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Comparative Biochemistry and Physiology, Part D 7 (2012) 191–200
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part D
journal homepage: www.elsevier.com/locate/cbpd
Identification of differentially expressed genes and quantitative expression of
complement genes in the liver of marine medaka Oryzias melastigma challenged with
Vibrio parahaemolyticus
Jun Bo a, e, John P. Giesy a, b, c, e, Rui Ye a, e, Ke-Jian Wang c, Jae-Seong Lee d, Doris W.T. Au a, e,⁎
a
State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, China
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Canada
c
State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian 361005, China
d
Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul, South Korea
e
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
b
a r t i c l e
i n f o
Article history:
Received 10 December 2011
Received in revised form 24 February 2012
Accepted 24 February 2012
Available online 3 March 2012
Keywords:
Suppression subtractive hybridization (SSH)
Complement system
Innate immunity
Gene expression
Fish
Immunotoxicology
a b s t r a c t
The innate immune system of fish is the primary defense against acute diseases. The marine medaka Oryzias
melastigma has been shown to be a potential marine fish model for ecotoxicology, but little is known about
the innate immune system of this small fish. In this study, suppression subtractive hybridization (SSH) was
used to identify differentially expressed immune genes in the liver of O. melastigma infected with Vibrio
parahaemolyticus. Among the 396 genes identified, based on NCBI BLAST search of the 1279 sequenced clones
in the SSH libraries, 38 (9.6%) were involved in the immune process. Besides, genes involved in biological regulations (5.6%); cellular metabolism (24.7%); general response to stimuli (4.8%); cellular component organization
(2.3%); signal transduction (2.5%) and transport process (2.8%) were also obtained. Ten complement component
genes involved in four activation pathways were quantified (using q-PCR) and exhibited different patterns of
transcription between the control and challenged individuals. The results reported upon here support the feasibility of developing O. melastigma as a marine model fish to understand the basic biological processes related to
immune function and for immunotoxicological research. Findings of this study established a genetic platform
for studying immune function using O. melastigma.
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
The immune system of teleosts, which responds rapidly to protect
fish from pathogen infection, is a potential target of environmental
xenobiotics (Inadera, 2006). In fish, the innate immune system is an
essential component in combating disease, and the acquired immune
system of fish is relatively underdeveloped compared to that of other
vertebrates, such as mammals and birds (Magnadottir, 2004). The
outcome of acute infections in fish appears to rely primarily on responses
of the innate immune system (Camp et al., 2000). There is a need to
advance current understanding of immune biology and toxicogenomics
in fish, in particular the innate immune system, which is crucial not only
for aquaculture management, but also for predictive ecotoxicology and
environmental risk assessment (Villeneuve and Garcia-Reyero, 2011).
The marine medaka Oryzias melastigma is one potential model
marine fish to be used in ecotoxicological studies (Kong et al., 2008;
⁎ Corresponding author at: Department of Biology and Chemistry, City University of
Hong Kong, Kowloon, Hong Kong, China Tel: + 852 3442 9710; fax: + 852 3442 0522.
E-mail address: bhdwtau@cityu.edu.hk (D.W.T. Au).
1744-117X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbd.2012.02.005
Shen et al., 2010; Wang et al., 2011). In particular, previous research
has demonstrated that O. melastigma is a potential model marine
fish for studying innate immune function (Bo et al., 2011). However,
there is little information available on genes that are most appropriate
for monitoring the function of the immune system, especially genes related directly to the innate immune system of the marine medaka. To
establish O. melastigma as a model marine fish for immunotoxicology,
characterization of the gene components and pathways for the innate
immune system of marine medaka is needed.
The innate immune system of vertebrates is independent of prior
exposure to any particular antigen. The complement system is a major
mediator of innate immune defense against infection via inflammation,
opsonization and cell lysis (Mayilyan et al., 2006), and it seems to play a
more pivotal role in body defense in fish. The complement system of
teleosts, like that of other vertebrates, can be activated through three
pathways. First, the classical complement activation pathway (CCP),
which is triggered by binding of antibody to the cell surface, can also
be activated by acute phase proteins or directly by viruses, bacteria
and virus-infected cells (Holland and Lambris, 2002; Gonzalez et al.,
2007). The alternative complement activation pathway (ACP), which is
independent of antibody, is activated directly by foreign microorganisms.
The lectin complement activation pathway (LCP) is elicited by binding of
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a protein complex consisting of mannose-binding lectins to mannans on
bacterial cell surfaces (Holland and Lambris, 2002; Whyte, 2007).
Recently, a fourth complement activation pathway, the “coagulation
system” complement activation has been proposed (Amara et al.,
2008). Findings by those authors suggest that various serine proteases
belonging to the coagulation system can activate the complement
cascade independently of the three previously established pathways.
All four pathways converge to the lytic pathway, which leads to opsonization or direct killing of the invaded microorganism.
In vertebrates, a diversity of plasma proteins that are made in the liver
serves as defense molecules in the innate immune system (Bayne et al.,
2001). In the work presented here, we extend knowledge of the immune
system to identify differentially expressed genes in the liver of O.
melastigma in response to bacterial challenge by use of suppression subtractive hybridization (SSH). Hepatic expression of ten innate immunerelated genes, which might be involved in complement activation
pathways, were investigated in male O. melastigma challenged with bacteria by use of quantitative real time PCR (q-PCR). The overall objective of
this study was to demonstrate that O. melastigma can serve as a potential
marine fish model for understanding the basic biological processes related to innate immune function and for immunotoxicological research.
following the manufacturer's instructions. For construction of the SSH
library, SMART PCR cDNAs were synthesized, amplified and digested
with Ras I from 1 μg of the total RNA for each group using the Super
SMART™ PCR cDNA Synthesis Kit (Clontech) according to the manufacturer's protocol.
2.5. Construction of the SSH library
SSH libraries were constructed according to previously established
methods (Chen et al., 2010). Briefly, genes that were up-regulated in
response to bacteria were compared with those in the liver of saline
controls in one run, while those genes that were up-regulated in the
saline control relative to the expression in livers of infected fish were
determined in a second SSH run. Construction of the SSH library was
performed using the PCR-Select™ cDNA Subtraction Kit (Clontech) by
using methods suggested by the manufacturer. The forward and reverse
SSH libraries were then plated on LB+Ampicillin (150 μL/mL, Sigma)
agar plates that had been pre-spread with 20 μL X-Gal (50 mg/mL,
Promega) and 100 μL IPTG (100 mM, Invitrogen) for screening of the
two libraries, respectively.
2.6. Identification of positive clones and DNA sequencing
2. Materials and methods
2.1. Experimental animals
Marine medaka (O. melastigma) were purchased from a commercial
hatchery in Taiwan. The State Key Laboratory in Marine Pollution (City
University of Hong Kong) has established a self-propagating population
of O. melastigma for more than 30 generations. Standard operating
procedures (SOPs) for large-scale culturing of O. melastigma have been
established. Fish were maintained in the laboratory in aerated 30‰
artificial seawater at 5.8 ± 0.2 mg O2 L − 1, 28± 2 °C in a 14-h light:
10-h dark cycle.
2.2. Preparation of bacteria
Vibrio parahaemolyticus is a curved, rod-shaped, Gram-negative,
bacterium found in brackish saltwater, which has caused great loss in
aquaculture (Cai et al., 2006). V. parahaemolyticus was purchased from
China General Microbiological Culture Collection Center, CGMCC, Beijing,
China, and cultured in LB broth at 28 °C with shaking at 200 rpm overnight, then the bacteria were collected by centrifugation (3000 g for
10 min at 4 °C) and suspended in sterile saline solution (0.65% NaCl) at
a concentration of 3×108 colony forming units (cfu)/mL.
2.3. Bacterial challenge and sampling for SSH
Adult O. melastigma (male, 5-month old, ~300 mg body weight) were
used in the experiment. During manipulations, fish were anesthetized
with 0.02% tricaine methanesulfonate (MS-222, Sigma-Aldrich). For the
bacterial challenge, a 2 μL stock bacterial suspension (6×105 cfu/fish)
or equal volumes of 0.65% NaCl for the control were administered into
the peritoneal cavity (nearby the caudal to the pelvic fins), using a 5 μL
Hamilton syringe equipped with an ultra-fine needle under a microscope.
A sublethal dose of bacteria was selected based on results of preliminary
experiments. After injection, fish were returned to the aquaria and
allowed to recover. Samples of the liver (n=6) were collected at the
end of 6 h, 24 h and 48 h post injection in the bacteria-challenged/salineinjected group. Samples of the liver were immediately frozen in liquid
nitrogen, and stored at −80 °C for total RNA extraction.
2.4. RNA isolation and cDNA synthesis
Total RNAs were extracted from a pooled sample of livers collected
at the three time-points by use of Trizol reagent (Invitrogen) and
The inserted fragment sizes of selected clones were determined by
use of PCR followed by 1% agarose gel electrophoresis separation. The
PCR reaction was performed using 1 μL bacterial culture, primers
pGEMT-F and pGEMT-R, and iTaq DNA polymerase (BioRed). PCR
amplification was conducted with the following cycles: 3 min at
94 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C, 90 s at 72 °C; and
3 min at 72 °C for the final extension. Selected clones were sequenced
by use of an ABI 3730 automated sequencer (Applied Biosystems,
USA) at the Beijing Genomics Institute (Beijing, China).
2.7. Sequence analysis
Sequences obtained were analyzed by use of DNASIS and DNAssist
2.0. Homology searches were performed using BLASTx and BLASTp programs, with default parameters against the non-redundant database, by
the National Center for Biotechnology Information (http://www.ncbi.
nlm.nih.gov/). The CD-Search service (Marchler-Bauer and Bryant,
2004) was used to identify the conserved domains (CD) present in
predicted protein sequences against NCBI's Conserved Domain Database
(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The best annotated hit from the similarity search was retained. Gene ontology (GO) annotation (Ashburner et al., 2000) based on BLAST analysis was performed
using AmiGO against the GO database (http://amigo.geneontology.org/
cgi-bin/amigo/go.cgi). The sequences were submitted to GenBank at
the NCBI and accession numbers were assigned.
Sequences were blasted against the Japanese medaka (Oryzias
latipes) genome database, and E values less than 10− 5 were considered
to be an orthologue between the two species. The non-redundant list of
gene symbols was further curated by the HUGO Gene nomenclature
committee. The gene symbol list was then input into the Database for
Annotation, Visualization and Integrated Discovery (DAVID) for enrichment analysis, Homo sapiens was chosen as the reference. The Kyoto
Encyclopedia of Genes and Genomes (KEGG) was used to determine
the placement of gene products in specific pathways.
2.8. Real time PCR for quantification of complement genes in O.
melastigma challenged with bacteria
Ten genes [complement component 1 q subcomponent-like 4 like
(C1q), complement component 3-2 (C3-2), complement component 4
(C4), complement component 5 (C5), complement component 8 (C8),
complement factor B (BF), complement factor H (HF), lectin, mannosebinding 2 (MBL2), mannose-binding lectin-associated serine protease
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J. Bo et al. / Comparative Biochemistry and Physiology, Part D 7 (2012) 191–200
(MASP) and C1 inhibitor] that are potentially involved in the complement
cascades were chosen for quantitative real time PCR (q-PCR) analyses.
Adult O. melastigma (male, 5-month old, ~300 mg body weight)
were anesthetized with 0.02% tricaine methanesulfonate (MS-222,
Sigma-Aldrich). Using a 5 μL Hamilton syringe equipped with an ultrafine needle under a microscope, 2 μL of stock bacterial suspension
(5.69 × 105 cfu) for the bacterial challenge or equal volume of 0.65%
NaCl for the control was administered into the peritoneal cavity. Fish
were sampled at 0, 6, 12, 24 and 48 h post injection from the bacterial
challenged and the vehicle control fish. Liver isolated from each fish
was frozen immediately in liquid nitrogen and stored at −80 °C for
total RNA extraction (n= 3, each replicate was pooled from 2 fish).
Total RNA was extracted using the TRIzol method, then reverse transcribed into cDNA using the One-Step TaKaRa Primescript™ RT Reagent
Kit (TaKaRa). Briefly, q-PCR assays were performed using the fluorescent dye Power SYBR Green PCR Master Mix and ABI 7500 System.
Gene-specific primers (Table 1) were designed with the Primer Express
Software v3.0. The 18S rRNA was used as the reference gene, and the qPCR was conducted as previously described (Bo et al., 2011). The
relative magnitudes of expression (fold change) of the tested genes,
were calculated using the relative expression software (ABI), based on
the 2 − ΔΔCT method (Livak and Schmittgen, 2001).
2.9. Statistical analysis
All results were presented as mean±standard deviation (SD), and
the statistical procedures were conducted using IBM SPSS Statistics
17.0. The magnitude of gene expression in bacterial-challenged fish was
expressed as the fold change relative to the value of the vehicle control.
If necessary, data were normalized by log-transform. Differences of relative gene expression among treatments were evaluated by one-way
ANOVA followed by Tukey's test. Differences were considered to be
significant when pb 0.05.
3. Results
3.1. Sequencing and analysis of clones from the SSH library
Two subtracted cDNA libraries (a forward and a reverse) were
generated from the livers of male O. melastigma challenged with V. parahaemolyticus. A total of 1278 clones were sequenced, and using the NCBI
Table 1
Primers used for quantitative real time PCR analysis.
Gene name
Primer sequence (forward and reverse)
(5′ → 3′)
Product length
(bp)
C1q
F: ATGGGCCAGCGTGGGACCT
R: GCTGGCCTGTGTGCCAGCTT
F: GGTCAAGAGTGAATGGAATGCCTA
R: CTAACAGAAACAAGATGGAGAGCC
F: GGCTGGAGTATGAGCAAGGCGG
R: TGGTCTTCTCGTCTCCGTTGCAGT
F: GGCGTGCACACCCTGAGCTT
R: TCACCTCCCGCCTCACTCCT
F: AGAAGCCCAAGGCCAACCCG
R: TGGTCCGAGGCAGAGGAGCG
F: GCCGCCAATCCCGGGAACAA
R: GGCGCCGGTGGTTTCCATGT
F: TGGCGGTGAGAGGGAGCACA
R: GGGTTCCCCAGCTGACCAATGC
F: GAGCGCGATCCTCTCACGCC
R: AGAGCACTCCGGCGTCACCA
F: CTGCAGCTTTGCCGCCATCG
R: GCAGCTGGCAGTGCTCCACA
F: GCTGGGTGGCCAACAAGACCA
R: GCTCCGTGCTGGGTGGAACC
F: CCTGCGGCTTAATTTGACCC
R: GACAAATCGCTCCACCAACT
235
C3-2
C4
C5
C8
HF
BF
MASP
MBL2
C1 inhibitor
18S
176
72
103
178
121
193
BLAST search among the clones, 396 translatable DNA sequences were
predicted to be proteins, and the putative amino acid sequences were
searched for conserved domains using CD-Search with creditable
expectation values (E-value ≤10− 5) (Table 2). These 396 genes were
categorized, using AmiGO against the Gene Ontology database, into
eight functional groups in association with different biological processes
as follows: 38 (9.6%) were involved in the immune system; 22 (5.6%) in
biological regulations; 98 (24.7%) were associated with cellular metabolic
processes; 19 (4.8%) as responses to stimuli; 9 (2.3%) cellular component
organization; 10 (2.5%) signal transduction; 11 (2.8%) transport processes, and 189 (47.7%) were unknown and thus not classified. Seventeen
genes [C1q, C3-2, C4, C5, C8, BF, HF, MBL2, MASP, C1 inhibitor, C1q-like
23 kDa protein, complement C1q-like protein 4 precursor, coagulation
factor X (F10), coagulation factor II (F2), kininogen 1 (KNG1), fibrinogen
beta chain (FGB), fibrinogen gamma chain (FGG)], which are involved
in the four complement pathways associated with primary defense
against bacterial infection, were identified in O. melastigma.
3.2. Analysis of expression pattern of the complement genes in liver using
q-PCR
Pathway analysis conducted with DAVID showed that the complement and coagulation cascades seemed to be more important than
other analyzed pathways, such as, the ribosome pathway, galactose
metabolism and systemic lupus erythematosus pathway, (Table 3).
Ten genes (C1q, C3-2, C4, C5, C8, BF, HF, MBL2, MASP and C1 inhibitor)
obtained from the SSH library were speculated to be involved in the
three complement activation pathways: classical pathway (C1q, C4,
C3-2), alternative pathway (BF, HF, C3-2) and lectin pathway (MBL2,
MASP, C4, C3-2) and involved in cell lysis (C5, C8) (Fig. 1).
Transcriptional profiles for the ten complement-related genes in the
liver of male O. melastigma were determined by q-PCR after being challenged with V. parahaemolyticus from 6 h to 48 h (Table 4). There was a
statistically significant, time-dependent up-regulation with respect to
the saline control group for the genes C3-2, C4 and HF. Expression of
C3-2 was small (0.9-fold and 1.1-fold, respectively) at 6 h and 12 h,
and it was 2.8-fold (p b 0.05) greater 24 h after bacterial challenge.
Expression of C4 was up-regulated (6.8- and 2.8-fold, respectively) 12
and 24 h after the bacterial challenge. Expression of HF was 2.4-fold
(p b 0.05) greater 48 h after infection. Conversely, transcription of
MASP was less at all the sampled points with the minimum transcription
level of 0.35-fold (p b 0.05) at 48 h. No significant changes were
observed for C5, C8, BF, MBL2 and C1 inhibitor. Expression of C1q was
not detectable in males at any of the exposure times.
4. Discussion
Most of the 38 immune-relevant genes (including complement
components, clotting molecules, anti-proteases, lectins, lysozyme,
antimicrobial peptide hepcidin etc.) identified by SSH are involved
in the acute phase response (APR). This suggests that a successful
bacteria-induced immune reaction was elicited and information
obtained from this study will provide useful insights into the immune
mechanism (especially for innate immune response) using O. melastigma as a potential marine fish model.
4.1. Immune process
107
100
113
69
134
The APR is a set of metabolic and physiological reactions occurring
in the host in response to tissue infection or injury and is a crucial
component of the innate immune response. The APR is best characterized by changes in concentrations of a group of plasma proteins
known as acute phase proteins (APPs) which are mainly synthesized
in the liver and serve as defense molecules in the innate immune system (Bayne et al., 2001; Peatman et al., 2007). The genes expressed as
components of the APR were obtained from the SSH library (Table 2)
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Table 2
Categorization of potential functional genes screened from the marine medaka O. melastigma SSH library according to GO annotation.
Gene name
GenBank accession no.
Species with homology to
Length (bp)
E-value (Blastp)
1. Immune processes (38 genes)
Alpha-1-antitrypsin
Alpha-2-macroglobulin
Antithrombin III
C1 inhibitor
C1q-like 23 kDa protein
Coagulation factor X
Coagulin factor II
Complement C1q-like protein 4 precursor
Complement component 1, q subcomponent-like 4 like
Complement component 4 (within H-2S)
Complement component C3-2
Complement component C5
Complement component C8 gamma chain
Complement factor B
Complement factor H precursor
Complement factor properdin
Complement regulatory plasma protein
C-type lysozyme
Ferritin heavy chain
Ferritin, middle subunit
Hepcidin-like precursor
Interleukin-1 receptor type II
Kininogen-1 precursor
Lectin, mannose-binding 2
Leukocyte cell-derived chemotaxin 2
Lipopolysaccharide-binding protein
LMP2
Mannose-binding lectin-associated serine protease-3b
Precerebellin-like protein
Proteoglycan 4
SAM domain and HD domain-containing protein 1
Secreted immunoglobulin domain 4
Serine/cysteine proteinase inhibitor
Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7
Skin mucus lectin
Serotransferrin precursor
Vitronectin protein
Warm-temperature-acclimation-related-65 kDa-protein-like-protein
HM137115
HM137129
HM137172
HQ144247
HM137123
HM137116
HM137108
HM137122
HM137118
HM137120
HM137119
HM137121
HM137124
HM137125
HQ144249
HQ144250
HM137126
GU980929
HM137113
HQ144243
HM562669
HM137114
HM137130
HM137110
HM137127
HM137117
HM137128
HM137111
HQ144248
HM137131
HM137112
HQ144244
HM137109
HQ144246
HM137133
JF437716
HQ144245
HM137132
Pseudopleuronectes americanus
Ctenopharyngodon idella
Takifugu rubripes
Larimichthys crocea
Neoditrema ransonnetii
Osmerus mordax
Larimichthys crocea
Salmo salar
Danio rerio
Oryzias latipes
Oryzias latipes
Oncorhynchus mykiss
Salmo salar
Oryzias latipes
Oncorhynchus mykiss
Danio rerio
Paralabrax nebulifer
Oryzias latipes
Chionodraco rastrospinosus
Anoplopoma fimbria
Pagrus major
Paralichthys olivaceus
Esox lucius
Danio rerio
Lates calcarifer
Paralichthys olivaceus
Oryzias latipes
Xenopus laevis
Oncorhynchus mykiss
Danio rerio
Danio rerio
Danio rerio
Xiphophorus hellerii
Danio rerio
Platycephalus indicus
Oryzias latipes
Oncorhynchus mykiss
Oryzias latipes
552
496
520
870
1001
630
874
735
649
584
1181
511
410
688
481
598
512
554
749
798
609
496
1163
262
480
362
618
787
908
521
428
760
638
525
534
710
374
936
2.00E−36
2.00E−64
2.00E−57
1.00E−99
7.00E−55
1.00E−41
1.00E−125
1.00E−41
3.00E−21
1.00E−80
9.00E−158
3.00E−34
2.00E−26
6.00E−78
1.00E−19
2.00E−27
1.00E−12
1.00E−62
3.00E−88
3.00E−90
1.00E−17
2.00E−49
8.00E−53
1.00E−41
3.00E−60
3.00E−43
3.00E−39
2.00E−23
2.00E−32
2.00E−55
5.00E−61
2.00E−66
1.00E−62
1.00E−15
5.00E−31
8.00E−110
1.00E−06
1.00E−362
2. Cellular metabolic processes (98 genes)
14 kDa apolipoprotein
14 kDa apolipoprotein
3-oxo-5-beta-steroid 4-dehydrogenase
40S ribosomal protein S17
40S ribosomal protein S18
40S ribosomal protein S8
60S acidic ribosomal protein P0
60S ribosomal protein L38
Acidic ribosomal protein P0
Adenylate kinase isoenzyme 2, mitochondrial
Aldehyde dehydrogenase 9A1
Aldolase B
Allantoicase
Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase
Alpha-N-acetylgalactosaminidase
AN1, ubiquitin-like, homolog
Apolipoprotein A-I
Apolipoprotein A-IV precursor
Apolipoprotein B
Apolipoprotein C-I
Atg12 protein
Beta-hydroxysteroid dehydrogenase type 3
Biotinidase
Biotinidase precursor
Biquitin-conjugating enzyme E2Q 1 (Ube2q1)
Carnitine acetyltransferase
Cathepsin F
Choriogenin H
Choriogenin L
ClpX caseinolytic protease X homolog
Cytochrome c oxidase subunit 5A, mitochondrial precursor
Cytochrome c oxidase subunit I
Cytochrome c oxidase subunit II
Cytochrome c oxidase subunit III
HM137237
JF437717
HM137207
HM137256
HM137229
HM137267
HM137214
HM137217
HM137286
JF437718
HM137226
HM137209
HM137258
HM137254
HM137239
HM137252
HM137242
HM137283
HM137228
HM137222
HM137251
HM137206
HM137230
HM137240
HM137272
JF437719
HM137210
HM137235
HM137269
HM137224
HM137247
HM137276
HM137277
JF437720
Epinephelus coioides
Perca flavescens
Anoplopoma fimbria
Salmo salar
Pagrus major
Anoplopoma fimbria
Anoplopoma fimbria
Salmo salar
Xenopus laevis
Salmo salar
Oryzias latipes
Poecilia reticulata
Danio rerio
Salmo salar
Salmo salar
Rattus norvegicus
Morone saxatilis
Bos taurus
Salmo salar
Solea senegalensis
Danio rerio
Oryzias latipes
Danio rerio
Perca flavescens
Danio rerio
Danio rerio
Paralichthys olivaceus
Oryzias latipes
Oryzias melastigma
Danio rerio
Salmo salar
Aplocheilus panchax
Anolis carolinensis
Oryzias latipes
701
391
784
503
759
251
1086
539
731
711
459
1121
935
535
1197
743
999
821
950
524
486
163
873
574
96
160
1190
1325
660
736
433
601
116
814
1.00E−39
3.00E−29
6.00E−133
2.00E−71
1.00E−29
3.00E−35
2.00E−148
3.00E−35
4.00E−94
1.00E−65
3.00E−79
0.00E+00
5.00E−95
1.00E−77
2.00E−121
2.00E−71
2.00E−61
6.00E−09
7.00E−60
2.00E−10
5.00E−48
8.00E−21
1.00E−41
5.00E−59
2.00E−11
3.00E−18
7.00E−137
4.00E−63
2.00E−113
1.00E−79
5.00E−61
1.00E−12
2.00E−11
2.00E−44
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195
Table 2 (continued)
Gene name
GenBank accession no.
Species with homology to
Length (bp)
E-value (Blastp)
Cytochrome c oxidase subunit VIa precursor
Cytochrome c oxidase, subunit Va
Cytochrome oxidase subunit I
Cytochrome P450 1A
Cytochrome P450 20A1
Cytochrome P450 2C33-like
Cytochrome P450, family 4, subfamily b, polypeptide 1
Cytosolic malate dehydrogenase A
Dolichyl-diphosphooligosaccharide-protein glycosyltransferase
Elongation factor 1 alpha
Elongation factor 1 gamma
Elongation factor 2
Endonuclease-reverse transcriptase HmRTE-e01
Epidermis-type lipoxygenase 3
Eukaryotic translation initiation factor 3, subunit 5 epsilon, 47 kDa
Eukaryotic translation initiation factor 3, subunit 6 interacting protein
Eukaryotic translation initiation factor 3, subunit 7 zeta
Eukaryotic translation initiation factor 4A, isoform 1A
Farnesyl pyrophosphate synthetase
F-box only protein 9
FK506-binding protein 5 (FKBP5)
Fructose-1,6-bisphosphatase 1
Glutamate dehydrogenase 1
Glyceraldehyde-3-phosphate dehydrogenase
Glyoxylate reductase/hydroxypyruvate reductase
Growth hormone receptor
Hepatic lipase
Histidine ammonia-lyase
Hydroxymethylglutaryl-CoA lyase
Hydroxysteroid (17-beta) dehydrogenase 4
Hypoxanthine phosphoribosyltransferase
Ribosomal protein L27a
Inositol polyphosphate-5-phosphatase A
Integral membrane protein 1
Inter-alpha (globulin) inhibitor H3
Ketodihydrosphingosine reductase
Lipocalin
Membrane-associated ring finger (C3HC4) 5
Methionyl aminopeptidase 2
Methyltransferase Mb3374
NADH dehydrogenase subunit 4
Ornithine aminotransferase
Peroxisomal trans-2-enoyl-CoA reductase
Phosphotriesterase-related protein
Protein-O-mannosyltransferase 2
Purine nucleoside phosphorylase
Ribosomal protein L13
Ribosomal protein L22
Ribosomal protein L23
Ribosomal protein L32
Ribosomal protein L37
Ribosomal protein L4
Sich73-252g14.4 protein
Similar to ribosomal protein L35a
Similar to Salivary gland secretion 1 CG3047-PA
TGF-beta-inducible nuclear protein 1
Transketolase
Ubiquitin
Ubiquitin and ribosomal protein S27a precursor
Ubiquitin carboxyl-terminal hydrolase isozyme L3
Ubiquitin carboxyl-terminal hydrolase isozyme L5
Ubiquitin-conjugating enzyme E2B
UDP-glucose 4-epimerase
UDP-glucose pyrophosphorylase 2
HM137205
HM137249
JF437721
JF437722
HM137245
HM137208
HM137281
HM137211
HM137266
HM137282
HM137246
HM137255
HM137221
HM137261
HM137231
HM137244
HM137259
HM137202
HM137223
HM137218
HM137264
HM137262
HM137275
HM137236
JF437723
HM137225
HM137268
JF437724
HM137233
HM137203
HM137241
JF437725
HM137260
HM137265
HM137199
HM137285
HM137238
HM137253
HM137248
HM137220
HM137215
HM137271
HM137250
HM137280
HM137273
HM137212
HM137201
HM137232
HM137227
HM137234
HM137219
HM137278
HM137274
HM137279
JF437726
HM137216
HM137257
HM137204
HM137270
HM137200
HM137284
HM137213
HM137263
HM137243
Thunnus obesus
Xenopus tropicalis
Enicmus brevicornis
Oryzias latipes
Salmo salar
Gadus morhua
Mus musculus
Oryzias latipes
Gallus gallus
Oryzias latipes
Oryctolagus cuniculus
Salmo salar
Heliconius melpomene
Salmo salar
Homo sapiens
Danio rerio
Danio rerio
Danio rerio
Salmo salar
Salmo salar
Salmo salar
Anoplopoma fimbria
Danio rerio
Dicentrarchus labrax
Osmerus mordax
Oryzias latipes
Pagrus major
Danio rerio
Takifugu rubripes
Fundulus heteroclitus
Solea senegalensis
Epinephelus coioides
Danio rerio
Bos taurus
Danio rerio
Danio rerio
Perca flavescens
Danio rerio
Danio rerio
Salmo salar
Oryzias latipes
Salmo salar
Salmo salar
Salmo salar
Danio rerio
Esox lucius
Solea senegalensis
Solea senegalensis
Danio rerio
Epinephelus coioides
Solea senegalensis
Solea senegalensis
Danio rerio
Macaca mulatta
Danio rerio
Anoplopoma fimbria
Salmo salar
Oncorhynchus mykiss
Ictalurus punctatus
Salmo salar
Salmo salar
Danio rerio
Oncorhynchus mykiss
Danio rerio
498
370
437
627
327
671
388
647
452
773
560
463
545
743
653
231
591
564
513
433
285
551
182
526
351
347
816
406
430
814
532
356
725
283
241
1008
176
864
508
933
487
290
578
567
582
832
249
401
679
483
181
152
537
499
225
603
640
508
348
707
228
829
366
159
2.00E−37
7.00E−23
2.00E−11
1.00E−08
1.00E−05
9.00E−64
1.00E−13
1.00E−45
5.00E−29
3.00E−68
2.00E−76
5.00E−60
1.00E−35
6.00E−110
3.00E−100
1.00E−36
3.00E−39
4.00E−66
3.00E−64
1.00E−43
1.00E−32
2.00E−84
6.00E−28
3.00E−93
2.00E−33
7.00E−55
2.00E−81
1.00E−69
4.00E−24
5.00E−139
1.00E−81
5.00E−48
2.00E−40
5.00E−40
3.00E−19
9.00E−164
6.00E−17
2.00E−93
3.00E−17
2.00E−20
1.00E−17
1.00E−42
2.00E−82
6.00E−84
8.00E−27
2.00E−95
2.00E−25
3.00E−35
3.00E−40
1.00E−70
1.00E−17
2.00E−20
4.00E−33
7.00E−32
4.00E−12
1.00E−70
6.00E−80
2.00E−79
7.00E−45
1.00E−77
3.00E−15
6.00E−42
6.00E−35
5.00E−17
3. Cellular component organization and cell adhesion (9 genes)
Acyl-CoA-binding protein
Coxsackie virus and adenovirus receptor precursor
Ependymin-1 precursor
G protein-coupled receptor 178
Mannose receptor C1-like protein
Myocilin
Nuclear receptor 2C2-associated protein
Stabilin-2 precursor
Stard10 protein
HM137178
JF437727
HM137179
HM137182
HM137180
HM137175
HM137176
HM137181
HM137177
Oryzias latipes
Danio rerio
Anoplopoma fimbria
Danio rerio
Danio rerio
Homo sapiens
Salmo salar
Salmo salar
Danio rerio
303
354
672
386
376
306
301
285
343
4.00E−34
3.00E−32
3.00E−16
8.00E−30
2.00E−06
3.00E−12
1.00E−31
2.00E−09
8.00E−11
(continued on next page)
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Table 2 (continued)
Gene name
GenBank accession no.
Species with homology to
4. Signal transduction (10 genes)
ADP-ribosylation factor 4
Canopy-1 precursor
Glucocorticoid receptor DNA binding factor 1
Insulin-like growth factor binding protein 1
Insulin-like growth factor-I
IQ motif containing GTPase activating protein 2
Muscle-specific beta 1 integrin binding protein 2
Proheparin-binding EGF-like growth factor
Ras-related protein Rac1
Retinal G protein coupled receptor
Length (bp)
E-value (Blastp)
HM137189
HM137185
HM137190
HM137186
HM137184
HM137187
HM137183
HM137191
JF437728
HM137188
Danio rerio
Salmo salar
Mus musculus
Perca flavescens
Cyprinus carpio
Xenopus laevis
Epinephelus coioides
Salmo salar
Brugia malayi
Danio rerio
481
447
609
531
302
437
312
664
150
671
3.00E−27
3.00E−40
3.00E−83
1.00E−54
1.00E−38
4.00E−45
3.00E−43
8.00E−17
1.00E−12
6.00E−40
5. Transport processes (11 genes)
ADP/ATP translocase 2
Alpha-type globin
Amisyn
Gga1 protein
NAC alpha (nascent polypeptide-associated complex)
Organic cation transporter like
Oxysterol binding protein-like 9
Solute carrier family 35, member D1
Solute carrier organic anion transporter family, member 2A1
Syntaxin 4
Transient receptor potential cation channel, subfamily M, member 7
JF437729
HM137196
JF437730
HM137195
JF437731
HM137192
JF437732
HM137194
HM137197
HM137198
HM137193
Danio rerio
Oryzias latipes
Homo sapiens
Danio rerio
Pagrus major
Danio rerio
Danio rerio
Homo sapiens
Bos taurus
Lateolabrax japonicus
Danio rerio
377
361
540
197
541
513
583
679
556
601
403
6.00E−48
3.00E−33
1.00E−50
9.00E−11
9.00E−83
4.00E−41
1.00E−103
2.00E−73
2.00E−51
7.00E−60
1.00E−38
6. Response to stimulus (19 genes)
Catalase
Cold-inducible RNA-binding protein
Fibrinogen beta chain precursor
Fibrinogen gamma polypeptide
Heat shock 70 kDa protein 4
Heat shock 70 kDa protein 5
Heat shock protein 90 beta
Liver angiotensinogen
Matrix metallopeptidase 13
Matrix metalloproteinase 9
Metallothionein
Mitochondrial uncoupling protein 3
Myoglobin
Peroxiredoxin 1
Peroxiredoxin 3
Peroxiredoxin 6
Plasminogen
Uncoupling protein 2
Vitellogenin 1
HM137160
HM137161
HM137173
HM137159
HM137164
HM137167
HM137157
HM137155
HM137168
HM137171
HM137170
HM137162
HM137165
HM137169
HM137174
HM137166
HM137163
HM137156
HM137158
Rachycentron canadum
Anoplopoma fimbria
Larimichthys crocea
Danio rerio
Cyprinus carpio
Danio rerio
Paralichthys olivaceus
Rhabdosargus sarba
Sparus aurata
Paralichthys olivaceus
Oryzias javanicus
Danio rerio
Tetraodon nigroviridis
Anoplopoma fimbria
Danio rerio
Salmo salar
Oryzias latipes
Oreochromis niloticus
Oryzias latipes
1174
455
493
681
483
292
148
1201
949
838
322
871
627
978
762
515
951
713
520
6.00E−155
4.00E−04
3.00E−71
1.00E−68
2.00E−47
2.00E−47
5.00E−21
4.00E−123
2.00E−105
1.00E−75
9.00E−21
5.00E−128
1.00E−41
2.00E−104
4.00E−104
2.00E−70
2.00E−159
6.00E−85
1.00E−83
7. Biological regulations (22 genes)
26S proteasome non-ATPase regulatory subunit 8
Activity-dependent neuroprotective protein
Acyl-CoA synthetase short-chain family member 2
Alanine-glyoxylate aminotransferase 2-like 1
Antileukoproteinase precursor
Apolipoprotein A-I-binding protein precursor
Apoptogenic 1 isoform 2
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
Ceruloplasmin
Cysteine-rich protein 2
Diablo-like protein
Heart-type fatty acid binding protein
Histone deacetylase 8
Imitation switch ISWI
Liver-basic fatty acid binding protein
Spen homolog, transcriptional regulator
TBT-binding protein
Thioredoxin domain containing 5
Transforming growth factor, beta receptor III
Whey acidic protein precursor
Yes-associated protein 1
Zinc finger protein 706
HM137135
HM137153
HM137134
HM137143
HM137138
JF437733
HM137148
HM137154
HM137147
HM137136
JF437734
HM137145
HM137144
HM137139
HM137142
HM137140
HM137149
HM137146
HM137141
HM137152
HM137151
HM137150
Osmerus mordax
Mus musculus
Danio rerio
Danio rerio
Salmo salar
Esox lucius
Mus musculus
Anoplopoma fimbria
Chionodraco rastrospinosus
Anoplopoma fimbria
Perca flavescens
Fundulus heteroclitus
Xenopus tropicalis
Xenopus laevis
Acanthopagrus schlegelii
Homo sapiens
Tetraodon nigroviridis
Danio rerio
Gallus gallus
Salmo salar
Danio rerio
Anoplopoma fimbria
887
393
1183
756
314
469
594
853
634
308
211
474
686
541
425
418
617
926
405
703
449
428
2.00E−58
1.00E−26
4.00E−84
3.00E−56
2.00E−13
1.00E−49
2.00E−35
1.00E−85
7.00E−90
2.00E−41
6.00E−16
3.00E−48
7.00E−38
4.00E−90
6.00E−55
2.00E−44
2.00E−22
1.00E−64
1.00E−09
9.00E−24
1.00E−32
1.00E−32
8. Unknown (189 genes)
which included complement components C1q, C3-2, C4, C5, C8, BF,
HF, MASP, MBL2, C1 inhibitor, properdin, alpha-2-macroglobulin,
alpha-1-antitrypsin, coagulation family (coagulation factors II, XI),
ferritin, LPS binding protein (LBP), α1-anti-trypsin, lysozyme, serine
protease inhibitors, antimicrobial peptide hepcidin, and ferritin,
among others, up-regulation of which is consistent with the early
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197
Table 3
Results of DAVID pathway analysis using the KEGG pathway database. The Y-axis is the Benjamini FDR p value (− log10) and the X-axis shows the different biological pathways.
stages of the innate immune system being involved in response to
invasion of microorganisms.
Hepcidin is an important antimicrobial peptide (AMP) of the innate immune system, and exists in various vertebrates including
fish (Krause et al., 2000; Park et al., 2001; Wang et al., 2009). Fish
hepcidins possess antibacterial activity in vitro and their expression
in liver is induced following bacterial challenge (Wang et al., 2009).
Our previous results show that the expression of mRNAs of hepcidins
in O. melastigma was rapidly and remarkably up-regulated after exposure
to V. parahaemolyticus. Such a response might play a role in innate defense during early developmental stages and post bacterial challenge
(Bo et al., 2011).
Lysozyme is an enzyme that disrupts bacterial cell walls by splitting
glycosidic linkages in the peptidoglycan layers. It acts directly on the
walls of Gram-positive bacteria, and on the inner peptidoglycan layers
of Gram-negative bacteria, after the complement and other enzymes
have disrupted the outer walls (Yano et al., 1996). Lysozyme exists in
most tissues and secretions of fish. In toxicological studies, lysozyme
levels have been most frequently examined as a sensitive response
(Reynaud and Deschaux, 2006; Sanchez and Porcher, 2009). The upregulation of transcription of lysozyme which was observed after
bacterial challenge is consistent with the results reported for fish due
to bacterial infection that has been previously reported (Ye et al.,
2010). The induced lysozyme activity of O. melastigma is probably
characteristic of antimicrobial infection in fish.
Serine proteinase inhibitors (serpins) are irreversible suicide inhibitors of proteases that regulate blood coagulation, prophenoloxidase activation, pathogen digestion, apoptosis, complement system
and cellular remodeling (Chen et al., 2010). Serpins have been widely
identified in mammals, insects, plants, microorganisms (Cao et al.,
2000), amphibians (Han et al., 2008), crustaceans (Homvises et al.,
2010) and teleosts (Cao et al., 2000). Serpins have also been shown
to participate in responses to bacterial and viral infections in crustaceans and teleost fishes (Chen et al., 2010; Donpudsa et al., 2010;
Homvises et al., 2010). Several serpins, such as serine proteinase inhibitor, serine/cysteine proteinase inhibitor and alpha1-antitrypsin
were isolated from the SSH library (Table 2). The greater expression
of serpins from O. melastigma challenged with bacteria indicates
that these proteinase inhibitors might function in protecting the
host from bacterial infection.
Kinetics of the APR are such that the quantities of specific transcripts
present in the cell starting an hour or less after the initiating stimulus
and lasting for several days thereafter reflect the ARP ‘status’ of the
organism (Bayne and Gerwick, 2001). It has been suggested that the
APR of fish could be a potential biomarker for environmental insults
(the presence of toxins, other pollutants, pathogens or parasites, or of
reporting other environmental perturbations) and as an index of fish
health status (Bayne and Gerwick, 2001). Molecules that are components of the APR that were obtained from the SSH library, will contribute to improvements in monitoring fish health and predicting the
impact of environmental stresses on fish populations.
4.2. Cellular metabolic process
Ninety eight genes that were involved in the cell metabolic process
were identified from the SSH library, and these genes constituted nearly
one-quarter of the total differentially expressed genes. It is interesting
that some genes, such as apolipoprotein and cathepsin, not only take
part in the cellular metabolic process but are also involved in the
immune process.
Four genes encoding apolipoprotein (apolipoproteins A-I, A-IV, B and
C-I) were up-regulated in the forward SSH library in response to bacterial infection. Apolipoproteins bind to lipids to form lipoproteins, which
transport the lipids through the lymphatic and circulatory systems. The
primary role of apolipoprotein A-I is reverse cholesterol transport, a
pathway by which cholesterol is transported from extra hepatic cells to
the liver for excretion. However, some researchers have demonstrated
that apolipoprotein A-I could play a role in innate defense against bacterial pathogens or virus in teleosts (Franca et al., 2006; Villarroel et al.,
2007; Johnston et al., 2008). Up-regulation of the apolipoprotein family
homology in this study might indicate a possible role of this family
being involved in anti-microbial infection.
Cysteine proteases, including the papain family, are a widespread
group of proteolytic enzymes that catalyze the hydrolysis of many
different proteins and play a role in intracellular protein degradation
and turnover (Ahn et al., 2009). Cathepsin F is a papain-like cysteine
protease that has been shown to play a role in innate defense in
Paralichthys olivaceus when stimulated by LPS (Ahn et al., 2009).
Other members of the cathepsin family such as cathepsin K, and cathepsin B in some fishes are also involved in innate immune response
(Zhang et al., 2008; Harikrishnan et al., 2010). In this study, greater
expression of cathepsin F gene transcripts post V. parahaemolyticus
challenge suggest a putative role in the immune defense against
bacterial infection. The dual functions of apolipoproteins and cathepsin F in O. melastigma, being involved in both metabolism and
immune defense against microbial infection, still need to be further
elucidated.
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Fig. 1. The speculated complement pathways in marine medaka O. melastigma. The stars indicate the genes that have been obtained from our SSH library.
Adapted from the KEGG pathway database.
4.3. Complement gene expression in bacteria-challenged O. melastigma
The complement system is a major part of innate immunity, which is
primarily involved in killing pathogenic microorganisms. In mammals,
the complement system is composed of more than 35 soluble and
membrane-bound proteins that recognize and clear microbes (Holland
and Lambris, 2002; Gonzalez et al., 2007). Fifteen components of
complement system sequences were obtained from our O. melastigma
SSH library. They are marked with stars in Fig. 1 which has formed a
hypothetical platform useful for further validation of complement and
coagulation pathway cascades for O. melastigma.
In mammals and teleosts, C3 is a central complement component and
is part of all the four complement activation pathways, and proceeds
through a lytic pathway that leads to formation of a membrane attack
complex (MAC) including C6–C9, which can directly lyse microbial
cells. Expression of C3-2 was induced gradually in O. melastigma from
6 h to 48 h following bacterial challenge.
In the classical pathway, no transcripts of C1q were detected in male
fish at any of the durations after infection (Table 3). C1 initiates CCP, and
CCP is triggered by binding of antibody to cell surfaces (Holland and
Lambris, 2002). In teleosts, the acquired immune system is not welldeveloped, and it takes longer to activate the CCP pathway. For example,
expression of the three different immunoglobulin genes (IgM, IgD and
IgZ) is significantly different between 2 and 8 weeks after stimulation
with the bacterium Flavobacterium columnare of mandarin fish
(Siniperca chuatsi) (Tian et al., 2009). In Epinephelus coioides, significantly greater expression of IgM gene transcripts was observed at 2, 4 and
5 weeks after infection with the bacterium Vibrio alginolyticus (Cui et
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199
Table 4
Transcriptional profiles for complement genes in V. parahaemolyticus challenged O. melastigma by q-PCR. Expression of genes in the liver was expressed as fold change relative to
the average value of the vehicle control. Data are expressed as mean ± SD (n = 3). ND indicates non-detectable expression.
Gene name
C1q
C3-2
C4
C5
C8
HF
BF
MASP
MBL2
C1 inhibitor
Time of post bacterial challenge (h)
6
12
24
48
ND
0.88 ± 0.32
0.95 ± 0.27
1.29 ± 0.55
0.71 ± 0.20
0.83 ± 0.28
0.76 ± 0.31
1.97 ± 1.42
0.58 ± 0.18
0.44 ± 0.15
ND
1.11 ± 0.48
6.83 ± 1.45⁎
0.95 ± 0.26
0.66 ± 0.23
1.34 ± 0.65
0.83 ± 0.44
1.49 ± 0.28
0.92 ± 0.41
1.54 ± 0.58
ND
2.76 ± 0.36⁎
2.84 ± 0.52⁎
1.16 ± 0.41
1.51 ± 0.62
1.28 ± 0.61
1.53 ± 0.54
0.47 ± 0.18
1.88 ± 1.37
1.77 ± 0.68
ND
1.72 ± 0.65
3.06 ± 1.63
1.53 ± 0.44
1.36 ± 0.40
2.42 ± 0.38⁎
1.76 ± 0.62
0.35 ± 0.06⁎
1.90 ± 1.0
2.14 ± 1.49
⁎ Indicates statistically significant at p b 0.05.
al., 2010). Therefore, it is not unexpected that no expression of C1q
mRNA was observed within 48 h after bacterial challenge. C4 can be
activated through the classical or lectin pathways (Boshra et al., 2006),
and in the present study expression of the C4 gene was significantly
up-regulated 12 h and 24 h following bacterial challenge. It takes longer
for activation of classical pathway after bacterial infection (Tian et al.,
2009; Cui et al., 2010), therefore it is possible that C4 is activated
through the lectin pathway ensuring early stages of response of
bacteria-challenged O. melastigma.
Activation of the alternative pathway is believed to be initiated by
spontaneous hydrolysis of the thiolester bond of C3. Hydrolyzed C3
binds to factor B (BF), making the latter susceptible to proteolytic
attack by factor D, leading to the formation of alternative pathway
C3 convertase from C3 and B. There was no significantly induced BF
from 6 h to 48 h after bacterial challenge. This result may indicate
that activation of alternative pathway is later at the early stages of
responses of O. melastigma to bacteria.
Expression of mRNA for C5 and C8 increase from 6 h to 48 h after bacterial challenge, although there was no statistically significant difference
between the challenged and control fish. Both C5 and C8 are downstream
molecules, which are involved in lysis of cells, and their responses were
detectable after 48 h.
Comparing the pattern of transcription of genes involved in the
three complement activation pathways, it is speculated that activation
of LCP occurs earlier than that of CCP and ACP in O. melastigma challenged with bacteria. However, the complement operating via the ACP
pathway was already competent in the response of hatched larvae of
Danio rerio to a challenge with LPS (Wang et al., 2008). It appears that
the role of the different complement activation pathways is different
due to the mode of exposure or the species studied.
In the lectin activation pathway, mannose-binding lectin (MBL)
binds to the surface of pathogens through mannose-binding lectinassociated serine protease (MASP), which results in opsonization and
antimicrobial protection (Fujita et al., 2004). The results of the current
study showed that expression of MASP was induced during the early
stage (6 h) and subsequently down-regulated in marine medaka after
bacterial challenge (48 h). These results are consistent with those
observed in common carp (Cyprinus carpio L.) after infection with the
parasite Ichthyophthirius multifiliis (Gonzalez et al., 2007). Hosts have
developed several systems including complement component systems
to prevent pathogens. Correspondingly, pathogens have also evolved
multi-strategies to survive. Host–pathogen interactions are a result of
mutual inhibition, evasion and adaptation strategies, and the coevolution of host cationic AMP and microbial resistance also has been
reported (Peschel and Sahl, 2006). Therefore, the depressed expression
of the MASP might be part of the bacterial counter-measure to avoid the
activation of the complement system.
Recently, the complement system has been used to assess immunomodulation in rainbow trout and humans after the host was exposed
by environmental contaminants 17 Beta-estradiol or DDT (bis [4chlorophenyl]-1,1,1-trichloroethane) (Dutta et al., 2008; Wenger et al.,
2011). The results of the present study support the feasibility of developing O. melastigma as an alternative model to understand the basic
biological processes related to immune function in marine fish, however
the immunomodulation of the complement system of O. melastigma
exposed to xenobiotics still needs to be further investigated.
Acknowledgments
This work was partially supported by the Area of Excellence Scheme
under the University Grants Committee of the Hong Kong Special
Administration Region, China (project no. AoE/P-04/2004), the State
Key Laboratory in Marine Pollution (City University of Hong Kong) and
the Hong Kong–France Research Collaboration Grant (No. 9231003) to
D.W.T. Au. Prof. J. Giesy was supported by the Canada Research Chair program, an at large Chair Professorship at the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, City University
of Hong Kong, the Einstein Professor Program of the Chinese Academy
of Sciences and the Distinguished Professor Program of King Saud
University.
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