HUAZHONG AGRICULTURAL UNIVERSITY

DEGREE RESEARCH PLAN AND PROPOSAL

NAME OF THE STUDENT: TRAN NGOC TUAN
STUDENT ID 2011308060001
COLLEGE: COLLEGE OF FISHERIES
MAJOR: AQUACULTURE
DEGREE: Ph.D.
RESEARCH AREA: FISH GENETICS AND BREEDING
THESIS TITLE:

cDNA LIBRARY CONSTRUCTION,

EST ANALYSIS AND EXPRESSION OF IMMUNERELATED GENES IN BLUNT-SNOUT BREAM,
MEGALOBRAMA AMBLYCEPHALA
AFTER
BACTERIA INFECTION
ADVISOR: PROFESSOR WANG WEIMIN
MEMBERS OF GUIDING GROUP:

DOCTOR GAO ZEXIA

PROFESSOR LIU HONG

06/12/2012

The Proposed Research Work, Its Originality and Creativeness

1.1 Work proposed
The blunt-snout bream (Megalobrama amblycephala) is herbivorous and distributes in
the middle portion of Yangtze River (Fishbase 2012). This species is a major freshwater
species and currently cultured in China because of many valuable characteristics, such
as comprising of high delicacy, high larval survival rate, natural foods feeding, fast
growth, tender flesh, high diseases resistance and so on (Tsao 1960; Zhou et al 2008).
The production of blunt-snout bream has increased in recent years, with 552900 tons in
2005 and 625789 tons in 2009, ranked sixth in whole Chinese freshwater fish
production (Ministry of Agriculture of the People's Republic of China 2006; Ministry
of Agriculture of the Peoples Republic of China 2010). The selection on this fish
species was started in 1986 from a population exploited from Yuni Lake, and after 6
generations the fish growth were improved up to 29% (Li and Cai 2000; Li and Cai
2003).
Since 1960s, many studies had been carried out on blunt-snout bream. The previous
researches mainly focused on the improvement on the cultured fish biomass, nutrient
requirement and genetic variations in cultured and wild population. However, studies
on blunt-snout bream diseases which caused great loss in cultured fish are still limited,
especially, on the response of immune-related genes to the pathogens. Therefore, a
detailed study on the expression of immune-related genes of blunt-snout bream is
necessary.
1.2 Originality and creativeness
1.2.1 Construction of cDNA library on the immune-related genes based on next
generation sequence by using PGM system in blunt-snout bream for the first time.
1.2.2 Measurement of the level of immune-related gene expression in blunt-snout
bream after bacterium infection for the first time.

1.

Literature Review
1.1 Overview on blunt-snout bream biological characteristics
Blunt-snout bream (M. amblycephala), also called Wuchang fish, is a member of
finfish Class of Actinopterygii, Order of Cypriniformes and Family of Cyprinidae.
According to The Fish Database of Taiwan (2012), the bream has small, laterally,
compressed and pointed head, short snout, large eyes and the mouth which is small,
protractile, slightly oblique and arched. The fish body compressed and high depth;
dorsal and ventral profile arched; keel on belly between pelvic fin origin and anal fin

origin. The scales are moderately large, cycloid; lateral line complete (L.l.:50-58);
dorsal fin rays (III, 7); pectoral fin rays (1+14-19); pelvic fin rays (1+8); anal fin rays
(3+27-32). Pectoral fin end close to pelvic fin origin; pelvic fin at lower side of body;
anal fin base long; caudal fin forked. Dark grayish dorsally, side and belly silvery
white. All fins are grayish.
The blunt-snout bream prefers to be benthopelagic and habitat in deep freshwater pools
(arrange of 5-20 m) with slow running or can be found in lakes, impoundments and
backwaters of rivers. This is a hypoxia-sensitive species (Shen et al 2010). In China,
the fish commonly distribute in the middle portion of Yangtze River; found in
Newshan Lake (a bay of Liangzi Lake with an area of 256 km2) and Yuli Lake
(Fishbase 2012).
Blunt-snout bream is basically native herbivorous freshwater finfish (Tsao 1960;
Ouyang et al 2001). In natural condition, the fish was defined mainly feed on aquatic
plants such as Vallisneria natans (68.3%), Hydrilla verticillata (33.7%), algae and
zooplankton (Tsao 1960). The protein requirement for fingerling was 35% and lipid 47% (Zhou et al 1997; Li et al 2010). However, protein demand was demonstrated in
correlation with temperature conditions; the optimum protein consumption for fish are
from 27-30.4% at 20oC and from 25.6-41.4% at 25-30oC (Shi et al 1988).
The bream rapidly growth in early period and slowly later in the life (Ouyang et al
2001), in which gaining of 16.4 cm (1 year), 30.7 cm (2 years), 38.8 (3 years), 41.8 cm
(4 years), 41.3 cm (5 years) and 45.5 cm (6 years) (Tsao 1960). The fish are matured
after 2 years with the relative fecundity average 232 egg/g (Ouyang et al 2001).
1.2 Studies on blunt-snout bream
Physiology
The oxy consumption of blunt-snout bream was low in bigger body weight and low
temperature of the water, i.e. the fish weighs of 10.3 g and 295 g consume of 364
mg/kg/h and 193.88 mg/kg/h, respectively (Ouyang et al 2001). This is a
benthopelagic species; dwelling in 5-20 m of water depth, however, the oxygen
consumption was relatively high. In the room temperature of 27oC just for a short time
of hypoxia (below 0.5 mg O 2/L) could cause the mortality of fish (Ouyang et al 2001).
The function of hypoxia-sensitive of fish was identified has an involved to the
expression of two genes, HIF-1 and HIF-2; though, these genes have a different
physiological function under normoxia and hypoxia situations (Shen et al 2010). The
spleen, kidney and liver were developmental places for red blood cells formation (Wu

1990). The activities of two antioxidized enzymes (the glutathione peroxidase (GSHPx) and superoxide dismutase (SOD)) of mitochondria were found in the liver of this
species and defined in being sensitive to the benzidine (1.5 to 2.0 mg/mL) (Dai et al
2003).
Nutrition and nutrient requirement
The non-animal ingredients were suitable for fish digestibility, showing with highest
digested proportion of 85.9% and soybean was more effective with 98.1% in digestion
(Zhou et al 2008). This study confirmed for the herbivorous feeding behavior of this
kind of fish (Tsao 1960) and for the effectiveness in decision of the lower cost diet in
culture this fish species because of protein sources is always play important role in
feed formulate (Wu et al 1995; Li et al 2011). However, the authors recently noticed
that the fish meal protein origin was highest digestibility of the blunt-snout bream
(weight of 712 g), followed by rapeseed and soybean meal (Cao et al 2012).
The protein and lipid requirement for optimum growth of the bream were different in
different period of life. However, the differences is not significantly large, i.e. for the
fingerling which consumes of the diet containing of 31% protein and 7% lipid (Li et al
2010), but for the yearling which were 30% protein and 6% lipid (Jiang et al 2012).
The protein consumption for the fingerling fish was recorded as a relationship to the
change of water temperature conditions which was at 20oC the optimum protein
demand for fish was from 27 to 30.4%, but at 25-30 oC this proportion was from 25.6 to
41.4% (Shi et al 1988). The requirement of bream to the inorganic salts was also
determined; the consumption of calcium, phosphorus, potassium, sodium, magnesium
and iron was 0.31-1.07%, 0.38-0.72%, 0.41-0.57%, 0.14-0.15%, 0.04% and 0.0240.048%, respectively (Shi et al 1997).
The enzymolysis ability in the intestine of fish was measured and resulted in highest in
the posterior intestine, followed by midgut and former bowel in feeding the fish by fish
meal including of diet (Cao et al 2012). Besides, digestive and metabolic enzyme
activities, liver composition and plasma biochemical parameters of blunt snout bream
fingerlings were profoundly determined in effects of dietary with protein and lipid
levels. Supplement of lipid level from 40 to 70 g/kg in diet promotes digestive enzyme
activities and reduces the proportion of protein catabolizing into energy. However,
subtraction of protein level from 350 to 310 g/kg stimulates of lipase activities (in
intestine and liver) and reduces of liver lipid content (Li et al 2011). Otherwise, higher
lipid level in diet formulate may affect to the response of fingerling blunt-snout bream

to the stress by the liver functions and non-immunity (Li et al 2012a). The fish oil
which was supplied into feed formulate was good for growth of bream and the
proportion of 4-6% should be added (Zhou et al 1997). However, to the bream
fingerling silver oil and the soybean oil were defined more effectively for growth than
other oils (Liu et al 1997; Wei and Chen 2003).
Replacement of trifolium for 20-40% in fish diet (15-30% protein) improved the fish
protein content and meat quality (Jia et al 1991). Besides, alfalfa meal was evaluated to
be suitable for the fish growth when supply of 100 g/kg bream feed (Hu et al 2005).
The bream diet which supplied with emodin (60 mg/kg) or Vitamin C (700 mg/kg)
may reduce the FCR and enhance the fish growth and non-specific immunity under the
stress conditions- crowded, temperature and bacterial infection (Ming et al 2011a;
Ming et al 2011b; Ming et al 2012a).
Genetics and breading
From 1960s, domestication of blunt-snout bream was started. In 1986, first selected
generation achieved according to the fish origin from Lake Yuni (the middle of
Yangtze River, China) (Li and Cai 2000; Li and Cai 2003). Surprisingly, the cultured
bream were recorded with earlier maturity, growth rate depression; thin and longer
body and disease susceptibility (Li and Cai 2003; Gao et al 2012). These were
explained by the depression of inbreeding in selected population even though the
growth rate was improved up to 29% (Li and Cai 2003).
The chromosomal number and karyotype formula of blunt-snout bream from Poyang
Lake was the first time classified as 2n= 48 and 2n= 18m+ 26sm+ 4st, respectively
(Ouyang 2000). Otherwise, in a study of Yin et al (1995) the karyotype of blunt-snout
bream is 2n=48, 26m + 18sm + 4st, NF=92. The tetraploidy bream were established
with the percentage of 93.2% of tetraploidy offspring rate by cross preproduction (Zou
et al 2004). Besides, the erythrocyte morphology in polyploidy fish were considered
and completely studied; in general, the differences of erythrocyte morphology in
polyploidy fish may cause a positive or negative response to the cumulative mortality
of cultured fish (Zou et al 2006). In comparison of the fertile autotetraploid (produced
by precise suppression of the first mitotic cleavage of the embryo by thermal shock)
and the interploid 3n (produced by mating of tetraploid females and diploid males) and
the diploid showed differences in quantity of erythrocyte, the diameter of erythrocytes
and nuclei, the erythrocyte surface area and the nuclear and erythrocyte volume (Zou
et al 2006). Moreover, interploid hybrids were spawned from the allotetraploid males

matted to blunt snout bream females which were high in fertilization and stable
survival after 60 days; the increase of growth rate of 7.5% in comparison between
interploid 3n and diploid blunt-snout bream during 180 days (Zou et al 2011).
Many studies on the genetic structure by using of molecular markers on blunt-snout
bream (mtDNA, RAPD, SRAP, microsatellite SSP, SNP) were conducted (Zhang,
2001; Li, 2008; Tang et al 2008; Li, 2010; Gao et al 2012). These markers were
developed for genome analysis to construct the gene-based linkage map and largescale expression analysis of bream or comparative genome analysis for the genus
Megalobrama fish species (Gao et al 2012). In addition, RAPD also was used as a
marker in non- and gynogenetic fry of bream in shocking to the cold temperature
(Zou et al 2001).
Disease infection and immunology
Aeromonas hydrophila was defined as a predominant pathogen infecting to the bluntsnout bream with septicaemia syndrome on the cultured fish in Zhejiang province,
China (Neilsen et al 2001). The A. hydrophila-infected blunt-snout bream syndrome
also was described as hemorrhage on the fish body, the histopathological changes
(vacuolar degeneration and necrosis in the dermis, inflammatory edema and
hemorrhage in the dermis and subcutis) and the lower number of red blood cells and
higher number of white blood cells in haematological physiological indexes (He et al
2006). An anti-viral galectin-like protein was isolated and characterized from bluntsnout bream serum with the amino acid sequence as Lys- Val- Ash- Leu- Asp- GluLys- Cys- Pro- Phe (He et al 2003).
Moreover, the two higher purified fusion protein of Ma-HSC70 and Ma-HSP70 were
also identified in blunt-snout bream; this is a basic for further study on molecular
structure and functions of two proteins and associated antibodies (Ming et al 2012b).
The HSC70 and HSP70 mRNA were higher expression level in the fish after A.
hydrophila infection (Ming et al 2011a).
Toll-like receptors (TLRs) play an important role in sensitivity with microbial infection
and stimulation of inflammatory and immune responses. The TLR3 was sequenced and
expressed from hepatopancreas, midgut, head kidney, heart, muscle, spleen and gill,
especially with highest expression in hepatopancreas and lest in heart and muscle of
the blunt-snout bream (Su et al 2009). TLR4 positive was observed throughout the
digestive tract of the blunt-snout bream, but the TLR4 positive cytoplasmic granular
structures were only observed in the midgut epithelial cells (Zhang et al 2009).

Moreover, the diet for cultured fish were supplied with emodin or Vitamin C may
enhance for higher activities of the serum TP, LSZ, AKP levels, liver SOD and CAT of
blunt-snout bream after A. hydrophila infection (Ming et al 2011a); high dietary lipid
reduces the resistance to A. hydrophila of blunt-snout bream in laboratory condition
(Li et al 2012a).
1.3 Studies on diseases resistance of fish caused by A. hydrophila
The bacterium A. hydrophila belong to the genus of Aeromonas is considered to be
almost synonymous with water and aquatic environments. The bacterium was known
as an opportunistic pathogen in freshwater aquatic habitats with heavily organic load
(Austin and Adams 1996) and in the stress condition (Peters et al 1988). A. hydrophila
has been associated with skin lesions, tail and fin rot, haemorrhagic septicaemia over
the body and tissue destruction, epizootic ulceration and necrosis in the liver and
kidney (Austin and Adams 1996; Janda and Abbout 2010).
A. hydrophila were recorded widely infecting to freshwater fish and marine fish
species (Austin and Adams 1996; Doukas et al 1998). This bacterium was isolated in
carp, Cyprinus carpio (Chiril et al 2008), goldfish, Carrassius auratus and Koi, C.
carpio koi (Citarasu et al 2011), and determined as a main pathogen infected to
rainbow trout, Salmo gairdneri (Peters et al 1988), walking catfish, Clarias batrachus
(Angka et al 1990), goldfish (Iqbal et al 1999), Nile tilapia, Oreochromis niloticus
(Ibrahem et al 2008).
Many previous studies have been conducted on the resistance to disease caused by A.
hydrophilla based on nonspecific immunity functions, including the interspecies
variations

among

silver

carp

(Hypophthalmichthys

molitrix),

bighead

carp

(Aristichthys nobilis), grass carp (Ctenopharyngodon idellus) and black carp

(Mylopharyngodon piceus); the intra-species variations among three strains of
common carp (wild C. carpio, mirror C. carpio specularis and jian common carp C.
carpio var. jian), three strains of crucian carp (Pengzhe crucian carp C. auratus var.
pengzesis, allogynogenetic crucian carp C. auratus ibelio and white crucian carp C.
auratus cuvieri), and two populations of soft-shelled turtle (Trionyx sinensis) from
Taihu lake of Yangtze River basin and Taiwan (Cai et al 2004).
The liver of Indian carp, Cirrhinus mrigala, which was infected to A. hydrophila, was
defined as a specific tissue of the transferrin gene expression due to transferrin mRNA
was detected only in liver RNA and to lesser extent in brain tissue (Sahoo et al 2009).
In another study, after families of Atlantic salmon were challenged by A. salmonicida ,

in lymphoid organs the pro-inflammatory genes (TLR5M, TLR5S, GATA3, IFN-g, IL17D) and pleiotropic cytokine gene IL-10, showed higher expression in the resistant
family (Zhang et al 2011). Besides, the IL-1 gene was suggested important in the
regulation of the Atlantic salmon, Salmo salar L. immune response against amoebic
gill disease (AGD) (Bridle et al 2006).
To rohu, Labeo rohita, MH class I and galactoside-binding soluble lectin were defined
as putative functions affecting innate and acquired immune response in a mixture of A.
hydrophila-susceptible and -resistant individuals (Robinson et al 2012). Otherwise,
some immune-related genes (IL-1b, iNOS, C3, CXCa and C-type and G-type
lysozymes genes) were up-regulated at 6-12h post-challenge and 2-microglobulin and
TNF genes were down- regulated after 48h post-challenge of Edwardsiella tarda
(Mohanty and Sahoo 2010). In other study with infection of A. hydrophila to ayu
(Plecoglossus altivelis), the results showed that the liver proteins and NKEF-B mRNA
were closely associated with A. hydrophila infection by the increase of expression in
many tissues at the early stage of bacterial infection (Chen et al 2009).
A double intraperitoneal injection experiment of the bacterium, A. hydrophila to
gilthead seabream (Sparus aurata L.), authors suggested that a relatively fast
elimination of the bacteria and recovery of fish during the secondary infection. This
based on the down-regulated of transcript levels of TLR, NCCRP-1, HEP, TCR, IgM,
MHC-II, C3 and CSF-1R and up-regulated of IL-1 genes during the first infection,
but mRNA expression levels were up-regulated during the re-infection (Reyes-Becerril
et al 2011). The prophenoloxidase (proPO) gene was determined to be related to the
immune response in red swamp crayfish, Procambarus clarkia, after challenge to A.
hydrophila, showing increase of expression level in tissue samples, especially highest
in haemocytes, but with no expression in ovary (Li et al 2012b). Furthermore, three Ltype lectin (ERGIC-53, VIP36 and VIP36-like) genes also confirmed involving to the
immune responses of channel catfish, Ictalurus punctatus, after E. ictaluri infection
(Zhang et al 2012).
Selective breeding has been carried out to improve resistance to infectious diseases in
aquaculture; the results demonstrated that a potential for genetic improvement of
disease resistance in aquatic animals against a specific pathogen is reality (Cai et al
2004). A study on the potential correlation of a variety of specific and nonspecific
immune parameters with aeromoniasis resistance in 13 different fullsib families of
rohu, L. rohita, was done in laboratory conditions. The results concluded that

bactericidal activity is more important in resistance against A. hydrophila infection

(Sahoo et al 2004). Besides, Sahoo et al (2011) confirmed that some of innate immune
parameters

(phagocytic

functional

activities

viz.,

respiratory

burst

and

myeloperoxidase activities and serum ceruloplasmin level) can be indirectly used as

marker traits for selection for improved resistance to aeromoniasis in rohu. In a while,
the serum lysozyme activity, haemagglutination titre and specific antibody titre were
raised in the E. tarda-infected rohu, L. rohita (Mohanty and Sahoo 2010). Moreover,
phagocytic activity of leukocytes, plasma lysozyme activity and specific antibody titre
were evaluated as more effective in the resistant families of common carp, C. carpio in
comparison to the sensitive ones (Ard et al 2010). Furthermore, complement and non2m-antiprotease activities play an important role in resistance against A. salmonicida
of Atlantic salmon, S. salar (Marsden et al 1996; Zhang et al 2011). In addition,
respiratory burst and peroxidase activity indicated a recovery of the immune system
against A. hydrophila of gilthead seabream (S. aurata L.) (Reyes-Becerril et al 2011).
Studiese on the genetic variation in the immune response of fish were conducted. In
Nile tilapia, O. niloticus, when a fully inbred clone susceptible to A. hydrophila was
crossed with a resistant full inbred clone, the resulting progeny exhibited intermediate
levels of resistance to that of their parents (Sarder et al 2001). In addition, hybrid
(female Nile tilapiamale blue tilapia) showed the highest resistance to the disease
caused by A. sobria in comparison to Nile tilapia, O. niloticus, and blue tilapia, O.
aureus (Cai et al 2004).

1 Objectives and Significance of the Proposed Research

3.1 Objectives
The main objective of this study is to study the expression of the important immunerelated genes in the larger context of the blunt-snout bream transcriptome following A.
hydrophila infection, and then confirm the expression pattern of selected genes of
interest by quantitative real-time reverse-transcription polymerase chain reaction (qRTPCR).
3.2 Significance
Our study will study the expression changes of immune-related genes in the challenged
fish in the transcriptome level and make a comparison with that in control fish. This
result may be applied for further fish selection of disease resistance generations.

4. Experimental Materials, Methods and Technical Approaches

4.1 Materials

Bacterium: A. hydrophila

Healthy blunt-snout bream fingerlings

Kits: TRIzol Reagent (Invitrogen), Turbo DNA-freeTM (Ambion), RNeasy Mini kit
(Qiagen), QIAquick PCR Purification Kit (Qiagen), Trimmer-2 kit (Evrogen), Ion
Total RNA-Seq Kit v2 (Invitrogen), Ion OneTouchTM 200 Template kit
(Invitrogen), Ion Sequencing kit (Invitrogen), Ion 318TM Chip (Invitrogen),
TaqMan Universal Master Mix II with UNG (Applied Biosystems).

4.2 Methods
4.2.1 Experimental fish and bacteria strain
Healthy fish fingerlings of blunt-snout bream was collected from the cultured pond in
E Zhou, Hubei province, China. Fish was cultured two weeks with the temperature of
the water about 27oC before challenge experiment. A total of 250 fish were randomly
divided to two groups (control and injected experiment) of 2 tanks for each.
The bacterial strain isolated from septicemia blunt-snout bream was cultured on the
tryptone soya broth, centrifuged, rinsed and diluted in sterile physiological saline. The
concentration of suspension was spectrophotometerically determined at 610 nm.
Experimental fish were intraperitoneally injected with 0.1 mL a suspension of 10 7 cfu
mL-1 live cell of A. hydrophila. The same volume of sterile physiological saline was
injected into control fish. The cumulative mortality of experimental fish was observed
during 5 days post-injection. Moribund fish with clinical signs were re-isolated and reidentificated the bacteria.
4.2.2 Sampling and RNA extraction
Tissues of 30 fish (muscle, hearth, kidney, spleen and liver) separately set into 3 pools
with 10 fish per pool from control and challenged fish were collected in 0h, 24h, 3 and
5 days post injection with bacterium. All of the samples were immediately stored into
sample protection solution (Takara) and stored at -80oC until total RNA extraction.
Total RNA of these samples was extracted using TRIzol Reagent (Invitrogen, USA)
according to the manufacturers instructions, following by RNA precipitation, RNA
washing and RNA resuspension.
The quality and quantity of total RNA was determined using a Nanodrop 2000
(Thermo Scientific, USA). Only RNA samples with a ratio of 260 of 280 from 1.9 to
2.1 and a ratio 260 of 230 from 2.0 to 2.5 were used for following analysis. The
samples were standardized to 200ng mL-1 and then equal volumes from each tissue
samples of different individuals were combined into a pool. The pools were DNasetreated with Turbo DNA-freeTM (Ambion) and purified with RNeasy Mini Kit (Qiagen,

USA) following the manufacturers instructions. RNA quality and quantity was
determined again at the end of the process.
4.2.3 cDNA synthesis and Ion PGM sequencing
The RNA pools were used for double-strand cDNA synthesis using SMART
technology (BD Biosciences Clontech), and then the cDNA was purified using the
QIAquick PCR Purification Kit (Qiagen).Full-length cDNA sample containing equal
amounts of cDNA from each library was normalized using the Trimmer-2 kit
(Evrogen, Russia) according to the manufacturers instructions, following by step to
step of cDNA precipitation, cDNA denaturation and hybridization and amplification of
normalized cDNA. The normalized cDNA was sequenced using the Ion PGM
sequencing system following to the manufacturers instruction.
4.2.4 Bioinformatics analysis
Short sequences (<100bp in length) or poor quality sequences were eliminated. High
quality ESTs were analyzed with computer software. All of the unique sequences were
subjected to the non-redundant (nr) protein and nucleotide database with BLASTx and
BLASTn program searching for functional annotation and sequence homology was
accepted as E-value < 1e-5. Unique sequences were clustered into categories using
Cluster of Orthologous Groups of proteins (COG). Gene Ontology (GO) annotions
were assigned using program Gopipe according to its uniprot accession number. In
addition, the unique sequences associated with the innate immune response were
identied by sequence alignment according to the previously public report (Duan et al
2012).
4.2.5 Quantitative real-time PCR
The qRT-PCR protocols were performed based on the StepOnePlus TM Real-Time PCR
system (Applied Biosystems). The TaqMan Universal Master Mix II (Applied
Biosystems) was used and followed by the manufacture protocol. Primers and probes
can be designed using Primer Express Software as described in the Primer Express
Software Version 3.0. The reactions contained 5 L TaqMan Universal Master Mix
with AMPerase UNG, 300 nM forward and reverse primer, 100 nM TaqMan probe, 3
L of cDNA and water up to a final volume of 10 L. All samples were run in
triplicate. The standard cDNA template was included in each plate in order to monitor
the reproducibility of the qRT-PCR assays. The cycling conditions were: initial
incubation steps at 50oC for 2 min (AmpErase UNG activation), and at 95oC for 10
min (hot-start DNA polymerase activation), followed by 40 cycles of 15 s at 95oC and

1 min at 60oC. The raw qPCR data were analysed with StepOne Software v2.0
(Applied Biosystems) (Holopainen et al 2012).
4.3 Technical approaches
Sampling and RNA extraction
cDNA synthesisIon PGM sequencing

EST analysis

Target gene determination

Gene expression level estimation by qRT-PCR
Gene expression

4.3 Pitfalls and alternatives

4.3.1 Experimental fish is not enough for sampling, so need to introduce more fish for
challenge test experiment.
4.3.2 Construction of cDNA library is not completed; need to prepare for more
samples.

5. The Knowledge and Technological Bases for the Proposed Research

I learned and practiced for the molecular technology, especially in the PCR technique
when I was in undergraduate and graduate study program.

6. Work completed or Progresses to date

I have finished for the challenged test and sampling tissues.

7. Expected Outcome
7.1 Research achievements (Germplasm, genes, knowledge etc)
-

cDNA library will be constructed.

Immune-related genes expression level will be measured.

7.2 Publications and patents

I will publish for 1 paper.

8. Timetable for the Proposed Research

-

08/2012-11/2012: searching and collecting for journals and determining the study

orientation.
12/2012-01/2013: challenge test, RNA extraction and cDNA synthesis.
03/2013-07/2013: expression of immune-related genes, writing and publishing papers.

9. References
1.

Angka SL. The Pathology of the walking catfish Clarias batrachus (L.), infected
intraperitoneally with Aeromonas hydrophila. Asian Fisheries Science, 1990, 3:343-351

2.

Ard L, Jeney Z, Adams A, Jeney G. Immune responses of resistant and sensitive

common carp families following experimental challenge with Aeromonas hydrophila.
Fish & Shellfish Immunology, 2010, 29:111-116

3.

Austin B, Adams C. Fish pathogens, In Austin B, Altwegg M, Gosling PJ, Joseph S eds.,
The genus Aeromonas. John. Wiley & Sons Ltd., West Sussex, England, 1996.197-243

4.

Bridle AR, Morrison RN, Cunningham PMC, Nowak BF. Quantitation of immune
response gene expression and cellular localisation of interleukin-1 mRNA in Atlantic
salmon, Salmo salar L., affected by amoebic gill disease (AGD). Veterinary Immunology
and Immunopathology, 2006, 114:121-134

5.

Cai WQ, Li SF, Ma JY. Diseases resistance of Nile tilapia (Oreochromis niloticus), blue
tilapia (Oreochromis aureus) and their hybrid (female Nile tilapia male blue tilapia) to
Aeromonas sobria. Aquaculture, 2004, 229:79-87

6.

Cao XL, Chen JJ, Liu HJ, Wang JL, Nie GX. Study of enzymolysis kinetics with four
kinds of protein feeds in Megalobrama amblycephala in vitro. Journal of Animal and
Veterinary Advances, 2012, 11(9):1378-1384

7.

Chen J, Wu HQ, Niu H, Shi YH, Li MY. Increased liver protein and mRNA expression of
natural killer cell-enhancing factor B (NKEF-B) in ayu (Plecoglossus altivelis) after
Aeromonas hydrophila infection. Fish & Shellfish Immunology, 2009, 26:567-571

8.

Chiril F, Fit N, Nads G, Negrea O, Ranga R. Isolation and characterization of an

Aeromonas hydrophila strain in a crap (Cyprinus carpio) toxemia focus. Bulletin
UASVM, Veterinary Medicine, 2008, 65(1):244-247

9.

Citarasu T, Alfred DK, Velmurugan S, Thanga VV, Kumaran T, Michael BM, Selvaraj T.
Isolation of Aeromonas hydrophila from infected ornamental fish hatchery during massive
disease outbreak. International Journal of Current Research, 2011, 2(1): 037-041

10. Dai J, Mei QM, Zhou PJ, Liu H. The influence of benzidine on the activities of the
antioxidative enzymes and their isoenzymes of the mitochondria. China Environmental
Science, 2003, 23(4):427-430
11. Doukas V, Athanassopoulou F, Karagouni E, Dotsika E. Aeromonas hydrophila infection
in cultured sea bass, Dicentrarchus labrax L., and Puntazzo puntazzo Cuvier from the
Aegean Sea. Journal of Fish Diseases, 1998, 21:317-320
12. Duan YF, Liu P, Li JT, Li J, Chen P. Immune gene discovery by expressed sequence tag
(EST) analysis of hemocytes in the ridgetail white prawn Exopalaemon carinicauda. Fish
& Shellsh Immunology, 2012, xxx:1-10
13. Fishbase, 2012. http://www.fishbase.org/summary/Megalobrama-amblyceph ala.html
(online: 08/10/2012)

14. Gao ZX, Luo W, Liu H, Zeng C, Liu XL, Yi SK, Wang WM. Transcriptome analysis and
SSR/SNP markers information of the blunt snout bream (Megalobrama amblycephala).
PLoS ONE, 2012, 7(8):e42637
15. He JH, Zou SX, Lu CP. Isolation and characterization of an anti-viral galectin-like protein
from Megalobrama amblycephala serum. Journal of Fisheries of China, 2003, 27(5):474479
16. He LJ, Liao LK, Yuan JF, Tang HY, Wu Q, Zhang GW. Pathological observation of
bacterial septicemia in Megalobrania amblycephala. Journal of Southwest Agricultural
University (Natural Science), 2006, 28(3):483-490
17. Holopainen R, Tapiovaara H, Honkanen J. Expression analysis of immune response genes
in sh epithelial cells following ranavirus infection. Fish & Shellsh Immunology, 2012,
32:1095-1105
18. Hu XF, Wang CZ, Zhang CM, He Y, Liu QW, Wang YF. Study on effect of alfalfa meal
on growth performance and quality of fish of bluntnose black bream (Megalobrama
amblycepnala). Jour. of Northwest Sci-Tech Univ. of Agri. and For. (Nat. Sci. Ed.), 2005,
33(11):49-56
19. Ibrahem, MD, Mostafa MM, Arab RMH, Rezk MA. Prevalence of Aeromonas hydrophila
infection in wild and cultured tilapia nilotica (O. niloticus) in Egypt. 8th International
Symposium on Tilapia in Aquaculture, 2008, Cairo, Egypt
20. Iqbal, MM, Tajima K, Ezura Y. Pathogenicity of motile Aeromonas species isolated from
fishes with Epizootic Ulcerative Syndrome (EUS) in Southeast Asian Countries. Bull.
Fac. Fish. Hokkaido Univ., 1999, 50(2):93-100
21. Janda JM, Abbout SL. The Genus Aeromonas Taxonomy, Pathogenicity, and Infection.
Clinical Microbiology Reviews, 2010, 23(1):35-73
22. Jia LZ, He XQ, Yang YX. A preliminary study on partial replacement of fish meal and
soybean cake meal by Trifolium in diets for blunt snout bream (Megalobrama
amblycephala) fingerlings. Acta Hydrobiologica Sinica, 1991, 15:91-93
23. Jiang YY, Li XF, Liu WB, Wu Y, Li GF, Zhu H. Effects of different protein and lipid
levels on the growth performance and body composition of blunt snout bream
(Megalobrama amblycephala) yearlings. Acta Hydrobiologica Sinica, 2012, 36(5):826836
24. Li HH. MtDNA sequence variation and genetic structure of the Megalobrama
amblycephala from Yuni Lake, Liangzi Lake and Poyang Lake. Freshwater Fish, 2008,
38(4):63-65

25. Li SF, Cai WQ. Genetic improvement of the herbivorous blunt snout bream
(Megalobrama amplycephala). Naga, WorldFish Center Quaterly, 2003, 26(1):20-23
26. Li SF, Cai WQ. Two-way selective response of Megalobrama amblycephala. Journal of
Fisheries of China, 2000, 24(3):201-205
27. Li XF, Jiang YY, Liu WB, Ge XP. Protein-sparing effect of dietary lipid in practical diets
for blunt snout bream (Megalobrama amblycephala) fingerlings: effects on digestive and
metabolic responses. Fish Physiol Biochem, 2011, DOI 10.1007/s10695-011-9533-9
28. Li XF, Liu WB, Lu KL, Xu WN, Wang Y. Dietary carbohydrate/lipid ratios affect stress,
oxidative status and non-specific immune responses of fingerling blunt snout bream,
Megalobrama amblycephala. Fish & Shellfish Immunology, 2012a, 33:316-323
29. Li Y. Genetic structure analysis of three wild Megalobrama amblycephala populations
and construction of genetic linkage map. (Ph D dissertation). Huazhong Agricultural
University, 2010
30. Li YH, Deng W, Yang KL, Wang WM. The expression of prophenoloxidase mRNA in red
swamp crayfish, Procambarus clarkii, when it was challenged. Genomics, 2012b, 99:355360
31. Liu W, Dai NH, Ren BG, Gong GM. Effects of different lipid diets on the growth of
juvenile Megalobrama amblycephala. Journal of Fisheries of China, 1997, 21:44-48
32. Marsden MJ, Freeman LC, Cox D, Secombes CJ. Non-specific immune responses in
families of Atlantic salmon, Salmo salar, exhibiting differential resistance to furunculosis.
Aquaculture, 1996, 146:1-16
33. Ming JH, Xie J, Xu P, Ge XP, Liu WB, Ye JY. Effects of emodin and vitamin C on growth
performance, biochemical parameters and two HSP70s mRNA expression of Wuchang
bream (Megalobrama amblycephala Yih) under high temperature stress. Fish & Shellfish
Immunology, 2012a, 32:651-661
34. Ming JH, Xie J, Xu P, Ge XP, Liu WB, Ye JY. Prokaryotic expression, purification and
identification of two HSP70S in Wuchang bream Megalobrama amblycephala.
Oceanologia Et Limnologia Sinica, 2012b, 43(1):185-191
35. Ming JH, Xie J, Xu P, Liu WB, Ge XP, Liu B, Zhou QL. Effects of emodin, Vitamin C
and their combination on crowding stress resistance of Wuchang bream (Megalobrama
amblycephala Yih). Acta Hydrobiologica Sinica, 2011a, 35(3):400-413
36. Ming JH, Xie J, Xu P, Liu WB, Zhou QL, Liu B, Xi BW. Effects of emodin, vitamin C
and their combination on biochemical parameters and two HSP70s mRNA expressions of
Wuchang bream (Megalobrama amblycephala) infected with Aeromonas hydrophil.

Journal of Fishery Sciences of China, 2011b, 18(3):588-601

37. Ministry of Agriculture of the Peoples Republic of China. Chinese fisheries yearbook.
Chinese Agricultural Press, Beijing, 2006
38. Ministry of Agriculture of the Peoples Republic of China. Chinese fisheries yearbook.
Chinese Agricultural Press, Beijing, 2010
39. Mohanty BR, Sahoo PK. Immune responses and expression profiles of some immunerelated genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection. Fish &
Shellfish Immunology, 2010, 28: 613-621
40. Nielsen ME, Hi L, Schmidt AS, Qian D, Shimada T, Shen JY, Larsen JL. Is Aeromonas
hydrophila the dominant motile Aeromonas species that causes disease outbreaks in
aquaculture production in the Zhejiang Province of China? Dis Aquat Org, 2001, 46:2329
41. Ouyang M, Chen DY, Yu X. Study on biology of Megalobrama amblycephala in Poyang
Lake. Acta Agriculturae Jiangxi, 2001, 13(1):47-50
42. Ouyang M. Study on the karyotype of Megalobrama amblycephala from Poyang Lake.
Acta Agriculturae Jiangxi, 2000, 2(2):61-64
43. Peters G, Faisal M, Lang T, Ahmed I. Stress caused by social interaction and its effect on
susceptibility to Aeromonas hydrophila infection in rainbow trout Salmo gairdneri. Dis.
Aquat. Org., 1988, 4:83-89
44. Reyes-Becerril M, Lpez-Medina T, Ascencio-Valle F, Esteban MA. Immune response of
gilthead seabream (Sparus aurata) following experimental infection with Aeromonas
hydrophila. Fish & Shellfish Immunology, 2011, 31:564-570
45. Robinson N, Sahoo PK, Baranski M, Mahapatra KD, Saha JN, Das S, Mishra Y, Das P,
Barman HK, Eknath AE. Expressed Sequences and Polymorphisms in Rohu Carp (Labeo
rohita, Hamilton) Revealed by mRNA-seq. Mar Biotechnol, 2012, DOI 10.1007/s10126012-9433-8
46. Sahoo PK, Meher PK, Mahapatra KD, Saha JN, Jana RK, Reddy PVGK. Immune
responses in different fullsib families of Indian major carp, Labeo rohita, exhibiting
differential resistance to Aeromonas hydrophila infection. Aquaculture, 2004, 238:115125
47. Sahoo PK, Mohanty BR, Kumari J, Barat A, Sarangi N. Cloning, nucleotide sequence and
phylogenetic analyses, and tissue-specific expression of the transferrin gene in Cirrhinus
mrigala infected with Aeromonas hydrophila. Comparative Immunology, Microbiology
and Infectious Diseases, 2009, 32:527-537

48. Sahoo PK, Rauta PR, Mohanty BR, Mahapatra KD, Saha JN, Rye M, Eknath AE.
Selection for improved resistance to Aeromonas hydrophila in Indian major carp Labeo
rohita: Survival and innate immune responses in first generation of resistant and
susceptible lines. Fish & Shellfish Immunology, 2011, 31:432-438
49. Sarderb MRI, Thompsona KD, Penmana DJ, McAndrewa BJ. Immune responses of Nile
tilapia (Oreochromis niloticus L.) clones: I. Non-specific responses. Developmental and
Comparative Immunology, 2001, 25:37-46
50. Shen RJ, Jiang XY, Pu JW, Zou SM. HIF-1and -2 genes in a hypoxia-sensitive teleost
species Megalobrama amblycephala: cDNA cloning, expression and different responses
to hypoxia. Comparative Biochemistry and Physiology, Part B, 2010, 157:273-280
51. Shi WL, Liu MZ, Lu MY, Huang FQ. Studies on requirement of Megalobrama
amblycephala for several main inorganic salts. Journal of Fisheries of China, 1997,
21(4):458-461
52. Shi WL, Shan J, Liu MZ, Yan H, Huang FQ, Zhou XW, Shen L. A study of the optimum
demand of protein by blunt snout bream (Megalobrama amblycephala). FAO library,
1988, Accession no: 289611
53. Su JG, Zhu ZY, Wang YP. cDNA cloning and characterization of toll-like receptor 3 in
bluntnose black bream, Megalobrama amblycephala. Acta Hydrobiologica Sinica, 2009,
33(5):986-993
54. Tang SJ, Li SF, Cai WQ. Development of microsatellite markers for blunt snout bream
Megalobrama amblycephala using 5-anchored PCR. Permanent Genetic Resources Note,
2008, doi: 10.1111/j.1755-0998.2009.02520.x
55. The Fish Database of Taiwan, 2012. http://fishdb.sinica.edu.tw/eng/speci es.php?
science=Megalobrama%20amblycephala (online: 08/10/2012)
56. Tsao WS. A biological study of Magalobrama amblycephala and M. terminalis of Liangtse Lake. Acta Hydrobiologica Sinica, 1960, 57-78
57. Wei XY, Chen SS. The influence of different fat sauce in dietary on blunt snout bream
growth and ingredients. Technical Research, 2003, 3:13-16
58. Wu JK, Yong WY, You WZ, Wen H, Liao CX, Wu DH. Apparent digestibility and
digestible energy of 12 feedstuffs for bluntnose black bream (Megalobrama
amblycephala Yih). Journal of Fisheries of China, 1995, 2(3):55-62
59. Wu WN. The formation of blood cell in Megalobrama amblycephala Yih. Journal of
Fisheries of China, 1990, 14(4):328-335

60. Yin HB, Fan ZT, Sun ZW, Pan F, Meng FR. The Karyotype and DNA content analysis of
blunt snout bream (Megalobrama amblycephala). Chinese Journal of Fisheries, 1995,
8(1):22-26
61. Zhang D. Study on genetic diversity of bluntnose black bream from Yunihu and Liangzi
lakes. J China Three Gorges Univ., 2001, 3:282-284
62. Zhang GY, Wang Y, Peng KM, Wang C, Tian G, Liu HZ. Strategic Localization of Tolllike Receptor 4 in the Digestive Tract of Blunt Snout Bream (Megalobrama
amblycephala). Anat. Histol. Embryol, 2009, 38:401-405
63. Zhang H, Peatman E, Liu H, Feng TT, Chen LQ, Liu ZJ. Molecular characterization of
three L-type lectin genes from channel catfish, Ictalurus punctatus and their responses to
Edwardsiella ictaluri challenge. Fish & Shellfish Immunology, 2012, 32: 598-608
64. Zhang ZB, Niu CJ, Storset A, Bgwald J, Dalmo RA. Comparison of Aeromonas
salmonicida resistant and susceptible salmon families: A high immune response is
beneficial for the survival against Aeromonas salmonicida challenge. Fish & Shellfish
Immunology , 2011, 31:1-9
65. Zhou WY, Yu CY, Liu JZ, Yang GH. Effects of quality and quantity of fat in diet on
growth of blunt snout bream (Megalobrama amblycephala). Fisheries Science &
Technology Information, 1997, 24(1):3-9
66. Zhou Z, Ren Z, Zeng H, Yao B. Apparent digestibility of various feedstuffs for bluntnose
black bream Megalobrama amblycephala Yih. Aquaculture Nutrition, 2008, 14:153-165
67. Zou SM, Li SF, Cai WQ, Yang HY, Jiang XY. Ploidy level, morphological characteristics
and performance of interploid hybrids by Megalobrama amblycephala allo-tetraploids
. Aquaculture Research, 2011, 42:1687-1693
68. Zou SM, Li SF, Cai WQ, Yang HY. Morphological differences of erythrocyte in
autotetraploid, interploid 3n of blunt snout bream, Megalobrama amblycephala. Journal
of Fishery Sciences of China, 2006, 13(6):891-896
69. Zou SM, Li SF, Cai WQ, Zhao JL, Yang HY. Establishment of fertile tetraploid
population of blunt snout bream (Megalobrama amblycephala). Aquaculture, 2004,
238:155-164
70. Zou SM, Li SF, Cai WQ, Zhao JL. Establishing gynogenetic groups of genetic improved
Megalobrama amblycephala and its genetic analysis. Journal of Fisheries of China, 2001,
25(4):311-316

10. Proposed Budget

Expenditures

Amount (Yuan)

Reasons

TRIzol Reagent

3,000

RNA extraction

Turbo DNA-freeTM Kit

1,400

Removing contaminated DNA

RNeasy Mini Kit

3,000

mRNA purification

1,000

cDNA purification

Trimmer-2 Kit

5,000

cDNA normalization

Ion Total RNA-Seq Kit v2

15,000

cDNA library construction

20,000

Enrichment of cDNA library

Ion Sequencing Kit

15,000

Shotgun sequencing

Ion 318TM Chip

13,000

Sequencing 300 Mb to 1 Gb

QIAquick PCR
Purification Kit

Ion OneTouchTM 200

Template Kit

TaqMan Universal
Master Mix II with UNG
Total

5,000

qRT-PCR to express of
immune-related genes
81,400 Yuan

Comments and Recommendation of Advisor:

The proposal contents are realistic and should be studied

The expected results will provide more information for subsequent studies on
diseases resistance selection for blunt-snout bream families in future

Advisor Signature:
Date:
Comments and recommendation of the Guiding Group:
-

The proposal plan will be performed basing on applying new gene technologies.

The proposal works can be carried out and completed.

The proposal plan should be remained and performed.

Leader of Guiding Group Signature

Date:
Members of Guiding Group
Name

Title

Major

Liu Hong

Professor

Aquaculture

Yan Ansheng

Professor

Aquaculture

Zhou Jie

Professor

Aquaculture

Cao Keju

Professor

Aquaculture

Xiong Bangxi

Professor

Aquaculture

Chen Xiaoxuan

Professor

Aquaculture

Gong Shiyuan

Professor

Aquaculture

Unit of Work

College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University
College of Fisheries,
Huazhong Agricultural University

Approval of the College:

Executive Dean Signature:

Stamp of College