Fish & Shellfish Immunology (2000) 10, 47–59
Article No. fsim.1999·0229
Available online at http://www.idealibrary.com on
Effects of sea lice (Lepeophtheirus salmonis Kröyer, 1837)
infestation on macrophage functions in Atlantic salmon
(Salmo salar L.)
AHMED MUSTAFA1*, CHRIS MACWILLIAMS1, NICOLE FERNANDEZ1,
KELLY MATCHETT1, GARY A. CONBOY2 AND JOHN F. BURKA1
1
Department of Anatomy and Physiology, and 2Department of Pathology and
Microbiology, Atlantic Veterinary College, University of Prince Edward
Island, Charlottetown, PEI CIA 4P3, Canada
(Received 1 December 1998, accepted after revision 7 June 1999)
Experiments were conducted to determine the e#ects of sea lice, Lepeophtheirus salmonis, on non-specific defence mechanisms in Atlantic salmon, Salmo
salar, by experimentally infesting hatchery-reared 1 and 2 year old postsmolts, S1 and S2, with laboratory grown infective copepodids at moderate to
high infection intensities ranging from 15–285 lice per fish. The e#ects of sea
lice-induced stress were investigated by measuring the blood levels of cortisol
and glucose as indicators of primary and secondary stress responses, and by
changes in macrophage respiratory burst activity and phagocytosis as indicators of tertiary stress responses as well as non-specific defence mechanisms.
Fish were sampled prior to sea lice infestation at day 0 and at days 3, 7, 14 and
21 post-infestation. Sea lice were at copepodid stage at day 3, at chalimus
stages at days 7 and 14, and at pre-adult stage at day 21. Blood levels of
cortisol and glucose were found to be significantly increased at day 21 in
fish-infested with the highest levels. Macrophage respiratory burst and phagocytic activities were found to be significantly decreased only at day 21. These
results indicate that sea lice do not suppress host defence mechanisms during
the earlier stages of infestation. They do have e#ects on the development of
chronic stress and on the host non-specific defence mechanisms soon after the
lice reach the pre-adult stage.
2000 Academic Press
Key words:
sea lice, Atlantic salmon, stress, macrophage, respiratory burst,
phagocytosis.
I. Introduction
The term sea lice refers to several species of marine ectoparasitic copepods of
the genera Lepeophtheirus and Caligus that commonly infest salmonids. Of
these, Lepeophtheirus salmonis is responsible for serious disease outbreaks
and high economic losses to salmon farmers throughout the northern hemisphere. Pre-adult and adult sea lice browse on the surface of salmon, eating
*Corresponding author
1050–4648/00/010047+13 $35.00/0
47
2000 Academic Press
48
A. MUSTAFA ET AL.
mucus, epidermal cells and blood, and eventually eroding the protective
surface of the fish (Mackinnon, 1997). In severe cases, the head of the salmon
can be eroded su$ciently to expose the underlying tissue and even the skull
roof (Egidius, 1985; Berland, 1993). These can lead to osmoregulatory problems
and predisposition to secondary bacterial infections and kill the fish (Wootten
et al., 1982; MacKinnon, 1997).
The life cycle of Lepeophtheirus salmonis consists of 10 stages. These stages
include two free-swimming nauplii, one free-swimming infectious copepodid,
four attached chalimus, two free-moving pre-adults, and one free-moving adult
(Kabata, 1972; Pike, 1989; Johnson & Albright, 1991). After the fourth
chalimus stage, the parasites are mobile and the cause of severe pathogenicity
due to their feeding activities on the fish (Grimnes & Jakobsen, 1996; Dawson,
1998).
The absence of completely e#ective and safe methods for treating sea lice
infections emphasises the need to develop alternate methods. The development of such methods is limited by deficiencies in the understanding of many
aspects of the basic biology of Lepeophtheirus salmonis, especially its e#ects
on host defence mechanisms. In all vertebrates, environmental stressors cause
neuroendocrine and autonomic changes that modulate both non-specific and
specific defence mechanisms, which are often considered the cause of higher
susceptibility of stressed individuals to disease (Ruis & Bayne, 1997). In
aquaculture, fish are exposed to stressors, such as transport, handling,
marking, grading etc., on a regular basis which elicit the release of corticosteroids from the interrenal cells and catecholamines from the chroma$n cells
by activating the hypothalamus-pituitary-interrenal axis (Schreck, 1996;
Barton & Iwama, 1991). Though the immune systems of fish are sophisticated
and complex, it is believed that these stress hormones can modulate
macrophage functions (Ruis & Bayne, 1997). Correlation between increased
production of cortisol and glucose and decreased macrophage functions in
salmonids have been well documented (Secombes, 1990; Schreck, 1996). With
respect to sea lice, Johnson & Albright (1992) showed that coho salmon
implanted with cortisol, by intraperitoneal injection in an oil-based pellet,
have a decreased inflammatory response and less epithelial hyperplasia
when infested with L. salmonis, and increased susceptibility to the parasite.
Mustafa & MacKinnon (1993) and Mustafa (1997) showed that Atlantic
salmon given cortisol implants acquired heavier infections with Caligus
elongatus. While cortisol implantation experimentally stimulates some of the
secondary e#ects of stress, there have apparently been no reports on the
assessment of stress on immune responses due to sea lice infestations and
development in salmonids, neither have any reports compared S1 and S2
Atlantic salmon smolts. Thus, until now it has been impossible to predict what
stages of the sea lice would have e#ects on host defence mechanisms,
especially in two di#erent age groups of Atlantic salmon smolts raised under
aquaculture conditions.
In this study, the e#ects of sea lice infestations on host stress levels (i.e.
plasma cortisol and glucose concentrations) and changes in the host nonspecific defence mechanisms (i.e. respiratory burst and phagocytic activities of
macrophages) have been examined along a sea lice development gradient
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
49
in two di#erent age groups of Atlantic salmon smolts under laboratory
conditions.
II. Materials and Methods
FISH AND THEIR MAINTENANCE
The experimental design consists of two studies. The first study used 300
2 year old Atlantic salmon smolts (S2), with a mean weight of 680 g and a mean
length of 37·2 cm; whilst the second study used 300 1 year old Atlantic salmon
smolts (S1), with a mean weight of 180 g and a mean length of 26·2 cm. In both
cases, Atlantic salmon smolts (Saint John river stocks) were obtained from a
Prince Edward Island hatchery and randomly assigned to two di#erent groups
(controls and tests) in four di#erent tanks (i.e. two control groups and two test
groups for each study). Fish were maintained in 1500 1 tanks and were
acclimated gradually to artificial seawater (Instant Ocean, Aquarium Systems, Mentor, OH, U.S.A.) over a 1 week period and were then maintained in
302 ppt at 101 C for a further 2 weeks prior to sea lice infestation.
Dissolved oxygen levels were monitored and maintained at ]8 ppm. The
photo-period was maintained at 14 h light: 10 h dark. Fish were fed daily to
satiation with the appropriate pelleted salmon diet to suit the fish size (Corey
Feed Mills, Frederiction, N. B., Canada) and cared for according to the
guidelines of the Canadian Council on Animal Care.
SEA LICE CULTURE
Infective copepodids were grown from egg-strings removed from ovigerous
sea lice collected from Atlantic salmon in aquaculture sites in the lower Bay
of Fundy. Egg-strings were placed in three, 20 l white plastic buckets with
sieved static sea water (27 ppt) collected from the same locality where the lice
were collected. The buckets were then placed in an environmental chamber at
a constant temperature of 102 C with gentle aeration supplied from
aquarium pumps and a 12 h light: 12 h dark cycle maintained by a timer. The
eggs hatched on day 1 and the maximum number of active copepodids were
obtained on day 12.
LABORATORY INFESTATION
The fish in the designated test tanks were infested with sea lice by adding
infective copepodids (30 000 in the first study and 20 000 in the second study) in
each of the two test tanks. During infestation the water level in all tanks was
lowered to one third of the normal and maintained with decreased water flow
but constant aeration. The water outlets were screened with 70 Nytex mesh
(Valox, Fredericton, N.B., Canada) and the room was kept dark. The control
tanks were treated similarly but no copepodids were added. After 21 h of
exposure, water circulation and light cycles were returned to normal.
SAMPLING
On day 0, i.e. prior to sea lice infestation, and days 3, 7, 14, and 21
post-infestation, 10 fish from each group were randomly sampled. Each fish
50
A. MUSTAFA ET AL.
was collected individually with a white 70 Nytex mesh-net and placed
immediately into a white plastic bucket containing a lethal dose of anaesthetic (MS-222; 200 mg l 1). Each fish was measured for length and weight
and bled to measure plasma levels of cortisol and glucose. Head kidneys from
each fish were then removed aseptically for macrophage assays. Fish were
then individually bagged and sea lice were counted later from each fish as well
as from each net and bucket corresponding to that particular fish.
PLASMA CORTISOL AND GLUCOSE ASSAYS
Plasma levels of cortisol and glucose of the representative fish from each
experimental group were analysed using validated and characterised radioimmunoassays (Coat-A-Count RIA and Glucose Oxidase Method) by the
Atlantic Veterinary College Diagnostic Services Unit.
ISOLATION OF MACROPHAGE CELLS
The head kidney samples that had been removed from fish were placed in
Leibovitz-15 medium (L-15) containing 2% foetal calf serum (FCS) on ice and
brought back to the laboratory. The samples were then macerated through a
coarse mesh in order to tease apart the larger fibres. The cells were centrifuged at 1000g, the supernatants discarded, and the cells then resuspended
in fresh L-15 containing 0·1% FCS. This process was repeated once again
and adjusted to obtain a cell concentration of 106 cells ml 1. Trypan blue
exclusion test was used to test for viability.
RESPIRATORY BURST ACTIVITY
The respiratory burst activity of phagocytic macrophage cells was measured
by the reduction of nitro-blue tetrazolium (NBT) by intracellular superoxide
radicals produced by leucocytes stimulated with phorbol myristate acetate
(PMA). Macrophage respiratory burst and phagocytic activity were measured
following the methods described by Secombes (1990) and Brown et al. (1996).
Briefly, aliquots of each sample (100 l) were dropped, in duplicate, onto glass
slides. The glass slides were then incubated in a moist chamber for 90 min at
15 C. The slides were then rinsed in a stream of phosphate bu#ered saline
(PBS), and incubated again for 15 min at 15 C with NBT (dissolved in L-15 at
1 mg l 1, and PMA added at 1 g ml 1). During this reaction NBT is reduced
by O 2 into an insoluble blue formazan. After incubation, the slides were
examined at 100 magnification to determine the proportion of activated
cells. At least 100 cells per sample were examined and the proportion of
activated cells was recorded.
PHAGOCYTIC ACTIVITY
The phagocytic activity of macrophage cells were evaluated using a microscopic counting technique as described by Mathews et al. (1990) and Brown
et al. (1996). This assay determined the proportion of phagocytic cells that
were able to take up opsonised formalin-killed bacteria, Yersinia ruckeri.
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
51
Briefly, a Y. ruckeri culture was grown in the laboratory and added to slides
containing attached macrophage cells that had been isolated from fish head
kidneys and incubated for 90 min at 15 C to yield a final particle to cell ratio
100:1. These slides were then incubated for another 60 min at 15 C. Following
incubation, the slides were washed with PBS, air dried, fixed in methanol and
stained with Di#-Quick (Leukostat Stain kit) for microscopic examination at
100 magnification. At least 100 cells were examined to determine phagocytic
capacity (the percentage of macrophage cells containing five or more bacteria)
(Enane et al., 1993).
CHEMICALS
MS-222, L-15, heparin, FCS, penicillin/streptomycin, trypan blue, NBT,
PMA were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A.
Di#-Quick-Leukostat Stain Kit was purchased from Fisher Scientific Ltd.,
Oshawa, Ont., Canada.
DATA ANALYSIS
The means and standard errors of the means were calculated for each assay.
Analyses were carried out using Student’s t-test, ANOVA and post-ANOVA
multiple comparison test after checking the data for normal distribution.
Di#erences were considered significant when P<0·05. All values shown in this
investigation are meanstandard error of means.
III. Results
Only copepodids were found on fish at day 3 post-infestation. On days 7 and
14 all lice found were at chalimus stages. On day 21, all lice were pre-adults.
Most of the copepodid and chalimus larvae were found attached to the gills
and fins and a few on the operculum and body surface. Pre-adults on the other
hand were mostly found on the body surface. During the first study, the
number of lice per fish ranged from 15–285, with the mean intensity of 106.
During the second study, the lice number ranged from 23–74, with the mean
intensity of 52. In both studies, the prevalence was 100%.
During the first study where S2 smolts were used, plasma cortisol concentrations increased following sea lice infestation and remained significantly
elevated (P<0·05) in the infested groups throughout the experiment, except on
day 14. The highest level of plasma cortisol was recorded on day 21 (Fig. 1).
During the second study where S1 smolts were used, plasma cortisol concentration increased significantly (P<0·05) in the infested groups on day 7
post-infestation and remained elevated for the rest of the experimental period
with the highest level on day 21 (Fig. 2).
Plasma glucose concentrations also increased with time post-infestation in
sea lice infested groups, both in the case of S2 and S1 smolts, with control
52
Plasma cortisol concentration (nmol l–1)
A. MUSTAFA ET AL.
250
Control
Infested
*
200
150
*
*
100
50
0
0
3
7
14
Day of sampling
21
Plasma cortisol concentration (nmol l–1)
Fig. 1. Plasma cortisol concentrations in control and sea lice infested Atlantic salmon
smolts (S2). *Significantly di#erent from controls.
100
Control
Infested
*
75
50
*
*
25
0
0
3
7
14
Day of sampling
21
Fig. 2. Plasma cortisol concentrations in control and sea lice infested Atlantic salmon
smolts (S1). *Significantly di#erent from controls.
groups remaining relatively constant (Figs 3 and 4, respectively). However,
glucose concentrations in both infested and control groups appeared to fall
within the ‘ normal ’ reference range values (3·65–7·40 mmol l 1) for Atlantic
salmon in seawater (Standards developed by Diagnostic Services at the
Atlantic Veterinary College).
In both studies, respiratory burst activity remained unchanged until day 14
between infested and control groups but a significant suppression (P<0·05)
was observed in the infested groups on day 21 (Figs 5 and 6). Phagocytic
capacity (>5 intracellular bacteria) showed a similar pattern to respiratory
burst activity with a significant suppression (P<0·05) on day 21 in the infested
groups compared to that of controls (Figs 7 and 8).
–1
Plasma glucose concentration (mmol l )
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
5
53
*
Control
Infested
*
4
*
3
2
1
0
0
3
7
14
Day of sampling
21
–1
Plasma glucose concentration (mmol l )
Fig. 3. Plasma glucose concentrations in control and sea lice infested Atlantic salmon
smolts (S2). *Significantly di#erent from controls.
6
5
Control
Infested
*
*
4
3
2
1
0
0
3
7
14
Day of sampling
21
Fig. 4. Plasma glucose concentrations in control and sea lice infested Atlantic salmon
smolts (S1). *Significantly di#erent from controls.
IV. Discussion
The prevalence of sea lice infestation in these studies was 100%. Between
day 0 and day 14, most sea lice were at copepodid and chalimus stages. During
these stages, most lice were recorded from gills and fins, and during mobile
pre-adult stages, most lice were recorded from body surfaces. These findings
are similar to those of Johnson & Albright (1991, 1992), Grimnes & Jakobsen
(1996), Dawson et al. (1997) and Dawson (1998). On the body surface, lice
were commonly found on the head, external operculum, areas between
dorsal and adipose fins, and peri-anal regions. In most fish, the head and the
54
Cells positive for respiratory burst (%)
A. MUSTAFA ET AL.
100
Control
Infested
75
*
50
25
0
0
3
7
14
Day of sampling
21
Cells positive for respiratory burst (%)
Fig. 5. Respiratory burst activity of macrophage cells isolated from head kidneys of
Atlantic salmon smolts (S2). *Significantly di#erent from controls.
100
Control
Infested
50
*
25
0
0
3
7
14
Day of sampling
21
Fig. 6. Respiratory burst activity of macrophage cells isolated from head kidneys of
Atlantic salmon smolts (S1). *Significantly di#erent from controls.
external opercular regions were preferred. The preference of these regions by
the mobile stages is likely feeding related, because these areas have thin
epidermis with no or fewer scales (Wootten et al., 1982; Jónsdóttir et al., 1992).
Details on the changes in blood parameters of S2 smolts in response to sea
lice have been submitted elsewhere (Bowers et al., 1999). Briefly, plasma
cortisol concentration, an indicator of primary stress, increased significantly
in the sea lice-infested fish, in both S2 and S1, with the highest levels on day 21
when most lice were at pre-adult stages. Plasma glucose concentration, an
indicator of secondary stress, also increased similarly with the highest level
on day 21. On the same day (i.e. day 21) macrophage respiratory burst and
–1
Phagocytic capacity (%) (>5 bacteria cell )
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
100
55
Control
Infested
75
*
50
25
0
0
3
7
14
Day of sampling
21
–1
Phagocytic capacity (%) (>5 bacteria cell )
Fig. 7. Phagocytic capacity of macrophage cells from control and sea lice infested
Atlantic salmon smolts (S2). *Significantly di#erent from controls.
75
Control
Infested
50
*
25
0
0
3
7
14
Day of sampling
21
Fig. 8. Phagocytic capacity of macrophage cells from control and sea lice infested
Atlantic salmon smolts (S1). *Significantly di#erent from controls.
phagocytic activities were also found to be significantly decreased for the lice
infested fish compared to those of controls. These findings are consistent with
the observations of Grimnes & Jakobsen (1996) and Dawson (1998) who
suggested that the pre-adult and adult stages of sea lice have greater impact
on the physiology and immunology of fish than the earlier stages. Bjørn &
Finstad (1997) also found similar results in trout infested with sea lice. In their
studies, a sudden increase in osmoregulatory disturbances and mortality
were observed among heavily infested sea trout after the lice reached the
preadult and adult stages, indicating a greater pathogenicity of preadult and
56
A. MUSTAFA ET AL.
adult lice than the chalimus stages. When salmon lice reach pre-adult
stage, they change from an immobile to mobile phase. Transformation of
chalimus to preadult induces significant change in louse activities and
distribution on the host and likely explains the sudden increase in the
pathogenicity.
The cortisol elevation on days 3 and 7 in the case of S2 smolts and on days
7 and 14 in the case of S1 smolts, even though significant compared to those of
controls, were at levels comparable to levels found in acutely stressed brown
trout (Pickering & Pottinger, 1989) and other teleost fish (Barton & Iwama,
1991). Plasma glucose concentrations, which similarly increased over controls
post infestation, were also within the normal range. The sedentary nature of
chalimus larvae may explain these results. During the chalimus stages, lice
stay attached to the gill lamellae and to scales of the fish, and since they
remain attached, the area of skin they feed on is localised and the impact
limited (Pike, 1989; Bjørn & Finstad, 1997).
Since fish are poikilothermic in nature, specific immune responses are often
delayed and smaller than in other vertebrates (Blazer, 1991). The first, and
often most important, responses of fish to infectious agents are therefore
nonspecific. These include various soluble and cellular factors, enzymes,
other proteins and cells for phagocytosis, particularly macrophages. During
phagocytosis, fish macrophages respond with an oxidative burst when they
encounter an appropriate stimulus. They convert molecular oxygen to a
number of highly reactive oxygen intermediates and damaging organic molecules (Bayne & Levy, 1991). Macrophage functions, as mentioned earlier, can
be a#ected by numerous environmental and physiological factors, especially
stress. In their studies with rainbow trout, Narnaware et al., (1994) showed a
depression of phagocytic activities within 3 h of an acute stress but could not
correlate the depression of phagocytosis with plasma cortisol concentrations.
They concluded that the depression due to acute stress was caused by
catecholamines, not corticosteroids which actually have e#ects only in the
longer term. This is consistent with the studies by Barton & Iwama (1991).
Persistence of stressors generally causes biological tolerance limits to be
exceeded, resulting in a maladaptation with adverse physiological and behavioral consequences. Prolonged increase in cortisol concentration can induce a
generalised immune suppression (Maule et al., 1989; Schreck, 1996; Ruis &
Bayne, 1997). Thus, the decrease in macrophage respiratory burst and phagocytic activities in the present studies could be a consequence of increased
cortisol concentrations over a long period. It is more likely that fish were
acutely stressed, perhaps due to a discomfort caused by chalimus larvae
(Bjørn & Finstad, 1997). During the third week stress became chronic as the
sealice increased size and matured into mobile stages, which eventually
suppressed the immune system, reducing the respiratory burst and phagocytic
activities of macrophage cells.
Atlantic salmon in nature usually take a minimum of 2 years to become a
smolt. In aquaculture, this period can be reduced to 1 year as a result of
environmental manipulation and optimum feeding with high energy diets.
There has been speculation that commercially raised S1 smolts would perform
less satisfactorily than naturally occurring S2 smolts when released in sea
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
57
water, due to their di#erences in physiology, behavior and genetics (Saunders,
1991). Such speculation contradicts opinions of other researchers who believe
that such di#erences are not important when fish are put in sea cages, fed well
and looked after during their marine phase. In the present studies, there
appeared to be no di#erence between the two age groups in terms of blood
parameters (i.e. plasma cortisol and glucose concentrations) or in macrophage
functions (i.e. respiratory burst and phagocytic activities). The only di#erence
in plasma cortisol concentrations on day 21, i.e. the lower cortisol concentration in S1 smolts, could be attributed, at least in part, to a lower infection
intensity of sea lice.
In conclusion, these studies indicate that sea lice induce a stress response
and immune suppression in their fish host, having greater e#ect during the
later stages of their life cycle when they are mobile and able to cause the most
damage. Other studies have found similar results and demonstrate that later
life stages are more detrimental physically and have a high physiological and
immunological impact on the fish (Pike, 1989; MacKinnon, 1997; Grimnes &
Jakobsen, 1996; Bjørn & Finstad, 1997). Further study will allow this information to be integrated into attempts to develop alternate methods to control
sea lice.
We thank Joanne Bowers, Pablo Gonzalez, Cheryl Wartman, Margaret Horne and Dr
David Sims for their help in collecting, counting and sampling the fish and sea lice. We
also thank Dr Laura Brown and Dr Simon Jones for useful discussion and advice
regarding macrophage assays. We are grateful to Dr Stewart Johnson and Dr Neil Ross
for their advice on the experimental design and for constructive comments on earlier
drafts of this manuscript. This research was funded by a grant from the NSERC/NRC
Partnership Program. The Salmon Health Consortium also provided useful advice and
financial support.
References
Barton, B. A. & Iwama, G. K. (1991). Physiological changes in fish from stress in
aquaculture with emphasis on the response and e#ects of corticosteroids.
Annual Reiew of Fish Diseases 1, 3–26.
Bayne, C. J. & Levy, S. (1991). The respiratory burst of rainbow trout, Oncorhynchus
mykiss (Walbaum), phagocytes is modulated by sympathetic neurotransmitters
and the ‘neuro’ peptide ACTH. Journal of Fish Biolology 38, 609–619.
Berland, B. (1993). Salmon lice on wild salmon (Salmo salar) in western Norway. In
Pathogens of wild and farmed fish: sea lice. (Boxshall, G. A. & Defaye, D., eds),
Ellis Horwood, West Sussex, UK. pp 179–187.
Bjørn, P. A. & Finstad, B. (1997). The physiological e#ects of salmon lice infection on
sea trout post smolts. Nordic Journal of Freshwater Research 73, 60–72.
Blazer, V. S. (1991). Piscine macrophage functions and nutritional influences: a review.
Journal of Aquatic Animal Health 3, 77–86.
Bowers, J. M., Mustafa, A., Speare, D. J., Conboy, G. A., Brimacombe, M., Sims, D. &
Burka, J. F. (1999). The e#ects of a single experimental challenge of sea lice,
Leophtheirus salmonis, on the stress response of Atlantic salmon, Salmo salar.
Journal of Fish Diseases (In press).
Brown, L. L., Iwama, G. K. & Evelyn, T. P. T. (1996). The e#ects of early exposure of
Coho salmon (Oncorhynchus kisutch) eggs to the p57 protein of Renibacterium
salmoninarum on the development of immunity to the pathogen. Fish & Shellfish
Immunology 6, 149–165.
58
A. MUSTAFA ET AL.
Dawson, L. H. J., Pike, A. W., Houlihan, D. F. & McVicar, A. H. (1997). Comparison of
the susceptibility of sea trout (Salmo trutta L.) And Atlantic salmon (Salmo salar
L.) To sea lice (Lepeophtheirus salmonis (Kröyer, 1837)) infections. ICES Journal
of Marine Sciences 54, 1129–1139.
Dawson, L. H. J. (1998). The physiological e#ects of salmon lice (Lepeophtheirus
salmonis) infections on returning post-smolt sea trout (Salmo trutta L.) In
western Ireland, 1996. ICES Journal of Marine Sciences 55, 193–200.
Egidius, E. (1985). Salmon lice, Lepeophtheirus salmonis. ICES identification leaflets for
disease and parasites of fish and shellfish. Leaflet No. 26. pp 1–4.
Enane, N. A., Frenkel, K., O’Connor, J. M., Squibb, K. S. & Zeliko#, J. T. (1993).
Biological markers of macrophage activation: application for fish phagocytes.
Immunology 80, 68–72.
Grimnes, A. & Jakobsen, P. J. (1996). The physiological e#ects of salmon lice infection
on post-smolt of Atlantic salmon. Journal of Fish Biology 48, 1179–1194.
Jónsdóttir, H., Bron, J. E., Wootten, R., & Turnbull, J. F. (1992). The histopathology
associated with the preadult stages of Lepeophtheirus salmonis on the Atlantic
salmon, Salmo salar L. Journal of Fish Diseases 13, 303–310.
Johnson, S. C. & Albright, L. J. (1991). Development, growth, and survival of
Lepeophtheirus salmnonis (Copepoda: Caligidae) under laboratory conditions.
Journal of the Marine Biology Association, U.K. 71, 425–436.
Johnson, S. C. & Albright, L. J. (1992). E#ects of cortisol implants on the susceptibility
and the histopathology of the responses of naive coho salmon Oncorhynchus
kisutch to experimental infection with Lepeophtheirus salmonis (Copepoda:
Caligidae). Diseases of Aquatic Organisms 14, 195–205.
Kabata, Z. (1972). Developmental stages of Caligus clemensi (Copepoda: Caligidae).
Journal of Fisheries Research Board of Canada 29, 1571–1593.
MacKinnon, B. M. (1997). Sea lice: a review. World Aquaculture 28, 5–10.
Mathews, E. S., Warinner, J. E. & Weeks, B. A. (1990). Assays of immune function in
fish macrophage. In: Techniques in Fish Immunology. (Stolen, J. C., Fletcher,
T. C., Anderson, D. P., Robertson, B. S., vanMuiswinkle, W. B. eds), SOS
Publications: NJ pp. 155–163.
Maule, A. G., Tripp, R. A., Kaattari, S. L. & Schreck, C. B. (1989). Stress alters immune
function and disease resistance in chinook salmon (Oncorhynchus tshawtscha).
Journal of Endocrinology 120, 135–142.
Mustafa, A. & MacKinnon, B. M. (1993). Sea lice and salmonids: preliminary observations on modulation of the e#ects of host stress using iodine. Bulletin of the
Aquaculture Association of Canada 93, 99–101.
Mustafa, A. (1997). Host factors important in determining infection intensity with the
sea louse, Caligus elongatus Nordmann, 1932, in Atlantic salmon, Salmo salar L.
and Arctic charr, Salvelinus alpinus (L.). Ph.D. Thesis, University of New
Brunswick, Fredericton, N. B., Canada, 175 pp.
Narnaware, Y. K., Baker, B. I. & Tomlinson, M. G. (1994). The e#ect of various stresses,
corticosteroids and adrenergic agents on phagocytosis in the rainbow trout,
Oncorhynchus mykiss. Fish Physiology and Biochemistry 13, 31–40.
Pickering, A. D. & Pottinger, T. G. (1989). Stress responses and disease resistance in
salmonid fish: e#ects of chronic elevation of plasma cortisol. Fish Physiology
and Biochemistry 7, 253–258.
Pike, A. W. (1989). Sea lice-major pathogens of farmed Atlantic salmon. Parasitology
Today 5, 291–297.
Ruis, M. A. W. & Bayne, C. J. (1997). E#ects of acute stress on blood clotting and yeast
killing by phagocytes of rainbow trout. Journal of Aquatic Animal Health 9,
190–195.3.
Saunders, R. L. (1991). Stresses encounter during production of Atlantic salmon smolts.
In Stress in salmonid aquaculture workshop proceedings. University of New
Brunswick, NB Department of Fisheries and Aquaculture and NB Research and
Productivity Council. pp. 12–17.
SEA LICE AND SALMON MACROPHAGE FUNCTIONS
59
Schreck, C. B. (1996). Immunomodulation: endogenous factors. In: Fish Physiology –
Vol 15. Iwama, G. K., Nakanishi, T., eds), Organisms pathogen and environment.
Academic Press, London, pp. 311–337.
Secombes, C. J. (1990). Isolation of salmonid macrophages and analysis of their killing
activity. In: Techniques in Fish Immunology (Stolen J. C., Fletcher, T. C.,
Anderson, D. P., Robertson, B. S., vanMuiswinkle, W. B. eds), SOS Publications.
NJ. pp. 137–154.
Wootten, R., Smith, J. W. & Needham, E. A. (1982). Aspects of the biology of the
parasitic copepods Lepeophtheirus salmonis and Caligus elongatus on farmed
salmonids, and their treatment. Proceedings of the Royal Society, Edinburgh. 81,
185–197.