Habitat-specific adaptation of immune responses
of stickleback (Gasterosteus aculeatus)
lake and river ecotypes
Jo ¨rn P. Scharsack1,*, Martin Kalbe1, Chris Harrod2and Gisep Rauch1,†
1Department of Evolutionary Ecology, and2Department of Evolutionary Genetics, Max-Planck-Institute for Limnology,
August-Thienemann-Strasse 2, 24306 Plo ¨n, Germany
Freshwater populations of three-spined sticklebacks (Gasterosteus aculeatus) in northern Germany are
found as distinct lake and river ecotypes. Adaptation to habitat-specific parasites might influence immune
capabilities of stickleback ecotypes. Here, naive laboratory-bred sticklebacks from lake and river
populations were exposed reciprocally to parasite environments in a lake and a river habitat. Sticklebacks
exposed to lake conditions were infected with higher numbers of parasite species when compared with the
river. River sticklebacks in the lake had higher parasite loads than lake sticklebacks in the same habitat.
Respiratory burst, granulocyte counts and lymphocyte proliferation of head kidney leucocytes were
increased in river sticklebacks exposed to lake when compared with river conditions. Although river
sticklebacks exposed to lake conditions showed elevated activation of their immune system, parasites could
not be diminished as effectively as by lake sticklebacks in their native habitat. River sticklebacks seem to
have reduced their immune-competence potential due to lower parasite diversity in rivers.
Keywords: ecological immunology; Gasterosteus aculeatus; ecotypes; parasites; immune response;
specific growth rate
Three-spined sticklebacks (Gasterosteus aculeatus) are a
powerful evolutionary model system due to the rapid and
repeated phenotypic divergence of freshwater forms from
a marine ancestor throughout the Northern Hemisphere
(Bell & Foster 1994). For example, a large-bodied benthic
form and a smaller, more slender limnetic form are present
in coastal lakes of British Columbia, Canada (McPhail
1984). Gene flow between stickleback ecotypes (or
ecomorphs) is limited even in close neighbouring
populations of the different ecotypes (Reusch et al. 2001;
Hendry & Taylor 2004). Strong assortative mating may
explain the limitedgene flow, and the reproductive isolation
betweenstickleback ecotypes is thought toreflect ecological
speciation (McKinnon et al. 2004; Olafsdottir et al. 2006;
Vines & Schluter2006).Thisecological speciationis driven
by environmental factors (e.g. climate, resources) and
interaction with other species (e.g. resource competition,
predation; Schluter 2001; Vamosi & Schluter 2004).
Interaction with habitat-specific pathogens (e.g. parasites)
and the underlying immunological capabilities of ecotype
hosts has yet to be fully considered as a potential factor in
the divergence of species.
Freshwater stickleback populations in northern
Germany are divided into lake and river ecotypes. Genetic
divergence between local lake and river ecotypes is higher
than that between populations of the same ecotype from
separate drainage systems(Reuschetal. 2001).Apparently,
the different ecotypes do not mate in the wild although
sexually mature lake and river sticklebacks will mate when
placed together in a tank in the laboratory (Rauch et al.
in the wild, as lakes in the region are connected by rivers,
permitting free movement between habitat types.
It is suggested that adaptation to habitat-specific
pathogens (e.g. parasites) is an advantage that might
contribute to the formation of a mating barrier between
the two stickleback ecotypes. Three-spined sticklebacks
are exposed to a diverse range of parasites, and single fish
can be infected by numerous different parasite species
(Chappell 1969; Wootton 1976; Zander et al. 1999; Kalbe
et al. 2002; Wegner et al. 2003a) and different genotypes of
the same species (Rauch et al. 2005). Species diversity of
abundant parasites in the study region is far greater in
lakes when compared with river habitats (Kalbe et al.
2002). Laboratory infections of lake and river sticklebacks
with a typical lake parasite, the eye fluke Diplostomum
pseudospathacaeum, showed that lake sticklebacks were
more resistant to the infection, demonstrating habitat
(parasite)- specific adaptation in the immune response of
stickleback ecotypes (Kalbe & Kurtz 2006).
Diversity of major histocompatibility complex (MHC)
genes could play a role in the variation in infection
resistance between the stickleback ecotypes. Investigations
on the influence of MHC genes on parasite load are
summarized and reviewed by Apanius et al. (1997). In
laboratory parasite infections, sticklebackswith intermedi-
ate numbers (5–6) of MHC class II beta alleles showed
lower infection rates than sticklebacks with high (9) or low
(3) numbers of alleles (Wegner et al. 2003b), indicating
that intermediate (optimal) MHC diversity conveys
resistance better than high MHC diversity. When
Proc. R. Soc. B (2007) 274, 1523–1532
Published online 10 April 2007
*Author for correspondence (firstname.lastname@example.org).
†Present address: Institute for Evolution and Biodiversity, University
of Mu ¨nster, Hu ¨fferstrasse 1, 48149 Mu ¨nster, Germany.
Received 14 February 2007
Accepted 23 March 2007
This journal is q 2007 The Royal Society
investigating the role of MHC class II beta in F2 hybrids of
lake and river sticklebacks after field exposure, Rauch et al.
(2006a) did not find that MHC genotype influenced
resistance to parasite infections. In contrast, genomic
background, independent of MHC genotype, explained a
significant percentage of the variation in parasite load
(Rauch et al. 2006a). In a laboratory experiment, it has
been shown that fishes can mount a genotype-specific
defence which works independently from the MHC-based
adaptive immune system (Rauch et al. 2006b). In the
present study, we aimed to identify functional immune
parameters that might be activated differentially in the two
ecotypes. Here, only pure family lines from lake and river
sticklebacks were investigated, as we were not only
interested in MHC-related immune defence, but also in
other defence components such as innate immunity.
The effects of host–parasite interaction can vary
between parasite species (Combes 2001; Hoole et al.
2003). Characteristics of the immune defence of the host
may depend on infection route, target organ and
pathogenicity of the invading parasite species (Buchmann
et al. 2001; Tully & Nolan 2002; Roberts et al. 2005;
Wiegertjes et al. 2005; Reite & Evensen 2006). A major
part of the innate immune defence of fish hosts against
macro parasites is the activation of granulocytes (Whyte
et al. 1989; Nie & Hoole 2000; Kurtz et al. 2004;
Scharsack et al. 2004). Production of oxygen radicals is a
key function of activated granulocytes (Verburg-van
Kemenade et al. 1996; Serada et al. 2005). In addition to
the innate line of defence (Jones 2001), fish hosts possess
adaptive immunity that produces specific antibodies
against parasite antigens (Roberts et al. 2005; Wiegertjes
et al. 2005). Clonal expansion of lymphocytes is a
fundamental part of the specific immune response of
fishes (Rijkers et al. 1980; Le Morvan-Rocher et al. 1995).
Lymphocyte proliferation is used as a measure for
activation of the specific immune system against parasites
in fish hosts (Hamers & Goerlich 1996; Nie et al. 1996;
Scharsack et al. 2000).
In the present study, laboratory-bred parasite-free lake
river parasites and parasite infection. After eight weeks of
ation, frequencies of granulocytes and respiratory burst
activity were analysed in head kidney leucocytes (HKL).
Additionally, growth performance was recorded as specific
growth rates (SGRs; Barber 2005). Variation in parasite
burden, immune defence and growth performance
between the stickleback ecotypes may contribute to our
understanding of the evolution of speciation and the
maintenance of mating barriers between lake and river
2. MATERIAL AND METHODS
(a) Experimental stickleback
Three-spined sticklebacks were caught from a river (Schwale)
and a lake (Vierer See) belonging to different drainage systems
in northern Germany in autumn 2002. To obtain sticklebacks
with a pure river and a pure lake genotype, respectively, six
crosses between wild-caught river and lake sticklebacks were
studying the role of MHC on parasite load, where the design
hybrid lines containing fish with a distinct river or lake MHC,
we also produced a second generation of the pure lines (for
results on parasite load of hybrid lines and breeding protocol,
for seven months in summer conditions at a density of 20 fish
per tank, before these fish were used in the experiment.
(b) Field exposure
TheF2fish wereexposed incages placedinthe riverand inthe
original capture locations of the parental generation. In the
river, cages were placed at intervals of 10 m in mid-channel
(approximate widthZ2 m and depthZ0.5 m). In the lake,
cages were placed approximately 10 m offshore (depth 1 m) at
intervals of 10 m along the shoreline. Cages were cylindrical
(1 m length and 40 cm diameter, placed horizontally) and
covered with 4 mm wire mesh (Rauch et al. 2006a).
In total, 120 sticklebacks were exposed, 20 fish from each
of the three river and the three lake families. Ten fish from
each family were placed in the river and 10 in the lake. To
expose fish to different within-river and within-lake con-
ditions, we randomly distributed two fish from each family
between two cages, repeated this procedure 10 times and
placed 10 cages in the river and 10 cages in the lake. This
procedure resulted in some cages containing no fish and other
cages containing two fish from a specific family. In addition,
we released 8–10 fish of the hybrid lines in each cage. For the
present study on the activation of immune cells, hybrid lines
were not used although they allowed for the control for an
MHC effect, because it was not clear whether other parts of
the immune system we were especially interested in, such as
the innate immunity, had a river or a lake origin. Prior to
release and after catching, we measured the length (total
length to the nearest millimetre) and mass (to the nearest
milligram) of every fish. Fish growth performance (both in
terms of length and weight) was compared between
experimental groups using SGRs, corrected for variation in
initial size following Barber (2005). Briefly, SGRs were
calculated using: SGRZ100!(lnsize at end)K(lnsize at start),
where sizeZlength (mm) or weight (mg). Prior to release,
each experimental fish was identified using five polymorphic
microsatellites to discriminate between them after the
experiment. The microsatellites Gac1097, Gac1125,
Gac4170, Gac5196 and Gac7033 developed by Largiader
et al. (1999) were used with the microsatellite analysis
protocols in Reusch et al. (2001).
(c) Parasite infection
Sticklebacks were collected eight weeks after release and at
the time of capture had not attained sexual maturity. Size and
weight of the fish were measured again. Individual identity
was determined with the microsatellite markers. Fish were
killed by immersion in an overdose of methane sulphonate
(MS 222; 1.5 g lK1) in tap water. All fish were examined for
parasite infection. Body surface and all inner organs including
eyes and gills were screened for parasites (for details on
parasite screening, see Kalbe et al. 2002). In total, 15 species
(two of which were present only on hybrid lines, not shown)
of macro parasites were recorded and counted on individual
stickleback, except for Trichodina sp. (table 1). For Trichodina
sp., number of parasites was estimated to be 10, 50 or 100 on
the visible parts of the fins. An estimation of Trichodina sp.
numbers was possible, as fish were not heavily infected and
numbers did not exceed 100 individuals per fish. Both
1524 J. P.Scharsack et al.Immune responses of stickleback ecotypes
Proc. R. Soc. B (2007)
Table 1. Class, species name, prevalence in per cent (P%), mean intensity (MI) of parasites per fish for lake and river sticklebacks after reciprocal exposure to the two habitats. (Note: in the
lake exposure, abundance of Diplostomum sp. per fish was significantly higher in river stickleback.?p!0.0031, significant difference between lake and river sticklebacks in the same habitat
exposure with Bonferroni correction for 16 tests;??, temporary ectoparasite, see text.)
lake stickleback (nZ25) river stickleback (nZ23)
lake stickleback (nZ22) river stickleback (nZ28)
larvae in copepod
Valipora campylancristrota trophic
larvae in copepod
cystacanth in isopod
larvae in copepod
larvae in copepod
larvae in various
Immune responses of stickleback ecotypes
J. P.Scharsack et al.
Proc. R. Soc. B (2007)
Trichodina sp. and Gyrodactylus sp. can reproduce on the fish.
infection intensity in the wild. For every stickleback, a relative
parasite load averaged over all parasite species present in the
number of parasites per fish from each species was recorded.
was divided by the maximal number of the respective parasite
species found in the habitat. Fish not infected by a parasite
species that was present in the habitat scored a zero for
this species. The parasite load of individual fish was calcu-
lated as the average of relative numbers of each parasite species
present in the respective habitat.
(d) Isolation of head kidney leucocytes
For immunological assays, leucocytes were isolated from the
head kidney of sticklebacks. All steps for leucocyte prep-
aration were performed on ice and only refrigerated media
and cooled centrifugeswere used. Cell suspensions from head
kidneys were prepared by forcing the tissues through a 40 mm
nylon screen (BD-Falcon, USA). Isolated HKL were washed
twice (48C, 10 min 550!g) with RPMI 1640 diluted with
10% (v/v) distilled water (R-90) and resuspended in a final
volume of 1 ml R-90 (Scharsack et al. 2004).
(e) Flow cytometric analysis of head kidney
Total cell numbers in HKL isolates were determined with the
standard cell dilution assay (SCDA, Pechhold et al. 1994) in a
modified form (Scharsack et al. 2004): washed cells (25 ml)
were transferred to individual flow cytometer tubes; 3!104
green fluorescent standard particles (4 mm, Polyscience,
USA) and propidium iodide (2 mg lK1, Sigma Aldrich)
were added to each tube. FSC/SSC characteristics of at
least 10 000 events were acquired in linear mode; fluor-
escence intensities at wavelengths of 530 and 585 nm were
acquired at log scale with a flow cytometer (FACSCalibur,
Becton and Dickinson, USA). Flow cytometric data were
analysed with the CELLQUEST PRO v. 4.02 software for
acquisition and analysis. Cellular debris with low FSC
characteristics was excluded from further evaluation. Standard
particles (green fluorescence positive) were discriminated from
viable HKL (propidium iodide negative, green fluorescence
negative). Total numbers of cells in individual samples were
calculated according to: N[vital cells]Zevents [vital cells]!
number [standard beads]/events [standard beads]. Total cell
suspensions to 1.25!106cells per ml, to have comparable cell
numbers for the subsequent respiratory burst assay.
Additionally, flow cytometric measurements of freshly
isolated HKL were used to determine proportions of
granulocytes (FSC/SSChigh) and lymphocytes (FSC/SSClow)
in individual HKL samples (Scharsack et al. 2004).
(f) Respiratory burst activity of head kidney
As one of the most important effector mechanisms of cell-
mediated innate immunity, the respiratory burst activity
of HKL was quantified in a lucigenin-enhanced chemi-
luminescence (CL) assay modified after Scott & Klesius
(1981), as described in Kurtz et al. (2004). In white 96 well
flat-bottomed microtitre plates, 160 ml of cell suspension
(2!105HKL per well) were added to 20 ml lucigenin solution
(2.5 g lK1PBS). Plates were incubated for 30 min at 188C to
allow uptake of lucigenin by the cells. Phagocytosis and
production of reactive oxygen species (ROS) was initiated by
the addition of 20 ml zymosan suspension (7.5 g lK1PBS) and
a microtitre plate luminometer (Berthold, Germany). Relative
luminescence (RLU) was evaluated for each sample using the
WINGLOW software. Maximum of respiratory burst activity
(RLU sK1, given in figure 3) calculated by the WINGLOW
software represents the peak of the activity curve recorded
during the 3 h of measurement.
(g) Cell cycle analysis
As a parameter for activation of the adaptive immune system,
we determined the relative number of lymphocytes in the
G2-M phase of the cell cycle after DNA labelling with
propidium iodide by means of flow cytometry. During the
cell cycle in the G0-1 phase, cells have a single set of
chromosomes and a constant content of total DNA. Cells
starting to proliferate enter the S (synthesis) phase charac-
terized by increasing amounts of DNA per cell. In the G2-M
phase, cells have completed DNA synthesis, are endowed
with a double set of chromosomes (DNA) and start to divide.
Accordingly, proliferating cells in the G2-M phase can be
distinguished from G0-1and S phase cells by their higher
DNA content. For cell cycle analysis, HKL were fixed with
ethanol (100 ml cell suspension as described above in 900 ml
ice cold ethanol 98%) and stored at 48C. Before measure-
ment, cells were centrifuged (550!g, 10 min, 48C) and
supernatant ethanol was removed. Cells were resuspended
with RNAse (500 mg lK1PBS) and incubated for 10 min at
room temperature to remove background labelling of RNA.
Propidium iodide (Sigma Aldrich) was added to a final
concentration of 7.5 mg lK1and cells were incubated again
for 10 min at room temperature. Individual samples were
measured for 3 min or up to 30 000 events with a Becton
Dickinson FACSCalibur flow cytometer. Red fluorescence
(propidium iodide) was measured in linear mode. Data were
evaluated with the CELLQUEST PRO v. 4.02 software. Cellular
debris (low scatter characteristics) and aggregated cells (high
scatter characteristics) were subtracted from further evalu-
ation. Doublet cells were subtracted from single cells as
described in Wersto et al. (2001). Lymphocytes were
identified according to their characteristic FSC/SSC profile.
Frequencies of lymphocytes in G0-1, S and G2-Mphases were
acquired by DNA content analysis of red fluorescence
intensity (propidium iodide labelling) of single cells from
the lymphocyte gate.
(h) Data analysis
The effect of habitat of exposure and origin on length and
weight increase, parasite species number, parasite load,
oxidative burst activity, proportion of granulocytes and
lymphocytes, and proportion of proliferating lymphocytes
were tested with ANOVA with the fixed factors habitat and
origin and their interaction and family effect nested within
origin. Family effect was controlled for in this design, but
family effect statistics are not shown, as drawing conclusions
from the nested factor statistics are not recommended in such
a split-plot design (Zar 1999). Cage effect was not included in
the full model as some family cage combinations were absent.
Cage effects were tested separately in a one-way ANOVA for
river and lake cages. Wherever necessary, data were log,
square-root or Box–Cox transformed in order to normalize
non-normally distributed data.
1526 J. P.Scharsack et al.Immune responses of stickleback ecotypes
Proc. R. Soc. B (2007)
To identify significant differences in infection intensities
for each parasite species in lake and river sticklebacks under
each experimental condition, we used student’s t-tests with
Bonferroni correction for multiple testing (p value set to
0.0031for 16 tests, table 1). Effects of habitat of exposure and
origin on fish length/weight increase, number of parasite
species per fish, parasite load, proportion of granulocytes,
respiratory burst activity and proportion of proliferating
lymphocytes were tested in a post hoc test using student’s
t-tests, with Bonferroni correction for multiple testing
(p value set to 0.0125 for four tests).
(a) Fish condition and growth performance
Of the 120 fish originally exposed to experimental
conditions, 98 were recovered—10 fish disappeared
(probably died or escaped) from the river cages while 12
fish were similarly unaccounted for in the lake. Fish grew
in terms of both length and weight during exposure to
experimental conditions, but there was significant vari-
ation in growth performance between experimental
groups (figure 1). Since stickleback SGR was negatively
correlated with individual size at the beginning of the
experiment (length: rZK0.56, nZ98, p!0.001; weight:
rZK0.63, nZ98, p!0.001), we followed Barber (2005)
and regressed SGR on initial body size to provide residual
SGR values (rSGR) and to correct growth rates for initial
body size. As might be expected by the close correlation
between length and weight in the sticklebacks (rZ0.95,
nZ98, p!0.001), variation in rSGRweight generally
follows that of rSGRlength(figure 1a,b). For both weight
(figure 1a) and length (figure 1b), a significant effect of
habitat (rSGRweight: F1,90Z7.01, pZ0.009; rSGRlength:
F1,90Z9.07, pZ0.003) but not of stickleback origin
(rSGRweight: F1,4Z1.0, pZ0.38; rSGRlength: F1,4Z0.97,
pZ0.38) was observed. The interaction term between
habitat and origin was significant (rSGRweight: F1,90Z
7.77, pZ0.006; rSGRlength: F1,90Z17.7, pZ0.0001).
Lake sticklebacks grew significantly better in their natural
environment when compared with river conditions
(rSGRweight: pZ0.0012; rSGRlength: p!0.0001). Interest-
ingly, we did not detect a significant difference in the
growth of river sticklebacks in the two habitats
(rSGRweight: pZ0.91; rSGRlength: pZ0.38). In the river
exposure, lake sticklebacks grew less than river stickle-
backs (rSGRlength: pZ0.0019). However, this was not
significant in terms of weight according to the Bonferroni
(rSGRweight: pZ0.03). In the lake exposure, no difference
in mean rSGRweightbetween lake and river sticklebacks
was observed (rSGRweight: pZ0.24). As a trend, mean
rSGRlength tended to be lower in river fish in lake
conditions relative to lake fish (rSGRlength: pZ0.02, not
significant with Bonferroni corrected pZ0.0125).
(b) Parasite infections
Sticklebacks exposed to the lake habitat were infected with
a higher number of parasite species than those exposed
to the river habitat (figure 2a; ANOVA: habitat: F1,90Z
habitat!origin interaction: F1,90Z0.30, pZ0.5851).
Sticklebacks in the lake habitat were infected with
(meanGs.e.) 2.96G0.20 parasite species and those in
the river habitat with 1.26G0.01 parasite species. Post hoc
comparisons showed that both lake and river sticklebacks
had a higher number of parasite species in the lake than in
the river (Bonferroni corrected pZ0.0125; figure 2a). For
every individual fish, a relative parasite load averaged over
all parasite species present in the respective habitat was
calculated (figure 2b). Between habitats, relative parasite
load was not significantly different, but lake and river
origin of sticklebacks had a significant influence on
parasite load (ANOVA: habitat: F1,90Z2.37, pZ0.1269;
origin: F1,4Z7.82,pZ0.0484;habitat!origin interaction:
F1,90Z2.63, pZ0.1082). In the lake exposure, stickle-
backs with river origin showed a significantly higher
parasite load when compared with lake sticklebacks in
this habitat (p!0.0125, post hoc t-test). No significant
difference in parasite load was detected between lake and
river sticklebacks exposed to the river habitat (figure 2b).
(% body length d–1)
n = 23
n = 22
n = 28
n = 25
(% body weight d–1)
Figure 1. Differences in relative growth performance (rSGR) in sticklebacks in terms of (a) weight and (b) length (meanCs.e.).
Lake sticklebacks had significantly reduced growth when exposed to river conditions when compared with lake conditions, in
terms of both length and weight (?p!0.0125, t-test, p value Bonferroni corrected for multiple tests).
Immune responses of stickleback ecotypes
J. P.Scharsack et al.
Proc. R. Soc. B (2007)
Mean (Gs.e.) number of parasites per fish in the lake
exposure was higher (pZ0.0025) in river sticklebacks
(59.17G6.24) than in lake sticklebacks (28.28G6.04)
(Bonferroni corrected significance threshold pZ0.0125).
Mean number of parasites per fish was not different in
lake sticklebacks (5.82G2.02) and river sticklebacks
(6.18G1.78) exposed to river conditions.
Two parasites (Gyrodactylus sp., Raphidascaris acus)
were found exclusively in sticklebacks exposed to the river
habitat (table 1), while three river parasite species were
also present in sticklebacks exposed to the lake. Parasite
infections of the sticklebacks specified for the single
parasite species are summarized in table 1. Highest
prevalence (100%) was observed for the eye fluke
Diplostomum sp. in river and lake sticklebacks exposed to
lake conditions. In sticklebacks exposed in the river, we
did not detect the presence of Diplostomum sp. Infection
intensities of Diplostomum sp. in the lake exposure were
significantly higher in sticklebacks with the river genotype
when compared with the lake genotype (p!0.0031;
table 1). Fish lice (Argulus foliaceus) are temporary
ectoparasites and can switch host rapidly, therefore
difference between lake and river sticklebacks have to be
interpreted with caution. All other parasite species did
not show significant differences between river and lake
sticklebacks within one exposure habitat. According to
Bonferroni correction for multiple testing, the p value for a
significant difference with the present data (table 1) was
set to p!0.0031 for the 16 tests performed.
(c) Immune response
activity of HKL was analysed. From all tested fish, HKL
responded to stimulation with zymosan with elevated
respiratory burst activity in a chemoluminescence assay.
Sticklebacks in the lake exposure, showed a higher
zymosan-inducedrespiratory burst activity when compared
with sticklebacks in the river exposure (ANOVA: habitat:
F1,90Z24.35, p!0.0001; origin: F1,4Z0.45, pZ0.5384;
habitat!origin interaction: F1,90Z0.01, pZ0.9366;
figure 3). River sticklebacks had a higher respiratory
burst activity in the lake when compared with the river
(p!0.0125, post hoc t-test, figure 3). Lake sticklebacks, as a
trend (pZ0.018) also showed elevated respiratory burst in
the lake when compared with the river (not considered
pZ0.0125). Respiratory burst activity was correlated to
pZ0.007). Correspondingly, sticklebacks in the lake
exposure had a higher proportion of granulocytes in HKL
isolates when compared with those in the river exposure
(ANOVA: habitat: F1,90Z6.60, p!0.0118; origin: F1,4Z
0.02, pZ0.8985; habitat!origin interaction: F1,90Z2.87,
pZ0.0935). River sticklebacks had a higher proportion of
granulocytes in the lake when compared with the river, but
not lake sticklebacks (p!0.0125, post hoc t-test, figure 4).
parasite species per fish
n = 23
n = 22
n = 28
n = 25
Figure 2. Parasite infections. (a) Number of parasite species per fish (meanCs.e.). Sticklebacks exposed in the lake had a higher
average number of parasite species than sticklebacks in the river. (b) Relative parasite load per fish. In the lake exposure, river
sticklebacks had higher parasite loads (?p!0.0125, t-test, p value Bonferroni corrected for multiple tests).
oxidative burst (RLUs–1)
Figure 3. Peak respiratory burst activity of head kidney
leucocytes in cultures with zymosan stimulation (meanC
s.e.). Sticklebacks exposed in the lake had a higher oxidative
burst activity than those in the river (?p!0.0125, t-test, p
value Bonferroni corrected for multiple tests;
significant trend pZ0.018).
1528J. P.Scharsack et al. Immune responses of stickleback ecotypes
Proc. R. Soc. B (2007)
To analyse the status of the adaptive, specific immune
response of exposed stickleback, frequencies of lympho-
cytesand their proliferation activity was tested bymeans of
flow cytometry. In contrast to oxidative burst activity and
percentage of granulocytes, the percentage of lymphocytes
in HKL isolates was lower in sticklebacks in the lake
habitat than in the river habitat (ANOVA: habitat:
F1,90Z4.82, p!0.0307; origin: F1,4Z0.04, pZ0.8482;
habitat!origin interaction: F1,90Z2.64, pZ0.1079).
Proportion of lymphocytes in HKL samples was nega-
tively correlated to proportion of granulocytes (rZ-0.589,
p!0.0001, nZ98). Correspondingly, river sticklebacks
displayed a trend (pZ0.0130, post hoc t-test, data not
shown) of a lower proportion of lymphocytes in the lake
when compared with the river, but not lake sticklebacks.
The proportion of proliferating lymphocytes (lympho-
cytes in G2-M phase of the cell cycle) was higher in
sticklebacks in the lake habitat than in the river habitat
(ANOVA: habitat: F1,90Z8.76, p!0.0039; origin: F1,4Z
6.79, pZ0.0592; habitat!origin interaction: F1,90Z0.56,
pZ0.4605). River sticklebacks exposed to lake conditions
exhibited a higher proliferation activity than in the river
(p!0.0125, post hoc t-test, figure 5). In the lake exposure,
proliferation (pZ0.0413, post hoc t-test, figure 5) when
compared with lake stickleback. In general, our results
supports the greater number of parasites species, showed
an increased activity in immune parameters. However,
habitat effects on immune parameters were most promi-
nent in river sticklebacks exposed to the lake.
(d) Cage effect
The effect of cage was small and did not influence length
increase, weight increase, number of parasite species,
parasite load, respiratory burst, percentage of granulo-
cytes, percentage of lymphocytes and proportion of
proliferating lymphocytes in the river habitat (pO0.0125,
p value Bonferroni corrected for multiple tests, statistical
with exception of proportion of proliferating lymphocytes
(one-way ANOVA: cage: F9,38Z3.03, pZ0.0080).
The results of the present study suggest divergence in
adaptation of immune systems to local conditions between
populations of different stickleback ecotypes. Patterns of
growth, parasite burdens and immune system activity in
sticklebacks translocated between lake and river habitats
with different levels of parasite diversity are consistent with
selection having favoured appropriate shifts in trade-offs
between growth and immunity in different habitats. At the
end of the exposure experiment, river sticklebacks that
naturally experience low parasite burdens had elevated
parasite burdens, relative to local lake stickleback when
exposed to a high parasite diversity lake habitat. This
occurred even though the river sticklebacks appeared to
mount a more vigorous immune defence.
All the sticklebacks recovered at the end of the
experiment had gained length and weight, but lake
sticklebacks exposed to river conditions grew significantly
less when compared with lake conditions while river
(figure 1a,b). This might indicate an increased special-
ization by lake sticklebacks towards prey resources more
typically available in lake rather than in river habitats (e.g.
zooplankton). On the contrary, river sticklebacks could be
more generally adapted to feed on different food
resources, e.g. prey from both the water column (drift)
and the benthos. In the studies of the evolutionary ecology
of sticklebacks, trophic specialization is well described
(Schluter 1993, 1995; Rundle et al. 2000; Reimchen &
Nosil 2001a) and variation in feeding efficiency on
particular prey resources is one factor identified in the
adaptive radiation of three-spined sticklebacks (Rundle
et al. 2000).
In the present study, food resources may have been
restricted in the enclosure under river conditions and river
sticklebacks may have outcompeted the sticklebacks of
lake origin, resulting in reduced growth of the latter. In the
lake cages, food constraints might have been reduced or
even absent. Nevertheless, adaptation to specific food
resources might contribute to the apparent reproductive
barrier between lake and river sticklebacks. Although the
growth of lake sticklebacks was limited in the river
exposure, parasite load and immune parameters were
not different from river sticklebacks that had been
similarly exposed. Limitation in nutritional resources
obviously did not result in stress and immunosuppression
well in bothhabitats
head kidney granulocytes (%)
Figure 4. Proportion of granulocytes in head kidney
leucocytes (meanCs.e.). River sticklebacks exposed in the
lake had a higher proportion of granulocytes in their head
kidneys than river sticklebacks in the river(?p!0.0125, t-test,
p value Bonferroni corrected for multiple tests).
proliferating lymphocytes (% G2-M)
Figure 5. Proliferation of head kidney lymphocytes (meanC
s.e.). River sticklebacks exposed in the lake had a higher
lymphocyte proliferation rate than river sticklebacks in the
river (?p!0.0125, t-test, p value Bonferroni corrected for
multiple tests;?), not significant trend pZ0.041).
Immune responses of stickleback ecotypes
J. P.Scharsack et al.
Proc. R. Soc. B (2007)
of lake sticklebacks exposed to an atypical habitat. Natural
(e.g. temperature, photoperiod, salinity) and artificial (e.g.
pollution) environmental factors can cause stress in fishes
that influences their immune system (Bly et al. 1997).
Many studies on the influence of environmental stress on
immune responses of bony fishes have demonstrated
immunosuppression (Clem et al. 1990; Rice et al. 1996;
Bly et al. 1997; Jokinen et al. 2000; Engelsma et al. 2003;
Prophete et al. 2006). In the present study, the higher
parasite burden of river sticklebacks in the lake exposure
cannot be explained by immunosuppression, as immune
parameters recorded here were elevated in river stickle-
backs in the lake exposure when compared with river
sticklebacks in their home habitat.
Parasite diversity described in a field study (Kalbe et al.
2002) in the two habitat types is reflected in the present
exposure experiment, indicating that individual immuno-
logical capabilities and not enclosure in cages are causative
for different parasite loads of individual sticklebacks. In
the lake exposure, river sticklebacks showed significantly
higher infections with Diplostomum sp. when compared
with lake sticklebacks (table 1). In laboratory infections of
lake and river sticklebacks with D. pseudospathacaeum,
river sticklebacks were shown to have a higher suscep-
tibility to the parasite (Kalbe & Kurtz 2006).
Summarizing the results of the parasite infections
during the exposure experiment, we have: (i) parasite
species richness was higher in the lake (figure 2a), (ii) river
sticklebacks in the lake exposure had higher parasite loads
when compared with lake sticklebacks in their home
habitat (figure 2b; table 1), and (iii) the parasite load of
lake and river sticklebacks exposed to river conditions was
not different (figure 2b; table 1).
In wild-caught stickleback, it is demonstrated that
specialization in pelagic versus benthic feeding results in
different infection rates of trophic transmitted parasites,
depending on the abundance of intermediate hosts (e.g.
Reimchen & Nosil 2001b,c). However, in the present
study, difference in parasite load between lake and river
sticklebacks in the lake exposure remains significant when
only non-trophically transmitted parasites, are tested (p!
0.0001), whereas parasite load of only trophically
transmitted parasites was not different (pZ0.84). This
shows that differences in overall parasite load between lake
and river sticklebacks in the lake exposure (figure 2) are
not due to trophic specialization of lake and river
The immune system was more active in sticklebacks
that underwent exposure to lake rather than river
conditions. In river sticklebacks, the respiratory burst
activity of HKL was significantly higher in the lake (for
lake sticklebacks as a trend pZ0.018; figure 3). In naive
and D. pseudospathacaeum-infected laboratory offspring of
lake and river sticklebacks, higher capacities for respir-
atory burst activity were detected in lake sticklebacks—
this was considered as an adaptation to higher parasite
abundances in lakes by Kalbe & Kurtz (2006). In the
present study, river sticklebacks were exposed to multiple
lake parasite species and able to mount a strong
respiratory burst response comparable to that of lake
Granulocytes are an important effector cell type of
respiratory burst (Verburg-van Kemenade et al. 1996;
Serada et al. 2005). Accordingly, river sticklebacks
exposed to lake conditions showed elevated proportions
of granulocytes in head kidneys when compared with river
sticklebacks in their home habitat (figure 4). The
proliferation of head kidney lymphocytes of river stickle-
backs was also significantly higher in the lake, indicating
increased activity of their specific immune system when
compared with the situation with exposure to river
conditions (figure 5). In summary, the immune par-
ameters we examined here showed clearest differences in
river sticklebacks exposed to the two different habitats.
Immune parameters were elevated in river sticklebacks in
the lake when compared with the river. Lake sticklebacks
showed a trend of increased activity respiratory burst
activity in the lake.
More parasite species are present in the lake. River
sticklebacks in the lake had higher parasite loads than lake
sticklebacks and exhibited stronger activation of their
immune system than in their native habitat. Thus, although
activity of their immune system was high, defence against
parasites was not as successful as that displayed by lake
a manner comparable to river stickleback.
Wegner et al. (2003a) investigated the diversity of
MHC genes in distinct lake and river stickleback
populations in northern Germany. They found almost
twice as many alleles of the MHC class II beta genes in
lake sticklebacks when compared with river sticklebacks,
suggesting that it is advantageous to maintain a higher
diversity of MHC class II in lake habitats with an
associated higher diversity of parasite species. In the
presentstudy, river sticklebacks exposed to lake conditions
invested heavily into immune defence, but still were less
successful defending themselves against the abundant
parasite fauna than lake sticklebacks. In laboratory
parasite infections, sticklebacks with sub-optimal diversity
of the MHC class II beta showed elevated respiratory
burst activity (Kurtz et al. 2004) and elevated expression
of MHC class II beta (Kurtz et al. 2006), suggesting a
compensatory upregulation of innate and adaptive
immune mechanisms to compensate deficiencies in
In the lake exposure, activity of both innate (respiratory
burst activity, figure 3) and adaptive immunity (lympho-
cyte proliferation, figure 5) responses was elevated in
river sticklebacks when compared with their home
habitat. Both, innate and adaptive immune mechanisms
might have been upregulated by river sticklebacks to
compensate for limitations in specialized (MHC based)
In the present study, two areas of specialization can be
defined, which could contribute to the development and
maintenance of a pre-zygotic reproductive barrier between
lake and river sticklebacks. First is growth potential, which
potentially limits the development of lake sticklebacks
under river conditions. This may reflect a cost of trophic
specialization in the lake ecotype. There is a strong
association between individual size and mortality risk in
fishes (Sogard 1997), and lake fish may face increased
predation risk if they enter river environments. Second is
adaptation to habitat-dependent parasite fauna which
seemed limited in river sticklebacks, possibly due to the
lower parasite diversity in their home habitat. These two
areas of specialization may reflect a trade-off between
1530 J. P.Scharsack et al.Immune responses of stickleback ecotypes
Proc. R. Soc. B (2007)
growth and immunity. River sticklebacks may be better
adapted to maintain growth in low-productivity river
habitats, but seem to have sacrificed their ability to resist
parasites when compared with lake sticklebacks. Our
results show that natural enemies may play an important
role in the development of divergent selection and
reproductive isolation in rapidly diverging taxa.
We would like to thank Prof. Manfred Milinski (MPIL-Ploen)
for the fruitful discussions on the project. We are grateful to
Prof. Per Jakobsen (University of Bergen) for help with the
interpretation of the results. Furthermore, we thank Gerhard
Augustin, Ulrich Breitenbach, Ilka Dankert, Harald Deiwick,
Roswithe Derner, Helga Luttmann and Gregor Thomas for
their great help in parasite screening and laboratory assistance.
We would like to thank two anonymous reviewers whose
comments greatly improved the manuscript.
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