggs, a heat shock to induce
triploidy (p=0.466, p=0.998, respectively; Fig-
ure 3). Of note, in the Bkt #1 trial, fish in both
diploid groups reached LE significantly later than
the triploid fish (p=0.003 for the 2n) vs. 3n
comparison, and p=0.002 for the 2n+ vs. 3n
comparison; Figure 3).
Discussion
Results of these trials comparing time to CLMax
of diploid and triploid rainbow trout and brook
trout indicate that ploidy has little if any general-
ized effect. Although the difference in time to LE
between ploidy groups was significant within some

Figure 1. Skeletal boxplots of time to LE (top panel) and
temperature at LE (bottom panel) for diploid and triploid
rainbow trout. Boxes represent the interquartile range, and lines
connect median values for comparison.
trials, the corresponding differences in tempera-
ture at LE were small in magnitude – the average
difference across the nine tests being 0.10°C, well
less than the average within-species standard
deviation (0.18°C) for this measure. Also, the dif-
ferences in time to LE (and temperature at LE)
were in both directions – the diploids reached LE
later than triploids in some trials, and sooner than
triploids in other trials. These observations could
possibly be interpreted as evidence of a ploidy 
block (trial) interaction. However, it is our feeling
that this was instead an effect of small differences
between groups within trials in physiological
characteristics unrelated to ploidy. Efforts were
made to subject the diploid and triploid fish within
each experimental group to a similar pre-trial
rearing environment – in the 6 – 12 month period
between initiation of feeding and conducting of the
thermal tolerance trials, the fish were reared either
in side-by-side tanks supplied by a common water
189
Figure 2. Skeletal boxplots of time to LE (top panel) and
temperature at LE (bottom panel) for diploid and triploid
brook trout. Boxes represent the interquartile range, and lines
connect median values for comparison.
source, or were pooled within a common tank.
Also, feeding, handling and tank cleaning practices
were similar between ploidy groups, in an attempt
to maintain similarity of rearing conditions prior
to testing. However, inevitable small differences
between ploidy groups in these conditions might
have had an effect on the general well-being of the
fish which was reflected in their response to the
thermal challenge. Because of the high sample
number per ploidy, the CLMax test has relatively
high power, which therefore permits statistical
discernment of differences which are very small in
magnitude.
A significant correlation between size and time
to LE was observed in the brook trout trials,
though this was not the case for the rainbow trout.
Galbreath et al. (2004) did not observe a consis-
tent effect for size (length and weight) or for con-
dition factor on time to LE in CLMax tests with
juvenile rainbow trout, brook trout and brown
190
Figure 3. Skeletal boxplots of time to LE and temperature at LE for brook trout (trial Bkt#1; upper panel) and rainbow trout (trial
Rbt#3a; lower panel). Boxes represent the interquartile range, and lines connect median values for comparison.
trout. Likewise, size had little or no correlation to
measures of CTMax in studies among same-age
fish by Dean (1973), Elliot, (1981), Baker &
Heidinger (1996), Benfey et al. (1997) and Carline
& Machung (2001). Nonetheless, Galbreath et al.
(2004) caution that it would be prudent to avoid
large differences in size within and between groups
when testing for relative thermal tolerance.
In two trials, Rbt #3a and Bkt #1, the heat shock
used to induce triploidy in newly fertilized eggs was
only partially successful. This provided the
opportunity to compare thermal tolerance of dip-
loids which had or had not received the heat shock,
and test whether the heat shock, in the absence of a
change in ploidy, had a persistent effect on per-
formance of the fish as juveniles. In both trials,
however, no difference was observed in time to
thermal maximum. Although deleterious effects
related to the heat or pressure shock used to pro-
duce triploid fish are well documented during the
incubation stages, rarely has continued poorer
performance in juvenile triploids been attributable
to enduring negative effects of the shock. One such
case was the study of Johnson et al. (2004) who
observed similar survival from initiation of feeding
to day 112 among pressure shocked triploids and
non-shocked diploids, but decreased survival of
heat shocked triploids, indicative of a deleterious
effect of heat shock. Curiously, however, growth
rate of the surviving heat shocked triploids was
similar to that of the other two groups. In studies
comparing performance of triploids produced ei-
ther by heat shock or by fertilization of eggs with
sperm from tetraploid males, neither Blanc et al.
(1987) nor Myers & Hershberger (1991) observed
differences in growth rate from initiation of feeding

to approximately 6 months, indicating lack of a
persistent negative effect attributable to the heat
shock. Neither study provided survival data for
this period.
In light of the larger size and reduced number of
erythrocytes in triploid fish relative to diploids
(Benfey 1999), numerous studies have attempted
to quantify differences in aerobic capacity and
cardiorespiratory physiology which could explain
potential performance differences related to a
change in ploidy. In large part, however, no sig-
nificant differences have been observed among
salmonids (Benfey & Sutterlin 1984, Oliva-Teles &
Kaushik 1987, Virtanen et al. 1990, Biron &
Benfey 1994, Stillwell & Benfey 1996, 1997, Sadler
et al. 2000b, Altimiras et al. 2002, Mercier et al.
2002). Having observed no difference in CTMax
attributable to ploidy, Benfey et al. (1997) ques-
tioned what might have been the causal factor(s)
behind increased mortality and poorer perfor-
mance observed anecdotally (see citations in the
Introduction). As no apparent differences in
aerobic capacity have been described, they specu-
lated that differences in ability to mobilize and/or
use energy stores during prolonged exposure might
provide an explanation. If such were the case, the
short duration of the CTMax protocols they used
could have precluded detecting differences related
to ploidy level. The CLMax tests described in the
present study involved withholding feed one to
2 days prior to commencement of the test, which
because of the slower heating rate (2 day)1) lasted
up to an additional 8 days. Under these more
chronic conditions of thermal stress, the fish were
obliged to use available energy stores to a much
greater extent, and yet still no consistent effect of
ploidy was observed.
Hyndman et al. (2003b) observed that triploid
brook trout exercised to exhaustion in warm
(19°C) water exhibited a reduced anaerobic po-
tential relative to diploids, associated with a re-
duced ability to clear muscle lactate. In fact, none
of the triploids in their experiment survived be-
yond 4 h following exercise, compared to zero
mortality among the diploids. Such a difference
attributable to ploidy would be critical to thermal
tolerance, for as temperature approaches lethal
limits, capacity to deliver sufficient oxygen to the
tissues in fish is accompanied by a time-limited
transition to mitochondrial anaerobiosis (Pörtner
191
2002). Nonetheless, even in the relatively chronic
conditions of the CLMax tests reported here, a
consistent difference in time to LE was not ob-
served between diploids and triploids.
In summary, CLMax tests were conducted on
juvenile diploid and triploid brook trout and
rainbow trout. Observed differences between
ploidy groups were small and did not indicate a
consistent effect of ploidy on thermal tolerance.
Nor was a consistent effect of size (length and
weight) or condition factor on time to lethal
maximum observed within the ranges tested. Re-
sults also indicated that the heat shock used to
induce triploidy had no persistent negative effect
on thermal tolerance in the juvenile trout.
Acknowledgements
The authors wish to express their sincere thanks to
Mr. Jack Jones at the Erwin National Fish
Hatchery and to Mr. John Murry at the Pisgah
Forest State Fish Hatchery, and to the other per-
sonnel at their respective hatcheries, for their
cooperation in obtaining fish and gametes used to
conduct this study.
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