Hydrobiologia 527: 63–75, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
63
Rapid carbon turnover during growth of Atlantic salmon (Salmo salar) smolts
in sea water, and evidence for reduced food consumption by growth-stunts
Timothy D. Jardine1,*, Deborah L. MacLatchy1, Wayne L. Fairchild2,
Richard A. Cunjak3 & Scott B. Brown4
1
Canadian Rivers Institute and Department of Biology, University of New Brunswick (Saint John), Saint John,
New Brunswick, Canada E2L 4L5
2
Fisheries and Oceans Canada, Moncton, New Brunswick, Canada E1C 9B6
3
Canadian Rivers Institute and Department of Biology, University of New Brunswick (Fredericton), Fredericton,
New Brunswick, Canada E3B 6E1
4
Environment Canada, National Water Research Institute, Burlington, Ont., Canada L7R 4A6
(*Author for correspondence: Tel.: +1-506-458-7148, Fax: +1-506-453-3583, E-mail: [email protected])
Received 2 September 2003; in revised form 28 January 2004; accepted 9 February 2004
Key words: stable isotopes, metabolism, muscle, liver, enrichment, growth-stunting
Abstract
Wild Atlantic salmon smolts were captured during spring out-migration in the Northwest Miramichi River,
New Brunswick, Canada, and placed on an isotopically distinct hatchery diet to determine the relative
contributions of growth and metabolic turnover to isotopic change. As expected for an ectothermic species,
growth explained a large amount of isotopic variation in changing stable carbon ratios of muscle tissue
(average r2 ¼ 0.46) for the 3 years of study. Turnover rates of muscle carbon in all 3 years in growing fish
(0.24–0.66 month)1) were higher than previously reported values for other ectothermic species, but there
was little evidence for isotopic change in non-growers (average r2 ¼ 0.041, p > 0.1). It is unlikely that nongrowers had consumed any of the hatchery diet over a 2-month period, thus preventing them from
acquiring the new carbon isotopic signature. This period of food deprivation resulted in nitrogen-15
enrichment in liver relative to muscle (p ¼ 0.003). It is advised that future isotope studies of metabolic
turnover rates in ectotherms be conducted on slow-growing animals over a long time period. This would
serve to avoid the obscuring effects of growth on isotopic change, and provide stronger comparisons to
endothermic tissue turnover rates.
Introduction
Diadromous salmonids such as Atlantic salmon
(Salmo salar) undergo a process known as parrsmolt transformation (PST), during which physiological and hormonal mechanisms throughout
migration facilitate a smooth transition from fresh
to salt water (McCormick et al., 1998). These
changes are accompanied by alterations in nutritional status. As migration proceeds, lipids are
catabolized to meet energy requirements (Sheridan
et al., 1985; Sheridan, 1989, 1994), reducing con-
dition factor (Leonard & McCormick, 2001) for a
short period before rapid growth at sea. The carbon turnover rate for Atlantic salmon is not
known, but the energy demands of PST and
migration may require higher tissue turnover rates
than have been documented in other fish species
(Hesslein et al., 1993; Herzka & Holt, 2000; Maruyama et al., 2001). Examination of turnover rates
in muscle tissue would help shed light on the
metabolic processes occurring within salmon
64
during this critical life stage. Earlier research has
shown a 50% increase in standard and active
metabolic rates following PST (Maxime et al.,
1989), while increases in activity in a variety of
enzymes also occurs in smolts (Leonard &
McCormick, 2001).
The transfer of animals to a diet with a distinct
stable carbon and/or nitrogen isotopic ratio
(13C/12C and 15N/14N) has been used to document
the incorporation of nutrients into various tissues
and to quantify the relative contribution of growth
and metabolic turnover in governing isotopic
change. Ectotherm tissue has been shown to
change primarily due to dilution of the original
ratio during growth (Fry & Arnold, 1982; Hesslein
et al., 1993; Maruyama et al., 2001), while metabolic requirements in endotherms drive observed
changes despite negligible growth in animal subjects (Tieszen et al., 1983; Hobson & Clark, 1992a,
b).
The traditional isotope studies mentioned
above have focused on experimental organisms
that either all gained weight or all lost weight
during the study. Studies on ectotherms tend to be
done on immature, rapidly growing animals (Fry
& Arnold, 1982; Hesslein et al., 1993; Maruyama
et al., 2001), while experiments on endothermic
species have focused on adult organisms that have
essentially completed growth (Tieszen et al., 1983;
Hobson & Clark, 1992a, b). We are currently
unaware of any comparisons of the rate of isotopic
change in response to a shift in diet in growers and
non-growers within the same group of animals.
In wild and hatchery-reared cohorts of salmon
smolts, a certain proportion of the population fails
to grow upon migration/transfer to a saline environment (Clarke & Nagahama, 1977; Woo et al.,
1978; Varnavsky et al., 1992). The mechanism
underlying this phenomenon has eluded researchers, and at present it is unclear whether growth
stunting occurs as a result of a lack of food consumption or some physiological deficiency associated with osmoregulation (Fryer & Bern, 1979;
Nishioka et al., 1982; Varnavsky et al., 1992; Duan
et al., 1995).
Stable nitrogen isotope analysis can also be
useful in interpreting the nutritional status of
organisms. Due to the preferential excretion of 14N
(Altabet & Small, 1990), periods of food deprivation result in 15N enrichment in a variety of tissues
in endotherms (Hobson et al., 1993). Similarly, a
strong enrichment in liver 15N has been documented in Atlantic salmon that fast during the
spawning migration and subsequent over-wintering (Doucett et al., 1999). Examination of stable
nitrogen ratios in muscle and liver of stunted
smolts fed a diet of known isotopic composition
could therefore provide answers as to whether
these fish are feeding normally.
In this study, the changes in carbon isotopic
composition of rapidly growing smolts fed a distinct hatchery diet were explored. We conducted
these analyses to estimate the relative contributions of growth and metabolic turnover to isotopic
change, and to estimate muscle tissue turnover
rates for Atlantic salmon. Further analyses were
conducted to clarify the reason for the observed
lack of growth in a portion of the population,
including the carbon isotopic ratios of fish that lost
weight during the experiment, and a comparison
of muscle and liver 15N/14N ratios.
Methods
Fisheries and Oceans Canada (DFO) installs a
trap to capture smolt out-migrants during the
spring in the main Northwest Miramichi River
(Fig. 1). This trap is installed annually (since 1998,
Chaput et al., 2002) below the confluence of the
Northwest and the Little Southwest Branches of
the River. Migratory smolts were collected at this
trap-net in fresh water in late May and early June.
Three years of collections were conducted [2000
(n ¼ 322), 2001 (n ¼ 384), and 2002 (n ¼ 116)].
Smolts were taken to the Miramichi Salmon
Conservation Centre (MSCC, South Esk, NB,
Canada) and tagged with streamer tags in 2000
(FLOY, Seattle, WA, USA) and passive integrated
transponders (PIT tags; Biomark, Boise, ID, USA,
11.5 mm, 125 kHz) in 2001 and 2002. The switch
was made to PIT tags after 2000 because some
smolts experienced open sores due to the streamer
tags. After tagging, smolts were caged in the
Miramichi River (Fig. 1) and a neighboring estuary (Tabusintac River, ~60 km northeast of Miramichi, not shown) for 4 days in tidal water (1 m
below surface, cage dimensions ¼ 65 cm · 40 cm ·
25 cm, 20 fish per cage), and returned to the
MSCC for placement in full strength salt water.
65
Figure 1. Map of the Miramichi River, New Brunswick, Canada. Fisheries and Oceans Canada installs a smolt trap every spring in the
Northwest Miramichi River. The trap is installed to estimate the population of out-migrant Atlantic salmon smolts from the main
Northwest Miramichi system. Smolts were caged for 4 days at the locations indicated (NW Miramichi, Miramichi estuary), and in the
Tabusintac estuary (~60 km northeast of Miramichi, not shown).
The fish were held in two 2846 l fiberglass tanks
with re-circulating water and bio-filters until the
end of the experiment. Krill (Argent Laboratories,
Richmond, BC, Canada) was briefly used as feed
during an acclimation phase (~1 week) before
switching to a pellet diet [3 mm floating, Corey
Feed Mills, Fredericton, NB, d13C ¼ )21.5&
(2000), )21.9& (2001), )23.3& (2002), d15N ¼
7.3& (2000), 7.6& (2001), 6.8& (2002)] for the
remainder of the study period (2–3 months) in all
3 years. All smolts caught in 2000 were fed to
satiation (twice daily until food remained on the
water surface). In 2001, the majority of smolts
were fed to satiation; a smaller group (n ¼ 55) was
placed directly (not caged in the estuary) into a
separate tank and fed a dietary level designed to
maintain a constant body weight (0.5% bw/day).
In 2002, all smolts (n ¼ 116) were placed directly
in salt water (not caged in the estuary) in a single
tank and given a maintenance diet (0.5% bw/day).
A group of smolts was sampled in each year at
the initiation of the experiment to establish initial
66
Table 1. Initial fork lengths (FL, in mm) and wet weights (wi, in grams), initial stable carbon ratios (di, in &), and expected equilibrium
stable carbon ratios (de, in &) after transfer to a hatchery diet, for migrating Atlantic salmon smolts captured in the Northwest
Miramichi River, 2000–2002 (di and wi are presented as mean ±1 standard deviation, n = number of individuals measured)
Year
FL (mm)
wi (g)
n
di
n
de
2000
133 ± 13
23.3 ± 6.7
64
)24.2 ± 1.3
6
)20.5
2001
131 ± 12
21.4 ± 5.9
84
)23.2 ± 2.0
11
)20.9
2002
134 ± 11
22.3 ± 5.8
55
)23.8 ± 1.9
23
)22.3
stable isotope ratios [2000 (n ¼ 6), 2001 (n ¼ 11),
2002 (n ¼ 23), Table 1]. In mid-July, a group of
fish was randomly selected and sacrificed with a
blow to the head [2000 (n ¼ 64), 2001 (n ¼ 75),
2002 (n ¼ 8)]. August samples were obtained in the
same manner in 2001 (n ¼ 9) and 2002 (n ¼ 8). A
September sample (n ¼ 15) was included in 2002.
Livers were immediately removed and flash frozen
in liquid N2; the remainders of the fish were frozen
(20 C) until selected samples were thawed for
isotopic analysis.
White muscle (removed from left side of
thawing fish, below dorsal fin, above lateral line)
and liver tissues were dried at 60 C for 48 h,
ground into a fine homogenate with a mortar and
pestle, and sent to the Stable Isotopes in Nature
Laboratory, University of New Brunswick, Fredericton, NB for stable isotope analysis. Samples
were combusted in a Carlo Erba NC2500 elemental analyzer and resultant gases delivered via
continuous-flow to a Finnigan Mat Delta Plus
isotope-ratio mass spectrometer (Thermo Finnigan, Bremen, Germany).
Isotope ratios (&) were measured and
expressed as
dX ¼ ½ðRsample =Rstandard Þ 1 1000;
where X is 13C or 15N and R is 13C/12C or 15N/14N.
The standard used for 13C measurements is PeeDee Belemnite carbonate (Craig, 1957), and
atmospheric nitrogen (Mariotti, 1983) is used for
15
N, values that are arbitrarily set at 0&. More
positive d values indicate enrichment of the heavier
isotope, while those with more negative ratios reflect depletion. Replicate standard lab samples
indicated an analytical error of ±0.2& for carbon
and ±0.3& for nitrogen.
Mathematical models used to describe changes
in stable carbon and nitrogen ratios as functions of
growth and metabolic turnover were first presented by Fry & Arnold (1982), and modified by
Hesslein et al. (1993) and Maruyama et al. (2001).
A mass-balance model in which tissue dilution is
the sole contributor to isotopic change following a
shift to a diet of a distinct isotopic composition
follows the function
dt ¼ de þ ðdi de Þ ðwi =wt Þ;
where di ¼ the initial d13C value prior to the diet
switch, wi ¼ the initial wet weight prior to the diet
switch, de ¼ the equilibrium d13C value attained
after equilibration to the new diet, wt ¼ wet weight
at the time (t) of sampling, and dt ¼ the d13C value
at the time (t) of sampling. These predicted values
(dt) are plotted on Figure 2 and labeled as ‘growth
only’. In the current study, di values were set using
the group of smolts sampled during tagging procedures (Table 1). These d13C values were considered representative of the initial isotope ratios of
migratory smolts. They showed wide variability
within years but were not different from year to
year. The added relevance of using wild fish for the
study outweighed the lower initial variability from
fish raised in a hatchery (T. Jardine, unpublished
data). Furthermore, differences between initial and
expected equilibrium values for wild fish in this
study (2000 diff. ¼ 3.7&, 2001 diff. ¼ 2.3&, 2002
diff. ¼ 1.5&) were considered sufficient to allow
quantitative interpretations. Equilibrium stable
carbon values (de) were estimated by adding a
fractionation factor of 1& to the year-specific
measured ratios of the pellet diet used in the
majority of this study. The estimated isotopic difference between an organism and its diet for carbon is 1& (DeNiro & Epstein, 1978; Rau et al.,
1983). A similar model describing nitrogen dilution with growth and turnover rates was not possible because of the poor correlation between d15N
67
Figure 2. Stable carbon ratios (&) vs. growth (% of initial weight) for Atlantic salmon smolts that gained weight during the study
period in A. 2000 (j), B. 2001 ()), and C. 2002 (m). Hatched lines indicate expected equilibration curves when growth alone is
controlling isotopic change, predicted from the equation dt ¼ de + (di ) de) · (wi/wt). Solid lines represent best-fit equations for the
observed data in the form y ¼ a + bxc. Initial smolt carbon ratios prior to the diet switch (in spring at the start of the experiment) are
indicated by the solid horizontal line labeled di, while the gray horizontal line labeled de indicates expected smolt equilibrium values (see
Methods).
68
and growth (see results), and the greater uncertainty associated with choosing a representative de
value (Adams & Sterner, 2000).
In order to estimate the contribution of metabolic turnover to carbon isotopic change, the
growth-metabolism model was used (Maruyama
et al., 2001) according to the following function:
dt ¼ de þ ðdi de Þ ðwi =wt Þ Ct ;
where t is the time (in months) after switching to
the hatchery diet and C is the proportion of the
initial carbon pool remaining after 1 month. The
value of C was solved by iteration (using Microsoft
Excel 97, improving prediction of dt) as described
by Maruyama et al. (2001), and accounts for the
more rapid than expected equilibration of a tissue
to the new diet, relative to growth alone.
Carbon half change period (t*, in months) was
calculated using the following equation (Maruyama et al., 2001):
t
¼
log 2
;
log G log C
where G is the average growth rate (month)1) of
smolts according to:
1t
wt
G¼
:
wi
Time-independent growth values are expressed as
percent of initial weight (Fry & Arnold, 1982)
using the formula
wt
% ¼ 100;
wi
while time-dependent growth rates are calculated
as specific growth rates (Ricker, 1979) by
wt
ðtÞ1 100:
SGRW ¼ ln
wi
For the purposes of this experiment, only fish that
gained weight (G > 1, % of initial weight > 100,
SGRW > 0) during the study period were analyzed with this approach.
Stable carbon ratios of fish that lost weight
over the course of the experiment (G < 1, % of
initial weight < 100, SGRW < 0) were plotted vs.
elapsed time (days since the diet was switched) in a
manner identical to that used by researchers
studying endotherms where weight gain was neg-
ligible (Tieszen et al., 1983; Hobson & Clark,
1992a, b).
The computer software SPSS 11.0 for windows
(SPSS, Chicago, IL) was used for all analyses.
Initial stable carbon ratios and wet weights among
years were tested non-parametrically (Kruskal–
Wallis) because within-year distributions exhibited
non-normality.
Despite being apparently fed to satiation, many
fish actually lost weight between capture in late
May and the summer sampling dates. Isotope ratios of all fish that gained weight, regardless of diet
regime, were regressed against individual growth
rates (% of initial weight) for each year. Stable
carbon ratios of all fish that lost weight, regardless
of diet regime, were regressed against elapsed time
(days since diet switch) for each year. Predicted
d13C values based on growth alone vs. observed
d13C and time vs. observed d13C were examined for
all 3 years.
Liver and muscle d15N values were compared
within individuals using paired sample t-tests. Pvalues are presented separately for fish that gained
weight during the study (G > 1, % of initial
weight > 100, SGRW > 0) and those that lost
weight during the study (G < 1, % of initial
weight < 100, SGRW < 0). This was done because
smolts that lose weight upon seawater-transfer
experience increased mortality (T. Jardine,
unpublished data).
Results
Smolts captured in the Northwest Miramichi
River had highly variable stable carbon ratios and
wet weights within years (Table 1). Average initial
stable carbon ratios and average initial weights
were similar among years (p ¼ 0.23 and p ¼ 0.21,
respectively).
The majority of smolts fed to satiation grew
rapidly after the switch to the hatchery diet
(average % of initial weight ¼ 182.5 ± 5.8 for
2000/2001) over a period of ca. 2 months. Smolts
on the maintenance diet grew more slowly (average % of initial weight ¼ 156.3 ± 12.5 for 2001/
2002). When all fish were combined, the fastest
overall growth (SGRW) occurred in 2000; fish in
2001 showed intermediate growth rates, and 2002
had the slowest growers (Table 2). This was a di-
69
Table 2. Average growth (SGRW, day)1), the proportion of the initial muscle carbon pool remaining after 1 month (C, dimensionless),
muscle carbon turnover rate (1 ) C, dimensionless) and muscle carbon half-change period (t*, in months) for Atlantic salmon smolts on
a hatchery diet, 2000–2002 (maintenance = 0.5% body weight day)1). Growth rates are presented as mean ± standard error
Year
Diet
SGRW
C
1)C
T*
2000
Satiation
1.0 ± 0.13
0.76
0.24
2.39
2001
Satiation
1.0 ± 0.07
–
–
–
Maintenance
0.3 ± 0.09
–
–
–
2002
All
0.8 ± 0.06
0.39
0.61
0.73
Maintenance
0.4 ± 0.16
0.34
0.66
0.64
rect result of the diet level used in the 3 years
(satiation in 2000, maintenance in 2002, combination in 2001).
For those smolts that gained weight between
date of capture and final sampling date, growth
explained a significant amount of stable carbon
variation in all 3 years (Fig. 2). Year 2000
(r2 ¼ 0.58, df ¼ 45, p < 0.005) was the most pronounced, compared with 2001 (r2 ¼ 0.36, df ¼ 74,
p < 0.005) and 2002 (r2 ¼ 0.45, df ¼ 16,
p < 0.005). Growth explained low amounts of
d15N variation in all 3 years (Fig. 3, 2000–
r2 ¼ 0.19, df ¼ 45, p < 0.005; 2001–r2 ¼ 0.32,
df ¼ 74, p < 0.005; 2002–r2 ¼ 0.22, df ¼ 16,
p < 0.005).
The slowest muscle carbon turnover (1 ) C)
occurred in 2000 (0.24 month)1), despite being the
year of fastest average growth; the calculated
carbon half-change period (t*) was 2.39 months.
Fish in 2001 had intermediate average growth
rates, turnover rates (0.61 month)1), and halfchange periods (0.73 months). Muscle tissue carbon turnover was highest in 2002 (0.66 month)1),
despite being the year of slowest average growth.
As a result, the calculated half-change period in
2002 was 0.64 months.
For non-growing smolts there was no correlation between the number of days since the diet
switch and d13C for all 3 years (Fig. 4; 2000 –
r2 ¼ 0.001, df ¼ 22, p ¼ 0.905; 2001 – r2 ¼ 0.119,
df ¼ 18, p ¼ 0.147; 2002–r2 ¼ 0.003, df ¼ 35,
p ¼ 0.753). After more than 2 months on the new
diet, d13C values in muscle tissue did not depart
from initial ratios, showing little evidence of
equilibration with the new diet signature. Two
non-growing fish reached expected equilibrium
values in 2001 (de ‡ )20.9, Table 1), while one fish
reached equilibrium in 2002 (de ‡ )22.3, Table 1).
Smolts sampled in July 2000 that gained weight
on the hatchery diet (>100% of initial weight) had
muscle 15N values that were enriched relative to
liver 15N values (Table 3, df ¼ 46, p < 0.001).
Smolts that lost weight during the study period
(<100% of initial weight), meanwhile, showed the
opposite pattern, with liver 15N values that were
enriched relative to muscle 15N values (Table 3,
df ¼ 16, p ¼ 0.003). Stable carbon ratios in liver
and muscle were not different in growing
(p ¼ 0.094) or non-growing smolts (p ¼ 0.535)
(Table 3).
Discussion
This paper supports the notion that changes in the
stable isotope ratios of ectothermic organisms are
strongly related to growth (Fry & Arnold, 1982).
Carbon turnover rates in this study, however, were
the highest ever recorded in an ectothermic
organism (Hesslein et al., 1993; Herzka & Holt,
2000; Maruyama et al., 2001), and may explain the
relatively low correlation between d13C and
growth. A unique element of this study was the
examination of isotope ratios in both growing and
non-growing subjects. The rapid metabolic turnover rates observed in growing fish were not seen
in non-growers. The reason for this lack of equilibration in non-growers likely is little or no consumption of the diet used in the study.
As expected for an ectothermic species, growth
explained a large proportion of observed stable
carbon variation in growing fish. These values,
however, were lower than those reported in previous studies. Prior research on a variety of fish
species attributed between 73 and 100% of observed variation to growth (Hesslein et al., 1993;
70
12.0
A
2000
11.0
10.0
15
δ N
2
r = 0.19
9.0
8.0
7.0
6.0
0
50
100
150
200
250
300
350
400
Growth as % of initial weight
12.0
B
2001
11.0
r2 = 0.32
15
δ N
10.0
9.0
8.0
7.0
6.0
0
50
100
150
200
250
300
350
400
Growth as % of initial weight
12.0
C
2002
11.0
r2 = 0.22
15
δ N
10.0
9.0
8.0
7.0
6.0
0
50
100
150
200
250
300
350
400
Growth as % of initial weight
Figure 3. Stable nitrogen ratios (&) vs. growth (% of initial weight) for Atlantic salmon smolts that gained weight during the study
period in A. 2000 (j), B. 2001 ()), and C. 2002 (m). Solid lines represent best-fit equations for the observed data in the form
y ¼ a + bxc.
71
Figure 4. Stable carbon ratio (&) vs. elapsed time (days since diet switch) for Atlantic salmon smolts that lost weight during the study
period in A. 2000 (j), B. 2001 ()), and C. 2002 (m). Initial d13C values (di) prior to the diet switch (in spring at the start of the
experiment) are included at t ¼ 0. Solid lines represent best-fit equations for the observed data in the form y ¼ a + bx. The horizontal
line labeled de indicates expected values for smolts in equilibrium with the hatchery diet.
72
Table 3. Stable carbon (d13C, &) and nitrogen (d15N, &) ratios in liver and muscle tissue of non-growing and growing wild Atlantic
salmon smolts in July 2000, after ~2 months on a hatchery diet (fed twice daily to satiation)
Liver
Muscle
p
Non-growers
d13C
)24.4 ± 0.9
)24.2 ± 1.4
0.535
Growers
d15N
d13C
8.4 ± 0.6
)21.5 ± 0.9
8.1 ± 0.6
)21.3 ± 1.4
0.003
0.094
d15N
7.6 ± 0.4
8.6 ± 0.4
<0.001
Ratios are presented as mean ± 1 standard deviation. Tissue differences were tested using paired t-tests.
Herzka & Holt, 2000; Maruyama et al., 2001). The
additive effects of rapid metabolic turnover account for the unexplained variation in d13C. In the
present study, higher turnover rates in Atlantic
salmon smolt muscle tissue were measured than
have been documented in the past in tissues of
other ectotherm species [0.03–0.06 month)1 in
broad whitefish, Coregonus nasus (Hesslein et al.,
1993), £0.15 month)1 in krill, Euphasia superba
(Frazer et al., 1997), not detectable in red drum,
Sciaenops ocellatus (Herzka & Holt, 2000), 0.01–
0.30 month)1 in migratory goby, Rhinogobius sp.,
(Maruyama et al., 2001)]. The high carbon turnover rates in salmon smolt muscle tissue are likely
a consequence of the rapid catabolism of lipid
stores to meet the energetic demands of osmoregulation in salt water, along with a general increase
in metabolism and enzymatic activity during parrsmolt transformation (Maxime et al., 1989;
Leonard & McCormick, 2001). Successfully
smoltified fish have been shown to contain decreased muscle lipid levels (Woo et al., 1978), due
to an increase in lipolysis during PST (Sheridan
et al., 1985; Sheridan, 1989, 1994).
The low correlation between d15N and growth
may be indicative of slower turnover of muscle
nitrogen compared to carbon, contrary to previous
work on Mysis relicta, where nitrogen turnover
occurred more rapidly (Johannsson et al., 2001).
It is also possible that our interpretation of
muscle carbon turnover rates is too liberal. The
expected equilibrium d13C used in this analysis (de,
Table 1) may have been lower than that which
actually occurs for smolt muscle tissue. Enrichment can be highly variable among species, life
stages, and tissues (DeNiro & Epstein, 1978), and
can often be greater than the assumed 1& standard enrichment used. For example, DeNiro &
Epstein (1978) showed a d13C range of )0.6 to
+2.7& enrichment in a variety of species relative
to their diet, while Rau et al. (1983) found increases of 0.73 and 1.38& at successive trophic
levels in an oceanic food web. In the current study,
substitution of an enrichment value of 2& resulted
in a decrease in the turnover rate in all 3 years:
2000 (0.24–0.05 month)1), 2001 (0.69–0.25
month)1), and 2002 (0.66–0.15 month)1), still
higher than most values reported in the literature
for fishes (Hesslein et al., 1993; Herzka & Holt,
2000).
To further examine possible turnover rates in
the absence of growth, the stable carbon ratios of
muscle tissue in fish that failed to grow during the
experiment were measured. In 2000, no smolts
showed evidence of equilibration with the hatchery
diet. In 2001 and 2002 (despite a non-significant
overall relationship) three fish (total) had values
indicative of equilibration with the hatchery diet.
It should be noted, however, that three of the fish
sampled initially in 2002 also had a value greater
than de, so a value greater than de later in 2002
does not necessarily imply equilibrium with the
hatchery diet.
The reasons for the observed pattern of isotopic stasis in non-growers are unclear, as high tissue
turnover rates evidenced in the growth-metabolism
model predicted a large amount of isotopic change
despite negligible growth. Based on the calculated
half-change periods (t*) for muscle tissue in
growing smolts, the length of the study period
(~2 months) should have been sufficient to observe
a shift from initial d13C values (di) towards expected equilibrium values (de). This was not the
case, as very little evidence of equilibration with
the new diet is apparent, particularly in 2000.
The first explanation for this phenomenon is
the potential misuse of predicted equilibrium d13C
values, as mentioned above. If enrichment relative
73
to the diet was in fact greater than 1&, calculated
metabolic turnover rates decrease, half-change
periods increase (t* ¼ 10.42, 2.33, and 4.01 months
for 2000, 2001, and 2002 respectively), and nongrowing fish would not be expected to approach
equilibration with the diet after ~2 months.
A second explanation for this lack of equilibration is the possibility that non-growing fish
were consuming no food. Stomachs of non-growing fish were empty (T. Jardine, personal observation); however, a quantitative assessment of gut
contents was not possible due to the limitations
associated with the technique (i.e. point-in-time
estimates). SIA provides a better measure of
nutrient uptake because it integrates temporal
variations in consumption by individual animals.
Frazer et al. (1997) and Herzka & Holt (2000)
conducted starvation experiments using stable
isotopes in two other ectotherm species, larval krill
and red drum. Starving krill exhibited no isotopic
change over a similar (~2 months) time frame as
the current study (Frazer et al., 1997), while a
short period of food deprivation resulted in no
isotopic change in red drum (Herzka & Holt,
2000). If a given fish in this study was consuming
little or no food, it is not possible for the isotopic
signature of the hatchery diet to become incorporated into the fish’s muscle tissue, regardless of
rapid tissue turnover rates. These so-called ‘stunted’ fish have been documented in smolt populations by other researchers; in most cases the
phenomenon was associated with premature seawater transfer (Clarke & Nagahama, 1977; Woo
et al., 1978).
The observed 15N enrichment in livers of smolts
that lost weight during the study also supports a
lack of food intake as the primary mechanism for
growth retardation. Growing smolts in this study
had muscle 15N values that were enriched relative
to liver. Doucett et al. (1999) reported a similar
enrichment for adult Atlantic salmon at the
beginning of the spawning migration. After
~8 months without food in that study (spawning
migration and over-wintering), kelts had enriched
15
N values in liver tissue relative to muscle, the
same effect observed in smolts that lost weight in
the current study. Doucett et al. (1999) hypothesized that the recycling of muscle protein to the
liver during nutritional stress was responsible for
the observed enrichment in liver 15N. This process
can serve to enrich the available protein pool due
to fractionation during transamination and
deamination (Macko et al., 1986). A similar
mechanism could account for observed liver 15N
enrichment in this study.
The evidence presented here supports starvation as the likely cause of growth reductions in
stunted smolts. Previous research had offered
mainly qualitative information on feeding rates in
stunts. Clarke & Nagahama (1977) stated that
stunted coho salmon (Oncorhynchus kisutch) ‘ate
little’ in seawater but regained condition after
being transferred back to fresh water. Woo et al.
(1978) noted a poor appetite in stunted coho
smolts, but also hypothesized that starvation was
not the root cause of stunting because storage
tissues in stunts contained reasonable amounts of
lipid and glycogen. Collie (1985) showed reduced
nutrient transport ability in coho stunts, which
supports the notion that digestive/metabolic effects
are also limiting growth. The first study on stunting in Atlantic salmon (Bjornsson et al., 1988)
found a cessation in growth to be accompanied by
what appeared to be a loss of appetite, although
the researchers were unclear as to whether the
stunts were feeding normally. The difficulty in
qualitatively observing feeding habits of individual
smolts within a large tank is due to the subjective
nature of visual observation. While stunted smolts
can at times be observed as being listless or moribund, the feeding performance of a particular
individual over time is nearly impossible to document.
Although strong empirical evidence of starvation, such as a study employing X-radiography
(McCarthy et al., 1993), is still lacking, the current
study provides a solid indication that starvation is
indeed occurring in these stunted smolts. What
remains unclear is whether the lack of food intake
was a result of inferior competitive ability or
simply a lack of desire to feed. Behavioral observations support the latter, as stunted fish generally
showed low activity levels, as was noted in past
studies (Bolton et al., 1987; Bjornsson et al., 1988).
However, dominance hierarchies do form within
salt-water tanks despite expected schooling
behavior (Handeland et al., 1998), so even a brief
setback in feeding rate could force individuals into
subordinate positions from which they do not recover. Also, compensative feeding mechanisms
74
(Damsgard & Arnesen, 1998) appeared to be absent in these groups of fish, as stunts failed to regain normal growth rates and eventually died.
Measurement of the neurohormones cholecystokinin, norepinephrine, and dopamine could be
useful in resolving this issue. These substances
have been implicated in regulating appetite in
many vertebrates, including fish (Silverstein &
Plisetskaya, 2000; de Pedro et al., 2001; Gelineau
& Boujard, 2001).
The results of this study indicate that the
influence of growth on isotope ratio in ectotherms
becomes greatest when growth is fastest, a
hypothesis that was put forth by previous
researchers (Hesslein et al., 1993; Maruyama et al.,
2001). It appears as though a true measure of ectothermic turnover rates can only be accomplished
by examining isotopic change in organisms that
have maintained a constant body weight over a
relatively long period of time. Maintaining a constant weight of a group of fish in captivity is difficult, particularly when the fish are in a juvenile
stage where rapid growth is extremely important
for normal development. The effects of metabolism in rapidly growing animals is obscured by
dilution during growth, while animals that lose
weight during a study period may not consume
enough of the new diet to adequately acquire a
signature indicative of equilibration.
Acknowledgements
A Toxic Substances Research Initiative grant to
SBB, DLM, and WLF, a DFO Environmental
Science Strategic Research Fund grant to WLF,
and Environment Canada provided funding for
this research. In-kind and financial support was
also received from the Science Branch (Diadromous Division) of DFO in Moncton, NB, the
Northumberland Salmon Protection Association,
and the Miramichi River Environmental Assessment Committee. The Atlantic salmon assessment
crew in Miramichi (G. Chaput, G. Atkinson,
S. Douglas, J. Hayward, D. Moore, J. Sheasgreen,
A. Astle, S. Clark, J. Dunnett, M. Frigault, A.
Hayward, S. Lavoie, B. Norton, M. Russell, B.
Silliker, and K. Underhill) was instrumental in
capturing smolts for the study. Staff at the SINLAB in Fredericton (A. McGeachy, C. Paton, and
M. Savoie) provided training in isotope techniques. M. Hambrook and his staff at the Miramichi Salmon Conservation Center admirably
conducted fish husbandry. L. Burridge, R. Doucett, R. Rochette, and two anonymous reviewers
supplied valuable comments in the preparation of
this manuscript.
References
Adams, T. S. & R. W. Sterner, 2000. The effect of dietary
nitrogen content on trophic level 15N enrichment. Limnology
and Oceanography 45: 601–607.
Altabet, M. A. & L. F. Small, 1990. Nitrogen isotopic ratios in
fecal pellets produced by marine zooplankton. Geochimica
et Cosmochimica Acta 54: 155–163.
Bjornsson, B. Th., T. Ogasawara, T. Hirano, J. P. Bolton &
H.A. Bern, 1988. Elevated growth hormone levels in stunted
Atlantic salmon, Salmo salar. Aquaculture 73: 275–281.
Bolton, J. P., G. Young, R. S. Nishioka, T. Hirano & H. A.
Bern, 1987. Plasma growth hormone levels in normal and
stunted yearling coho salmon, Oncorhynchus kisutch. Journal
of Experimental Zoology 242: 379–382.
Chaput, G., P. Hardie, J. Hayward, D. Moore, J. Sheasgreen &
NSPA, 2002. Migrations and biological characteristics of
Atlantic salmon (Salmo salar) smolts from the Northwest
Miramichi River, 1998 to 2000. Canadian Technical Report
of Fisheries and Aquatic Sciences No. 2415. 70pp.
Clarke, W. C. & Y. Nagahama, 1977. Effect of premature
transfer to seawater on growth and morphology of the
pituitary, thyroid, pancreas, and interrenal in juvenile Coho
salmon (Oncorhynchus kisutch). Canadian Journal of Zoology 55: 1620–1630.
Collie, N. L., 1985. Intestinal nutrient transport in coho salmon
(Oncorhynchus kisutch) and the effects of development,
starvation, and seawater adaptation. Journal of Comparative Physiology 156B: 163–174.
Craig, H., 1957. Isotopic standards for carbon and oxygen and
correction factors for mass-spectrometric analysis of carbon
dioxide. Geochimica et Cosmochimica Acta 12: 133–149.
Damsgard, B. & A. M. Arnesen, 1998. Feeding, growth, and
social interactions during smolting and seawater acclimation
in Atlantic salmon, Salmo salar L. Aquaculture 168: 7–16.
DeNiro, M. J. & S. Epstein, 1978. Influence of the diet on the
distribution of carbon isotopes in animals. Geochimica et
Cosmochimica Acta 42: 495–506.
De Pedro, N., M. J. Delgado & M. Alonso-Bedate, 2001.
Fasting and hypothalamic catecholamines in goldfish.
Journal of Fish Biology 58: 1404–1413.
Doucett, R. R., R. K. Booth, G. Power & R. S. McKinley,
1999. Effects of the spawning migration on the nutritional
status of anadromous Atlantic salmon (Salmo salar): insights
from stable isotope analysis. Canadian Journal of Fisheries
and Aquatic Sciences 56: 2172–2180.
Duan, C., E. M. Plisetskaya & W. W. Dickoff, 1995. Expression
of insulin-like growth factor I in normally and abnormally
75
developing Coho salmon (Oncorhynchus kisutch). Endocrinology 136: 446–452.
Frazer, T. K., R. M. Ross, L. B. Quetin & J. P. Montoya, 1997.
Turnover of carbon and nitrogen during growth of larval
krill, Euphasia superba Dana: a stable isotope approach.
Journal of Experimental Marine Biology and Ecology 212:
259–275.
Fry, B. & C. Arnold, 1982. Rapid 13C/12C turnover during
growth of brown shrimp. Oecologia 54: 200–204.
Fryer, J. N. & H. A. Bern, 1979. Growth hormone binding to
tissues of normal and stunted juvenile Coho salmon, Oncorhynchus kisutch. Journal of Fish Biology 15: 527–533.
Gelineau, A. & T. Boujard, 2001. Oral administration of cholecystokinin receptor antagonists increase feed intake in
rainbow trout. Journal of Fish Biology 58: 716–724.
Handeland, S. O., A. Berge, B. Th. Bjornsson & S. O. Stefansson, 1998. Effects of temperature and salinity on osmoregulation and growth of Atlantic salmon (Salmo salar L.)
smolts in seawater. Aquaculture 168: 289–302.
Herzka, S. Z. & G. J. Holt, 2000. Changes in isotopic composition of red drum (Sciaenops ocellatus) larvae in response to
dietary shifts: potential applications to settlement studies.
Canadian Journal of Fisheries and Aquatic Sciences 57: 137–
147.
Hesslein, R. H., K. A. Hallard & P. Ramlal, 1993. Replacement
of sulfur, carbon, and nitrogen in tissue of growing broad
whitefish (Coregonus nasus) in response to a change in diet
traced by d34S, d13C, and d15N. Canadian Journal of Fisheries and Aquatic Sciences 50: 2071–2076.
Hobson, K. A. & R. G. Clark, 1992a. Assessing avian diets
using stable isotopes I: turnover of 13C in tissues. The
Condor 94: 181–188.
Hobson, K. A. & R. G. Clark, 1992b. Turnover of 13C in cellular and plasma fractions of blood: implications for nondestructive sampling in avian dietary studies. The Auk 110:
638–641.
Hobson, K. A., R. T. Alisauskas & R. G. Clark, 1993. Stablenitrogen isotope enrichment in avian tissues due to fasting
and nutritional stress: implications for isotopic analyses of
diet. The Condor 95: 388–394.
Johannsson, O. E., M. F. Leggett, L.G. Rudstam, M. R. Servos, M.A. Mohammadian, G. Gal, R. M. Dermott & R. H.
Hesslein, 2001. Diet of Mysis relicta in Lake Ontario as revealed by stable isotope and gut content analysis. Canadian
Journal of Fisheries and Aquatic Sciences 58: 1975–1986.
Leonard, J. B. K. & S. D. McCormick, 2001. Metabolic enzyme
activity during smolting in stream- and hatchery-reared
Atlantic salmon (Salmo salar). Canadian Journal of Fisheries and Aquatic Sciences 58: 1585–1593.
Macko, S. A., M. L. Fogel Estep, M. H. Engel & P. E. Hare,
1986. Kinetic fractionation of stable nitrogen isotopes during
amino acid transamination. Geochimica et Cosmochimica
Acta 50: 2143–2146.
Mariotti, A., 1983. Atmospheric nitrogen is a reliable standard
for natural 15N abundance measurements. Nature 311: 251–
252.
Maruyama, A., Y. Yamada, B. Rusuwa & M. Yuma, 2001.
Change in stable nitrogen isotope ratio in the muscle tissue
of a migratory goby, Rhinogobius sp., in a natural setting.
Canadian Journal of Fisheries and Aquatic Sciences 58:
2125–2128.
Maxime, V., G. Boeuf, J. P. Pennec & C. Peyraud, 1989.
Comparative study of the energetic metabolism of Atlantic
salmon (Salmo salar) parr and smolts. Aquaculture 82: 163–
171.
McCarthy, I. D., D. F. Houlihan, C. G. Carter & K. Moutou,
1993. Variation in individual food consumption rates of fish
and its implications for the study of fish nutrition and
physiology. Proceedings of the Nutrition Society 52: 427–
436.
McCormick, S. D., L. P. Hansen, T. P. Quinn & R. L. Saunders, 1998. Movement, migration, and smolting of Atlantic
salmon (Salmo salar). Canadian Journal of Fisheries and
Aquatic Sciences 55: 77–92.
McCormick, S. D., R. A. Cunjak, B. Dempson, M. F. O’Dea &
J. B. Carey, 1999. Temperature-related loss of smolt characteristics in Atlantic salmon (Salmo salar) in the wild.
Canadian Journal of Fisheries and Aquatic Sciences 56:
1649–1658.
Nishioka, R. S., H. A. Bern, K. V. Lai, Y. Nagahama & E. G.
Grau, 1982. Changes in endocrine organs of Coho salmon
during normal and abnormal smoltification – an electron
microscope study. Aquaculture 28: 21–38.
Rau, G. H., A. J. Mearns, D. R. Young, R. J. Olson, H. A.
Scafer & I. R. Kaplan, 1983. Animal 13C/12C correlates with
trophic level in pelagic food webs. Ecology 64: 1314–1318.
Ricker, W. E., 1979. Growth rates and models. In Hoar, W. S.,
D. J. Randall & J. R. Brett (eds), Fish Physiology. Vol. III.
Bioenergetics and Growth. Academic Press, New York:
677–743
Sheridan, M. A., N. Y. S. Woo & H. A. Bern, 1985. Changes in
the rates of glycogenesis, lipogenesis, and lipolysis in selected
tissues of the coho salmon (Oncorhynchus kisutch) associated
with parr-smolt transformation. Journal of Experimental
Zoology 236: 35–44.
Sheridan, M. A., 1989. Alterations in lipid metabolism
accompanying smoltification and seawater adaptation of
salmonid fish. Aquaculture 82: 191–203.
Sheridan, M. A., 1994. Regulation of lipid metabolism in poikilothermic vertebrates. Comparative Biochemistry and
Physiology 107B: 495–508.
Silverstein, J. T. & E. M. Plisetskaya, 2000. The effects of NPY
and insulin on food intake regulation in fish. American
Zoologist 40: 296–308.
Tieszen, L. L., T. W. Boutton, K. G. Tesdahl & N. A. Slade,
1983. Fractionation and turnover of stable carbon isotopes
in animal tissues: Implications for d13C analysis of diet.
Oecologia 57: 32–37.
Varnavsky, V. S., T. Sakamoto & T. Hirano, 1992. Stunting of
wild Coho salmon (Oncorhynchus kisutch) in seawater: patterns of plasma thyroid hormones, cortisol, and growth
hormones. Canadian Journal of Fisheries and Aquatic Sciences 49: 458–461.
Woo, N. Y. S., H. A. Bern & R. S. Nishioka, 1978. Changes in
body composition associated with smoltification and premature transfer to seawater in Coho salmon (Oncorhynchus
kisutch) and King salmon (O. tschawytscha). Journal of Fish
Biology 13: 421–428.