Chronic exposure to the parasite Enteromyxum
leei (Myxozoa: Myxosporea) modulates the
immune response and the expression of
growth, redox and immune relevant genes
in gilthead sea bream, Sparus aurata L.
Ariadna Sitja `-Bobadillaa,*, Josep Calduch-Ginerb, Alfonso Saera-Vilab,
Oswaldo Palenzuelaa, Pilar A´lvarez-Pelliteroa, Jaume Pe ´rez-Sa ´nchezb
aFish Pathology Group, Department of Marine Species Biology, Culture and Pathology, Instituto de Acuicultura de Torre de
la Sal (Consejo Superior de Investigaciones Cientı ´ ficas), Torre de la Sal, s/n. 12595 Ribera de Cabanes, Castello ´n, Spain
bFish Nutrition and Growth Endocrinology Group, Department of Marine Species Biology, Culture and Pathology, Instituto
de Acuicultura de Torre de la Sal (Consejo Superior de Investigaciones Cientı ´ ficas), Castello ´n, Spain
Received 31 October 2007; revised 25 January 2008; accepted 29 January 2008
Available online 8 February 2008
GH receptors, IGF-I;
bream producing a slow-progressing disease, which may end in the death of fish. The present
work aimed to better know the host immune response and the underlying molecular mecha-
nisms, which may help to understand why some individuals seem to be refractory to the dis-
ease. Three main aspects involved in fish health and welfare (immune, growth and redox
status) were studied in fish exposed to E. leei-contaminated effluent, in comparison with
control animals (not exposed to the disease). After chronic exposure (113 days), prevalence
of infection was 67.8%. Among exposed fish, parasitized and non-parasitized fish exhibited
clear differences in some of the measured innate immune factors (respiratory burst, serum
peroxidases, lysozyme and complement), and in the expression of immune, antioxidant and
GH-related genes. The respiratory burst of parasitized fish was significantly higher, and serum
peroxidases and lysozyme were significantly decreased both in parasitized and non-parasitized
fish. The gene expression of GHR-I, GHR-II, IGF-I and IGF-II was measured in head kidney (HK)
samples, and that of interleukin (IL)-1b, tumour necrosis factor (TNF)-a, a-2M, GR, GPx-1 and
GRP-75 was measured in intestine and HK samples, by rtqPCR. Parasitized fish exhibited
a down-regulation of IL-1b, TNF-a and GPx-1 in the intestine, and GHR-I and IGF-I were also
down regulated in HK. a-2M and GRP-75 were over-expressed in the intestine of parasitized
The myxosporean parasite Enteromyxum leei invades the intestine of gilthead sea
* Corresponding author. Tel.: þ34 964 319500; fax: þ34 964 319509.
E-mail address: firstname.lastname@example.org (A. Sitja `-Bobadilla).
1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fsi
Fish & Shellfish Immunology (2008) 24, 610e619
animals. Non-parasitized fish had increased transcripts of GHR-I and IGF-I with respect to
control animals, which could furnish their immunocytes with an advantage to combat the
parasite. The expression of GHR-II and IGF-II was not altered by the parasite challenge.
ª 2008 Elsevier Ltd. All rights reserved.
Gilthead sea bream (Sparus aurata L.) is the main aquacul-
tured species in the Mediterranean area, with more than
90,000 Mt produced in 2005 . However, the massive es-
tablishment of sea cages has increased the incidence of
transmissible parasites [2,3] and some of them, such as
the myxosporean Enteromyxum leei , have emerged as
serious risks to production . The Myxozoan phylum in-
cludes more than 2180 species and most of them are fish
parasites . Research in this group of multicellular organ-
isms is held back by the lack of in vitro cultures and the
difficulty to set up experimental transmission models. How-
ever, E. leei is an excellent model to study hosteparasite
interactions because the parasite is fish-to-fish transmitted
either orally, by cohabitation with infected animals, or by
contact with waters coming from infected tanks .
In gilthead sea bream, this enteromyxosis progresses
slowly under laboratory conditions  in comparison with
other more susceptible hosts, such as sharpsnout sea bream
(Diplodus puntazzo) . Infected fish may become emaci-
ated and finally die with a cachectic syndrome, and no
treatments or prophylactic control measures are available.
Therefore, characterization of the fish immune system and
its regulation is crucial for the development of vaccines and
other prevention strategies based on selection of disease-
resistant strains. Some aspects of the humoral and cellular
immune responses against this myxosporean have been
studied in gilthead sea bream [10,11] and sharpsnout sea
bream [9,12], but no information is available on the molec-
ular mechanisms involved in such immune defence, the
hosteparasite interaction, or the parasite invasion mecha-
nisms. Field and experimental data suggest that some gilt-
head sea bream stocks are partially resistant to the disease
[8,13]. Thus, it is of interest to know the differences in the
immune response between those fish which seem to be
resistant to the parasite and those which get infected.
Both innate and adaptive immune effectors against para-
sites exist in teleosts [14e16]. A functional parallelism
between fish and mammalian systems, including the gut as-
sociated lymphoid tissue (GALT), is increasingly confirmed
by recent findings [17e18]. Innate and adaptive immune
responses have also been studied for a few myxosporoses
Immune-neuroendocrine interactions in fish, as in mam-
mals, have become evident in recent decades [24,25].
Growth hormone (GH) is among such endocrine factors
modulating the fish immune system. In gilthead sea bream,
GH  and insulin-like growth factor I (IGF-I)  recep-
tors have been detected in different immunocytes, GH
expression has been reported in the head kidney (the equiv-
alent to mammalian bone marrow) , and phagocyte ac-
tivating action of GH and IGF-I has also been documented
. However, the immune-related systems of fish are
not clearly understood at the molecular level, and their in-
volvement in fish defence against parasites is just starting
to be studied.
Another important aspect of fish health depends on the
maintenance of cellular homeostasis and integrity by the
redox system and heat-shock proteins (HSPs). Mammalian
HSPs are known to perform protein-stabilizing functions
and to play a role in different immunological aspects .
Diseases, including parasitosis, can induce the expression
of HSPs both in mammals and fish [31,32]. GRP-75 (also
known as mortalin) is a member of the HSP70-family of
chaperons involved in regulating glucose responses, antigen
processing and cell mortality in mammalian models ,
and its implication in haematopoiesis has been demon-
strated in fish . The redox system in fish has almost
the same enzymatic routes as in mammals and the regula-
tion of glutathione peroxidases (GPx) and reductases (GR)
has recently been reviewed . Interestingly, the signal-
ling events involved in the phosphorylation cascades
induced by interleukin (IL)-1b and tumour necrosis factor
(TNF)-a are also subject to redox regulation .
Therefore, the aim of this work was to explore different
features related to fish health and welfare (immune,
growth and redox status), and their involvement in the
defence against an important parasitic disease. Previous
studies have examined the in vivo expression profiles of
genes encoding fish cytokines under diverse disease condi-
tions, but none has tried to relate those profiles with other
immune factors and immune, redox and GH-related genes
in fish exposed to a parasite challenge. Thus, the probable
differences between fish which become infected and those
which do not after a long-term parasite challenge were
studied in terms of gene expression profile and innate im-
munity. For such purposes, naı ¨ve gilthead sea bream were
exposed to E. leei-contaminated effluent until the infection
was clearly established in some animals, and others
remained still uninfected. A single end-point sampling
was chosen in order to obtain data from a high number of
infected fish in a situation of chronic infection, in an effort
Materials and methods
Experimental design and sampling procedure
Naı ¨ve gilthead sea bream were obtained from a commercial
fish farm with no previous records of enteromyxosis. Upon
arrival, they were checked for the absence of the parasite
(see below) and acclimated to the experimental conditions
two weeks before the beginning of the experiment. Fish
(n Z 132; average weight 134 g) were divided into two
groups, control (CTRL) and recipient (R). Each group was
E. leei modulates gilthead sea bream gene expression611
split in two replicate 200-L fibre-glass tanks. A first sam-
pling (day 0) of 6 fish from each group was conducted.
Then, R fish were exposed to E. leei-contaminated effluent
as previously described . Briefly, R tanks were set to re-
ceive exclusively the effluent water from another tank con-
taining 24 fish coming from an infected stock (donors Z D;
average weight 127.3 g; prevalence of infection 54%),
resulting in a D/R ratio Z 0.8. CTRL fish were kept under
the same conditions of water flow and oxygen concentra-
tion, but without receiving E. leei-contaminated water.
Over the course of the study, day length followed
natural changes and water was heated in order to keep
temperature always above 18?C (range: 18e23?C, with
a maximum day-to-day variation of 1?C). Sea water
(37.5& salinity) was pumped from ashore (open system),
5 mm-filtered and UV irradiated. Water flow was 10 L min?1,
and oxygen content of outlet water remained higher than
85% saturation. All fish were fed daily a commercial dry pel-
let diet at about 1% of body weight. Disease signs and daily
mortalities were recorded throughout the experiment.
After the sampling before exposure to the parasite (day
0), the progression of the infection was monitored by
sampling both CTRL and R fish at 27, 50, 90 and 113 days
post exposure (p.e.). At each sampling, an equal number
of fish from each replicate was randomly sized and weighed
(see Fig. 1 for the total number of fish sampled at each sam-
pling point). At day 90 p.e., fish from both CTRL and R
groups were non-lethally sampled for PCR (see below). At
the remaining samplings (27, 50 and 113 p.e.), fish were
killed by over-exposure to anaesthetic (3-aminobenzoic
acid ethyl ester, 100 mg L?1). One piece of the posterior
intestine was fixed in 10% buffered formalin for diagnosis
of the disease in histological sections, and another piece
was processed for PCR detection of the parasite (see
below). In addition, only at the last sampling (113 days
p.e.), blood and tissue samples were taken for immunolog-
ical and gene-expression analyses. One blood aliquot was
immediately used to measure the respiratory burst activity.
The remaining blood was allowed to clot overnight at 4?C,
centrifuged at 3000 ? g for 20 min at 4?C, and serum ali-
quots were stored at ?80?C until used in immune assays.
Head kidney (HK) and posterior intestine were rapidly ex-
cised, frozen in liquid nitrogen, and stored at ?80?C until
RNA extraction and analysis. Immunological data were ob-
tained from all fish sampled at this time and gene expres-
sion analyses were calculated for 8 CTRL and 16 R fish
Parasite diagnosis was performed by histology and/or PCR.
For histological examination, fixed intestinal portions were
embedded in Technovit resin (Kulzer, Heraeus, Germany),
1 mm-sectioned and stained with toluidine blue. For PCR,
two types of samples were taken. Non-lethal (NL) sampling
was performed by probing the rectum with a cotton swab,
and lethal (L) samples consisted of small tissue portions
taken from the posterior intestine. Sampling and PCR diag-
noses were carried out as described in , with primers
specific for E. leei rDNA. This procedure has been validated
against a gold standard (histological observation of the
whole digestive tract), resulting in a high sensitivity
(0.96) and specificity (1) (O. Palenzuela, unpublished
data). NL-PCR was applied to evaluate the parasitic status
of D fish, R fish upon arrival to the experimental facilities,
and also of R fish at 90 days p.e. to adjust the timing of the
last sampling and to ensure enough infected fish for com-
parative analyses. The prevalence of infection at each sam-
pling point was calculated considering any positive fish
detected by PCR and/or histology. The intensity of infec-
tion was evaluated in the histological sections and scored
as high, medium or low according to the number of parasitic
stages per observation field at 120?: low Z scattered
stages in some fields, medium Z more than 10 stages in
high67.8178.3 (28)180.1 (14)113
n.a.53.6172.9 (28)170.3 (28)
medium18.8155.8 (16)148.3 (16)50
low12.5141.2 (16)139.3 (16)27
-0134.5 (6)134.0 (6)0
infection in R
bream exposed to Enteromyxum leei. Panels A and B show histological sections of intestines of parasitized R fish at 27 and 113 days
post-exposure (p.e.), respectively. Arrows point to parasite stages.aThe number in parentheses indicates the number of sampled
fish.bMI was calculated only from data obtained from histological diagnose, thus it cannot be calculated (n.a.) at 90 days p.e.,
when fish were diagnosed only by NL-PCR.
Fish weight, mean prevalence (P) and mean intensity (MI) of infection of control (CTRL) and recipient (R) gilthead sea
612A. Sitja `-Bobadilla et al.
all fields; high Z more than 30 stages in all fields. The mean
intensity of infection was calculated only for fish found to
be parasitized by histology.
Induction of the respiratory burst (RB) activity in blood
leucocytes was measured directly from heparinized blood,
following the method described in , with some modifi-
cations. Briefly, 100 ml of diluted blood (1:25) in HBSS
(Hanks’ Balanced Salt Solution, pH 7.4) were dispensed in
white flat-bottomed 96-well plates and incubated with
100 ml of a freshly prepared luminol suspension (2 mM lumi-
nol in 0.2 M borate buffer pH 9.0, with 2 mg ml?1PMA) for
1 h at 24e25?C. Luminol-amplified chemiluminescence
was measured every 3 min with a plate luminescence
reader for generation of kinetic curves. Each sample was
run in duplicate and read against a blank in which no blood
was added. The integral luminescence in relative light units
(RLU) was calculated.
Total serum peroxidases (PO), which include myeloper-
oxidase, were chosen as a measure of the oxidizing capacity
of the plasma. They were measured following the pro-
cedure described in . Briefly, 15 ml of serum were mixed
in flat-bottomed well plates with 135 ml of HBSS-plus (HBSS,
without Ca2þand Mg2þ, 0.1% NaCl and antimycotic/antibi-
otic mixture) and 50 ml of 3,30,5,50-tetramethylbenzidine
hydrochloride (TMB). After 2 min of incubation, the reac-
tion was stopped with 25 ml of 1 N H2SO4, and the optical
density was read at 450 nm. Wells in which no serum was
added were run as blanks.
Serum lysozyme (LY) was measured by a turbimetric
assay adapted to 96-well microplates, as previously de-
scribed . The lytic capacity of the serum by the alterna-
tive complement pathway (ACP) was determined using
sheep red blood cells (SRBC) as targets, as in , with
the only exception of using 2.85 ? 108SRBC ml?1. The dilu-
tion corresponding to 50% haemolysis/ml was expressed as
RNA extraction and RT procedure
RNA was extracted from samples of posterior intestine and
HK using the ABI PRISM? 6100 Nucleic Acid PrepStation
(Applied Biosystems, Foster City, CA, USA). Briefly, HK and
25 mg ml?1with a guanidine-detergent lysis reagent. The
reaction mixture was treated with proteinase K, and RNA
purification was achieved by passing the tissue lysate
(0.5 ml) through a purification tray containing an applica-
tion-specific membrane. Wash solutions containing DNase
were applied, and total RNA was eluted into a 96-well
PCR plate. The RNA yield was 40e50 mg with absorbance
measures (A260/280) of 1.9e2.1. Reverse transcription
(RT) with random decamers was performed with the
High-Capacity cDNA Archive Kit (Applied Biosystems). For
this purpose, 500 ng total RNA were reverse transcribed
in a final volume of 100 ml. RT reactions were incubated
for 10 min at 25?C and 2 h at 37?C. Control reactions
were run without reverse transcriptase and were used as
negative controls in PCR assays.
The expression of genes related to growth (GH receptor I
(GHR-I), GH receptor II (GHR-II), IGF-I and IGF-II), was
measured in HK samples, and those related to immune
response (IL-1b, TNF-a, a-2-macroglobulin (a-2M)), and
antioxidant defences (glutathione reductase (GR), glutathi-
one peroxidase-1 (GPx-1) and mortalin (GRP-75)) were
measured in both intestine and HK samples. Transcript
measurements were made by real-time quantitative PCR
Rad, Hercules, CA, USA) as previously described . RTre-
actions were conveniently diluted (1:7.5; 1:75; 1:375), and
7.5 ml were used for PCR reactions in 25-ml volume. Each
PCR-well contained a SYBR Green Master Mix (Bio-Rad) with
specific primers at a final concentration of 0.3e0.9 mM (see
Table 1). b-Actin was used as housekeeping gene and the
efficiency of PCR reactions for target and reference genes
amount of product in a particular sample was determined by
interpolation of the cycle threshold (Ct) value. The specific-
ity of reaction was verified by analysis of melting curves and
by electrophoresis and sequencing of PCR amplified prod-
ucts. Reactions were performed in triplicate and fluores-
cence data acquired during the extension phase were
normalized to b-actin by the delta-delta method , using
data in CTRL fish as reference values. No changes in b-actin
Differences in immune and gene expression data between
CTRL, non-infected R and infected R fish were analysed by
One-way analysis of variance (ANOVA) followed by Stu-
denteNewmaneKeuls test or by Dunn’s method. The
significance level was set at P < 0.05. When the tests of
normality or equal variance failed, a KruskaleWallis one-
way ANOVA on Ranks followed by Dunn’s method was
applied instead. All the statistical analyses were performed
using Sigma Stat software (SPSS Inc., Chicago, IL, USA).
Progression of the infection
Fig. 1 shows the progressive increase in the prevalence and
the mean intensity of infection by Enteromyxum leei in R
fish. At the last sampling (113 days p.e.), R fish reached
a 67.8% prevalence with a high mean intensity of infection.
CTRL fish were non-parasitized. Data on immune factors
and gene expression were analysed according to the para-
sitic status at this time point, and fish were classified in
threecategories: CTRL(n Z 14),
(n Z 9) and parasitized R (n Z 19) fish.
At the first samplings, most parasite stages were primary
cells, sometimes harbouring secondary cells, located in the
basal part of the gut epithelium (Fig. 1A), and no cellular
reaction was detected. By contrast, at the last sampling,
most fish with high intensity of infection harboured sporo-
blasts and/or mature spores, located within the epithelium
E. leei modulates gilthead sea bream gene expression 613
(Fig. 1B). At 113 days p.e., the observation of the histolog-
ical sections of most infected fish revealed a clear damage
to the intestinal architecture. Parasitic stages occupied
extensive areas of the epithelium, with partly dislodged en-
terocytes displaying hypertrophied nuclei and eosinophilic
granular cell infiltration in the mucosa and submucosa of
the intestine. Rodlet cells were also abundant.
There were no significant differences in body weight
between CTRL and R fish during the experimental period
(Fig. 1), or at the last sampling between the three fish cate-
ciated syndrome, typical of the terminal stage of this
enteromyxosis, which usually appears after more than
factor [(weight length?3) ? 100] of non-parasitized R fish was
significantly higher (2.525 ? 0.048) than that of CTRL
(2.438 ? 0.040) fish. No CTRL or R fish died during the trial.
For the three fish categories, mean values were calculated
for each of the four immunological factors measured.
Parasitized fish exhibited the highest RB values, being
significantly higher than those of non-parasitized R ones
(Fig. 2A). By contrast, serum PO (Fig. 2B) and LY (Fig. 2C)
were significantly lower in all fish exposed to the infection,
regardless of their parasitic status. ACP was somewhat
higher in R parasitized fish but the high individual variability
made impossible any statistical significance (Fig. 2D).
The relative expression of the studied genes in parasitized
and non-parasitized R fish (both exposed to the infection)
was analysed with respect to CTRL animals (not exposed to
the infection), and differences among both types of R fish
were also analysed. The data on the expression of immune
and anti-oxidant related genes in the HK of parasitized R
and non-parasitized R fish are shown in Fig. 3A and B, re-
spectively. Relative to CTRL fish, no significant changes
were found in the HK of non-parasitized R fish. The expres-
sion of GPx-1 in parasitized R fish was significantly down-
regulated relative to CTRL and non-parasitized R fish.
Fig. 4 shows the expression profile of the same genes in
the intestine. In this tissue, parasitized-R fish exhibited
a significant up-regulation of GRP-75 and a-2M, whereas
TNF-a, GPx-1 and IL-1b were significantly down-regulated
with respect to CTRL fish, and the two latter genes also
with respect to non-parasitized R fish (Fig. 4A). Of note,
no significant differences were observed in non-parasitized
R fish vs. CTRL ones (Fig. 4B). Concerning the expression of
GH-related genes in HK, IGF-I and GHR-I were significantly
down-regulated in parasitized R fish (Fig. 5A), whereas
both genes were up-regulated in non-parasitized R fish
(Fig. 5B). No significant differences were observed in the
expression of GHR-II and IGF-II among the three fish
After 113 days of exposure to parasite-containing effluent,
two categories of R fish were obtained: non-parasitized (ex-
posed, but not infected) and parasitized (exposed and in-
fected by E. leei) fish, which were compared with CTRL
animals (not exposed to the parasite). This exposure time
(113 days) was chosen to ensure a high prevalence of infec-
tion. Data gathered in previous experiments over several
years have shown that E. leei experimental infection by
Gilthead sea bream primer sequences used for real-time PCR
Gene Accession no.Primer sequencePosition
GCG ACC TAC CTG CCA CCT ACA CC
TCG TCC ACC GCC TCC AGA TGC
CAG GCG TCG TTC AGA GTC TC
CTG TGG CTG AGA GGT GTG TG
GCC AAA CTC GGT GCC TCT CCT ACT GC
CTG CCC TGT GAG CCA TCT GAC AAT CGG
GAA GGT GGA TGT GAA TGG AAA AGA TG
CTG ACG GGA CTC CAA ATG ATG G
TGT TCA GCC ACC CAC CCA TCG G
GCG TGA TAC ATC GGA GTG AAT GAA GTC TTG
TCC GGT GTG GAT CTG ACC AAA GAC
TGT TTA GGC CCA GAA GCA TCC ATG
ACC TGT CAG CCA CCA CAT GA
TCG TGC AGA TCT GGG TCG TA
GAG TGA ACC CGG CCT GAC AG
GCG GTG GTA TCT GAT TCA TGG T
TGT CTA GCG CTC TTT CCT TTC A
AGA GGG TGT GGC TAC AGG AGA TAC
TGG GAT CGT AGA GGA GTG TTG T
CTG TAG AGA GGT GGC CGA CA
TCC TGC GGA ATC CAT GAG A
GAC GTC GCA CTT CAT GAT GCT
614A. Sitja `-Bobadilla et al.
exposure to water effluent is quite effective and consistent
, and therefore the infective pressure during the current
experiment was high enough to ensure a homogeneous and
continuous contact of the parasite with all R fish. Thus, in
order to understand why some exposed R fish were not par-
asitized, the differences in the immune response between
these fish categories, several cellular and humoral innate
factors were measured, and the expression of several
immune relevant genes was determined by rtqPCR in an im-
mune-competent organ (HK) and at the site of the infection
The RB of blood leucocytes was the only immune factor
significantly increased in parasitized R fish, with respect to
non-parasitized ones. This immune factor was also signifi-
cantly increased in sharpsnout sea bream parasitized by
E. leei at 20 days p.e.  and in turbot parasitized by the
related species E. scophthalmi . The increase observed
in the current study could be due to an increase in the num-
ber of cells involved in the RB (probably mobilized from HK
to combat the parasite at the intestine), or to an enhanced
activity of such cells. This mobilization would be in accor-
dance with the decrease in the percentage of HK acido-
philic leucocytes at 107 days p.e. reported in gilthead sea
bream exposed to E. leei by cohabitation .
Recognition molecules, like PO, LY and complement can
partake in direct fish pathogen elimination [16,42]. In pre-
vious studies of E. leei-exposed fish, serum PO was elevated
in sharpsnout sea bream from 5 to 55 days p.e. and in
gilthead sea bream at 10 days p.e. , though a depletion
was noticed later on, from 22 to 108 days p.e . There
are no data on how E. leei infection affects LY activity. In
sharpsnout sea bream, no LY could be detected in either in-
fected or healthy animals  and inconsistent patterns
have been reported in sparids infected by myxosporeans
or in other fish affected by other enteromyxosis [9,23,43].
In the present study, both serum PO and LY were signifi-
cantly depleted in exposed fish. Taking all this together,
it appears that in gilthead sea bream, PO could be initially
increased in response to the parasite exposure, but quickly
consumed to fight it, and values do not recover even in non-
parasitized R fish.
a-2M is a versatile anti-protease capable of trapping and
functionally silencing all classes of microbial and parasite
proteases , which is expressed in gilthead sea bream
early in the fish development in liver and muscle .
The association of a-2M with resistance to the fish hemofla-
gellate Cryptobia salmositica  has been demonstrated.
Supporting this, we found herein that a-2M was up-
regulated in the intestine (but not significantly in HK) of
parasitized gilthead sea bream, which suggests a role in
counteracting the putative action of parasite proteases at
the local level. Proteases are in fact involved in the patho-
genicity of parasites , and their presence in several
myxosporeans has been documented [48,49]. Thus, it is hy-
pothesized that proteases are also involved in E. leei host
invasion, as the parasite breaches the epithelium and
locates through enterocytes. Furthermore, the modulation
of circulating and expression levels of antiproteases by fish
PO (O.D. 450 nm)
IRLU after PMA stimulation
serum lysozyme (B), serum peroxidases (C), and serum complement activity of alternative pathway (D) of control (CTRL), non-par-
asitized recipient (R-NON PAR) and parasitized recipient (R-PAR) gilthead sea bream after 113 days of exposure to Enteromyxum
leei. Different letters indicate statistically significant differences at P < 0.05.
Mean ? SEM of the respiratory burst (expressed as the integral of relative light units, IRLU) of blood leucocytes (A),
E. leei modulates gilthead sea bream gene expression 615
parasitic infections has recently been reported. Increased
serum total antiproteases and serum a-2M were found in
E. leei-parasitized sharpsnout sea bream  and E. scoph-
thalmi-parasitized turbot , respectively. Similarly, a-2M
was significantly over-transcribed in grass carp parasitized
by the copepod Sinergasilus major  and in carp intra-
peritoneally injected with Trypanoplasma borreli . Ad-
ditionally, the increased expression of a-2M may also be in
response to host proteases released from injured tissues.
IL-1b and TNF-a are pro-inflammatory cytokines mainly
produced and released by activated leucocytes , which
have been cloned and sequenced in gilthead sea bream
[53,54]. In the present work, the expression of both cyto-
kines remained unaltered in non-parasitized R fish, but in
parasitized R fish it was significantly down-regulated by
the infection at the intestine. In a preliminary study ,
TNF-a was also significantly down-regulated in the HK of
E. leei-exposed gilthead sea bream starting on day 38
p.e., and IL-1b expression was increased at the first stages
(10 days) after exposure. Most studies on the effect of a par-
asitic infection in fish have focused on short-term infection
models and have detected over-transcription of both cyto-
kines after challenge with Myxosporea , Monogenea
[56,57], Copepoda  and Protozoa [51,59,60e62], with
a subsequent return to normal levels.
There is scarce information on suppressed cytokine
expression in piscine-parasite models, but this phenomenon
is well documented in parasite murine models [63,64]. Sev-
eral mechanisms could be involved in the down-regulation
of pro-inflammatory cytokines with the progress of parasite
infection. First, a-2M may inhibit the synthesis of certain
cytokines following cell activation (see ), and thus the
concurrent increased a-2M transcription in parasitized R
fish could be responsible for the parallel down-regulation
of IL-1b and TNF-a. Second, parasitized R fish (after
113 days of exposure to E. leei) might just be switching to
an anti-inflammatory phase to avoid excessive tissue injury
provoked by reactive oxidative species (ROS) generated by
activated leucocytes during a prolonged pro-inflammatory
Glutathione peroxidases are part of the enzymatic
antioxidant defence of fish, and their activity and/or
expression can be modulated by natural and anthropogenic
factors. Thus, the observed reduction in the transcription
of GPx-1, both in the intestine and the HK of parasitized R
gilthead sea bream, could render them in a state of
oxidative stress if the production of ROS is maintained
high (see ). However, if the observed high blood RB is
considered as the delayed effect of the initial pro-inflam-
matory phase, the down-regulation of GPx-1 could be
regarded as another indicator of the switching to an anti-
inflammatory phase. The effect of parasites in piscine
GPx-1 is mostly unknown, but mammals infected by some
protozoans exhibited reduced erythrocyte GPx-1 .
Relative Gene Expression
Relative Gene Expression
expression of inflammatory cytokines, anti-proteases, and an-
tioxidant enzymes and chaperones in the head kidney of para-
sitized (A) and non-parasitized (B) R gilthead sea bream after
113 days of exposure to Enteromyxum leei. Data in control
fish were used as arbitrary reference values in the normaliza-
tion procedure (values >1 or <1 indicate increase or decrease
with respect to reference values). Continuous line inside the
box is the median; non-continuous line inside the box is the
mean. Each value is the result of triplicate determinations in
8 animals. Statistically significant differences (P < 0.05) with
respect to control group are indicated (*). In plot A, statisti-
cally significant differences (P < 0.05) between parasitized
and non-parasitized animals are also indicated (þ).
Boxewhisker plots representing the relative gene
Relative Gene Expression
Relative Gene Expression
expression of inflammatory cytokines, antiproteases, and anti-
oxidant enzymes and chaperones in the intestine of parasitized
(A) and non-parasitized (B) R gilthead sea bream after 113 days
of exposure to Enteromyxum leei. See legend to Fig. 3 for ex-
planation of symbols and data handling.
Boxewhisker plots representing the relative gene
616A. Sitja `-Bobadilla et al.
GRP-75 was also differentially expressed in parasitized R
gilthead sea bream, with a clear over-transcription in
intestine. In mammals, GRP-75 is an essential protein that
has been implicated in multiple functions . Collective
data have provided evidence that a relationship exists be-
tween other HSPs and disease in fish [32,70,71]. In general,
an over-expression of HSPs is known to have deleterious
consequences , and particularly that of mortalin is
known to contribute to human carcinogenesis . The
biological meaning of the observed up-regulation can only
be hypothesized, since there is almost no information on
the particular regulation of GRP-75 in fish . It is known
that GRP-75-defective mutant zebrafish suffers a serious
blood developmental defect resulting in ineffective haema-
topoiesis . Thus, it is conceivable that the over-expres-
sion of GRP-75 in the intestine of parasitized R gilthead sea
bream may represent some adaptive response to restore
cellular homeostasis, in the intestinal tissue destroyed by
parasite invasion and the host immune reactive products.
This hypothesis would be in accordance with recent evi-
dence of the importance of HSPs to adapt to pathogenic
conditions in the digestive tract (reviewed in ). In any
case, further transcriptional and functional analyses are
needed to understand cell and tissue GRP-75 function and
regulation in fish.
This is the first work to address the interaction of
neuroendocrine and immune systems with a gene-expres-
sion approach during a long-term parasite challenge. The
expression of GH-related genes in parasitized fish exhibited
an opposite regulation to that of non-parasitized animals.
Theconcurrent down-regulationofIGF-Iand GHR-I
observed in the HK of parasitized R fish has not been
reported before in piscine models, and it could be consid-
ered a case of impaired GH function in immune-related
organs. In humans and rodents an important complication
of sepsis, trauma and chronic inflammatory diseases is the
hepatic GH resistance, characterized by normal GH secre-
tion with impaired production of hepatic target genes
including IGF-I , and some of the mechanisms involved
have been elucidated . However, the means by which
these genes were down-regulated in E. leei parasitized
fish remains to be established. On the other hand, the up-
regulated expression of GHR-I and IGF-I in the HK of non-
parasitized R fish could indicate a higher capacity to effec-
tively combat parasites. Supporting this, specific binding
sites for GH were characterized in gilthead sea bream HK
neutrophils and macrophages , and experimental evi-
dence indicates that GH usually acts in fish as an immunos-
timulant , though the underlying molecular mechanisms
are poorly understood. Of note, in the present study, the
HK expression of GHR-II and IGF-II was not altered in both
parasitized and non-parasitized R fish. This is in accordance
with the recent notion that GHR-I and II have evolved as
duplicated GHR subtypes having different patterns of tissue
distribution, post-receptor signalling and hormonal tran-
scriptional regulation [79,80].
In conclusion, although some immune parameters were
affected in the same direction by all the fish exposed to the
parasite (LY, PO), those fish which became parasitized
exhibited a clearly different gene-expression profile than
those which did not, particularly at the site of infection
(intestine). This on-site regulation indicates the magnitude
of the hosteparasite interaction and the involvement of
intestinal cells in the host defence. Further studies are
needed to analyse in depth the differential expression of
the studied genes, and a microarray approach is currently
underway to find other genes that may be differently
expressed in R fish which become parasitized and in those
which do not.
This work was partially funded by the UE-VI FP project:
‘‘Combined genetic and functional genomic approaches for
stress and disease resistance marker assisted selection in
fish and shellfish’’ (AQUAFIRST, contract no. SSP98-CT-
2004-513692). The authors are grateful to M.A. Gonza ´lez
and M.C. Ballester for excellent technical assistance.
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E. leei modulates gilthead sea bream gene expression619