Journal of Fish Biology (2010) 76, 2318–2341
doi:10.1111/j.1095-8649.2010.02636.x, available online at www.interscience.wiley.com
Effects of the sea louse Lepeophtheirus salmonis
on temporal changes in cortisol, sex steroids, growth
and reproductive investment in Arctic charr
Salvelinus alpinus
H. Tveiten*†, P. A. Bjørn*, H. K. Johnsen‡, B. Finstad§
and R. S. McKinley
*Nofima Marin, N-9291, Tromsø, Norway, ‡Norwegian College of Fishery Science, University
of Tromsø, N-9037 Tromsø, Norway, §Norwegian Institute of Nature Research, N-7485
Trondheim, Norway and Centre for Aquaculture and the Environmental Research,
The University of British Columbia, West Vancouver, BC, V5Z 1M9 Canada
(Received 15 May 2009, Accepted 3 February 2010)
Groups of mature (5+ year old) Arctic charr Salvelinus alpinus held in sea water were exposed
for 34 days to either a high (mean ± s.e. 0·15 ± 0·01 sea lice Lepeophtheirus salmonis g−1
fish mass) (HI), medium (0·07 ± 0·00 sea lice g−1 fish mass) (MI) or no [control (C)] sea-lice
infection during early stages of gonad development (June to July). Infection with sea lice resulted
in increased plasma cortisol concentrations and this was related to intensity of infection; females
tended to have higher cortisol concentrations than males at high infection intensities (HI group:
female c. 130 ng ml−1 ; male c. 80 ng ml−1 ). Plasma osmolality (C c. 330, MI c. 350 and HI c. 415
mOsm) and chloride concentrations (C c. 135, MI c. 155 and HI c. 190 mM) increased significantly
with infection intensity, indicating osmoregulatory problems in infected fish. A strong positive
relationship between plasma osmolality and cortisol concentration was recorded. Plasma sex-steroid
concentrations were influenced negatively by sea-lice infection, particularly in the HI group, and
were inversely related to plasma cortisol concentrations. The most heavily infected fish postponed
the initiation of reproductive development until exposed to fresh water and timing of ovulation
tended to be delayed in these fish. Growth rate and condition were negatively influenced by sea-lice
infection and growth rate was inversely related to plasma cortisol concentrations. Sea-lice infection
resulted in mortality among females in the HI group, and the proportion of maturing females was
lower in the MI group (46%) than in the controls (85%). Egg production in the MI and HI groups
was c. 50 and 30% of the C group. Egg size, embryonic survival and fry mass did not differ
across groups. Sea lice influence reproductive development and egg production in S. alpinus, and
consequently these parasites may influence populations via sublethal effects on broodfish, affecting
© 2010 The Authors
growth and condition, and their reproductive output.
Journal compilation © 2010 The Fisheries Society of the British Isles
Key words: condition; ectoparasite; egg production; growth; stress.
INTRODUCTION
In fishes, stress may have the potential to affect reproductive development, investment and gamete quality (Schreck et al., 2001). There is evidence that physiological
†Author to whom correspondence should be addressed. Tel.: +47 77629063; fax: +47 77629100;
email: [email protected]
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S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
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stress exerts inhibitory effects on reproductive processes in teleosts through modified endocrine function (Pankhurst & Van Der Kraak, 1997), mediated specifically
through changes in plasma sex-steroid concentrations (Pankhurst & Van Der Kraak,
2000). Also, studies involving in vivo administration of cortisol do show inhibitory
effects of cortisol on reproductive endocrine processes and development (Carragher
et al., 1989; Foo & Lam, 1993). As in mammals, an elevation of plasma cortisol
is the main indicator of stress in fishes (Wendelaar-Bonga, 1997). Cortisol is the
main corticoid steroid produced by the interrenals and plays the role of both gluco
and mineralo-corticosteroid as seen in higher vertebrates (Mommsen et al., 1999),
and stress may influence growth physiology and energy accumulation (Barton, 1997).
Since cortisol may have wide-ranging metabolic effects (Pankhurst & Van Der Kraak,
1997; Mommsen et al., 1999), it is difficult to discern whether there is a direct
effect of cortisol on steroid synthesis or indirect effect through affecting nutritional
status of the fishes which is important for the initiation or progression of reproductive development (Leatherland, 1999). Pankhurst & Van Der Kraak (2000) provided
compelling evidence that cortisol does have an effect on plasma sex-steroid concentrations without altering nutritional status and, thus, may act at different levels to
inhibit reproductive development.
In salmonid, infection with the sea louse Lepeophtheirus salmonis induces an
integrated stress response. In sea trout (anadromous brown trout) Salmo trutta L. for
example, plasma cortisol concentrations are elevated within a few days of infection,
when the sea lice still are at an early stage of development, without inducing any
osmoregulatory problems (Bjørn & Finstad, 1997; Finstad et al., 2000). A severe
osmoregulatory problem, which is associated with an additional increase in plasma
cortisol concentrations, occurs when the sea lice reach their pre-adult and adult stages,
usually 20–25 days post-infection (Bjørn & Finstad, 1997; Finstad et al., 2000).
Thus, the stress response to sea-lice infection may be considered to be chronic in its
nature and no acclimation seems to occur. Under experimental conditions, reduced
growth and fish mortality may be induced by high sea-lice infection (0·5–1·5 sea
lice g−1 fish) (Bjørn & Finstad, 1997), which is likely also to occur under natural
conditions (Birkeland, 1996; Bjørn et al., 2001). Although increased mortality would
obviously reduce reproductive output in a population, the long-term, sublethal, stress
effect of sea-lice infection on growth and reproductive processes is little studied.
Wild, anadromous Arctic charr Salvelinus alpinus (L.) undertake annual migrations to sea during the summer months, and usually spend 40–50 days in sea water
before returning to fresh water to overwinter (Jobling et al., 1998). In winter, food
intake is very limited but during their short sea migration the fish grow rapidly
and accumulate body energy at a high rate which is subsequently allocated to
meet energy demand for reproduction and metabolism during overwintering (Jobling
et al., 1998).
During its short sea migration, S. alpinus may be heavily infected by sea-lice
(Bjørn et al., 2001). Although the physiological response to sea-lice infection is little studied in S. alpinus, the sea lice appear to have a similar developmental rate,
host distribution and pathogenicity to that found in sea trout S. trutta (Bjørn &
Finstad, 1998). Sea-lice infection, by inducing physiological stress to the fish, may
therefore have the potential to influence growth performance, reproductive development and gamete investment in S. alpinus. To that end, the effect of different
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
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H. TVEITEN ET AL.
sea-lice infection intensities on stress physiology, reproductive endocrine homeostasis and how these relate to growth, reproductive development, gamete production
and embryonic survival in S. alpinus were studied.
MATERIALS AND METHODS
FISH AND HOLDING CONDITIONS
The study was conducted at the Aquaculture Research Station, Kårvika, northern Norway
(70◦ N) during the period April 2000 to May 2001. The fish used in the study were mature
5+ year-old (Hammerfest strain) S. alpinus of the 1995 year class. On 17 November 1999,
all fish were anaesthetized in benzocaine (50 ppm) in order to remove gametes and confirm
reproductive status. To simulate feeding conditions experienced by wild S. alpinus (Dutil,
1986; Boivin & Power, 1990), fish were not fed during winter (until early June). Fish was
then fed (Skretting, 6 mm; www.skretting.no) until 27 July by means of automatic disc
feeders. Feed supply was in excess based on calculations of feed requirement using a growth
model for S. alpinus (Jobling, 1983). On 26 April 2000, three groups of fish with 33–34
fish in each group were established by selecting fish from a group of c. 300. The fish were
weighed (M, mean + s.e. 731 ± 40 g), fork length, LF , measured (42·5 ± 0·8 cm) and
tagged individually with FT-69 fingerling tags (Floy Tag & Mfg.; www.floytag.com), and
randomly distributed into three circular tanks (500 l). Until 26 April, fish were held at ambient
photoperiod, thereafter all groups were exposed to a simulated natural photoperiod according
to Johnsen et al. (2000). Briefly, each tank was shielded by a black, light-proof plastic canopy,
and lighting was provided at an intensity of c. 80 lx at the water surface. Photoperiod was
controlled by automatic timers without a twilight period.
Tanks were supplied with fresh water of ambient temperature (0·1–3·2◦ C) until 16 June.
Then, during a 3 day period, the fish were gradually transferred to full strength sea water
(salinity 33). On 23 June, two of the groups were infected with either a medium or a high
dose of sea-lice copepodids, and all groups were then maintained in sea water for 34 days
(27 July) which approximate the length of a natural sea migration of this strain of S. alpinus
(Jobling et al., 1998). When kept in sea water, temperature increased slowly from 6·6 to
8·1◦ C. In June, wild fish of this strain of S. alpinus migrate to sea (Rikardsen et al., 1997)
and no mortality was recorded after transfer. After residence in sea water (on 27 July), fish
were transferred, during a 3 day period, back to fresh water at ambient temperature (10·2◦ C).
From August to the beginning of the spawning season (20 October), temperature decreased
slowly from c. 10 to 5◦ C. Since temperature may influence timing of ovulation and egg
quality in salmonids, including S. alpinus (Gillet, 1991; Taranger et al., 2000), all tanks were
maintained at a temperature close to 5◦ C throughout the spawning season.
SEA-LICE INFECTION
On 23 June, two of the groups were infected with either a medium (MI) or a high (HI)
dose of sea lice copepodids, whereas control fish were sham infected. Infection was performed
according to Bjørn & Finstad (1998) with either c. 120 or 240 copepodid larvae per fish in
the MI and HI groups, respectively, to attain levels of c. 50 and 100 lice per fish based on
65% infection success and 65% lice survival. This procedure resulted in 100% prevalence
of sea lice-infected fish and a final (end of seawater residence) sea-lice infection intensity of
72 ± 4 and 140 ± 5 sea lice per fish (mean ± s.e.), which was equivalent to 0·07 ± 0·00
and 0·15 ± 0·01 sea lice g−1 (mean ± s.e.) of fish in the MI and HI groups, respectively.
Calculation of final sea-lice infection intensity in the HI group was based on recordings from
surviving fish. As predicted from previous experiments (Bjørn & Finstad, 1998), the sea lice
had reached pre-adult stages (c. 260 degree-days of development) at the end of seawater
residence.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
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SAMPLING
All fish from each of the three groups were sampled for blood, LF and M on 26 April,
19 May, 16 June, 27 July, 9 September, 21 September, 17 October and 29 November. To
minimize handling stress fish were sedated with benzocaine (70 ppm) by adding it to the
tank through a tube, carefully placed in the tank through a hole in the canopy wall. Water
supply was then closed, and the fish were left undisturbed until immobilized. All fish were
immobilized within 3 min and was then transferred to an oxygenated sedation bath of lower
benzocaine concentration (35 ppm) and maintained there until sampling. The whole sampling
procedure was terminated within 22–25 min. Under normal conditions (in fresh water), this
sampling procedure results in cortisol levels c. 20 ng ml−1 , 20–25% lower than that recorded
when conventional netting is used (pers. obs.).
At each sampling, the fish were weighed (c. 0·5 g) and LF measured (c. 0·1 cm). Condition
factor (K) was calculated as: K = 100 M LF −3 (M in g and LF in cm). Mass data were
used to calculate specific growth rates (G) for individual fish according to the formula:
G = 100 (lnM2 − lnM1 ) (t2 − t1 )−1 , where M1 and M2 are fish masses at time (days) t1 and
t2 , respectively.
Blood (1–2 ml) was sampled from the caudal vasculature using vacutainer tubes (Vacutainer; www.bd.com) containing 57·2 U.S.P. units Li+ -heparin. The samples were held on ice
until centrifugation at 3800 g for 10 min at 0–2◦ C and plasma was stored at −80◦ C until
assayed for cortisol and sex steroids.
P L A S M A A N A LY S E S
Plasma concentrations of oestradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11KT) were measured by means of radioimmunoassay (RIA), according to Schulz (1985).
Validations of the assays for S. alpinus plasma, and cross-reactivities of the E2 and T antiserum, have been previously examined (Frantzen et al., 2004). Cross-reactivities of the 11-KT
antiserum are given by Schulz (1985).
Briefly, steroids were extracted from 200 μl plasma with 4 ml diethyl ether under vigorous
shaking for 4 min. The aqueous phase was frozen in liquid nitrogen, whereas the organic phase
was transferred to a glass tube, evaporated in a water bath at 45◦ C and then reconstituted
by addition of 600 μl assay buffer and then assayed for E2, T and 11-KT.
Plasma concentrations of cortisol were measured using the same RIA procedure as described for measurement of sex steroids. The detection limit for the assay was 0·6 ng ml−1 .
The cortisol antiserum, raised in New Zealand white (NZW) rabbits, gave 14·8% crossreaction with 17,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol), 8·8% with 17,21dihydroxy-4-pregnene-3,11,20-trione (cortisone), 4·7% with 11β,17-dihydroxy-4-pregnene3,20-dione (21-deoxycortisol), 1·9% with 5β-pregnane-3α,17,20β-triol, 1·2% with 5βpregnane-3α,17,20β-triol sulphate, 1·0% with 3β-hydroxy-5-pregnen-20-one (pregnenolone),
0·7% with 5β-pregnane-3β,17,20β-triol, 0·6% with 17,20β,21-trihydroxy-4-pregnen-3-one,
0·6% with 17,20α-dihydroxy-4-pregnen-3-one and <0·1% with 4-pregnene-3,20-dione (progesterone), 17-hydroxy-4-pregnen-3,20-dione (17-hydroxyprogesterone), 17,20β-dihydroxy4-pregnen-3-one (17,20β-P) 17β-hydroxy-4-androstene-3,11-dione (11-ketotestosterone), 17β-hydroxy-4-androsten-3-one (testosterone) and 1,3,5(10)-estratriene-3,17β-diol
(oestradiol-17β).
To validate the cortisol assay for S. alpinus, a plasma pool was divided into four aliquots,
two of which were stripped of endogenous steroids by charcoal treatment [45 mg charcoal (Sigma-Aldrich; www.sigmaaldrich.com) and 4·5 mg Dextran T 70 (Pharmacia Biotech;
www.apbiotech.com) per ml plasma, incubated for 1 h at room temperature under continuous shaking]. Half of stripped and untreated plasma samples were then spiked with 150 ng
cortisol (Sigma-Aldrich) per ml plasma to give four preparations (untreated, untreated and
spiked, stripped, and stripped and spiked). To test extractions of steroids, these preparations
were subjected to ether extraction as described above. Products resulting from the different
treatments were then assayed by the cortisol-RIA at two dilutions. Steroid recovery after
ether extraction was 87%. Cortisol values were corrected for recovery losses. The inter and
intra-assay coefficients of variation (c.v.) for the cortisol assay were 12·7% (n = 28) and
© 2010 The Authors
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H. TVEITEN ET AL.
7·7% (n = 9), respectively. Analysis of plasma cortisol concentrations was carried out for
samplings before (16 June), at the end (27 July) and after (21 September) sea-lice infection.
Osmolality [mosmol kg−1 (mOsm)] was measured by Fiske ONE-TEN Osmometer (Fiske
Associates; www.aicompanies.com), and chloride (Cl− ) concentrations were determined
using a Corning 925 chloride titrator (CIBA Corning Diagnostics; www.novartis.co.uk). Analysis of plasma osmolality and Cl− concentrations was carried out for the same samplings as
indicated for cortisol.
There was not always sufficient plasma to carry out all the analysis indicated above, and
the number of fish analysed for the different variables may deviate slightly from the total
number of fish in the different groups.
GAMETE COLLECTION
From 3 October onwards, the fish were anaesthetized and examined for spermiation and
ovulation at 5–7 days intervals. Spermatocrit (S) was assessed from milt transferred to haematocrit tubes (75 mm × 1·2 mm), spun at 4500 g for 10 min and calculated according to the
formula: S = 100HC HT−1 , where HC is the height of the cell layer in the haematocrit tube
and HT is the total height of the seminal fluid including the cell layer. Spermatocrit was measured at the beginning (just after the first ovulating female), mid (c. 50% ovulated females)
and at the end (all females had ovulated) of the spawning season.
Eggs were collected from ovulated females and the total volume was recorded. The number
of eggs in a sample of 25 ml was counted, and total fecundity (total number of eggs)
and relative fecundity (number of eggs kg−1 ) were calculated. Egg diameters were determined as the average from measurement of 75–100 freshly ovulated eggs from each female.
Eggs were fertilized with milt (in excess) pooled from several males from the same group.
Fertilized eggs were then incubated, in triplicate samples (c. 1200 eggs), in an upwelling
incubator. Each incubator (diameter. 12 cm) had a perforated bottom plate through which
water was delivered at a rate of c. 0·5 l min−1 at a temperature of 3·95 ± 0·02◦ C
(mean ± s.e.).
Fertilization rate was estimated by examining at least 75 eggs c. 2 days after fertilization.
Eggs were cleared in a solution of glacial acetic acid and saline (1:20 v/v), examined under
a binocular microscope, and cleaved eggs were classified as fertilized. Eggs from 11, six and
four females in the control, MI and HI groups, respectively, were fertilized and incubated.
Dead eggs were removed and counted at regular intervals during incubation. Egg survival to
the eyed stage was determined after 300 ± 8 degree-days (mean ± s.e.) of incubation, and
the proportion of eggs that survived to the eyed stage was assessed in relation to the number
of fertilized eggs.
Egg production by each female was assessed as relative egg mass (MRE ) calculated according to the formula: MRE = 100ME MB−1 , where ME is the wet mass of the eggs with ovarian
fluid and MB is the mass of the female after stripping.
F I R S T F E E D I N G A N D E A R LY G R O W T H
Approximately 100 degree-days after hatching, 500 fry from each female in each group
were transferred to first-feeding trays (10 cm × 15 cm; water depth 5 cm), and feeding
was initiated at 165 ± 8 degree-days (mean ± s.e.) after hatch when c. 0·33 of the yolk sac
remained. First feeding was carried out at 4·05 ± 0·03◦ C (mean ± s.e.) and under conditions
of continues dim light and food supply (Skretting, Nutra Starter, particle size was increased
gradually from 0·3 to 1·0 mm). At hatch, and 3 and 6 weeks after initiation of first feeding,
fry LF (mm) and M (mg) were recorded (n = 10) for each batch.
The experiments and procedures described here have been conducted in accordance with
the laws and regulations controlling experiments and procedures with live animals in Norway,
i.e. the Animal Welfare Act of 20 December 1974, No. 73, chapter VI, sections 20–22 and
the Regulation on Animal Experimentation of 15 January 1996.
© 2010 The Authors
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S TAT I S T I C A L A N A LY S E S
All data were first tested for normal distribution by the non-parametric Kolmogorov–
Smirnov test (Lilliefors distribution). A two-way ANOVA was used to investigate possible effects of time, sea-lice infection and their interaction on the variables studied. When
appropriate, data were square-root, log10 or arcsin transformed to obtain normality. When the
two-way ANOVA revealed significant effects, a one-way ANOVA, followed by a Tukey post
hoc test, was used to identify where differences occurred between groups at each sampling
date and within groups between sampling dates. All values are given as mean ± s.e. Possible
relationships between variables were assessed using Pearson correlation analysis (Zar, 1999).
Proportions of maturing fish were compared using χ 2 analyses of frequency data (Zar, 1999).
A probability level of P < 0·05 was considered significant in all tests. All computations were
performed with Systat 9.2 (www.systat.com).
RESULTS
SEA-LICE INFECTION
At the end of seawater residence, sea-lice infection intensities differed significantly
between all treatment groups (Fig. 1) but did not differ between male and female
fish within treatments (P > 0·05).
M O RTA L I T Y A N D M AT U R AT I O N R AT E S
Sea-lice infection had a significant effect on fish survival. In the HI group, 40%
of the fish died during the period of sea-lice infection, whereas corresponding value
in the control and MI groups was 6% (Fig. 2). In the control group, for unknown
reasons, another five fish died after re-entry to fresh water, with mortality reaching
a total of 21% in this group. The proportions of maturing and not re-maturing fish
was also significantly affected by lice infection, with only about half the number of
the females re-maturing in the MI group compared with that of the control (Table I).
In the HI group, although not statistically different, the total number of maturing
females was reduced by what appeared to be sex-specific mortality (Table I). This
resulted in a skewed female-to-male ratio (30 v. 70%) in the HI group, although not
statistically different from that of the other groups (P > 0·05). There was no effect
of sea-lice infection on the proportion of maturing males (Table I).
Table I. Frequency (%) of sexual maturation in male and female Salvelinus alpinus exposed
to no (control), medium and high sea-lice infection for 34 days during gonad recrudescence
(23 June to 27 July). Number in parentheses indicates number of fish. Different superscript
lowercase letters within columns indicate significant differences (P < 0·05) between groups
Category of fish
Treatment
Control
Medium infection
High infection
Mature females
Immature females
Mature males
Immature males
84·6 (11)a
46·2 (6)b
66·7 (4)a
15·4 (2)a
53·8 (7)b
33·3 (2)a
92·3 (12)a
94·2 (16)a
85·7 (12)a
7·7 (1)a
5·8 (1)a
14·3 (2)a
© 2010 The Authors
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H. TVEITEN ET AL.
C O RT I S O L A N D S E X S T E R O I D S
There was a significant effect of sea-lice infection on plasma cortisol concentrations, and plasma cortisol concentrations increased in a dose–response-like manner
(Fig. 1), with cortisol tending although not significantly (P > 0·05) to be higher in
females than in males at high infection intensity. The two-way ANOVA revealed,
however, a significant overall higher plasma cortisol concentration in maturing females compared with maturing males. Also at the individual level, there was a significant positive relationship between sea-lice infection intensity and plasma cortisol
concentrations with this relationship being stronger in maturing females (R 2 = 0·86,
n = 16) than in maturing males (R 2 = 0·33, n = 40).
Before seawater transfer and sea-lice infection, cortisol concentrations were
15–20 ng ml−1 in all groups both in males and females [Fig. 3(a), (b)]. In the control group, plasma cortisol concentrations did not change statistically with time,
and both in maturing males and females these concentrations were largely unaffected by seawater exposure and season [Fig. 3(a), (b)]. In the MI group, sea-lice
exposure resulted in male and female plasma cortisol concentrations of c. 60 and
70 ng ml−1 , respectively [Fig. 3(a), (b)], with female values being significantly higher
and lower, respectively, compared with corresponding values in the control and HI
200
R2 = 0·33 (male)
c (P = 0·056)
180
R2 = 0·86 (female)
160
C
Cortisol (ng ml−1)
140
120
bc
100
b
80
C
ab
B
60
B
40
a
a A
A
20
0
0
0·05
0·1
0·15
0·2
−1
Sea-lice infection (sea lice g fish) 27 July (end of seawater exposure)
Fig. 1. Relationship between mean ± s.e. sea-lice infection intensity and plasma cortisol concentrations in male
( , , ) and female ( , , ) Salvelinus alpinus exposed to no [control ( , )], medium [MI ( , )]
and high [HI ( , )] sea-lice infection during gonad recrudescence (23 June to 27 July). Differences
in plasma cortisol concentrations are indicated by different lowercase letters (P < 0·05). Differences in
sea-lice infection intensity are indicated by different uppercase letters (P < 0·05). Fish were sampled at
the end of sea-lice and seawater exposure (fish still in sea water). Strength of relationships (R 2 ) between
variables is calculated from individual data.
© 2010 The Authors
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S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
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50
Cumulative mortality (%) 27 July
b
40
30
20
10
0
a
a
Control
MI
Treatment
HI
Fig. 2. Per cent mortality in groups of Salvelinus alpinus exposed to no (control), medium (MI) and high (HI)
sea-lice infection during gonad recrudescence (23 June to 27 July). Different lowercase letters above
bars indicate significant differences (P < 0·05) between treatments.
groups. Cortisol values in MI males did not differ significantly from other groups,
but only marginally (P > 0·05) differ from males in the control group. Plasma cortisol concentration in females that did not re-mature in the MI group was 48·5 ±
10·8 ng ml−1 , values being not different from that of maturing females in the same
group. In the HI group, male and female plasma cortisol concentrations were close to
80 and 130 ng ml−1 , respectively, at the end of sea-lice infection, and female values
was significantly higher compared with corresponding values in the control and MI
groups [Fig. 3(a), (b)]. Cortisol values in HI males were significantly different from
that of the control, but did not differ from male MI values. In the autumn (September), plasma cortisol concentrations were only slightly higher than those recorded
before lice infection [Fig. 3(a), (b)] and did not tend to differ between sexes.
In late spring, low sex-steroid concentrations were recorded in all groups [Figs 4(a)
and 5(b)]. From June onwards, plasma sex-steroid concentrations rose rapidly in
both male and female fish in the control group. More specifically, in males T peaked
in September, whereas peak values of 11-KT were recorded in October [Fig. 4(a),
(b)]. Thereafter, sex-steroid concentrations decreased throughout the spawning season
[Fig. 4(a), (b)]. In female fish, E2 reached highest concentrations during September,
and then declined c. 1 month earlier than did T to reach minimal concentrations in
November [Fig. 5(a), (b)]. Sea-lice infection had a significant influence on temporal
changes in plasma sex-steroid concentrations both in maturing males and females.
Elevation in plasma sex-steroid concentrations, both in the MI and HI groups, was
significantly delayed compared with the control group, the exception being E2 in the
MI group [Figs 4(a) and 5(b)]. After transferring back to fresh water, concentrations
rose rapidly in the MI and HI groups, and timing of peak plasma sex-steroid concentrations were similar in all groups [Figs 4(a) and 5(b)]. In the HI group, however,
sex-steroid concentrations did not fully reach the levels of those in the control and MI
groups, of which was particularly evident for 11-KT in male fish [Figs 4(a) and 5(b)].
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H. TVEITEN ET AL.
100
(a)
b B
80
ab
60
B
40
A
a
a
A
A
a
20
a A
A
A
a
a
Cortisol (ng ml−1)
a
A
0
Pre infection
180
End infection
Post infection
(b)
160
c
140
B
120
100
b
80
B
60
a
40
a A
a
A
a A
a
A
A
a A
a
A
20
0
Pre infection
End infection
Time of sampling
Post infection
Fig. 3. Plasma cortisol concentrations in maturing (a) male and (b) female Salvelinus alpinus before (16 June,
in fresh water), at the end (27 July, in sea water) and post (21 September, in fresh water) sea-lice
infection. The fish was exposed to no (control, ), medium ( ) and high ( ) sea-lice infection during
gonad recrudescence (23 June to 27 July). Differences between treatments within sampling dates are
indicated by different lowercase letters (P < 0·05). Differences across sampling dates within treatments
are indicated by different uppercase letters (P < 0·05).
At the end of sea-lice infection, there was a significant negative relationship
between plasma concentrations of T and cortisol both in male and female fish (Fig. 6).
Taking the data together, this negative relationship was apparent also at the individual level, but stronger for females (R 2 = 0·65, n = 17) than for males (R 2 = 0·40
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2327
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
50
(a)
Breeding period
Sea-lice and seawater exposure
Testosterone (ng ml−1)
40
a
a
b
a
a
30
a
a
a
a
20
a
c
a
a
10
0
21/4
90
b
a
a
a
a
a
a
21/5
a
a
a
20/6
a
20/7
19/8
18/9
18/10
(b)
17/11
17/12
Breeding period
Sea-lice and seawater exposure
11-ketotestosterone (ng ml−1)
80
b
70
b
60
50
b
40
30
20
10
0
21/4
a
a
a
a
a
a
21/5
a
a
a
20/6
a
ab
a
a
a
a
a
a
a
c
b
a
20/7
19/8
18/9
Date (day/month)
18/10
7/11
17/12
Fig. 4. Temporal changes in plasma (a) testosterone and (b) 11-ketotestosterone concentrations of male Salvelinus alpinus exposed to no [control ( )], medium ( ) and high ( ) sea-lice infection during gonad
recrudescence (23 June to 27 July). Different letters indicate significant differences (P < 0·05) within
sampling dates. Horizontal bars indicate timing of sea-lice and seawater exposure, and the breeding
period, respectively.
n = 41). Similar relationships were obtained for the other sex steroids measured
(female E2: R 2 = 0·57 and male 11-KT: R 2 = 0·41).
P L A S M A O S M O L A L I T Y A N D C H L O R I D E C O N C E N T R AT I O N S
Plasma osmolality and chloride concentrations are shown in Fig. 7(a), (b), respectively. There was no overall effect of sex on plasma osmolality and chloride concentrations, neither was there any interaction between sex and treatment. In fresh water,
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2328
H. TVEITEN ET AL.
160
(a)
Sea-lice and seawater exposure
Breeding period
140
a
Testosterone (ng ml−1)
120
a
100
80
a
60
a
b
ab
40
a
a
a
20
0
21/4
60
a
a
a
a
a
a
21/5
20/6
b
a
a
a
a
a
a
a
a
20/7
19/8
18/9
18/10
17/11
17/12
(b)
Breeding period
Sea-lice and seawater exposure
50
Oestradiol (ng ml−1)
b
40
a
a
a
ab
a
30
20
b
a
b
a
a
10
0
21/4
a
a
a
a
a
a
21/5
a
a
a
20/6
a
a
a
a
20/7
19/8
18/9
Date (day/month)
18/10
17/11
17/12
Fig. 5. Temporal changes in plasma (a) testosterone and (b) oestradiol-17β concentrations of maturing female
Salvelinus alpinus exposed to no [control ( )], medium ( ) and high ( ) sea-lice infection during gonad
recrudescence (23 June to 27 July). Different letters indicate significant differences within sampling dates
(P < 0·05). Horizontal bars indicate timing of sea-lice and seawater exposure, and the breeding period,
respectively.
just before sea-lice infection, plasma osmolality and chloride concentrations were
c. 310 mOsm and 140 mM, respectively, in all groups. In the control group, seawater exposure resulted in a slight, but significant, increase in plasma osmolality (c.
330 mOsm), whereas chloride concentrations remained largely unaffected compared
with freshwater values [Fig. 7(a), (b)].
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
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S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
30
R2 = 0·40 (male)
R2 = 0·65 (female)
Testosterone (ng ml−1)
25
20
15
10
5
0
0
50
100
150
200
Cortisol (ng ml−1) 27 July (end of seawater exposure)
Fig. 6. Relationship between plasma cortisol and testosterone concentrations in male ( , , ) and female
( , , ) Salvelinus alpinus exposed to no [control ( , )], medium ( , ) and high ( , ) sea lice
during gonad recrudescence (23 June to 27 July). Fish were sampled at the end of sea-lice and seawater
exposure (fish still in sea water). Strength of relationships (R 2 ) between variables is calculated from
individual data.
Sea-lice infection resulted in a significant increase in plasma osmolality and chloride concentrations. The effect was most apparent in the HI group where plasma
osmolality and chloride concentrations reached levels of c. 410 mOsm and 200 mM,
respectively [Fig. 7(a), (b)]. Also in the MI group, plasma osmolality (c. 350 mOsm)
and chloride concentrations (c. 155 mM) were influenced by sea-lice infection and
was statistically higher and lower, respectively, compared with the control and HI
groups [Fig. 7(a), (b)]. Plasma osmolality and chloride concentration in females that
did not re-mature in the MI group were 338·1 ± 5·3 mOsm and 144·3 ± 1·4 mM,
respectively, values not being significantly different from that of maturing fish in the
same group. Osmolality and chloride values returned to pre-infection values after
transfer to fresh water [Fig. 7(a), (b)].
Also at the individual level there was a strong positive relationship between sealice infection intensity and plasma osmolality (Fig. 8), with no apparent difference
between males (R 2 = 0·67, n = 35) and females (R 2 = 0·71, n = 18). Furthermore,
plasma cortisol concentration was significantly and positively related to plasma osmolality (Fig. 9). At the individual level, this relationship was stronger for females
(R 2 = 0·77, n = 17) than for males (R 2 = 0·47, n = 37).
G ROW T H A N D C O N D I T I O N
After initiation of feeding in early June, growth rate and condition increased
rapidly in all groups. Growth and condition were, however, significantly affected in
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2330
H. TVEITEN ET AL.
440
(a)
c
420
B
Osmolality (mOsm)
400
380
360
b
340
a
a A
320
a A
a
B
B
A
a
A
a
A a
A
300
280
Pre-infection
210
End-infection
Post-infection
(b)
200
c
B
Chloride (mmol l−1)
190
180
170
160
150
b
a
A
a A
a
A
a
B
A
a A a A a
A
140
130
Pre-infection
End-infection
Time of sampling
Post-infection
Fig. 7. Plasma (a) osmolality and (b) chloride concentrations in maturing male and maturing female Salvelinus
alpinus before (16 June, in fresh water), at the end (27 July, in sea water) and after (21 September, in
fresh water) sea-lice infection. The fish was exposed to no [control ( )], medium ( ) and high ( ) sealice infection during gonad recrudescence (23 June to 27 July). Differences between treatments within
sampling dates are indicated by different lowercase letters (P < 0·05). Differences across sampling dates
within treatments are indicated by different uppercase letters (P < 0·05).
both sea-lice-infected groups, with the effect being most pronounced in the HI group
[Fig. 10(a)–(d)]. Before sea-lice infection, K in females that did not re-mature in the
MI group was significantly lower compared with that of their maturing counterparts
[Fig. 10(d)]. During late summer and autumn, after withdrawal of food, it appeared,
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2331
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
480
460
R2 = 0·68
Osmolality (mOsmol)
440
420
400
380
360
340
320
300
0·00
0·05
0·10
0·15
0·20
0·25
0·30
−1
Sea-lice infection (sea lice g fish 27 July (end of seawater exposure)
Fig. 8. Relationship between sea-lice infection intensity and plasma osmolality in maturing male and maturing
female Salvelinus alpinus exposed to no [control ( )], medium ( ) and high ( ) sea-lice infection
during gonad recrudescence (23 June to 27 July). Fish were sampled at the end of sea-lice and seawater
exposure (fish still in sea water).
however, that K decreased at a slower rate in the sea-lice-infected groups, and
at the end of the experiment there were no differences in K between treatments
[Fig. 10(b), (d)]. Growth rate during seawater and sea-lice exposure was significantly
and negatively related to plasma cortisol concentrations (Fig. 11). This relationship
was also apparent at the individual level but again being stronger for females (R 2 =
0·75, n = 17) than for males (R 2 = 0·25, n = 40).
OV U L AT I O N A N D G A M E T E P R O D U C T I O N
Eleven, six and four females ovulated and were stripped of eggs in the control, MI
and HI groups, respectively. In the control group, the first ovulation was recorded on
20 October, c. 10 days earlier than in the MI and HI groups, but median ovulation
time did not differ significantly between groups (Fig. 12). Fertilization rates were
between 98 and 100% and did not differ between treatments.
Low number of maturing females in the MI and HI groups resulted in reduced
reproductive output in sea-lice-infected fish, and egg production in the MI and HI
groups was only c. 50 and 30%, respectively, of that in the control group (Table II).
Relative and absolute fecundity also tended to be reduced among HI fish, although
values were not statistically different (Table II). Egg size and mortality until hatch
were very similar between treatments, as well as fry mass at hatch and after 6 weeks
of start feeding (Table II).
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
3756 ± 470
3500 ± 357
3082 ± 839
840 ± 131 11·2 ± 2·1 4173 ± 705
Relative
Absolute
fecundity fecundity (eggs
(eggs kg−1 )
female−1 )
1014 ± 100 15·2 ± 1·7 4496 ± 361
962 ± 87 13·4 ± 1·5 4412 ± 335
Relative
egg mass
(%)
*Total egg production was not subject to statistical evaluation.
Control
Medium
infection
High
infection
Fish mass
before
stripping
(g)
4·25 ± 0·06
4·30 ± 0·03
4·24 ± 0·09
Egg
diameter
(mm)
12 327
41 332
20 998
5·0 ± 3·4
5·7 ± 1·9
5·4 ± 2·0
39·6 ± 1·9
39·9 ± 0·8
39·4 ± 0·8
Total egg
Egg
Larval mass
production*
mortality to
at hatch
(number of eggs) hatch (%)
(mg)
52·5 ± 5·0
54·7 ± 2·3
48·8 ± 1·4
Larval mass 6
weeks after
start feeding
(mg)
Table II. Indices of reproductive output, egg and embryonic quality in female Salvelinus alpinus exposed to no (control), medium and high sea-lice
infection for 34 days during gonad recrudescence (23 June to 27 July). Values are mean ± s.e. There were no significant differences between
treatments (P > 0·05)
2332
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2333
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
Spermatocrit tended to be reduced in sea-lice-infected fish compared with control
fish at the beginning of the spawning season (control: 13·1 ± 1·1; MI: 9·5 ± 1·0;
HI: 10·6 ± 1·0), but values were not statistically different. In sea-lice-infected fish,
spermatocrit increased somewhat with time and at the end of the spawning season
only small differences in spermatocrit were recorded between treatments (control:
13·0 ± 1·8; MI: 13·1 ± 1·4; HI: 11·4 ± 1·0).
DISCUSSION
The present study has shown that sea-lice infection during early stages of gonad
growth of S. alpinus has the potential to elicit a significant reduction in reproductive
output. In sea-lice-infected fish, reproductive output was reduced mainly via the
number of maturing females and increased fish mortality, whereas quality of the
gametes that were produced appeared to be little influenced. The observed effects of
sea lice appear to be mediated through influences on stress physiology, alterations in
reproductive endocrine homeostasis and fish body condition at the time of infection.
Compared with studies on other salmonid species, the relative sea-lice infection
intensity (sea lice g−1 fish) used in this study is considered to be within the subclinical range where physiological effects are likely to occur, whereas in absolute
terms (sea lice fish−1 ) they may be considered as clinical infections (Wagner et al.,
2008). Infection intensities are also comparable with those that are encountered under
180
160
R2 = 0·47 (male)
R2 = 0·77 (female)
Cortisol (ng ml−1)
140
120
100
80
60
40
20
0
300
350
400
450
Osmolality (mOsmol) 27 July (end of seawater exposure)
500
Fig. 9. Relationship between plasma osmolality and cortisol concentrations in male ( , , ) and female
( , , ) Salvelinus alpinus exposed to no [control ( , )], medium ( , ) and high ( , ) sea-lice
infection during gonad recrudescence (23 June to 27 July). Fish were sampled at the end of sea-lice and
seawater exposure (fish still in sea water). Strength of relationships (R 2 ) between variables is calculated
from individual data.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
9/7
8/8
7/9
a
a
a
14/6
b
ab
a
14/7
a
a
b
13/8
a
ab
b
12/9
7/10
b
ab
a
Sea-lice and seawater exposure
15/5
9/6
c
b
a
ab
a
b
6/12
b
b
a
a
ab
b
11/11
9/7
8/8
7/9
b
b
a
15/5
a
a
a
a
ab
ab
0·85
0·95
14/6
b
14/7
13/8
ab
a
1·15
1·05
b
1·25
a
b
b
12/9
a
a
a
a
a
a
6/12
12/10
a
b
ab
11/11
a
a
a
11/12
Breeding period
6/11
a
a
a
Breeding period
7/10
b
ab
a
Sea-lice and seawater exposure
9/6
a
c
a
a
a
(d)
b
ab
ac
c
bc
b
c
Sea-lice and seawater exposure
1·35
1·45
b
ab
a
(c)
−0·8
10/5
−0·6
−0·4
−0·2
0
0·2
0·4
0·6
0·8
1
1·2
1·4
1·6
0·75
15/4
11/12
Date (day/month)
a
a
a
Breeding period
12/10
6/11
b
ab
a
Breeding period
Fig. 10. Temporal changes in (a), (c) specific growth rate in mass (Gw ) of maturing (a) male and (c) female and (b), (d) condition factor (K) of maturing (b) male and
(d) female Salvelinus alpinus exposed to no [control ( )], medium ( ) and high ( ) sea-lice infection during gonad recrudescence (23 June to 27 July). (c), (d), Gw
and K of not re-maturing females before and after sea-lice exposure in the medium group are indicated ( ). Horizontal bars indicate timing of sea-lice exposure and the
breeding period, respectively. Differences between treatments within sampling dates are indicated by different lowercase letters (P < 0·05).
a
a
a
(b)
0·8
15/4
0·9
1
1·1
1·2
1·3
1·4
1·5
b
b
a
b
b
a
a
b
c
Sea-lice and seawater exposure
(a)
−0.4
10/5
−0·2
0
0·2
0·4
0·6
0·8
1
1·2
1·4
Gw (% day−1)
K
Gw (% day−1)
K
2334
H. TVEITEN ET AL.
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2335
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
1·6
R2 = 0·25 (male)
R2 = 0·75 (female)
1·4
Gw (% day−1 16/06 to 27/07)
1·2
1
0·8
0·6
0·4
0·2
0
0
50
100
150
200
Cortisol (ng ml−1) on 27 July (end of seawater exposure)
Fig. 11. Relationship between plasma cortisol at the end of sea-lice exposure (27 July) and specific growth
rate in mass (Gw 16 June to 27 July) of maturing male ( , , ) and female ( , , ) Salvelinus
alpinus exposed to no [control ( , )], medium ( , ) and high ( , ) sea-lice infection during gonad
recrudescence (23 June to 27 July). Strength of relationships (R 2 ) between variables is calculated from
individual data.
natural conditions (Bjørn et al., 2001). The different sea-lice intensities used can
therefore be considered as relevant for studying physiological effects of sea-lice
infection also in S. alpinus, even though few studies have been carried out for this
species. Generally, however, the present observations corroborate earlier studies on
Atlantic salmon Salmo salar L. and brown trout S. trutta (Bjørn & Finstad, 1997;
Finstad et al., 2000; Bjørn et al., 2001; Fast et al., 2006) and indicate that S. alpinus
display similar stress-related physiological changes and suits of responses to sea-lice
infection to that of other salmonids.
Sea-lice infection resulted in a reduction in egg production by 50 and 70% in the
MI and HI groups, respectively, compared with that of the control. In the HI group,
reduced egg production was caused mainly by a combination of increased mortality
and a reduced proportion of maturing females, whereas in the MI a reduction in
proportion of maturing females was the main cause. Since, as far as is known, there
are no previous reports on the effect of sea-lice infection on reproductive performance
in any salmonid species, comparison is difficult, but a reduction in egg production
by 50 and 70% after sea-lice exposure must be considered to be substantial.
There is compelling evidence that cortisol has an inhibitory effect on plasma sexsteroid concentrations (Pankhurst & Van Der Kraak, 2000), and, thus, this steroid
may influence or inhibit reproductive development. The close negative association
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2336
H. TVEITEN ET AL.
100
Cumulative ovulation (%)
80
60
40
20
0
15/10
20/10
25/10
30/10
4/11
Date (day/month)
9/11
14/11
19/11
Fig. 12. Influence of sea-lice infection during gonad recrudescence (23 June to 27 July) on cumulative percentage of ovulated female Salvelinus alpinus over time. Fish were exposed to no [control ( )], medium
( ) and high ( ) sea-lice infection during gonad recrudescence (23 June to 27 July).
between plasma cortisol and sex-steroid concentrations especially that of T, found in
the present study, supports this notion. It may therefore be hypothesized that elevated
cortisol concentrations in sea-lice-infected fish have suppressed plasma sex-steroid
concentrations to the level that reproductive development was inhibited as seen in
the MI and HI groups.
There are, however, a few problems with the latter interpretation. Although information about the temporal changes in cortisol concentration during sea-lice exposure
in the different groups is lacking, cortisol concentrations in females that did not remature in the MI group were the same as maturing fish at the end of sea-lice exposure.
Neither osmolality nor chloride concentrations differ. Also, re-maturing females in
the HI group had much higher plasma osmolality, cortisol and chloride concentrations than non-maturing females in the MI group. It is therefore possible that high
plasma cortisol or osmoregulatory problems may not alone have resulted in suppression of ovarian development. Since there is evidence that energy acquisition and
nutritional status are important for mediating cessation or triggering of reproductive
development in salmonids (Rowe & Thorpe, 1990; Rowe et al., 1991; Kadri et al.,
1996; Silverstein et al., 1998; Shearer & Swanson, 2000), condition of the fish may
also be taken into account. Although there was no difference in female condition
between groups before sea-lice infection (P > 0·05), sea-lice-infected females that
did not re-mature had significantly lower condition before sea-lice infection than
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
2337
their maturing counterparts (i.e. they were from the lower part of the distribution)
[Fig. 10(d)]. Since there were no differences in growth rate, osmolality, plasma cortisol or chloride concentrations in females with high and low K, the reason for the
different effects of sea-lice infection on maturation may be related to differences in
nutritional status.
Replenishment of somatic reserves, which seems necessary before gonad growth
can be initiated (Rowe et al., 1991), together with increased energy requirements
for maintaining osmoregulatory functions may therefore have resulted in limited
energy available for reproductive growth in low K individuals. A rapid increase in
plasma sex-steroid concentrations in maturing high K females (even in the HI group)
after loss of the stressor (fish devoid of food), indicating that overall energy stores
were monitored as sufficient for reproductive development, supports this notion.
These observations, however, also suggest that endocrine signalling of nutritional
status (which are likely to be multi-factorial) from the somatic to the reproductive
neuroendocrine axis may be influenced (blocked) or differently translated when the
fish are under stressful conditions.
Although dead fish were not sexed, it appears that most of the fish that died were
females which also had the lowest condition before sea-lice infection. In line with the
observed female-specific reduction in maturation rate and survival, females appear
to have a stronger and more closely regulated physiological response to sea-lice
infection compared with that of males. In females, there was always a stronger (R 2
of 0·65–0·86) relationship than for males (R 2 of 0·25–0·47), between cortisol and
all the variables investigated (infection intensity, osmolality, growth rate and sex
steroids). In studies on brown trout S. trutta and O. mykiss, significant differences
in plasma cortisol concentrations between males and females in response to stress
have also been detected, with females displaying higher concentrations than males
(Campbell et al., 1992, 1994; Pottinger & Carrick, 1999; Clements et al., 2002).
The reason for this is not clear but may be related to sexual differences in plasma
sex steroids as indicated for mammals (McQuillan et al., 2003). Sexual differences
in susceptibility to stress may also explain why the proportion of maturing males
was not influenced by sea-lice infection. On the other hand, before sea-lice infection
K was somewhat higher in maturing males than in maturing females [Fig. 10(b),
(d)], and a possible difference in nutritional status may also have influenced the
sex-specific effect of sea-lice exposure on maturation. Definitively, further studies
on the influence of stress on reproduction should take both sex and body conditions
into consideration.
In the fish that did mature, reproductive physiology was also influenced by sea-lice
exposure. First, in the control group, temporal changes in plasma sex-steroid concentrations, both in males and females, were very similar to what has been recorded
previously for this strain of S. alpinus when fish are held in fresh water (Tveiten
et al., 1998; Frantzen et al., 2004). In sea-lice-exposed fish, however, plasma sexsteroid concentrations were reduced, and in females the effect was found to be more
evident for T than for that of E2. In the MI group, where cortisol was moderately
elevated, plasma T concentrations were depressed compared with controls, whereas
those of E2 were not. Unaffected E2 concentrations, despite depressed plasma T
concentrations may, however, indicate that T synthesis was sufficient (T should be
available for aromatization) for ample E2 synthesis. This may indicate that cortisol
exerts its effect, in part, on suppression of pathways leading to the production of T,
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2338
H. TVEITEN ET AL.
rather than on aromatase activity. This is in agreement with results from studies
of O. mykiss (Pankhurst & Van Der Kraak, 2000) and with what was observed in
the HI group, where a further reduction in plasma T concentrations was associated
with a significant reduction also in E2. Also, after return to fresh water, plasma
T concentrations were significantly lower in the HI group without the effect being
that apparent for E2. Furthermore, the close negative relationship between plasma
cortisol and sex-steroid concentrations together with the rapid increase in sex-steroid
concentrations after re-entry to fresh water may indicate a direct effect of stress
and cortisol on sex steroid synthesis. It appears that the delay in commencement of
gonad development induced by sea-lice infection tended to delay, but did not greatly
influence, the timing of ovulation.
In male fish also, plasma sex-steroid concentrations were reduced by sea-lice
infection, but apparently not to the extent that reproductive development was compromised in terms of proportions of maturing individuals. In the sea-lice-infected
groups there was a tendency towards reduced density of spermatozoa during the first
part of the spawning season. If this reduction would cause any decline in fertilization
success under natural condition, where spermatozoa density may be a limiting factor,
is not known.
Gamete quality and reproductive investment was little affected in the sea-liceinfected fish. Studies on brown trout S. trutta and O. mykiss showed that prolonged
stress, associated with elevated plasma cortisol concentrations, had a significant effect
on egg and progeny survival (Campbell et al., 1992, 1994). Based on sex steroid
profiles, stress in the present experiment was applied during early stages of gonad
growth (Frantzen et al., 1997; Tveiten et al., 1998), which is different from studies
on S. trutta and O. mykiss (Campbell et al., 1992, 1994) where stress was maintained throughout gonad development. The apparent lack of stress on gamete quality
in the present study may be due to the fact that in sea-lice-infected fish gonad
development was delayed until after return to fresh water and, thereby, loss of the
stressor.
Although sea-lice infection intensities in the range used in this study are encountered in sea trout S. trutta and S. alpinus populations in the wild (Bjørn et al., 2001),
extrapolation of the present results to what may occur under natural conditions is
difficult. Seen in the light that condition at the time of sea-lice exposure may be
critical for initiation and progression of maturation, there may be some reason for
concern. For example, in the wild, K of descending S. alpinus of the Hammerfest
strain is c. 0·7 (Rikardsen et al., 1997; Rikardsen & Elliott, 2000), which is actually
slightly below the condition in not re-maturing females in the present study. Sea-lice
infection in the wild may therefore elicit a larger reduction in maturing fish, or there
may be a reduction in maturation to lower infection intensities, than seen in this
study. A similar scenario of lost feeding opportunity as indicated in this study may
also apply under natural conditions, where sea lice-infected fish re-enter fresh water
at an earlier stage than non-infected fish, losing opportunity for energy accumulation as seen in sea trout S. trutta (Tully et al., 1993; Birkeland, 1996; Birkeland &
Jakobsen, 1997).
Finally, the apparent female-specific effect of sea-lice infection on maturation
and mortality would certainly also have implications for the long-term reproductive
potential of a given population via a continuous decrease in female to male ratio
among spawning fish.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
2339
This study forms part of project no. 149187/730 supported by the Norwegian Research
Council. The technical assistance of A. B. Tennøy, B.-S. Sæther, M. Frantzen, J. Espen Tau
Strand, M. Johnsen and T. Lexau Hanebrekke is highly appreciated. R.S.M acknowledges
financial support from AquaNet project EI 4 – Risks and consequences of sea lice infestations.
Thanks to R. W. Shulz, University of Utrecht, The Netherlands for providing the 11-KT
antiserum.
References
Barton, B. A. (1997). Stress in finfish: past, present and future – a historical perspective. In
Fish Stress and Health in Aquaculture (Iwama, G. K., Pickering, A. D., Sumpter, J. P.
& Schreck, C. B., eds), pp. 1–33. Cambridge: Cambridge University Press.
Birkeland, K. (1996). Consequences of premature return by sea trout (Salmo trutta) infested
with the salmon louse (Lepeophtheirus salmonis Krøyer): migration, growth, and mortality. Canadian Journal of Fisheries and Aquatic Sciences 53, 2808–2813.
Birkeland, K. & Jakobsen, P. J. (1997). Salmon lice, Lepeophtheirus salmonis, infestation as
a causal agent of premature return to rivers and estuaries by sea trout, Salmo trutta,
juveniles. Environmental Biology of Fishes 49, 129–137.
Bjørn, P. A. & Finstad, B. (1997). The physiological effects of salmon lice infection on sea
trout post smolts. Nordic Journal of Freshwater Research 73, 60–72.
Bjørn, P. A. & Finstad, B. (1998). The development of salmon lice (Lepeophtheirus salmonis)
on artificially infected post smolts of sea trout (Salmo trutta). Canadian Journal of
Zoology 76, 970–977.
Bjørn, P. A., Finstad, B. & Kristoffersen, R. (2001). Salmon lice infection of wild sea trout
and Arctic char in marine and freshwaters: the effects of salmon farms. Aquaculture
Research 32, 947–962.
Boivin, T. G. & Power, G. (1990). Winter condition of anadromous Arctic charr (Salvelinus
alpinus) in eastern Ungava Bay, Quebec. Canadian Journal of Zoology 68, 2284–2289.
Campbell, P. M., Pottinger, T. G. & Sumpter, J. P. (1992). Stress reduces the quality of
gametes produced by rainbow trout. Biology of Reproduction 47, 1140–1150.
Campbell, P. M., Pottinger, T. G. & Sumpter, J. P. (1994). Preliminary evidence that chronic
confinement stress reduces the quality of gametes produced by brown and rainbow
trout. Aquaculture 120, 151–169.
Carragher, J. F., Sumpter, J. P., Pottinger, T. G. & Pickering, A. D. (1989). The deleterious
effects of cortisol implantation on reproductive function in two species of trout, Salmo
trutta L. and Salmo gairdneri Richardson. General and Comparative Endocrinology
76, 310–321.
Clements, S. P., Hicks, B. J., Carragher, J. F. & Deudal, M. (2002). The effect of a trapping
procedure on the stress response of wild rainbow trout. North American Journal of
Fisheries 22, 907–916.
Dutil, J. D. (1986). Energetic constraints and spawning interval in the anadromous Arctic
charr (Salvelinus alpinus). Copeia 1986, 945–955.
Fast, M. D., Muise, D. M., Easy, R. E., Ross, N. W. & Johnson, S. C. (2006). The effects of
Lepeophtheirus salmonis infections on the stress response and immunological status
of Atlantic salmon (Salmo salar). Fish and Shellfish Immunology 21, 228–241.
Finstad, B., Bjørn, P. A., Grimnes, A. & Hvidsten, N. A. (2000). Laboratory and field investigations of salmon lice (Lepeophtheirus salmonis, Krøyer) infestation on Atlantic salmon
(Salmo salar L.) post-smolts. Aquaculture Research 31, 795–803.
Foo, J. T. W. & Lam, T. J. (1993). Retardation of ovarian growth and depression of serum
steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation. Aquaculture 115, 133–143.
Frantzen, M., Johnsen, H. K. & Mayer, I. (1997). Gonadal development and sex steroids in
a female Arctic charr broodstock. Journal of Fish Biology 51, 697–709.
Frantzen, M., Damsgård, B., Tveiten, H., Moriyama, S., Iwata, M. & Johnsen, H. K. (2004).
Effects of fasting on temporal changes in plasma concentrations of sex steroids, growth
hormone and insuline-like growth factor I, and reproductive investment in Arctic charr
(Salvelinus alpinus). Journal of Fish Biology 65, 1526–1542.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
2340
H. TVEITEN ET AL.
Gillet, C. (1991). Egg production in an Arctic charr (Salvelinus alpinus L.) brood stock:
effects of temperature on the timing of spawning and the quality of eggs. Aquatic
Living Resources 4, 109–116.
Jobling, M. (1983). Influence of body weight and temperature on growth rates of Arctic charr,
Salvelinus alpinus (L.). Journal of Fish Biology 22, 471–475.
Jobling, M., Tveiten, H. & Hatlen, B. (1998). Cultivation of: an update. Aquaculture International 6, 181–196.
Johnsen, H. K., Eliassen, R. A., Sæther, B. S. & Larsen, J. S. (2000). Effects of photoperiod
manipulation on development of seawater tolerance in Arctic charr. Aquaculture 189,
177–188.
Kadri, S., Mitchell, D. F., Metcalfe, N. B., Huntingford, F. A. & Thorpe, J. E. (1996). Differential patterns of feeding and resource accumulation in maturing and immature
Atlantic salmon, Salmo salar. Aquaculture 142, 245–257.
Leatherland, J. F. (1999). Stress, cortisol and reproductive dysfunction in salmonids: fact or
fallacy. Bulletin of the European Association of Fish Pathologists 19, 254–257.
McQuillan, H. J., Lokman, P. M. & Young, G. (2003). Effects of sex steroids, sex, and sexual
maturity on cortisol production: an in vitro comparison of chinook salmon and rainbow
trout interrenals. General and Comparative Endocrinology 133, 154–163.
Mommsen, T. P., Vijayan, M. M. & Moon, T. W. (1999). Cortisol in teleosts: dynamics,
mechanisms of action, and metabolic regulation. Reviews in Fish Biology and Fisheries
9, 211–268.
Pankhurst, N. W. & Van Der Kraak, G. (1997). Effects of stress on reproduction and growth
of fish. In Fish Stress and Health in Aquaculture (Iwama, G. K., Pickering, A. D.,
Sumpter, J. P. & Schreck, C. B., eds), pp. 73–93. Cambridge: Cambridge University
Press.
Pankhurst, N. W. & Van Der Kraak, G. (2000). Evidence that acute stress inhibits ovarian
steroidogenesis in rainbow trout in vivo through the action of cortisol. General and
Comparative Endocrinology 117, 225–237.
Pottinger, T. G. & Carrick, T. R. (1999). Modification of the plasma cortisol response to
stress in rainbow trout by selective breeding. General and Comparative Endocrinology
116, 122–132.
Rikardsen, A. H. & Elliott, J. M. (2000). Variations in juvenile growth, energy allocation and
life-history strategies of two populations of Arctic charr in North Norway. Journal of
Fish Biology 56, 328–346.
Rikardsen, A. H., Svenning, M.-A. & Klemetsen, A. (1997). The relationships between
anadromy, sex ratio and parr growth of Arctic charr in a lake in North Norway.
Journal of Fish Biology 51, 447–461.
Rowe, D. K. & Thorpe, J. E. (1990). Suppression of maturation in male Atlantic salmon
(Salmo salar L.) parr by reduction in feeding and growth during spring month. Aquaculture 86, 291–313.
Rowe, D. K., Thorpe, J. E. & Shanks, A. M. (1991). Role of fat stores in the maturation of
male Atlantic salmon (Salmo salar) parr. Canadian Journal of Fisheries and Aquatic
Sciences 48, 405–413.
Schreck, C. B., Contreras-Sanchez, W. & Fitzpatrick, M. S. (2001). Effects of stress on fish
reproduction, gamete quality, and progeny. Aquaculture 197, 3–24.
Schulz, R. (1985). Measurements of five androgens in the blood of immature and mature
rainbow trout, Salmo gairdneri (Richardson). Steroids 46, 717–726.
Shearer, K. D. & Swanson, P. (2000). The effect of whole body lipid on early sexual maturation of 1+ age male Chinook salmon (Oncorhynchus tshawytscha). Aquaculture 190,
343–367.
Silverstein, J. T., Shearer, K. D., Dickhoff, W. W. & Plisetskaya, E. M. (1998). Effect of
growth and fatness on sexual development of Chinook salmon (Oncorhynchus
tshawytscha) parr. Canadian Journal of Fisheries and Aquatic Sciences 55, 2376–2382.
Taranger, G. L., Stefansson, S. O., Oppedal, F., Andersson, E., Hansen, T. & Norberg, B.
(2000). Photoperiod and temperature affects gonadal development and spawning time
in Atlantic salmon (Salmo salar L.). In Proceedings of the 6th International Symposium
on the Reproductive Physiology of Fish (Norberg, B., Kjesbu, O. S., Taranger, G. L.,
Andersson, E. & Stefansson, S. O., eds), p. 345. Bergen: John Grieg A/S.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341
S E A L I C E A F F E C T S A LV E L I N U S A L P I N U S R E P RO D U C T I O N
2341
Tully, O., Poole, W. R. & Whelan, K. R. (1993). Infestation parameters for Lepeophtheirus
salmonis (Krøyer) (Copepoda: Caligidae) parasitic on sea trout, Salmo trutta L., off the
west coast of Ireland during 1990 and 1991. Aquaculture and Fisheries Management
24, 545–555.
Tveiten, H., Mayer, I., Johnsen, H. K. & Jobling, M. (1998). Sex steroids, growth and condition of Arctic charr broodstock during an annual cycle. Journal of Fish Biology 53,
714–727.
Wagner, G. N., Fast, M. D. & Johnson, S. C. (2008). Physiology and immunology of Lepeophtheirus salmonis infections of salmonids. Trends in Parasitology 24, 176–183.
Wendelaar-Bonga, S. E. (1997). The stress response in fish. Physiological Reviews 77,
591–625.
Zar, J. H. (1999). Biostatistical Analysis, 4th edn. New Jersey, NJ: Prentice-Hall, Inc.
© 2010 The Authors
Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 2318–2341