Beruflich Dokumente
Kultur Dokumente
Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep
Institute for Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Greece
BioMar Hellenic SA, Block no 6, Str no 3-5, 2nd Industrial Zone of Volos, Greece
a r t i c l e
i n f o
Article history:
Received 24 August 2015
Received in revised form
11 November 2015
Accepted 25 December 2015
Keywords:
Sample size
Gene expression
Fish meal replacement
Immune response
Gilthead sea bream
a b s t r a c t
One of the main concerns in gene expression studies is the calculation of statistical signicance which in
most cases remains low due to limited sample size. Increasing biological replicates translates into more
effective gains in power which, especially in nutritional experiments, is of great importance as individual
variation of growth performance parameters and feed conversion is high. The present study investigates
in the gilthead sea bream Sparus aurata, one of the most important Mediterranean aquaculture species.
For 24 gilthead sea bream individuals (biological replicates) the effects of gradual substitution of sh meal
by plant ingredients (0% (control), 25%, 50% and 75%) in the diets were studied by looking at expression
levels of four immune-and stress-related genes in intestine, head kidney and liver. The present results
showed that only the lowest substitution percentage is tolerated and that liver is the most sensitive
tissue to detect gene expression variations in relation to sh meal substituted diets. Additionally the
usage of three independent biological replicates were evaluated by calculating the averages of all possible
triplets in order to assess the suitability of selected genes for stress indication as well as the impact of
the experimental set up, thus in the present work the impact of FM substitution. Gene expression was
altered depending of the selected biological triplicate. Only for two genes in liver (hsp70 and tgf) signicant
differential expression was assured independently of the triplicates used. These results underlined the
importance of choosing the adequate sample number especially when signicant, but minor differences
in gene expression levels are observed.
2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
For gene expression studies today, a wide range of methods is
available. Each of the methods comprises advantages as well as
disadvantages. For low throughput expression analysis the use of
quantitative PCR (qPCR) has been widely applied and the technique has become an important technology in the area of functional
genomics (Vandesompele et al., 2002). In order to obtain statistical signicance in qPCR (i) PCR efciency, (ii) selection of suitable
reference genes as well as (iii) the number of biological replicates
are of importance (Bustin, 2008). While in most qPCR publications
the PCR efciency as well as the selection of appropriate reference
genes are investigated (e.g., De Santis et al., 2011), the number
of required biological replicates are not discussed (Karlen et al.,
Corresponding author at: Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research Thalassocosmos, Gournes Pediados
P.O.Box 2214, 71003 Heraklion, Crete, Greece.
E-mail address: sarris@hcmr.gr (E. Sarropoulou).
1
Present address: Institute of Animal Science, Agricultural Research Organization,
P.O. Box 170, Bet Dagan 50250, Israel.
http://dx.doi.org/10.1016/j.aqrep.2015.12.004
2352-5134/ 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
83
Table 1
Diet composition of the experimental diets.
Ingredients (g/kg)
Feed code
Control
RH30
RH31
RH32
200.00
10.00
40.00
150.00
100.00
162.00
60.22
150.00
40.00
79.00
6.78
140.00
10.00
40.00
150.00
120.00
196.00
65.03
150.00
40.00
80.00
12.97
90.00
10.00
40.00
150.00
135.00
235.00
64.58
150.00
40.00
81.00
18.42
40.00
10.00
41.00
150.00
160.00
265.00
65.49
150.00
41.00
81.00
23.51
Nutrients
Moisture (%)
Protein (%)
Fat (%)
Ash (%)
Cellulose (%)
Starch (%)
Digestible energy (MJ/kg)
9.14
44.00
19.00
5.48
4.21
5.91
17.13
7.77
44.00
18.82
5.33
4.32
6.58
17.15
6.87
44.00
18.57
5.16
4.40
7.00
17.12
5.69
44.00
18.40
5.03
4.54
7.43
17.14
RH = Research Hellas.
in the end of the experiment sh were harvested and the total population was weighed. The duration of the trial was 236 days (9th
September4th May) and temperature, oxygen concentration and
mortalities were recorded daily. Average water temperature was
18.9 C and the range was 14.627.1 C. Feeding rate was predicted
by feeding tables according to the temperature and estimated
biomass increase. The diets were produced by BioMar Hellenic S.A.
(Volos, Greece) and labeled as RH (Research Hellas). Standard
bream diet (Eco Sigma 463 4.5 mm) was used as the control diet
(20% FM) and compared to three experimental diets with decreasing levels of shmeal, a diet with 15% FM (RH30), a diet with 10%
FM (RH31) and a diet with 5% FM (RH32). Thus, the experimental
diets corresponded to 25% (RH30), 50% (RH31) and 75% (RH32) of
FM substitution compared to the control diet. Main ingredients are
shown in Table 1. The experimental period was 6 months and sh
reached the size of 350 g prior to tissue sampling. At the end of the
experiment out of 24 sh per dietary treatment (in total 96) the
distal intestine, head kidney and liver were removed and stored
in RNAlater at 80 C (in total 288 samples) for gene expression
studies.
2.2. RNA extraction and reverse transcription
2. Methods
Animal care was carried out according to the Guidelines for the
treatment of animals in behavioral research and teaching.
The experiment was held in triplicate groups of sea cages
(6 6 6 m) in the sh farm of Platia (Saronic Gulf). In each cage
5.000 graded sh (A-class) were placed after hand-counted and
weighed in the beginning and at the end of the experiment. Average
initial weight of the sh was approximately 125 g. To estimate sh
weight 10% of the population was weighed in the beginning, while
84
Table 2
Gene annotation.
Gene
Abbreviation
Accession #
Reference
Kegg pathway
HSP70
EU805481
Present study
Beta-2 microglobulin
B2M
FM148172
Present study
TGF-1
AF424703
Cathepsin S
CatS
EST database
Present study
IGF1
EF563837.1
ko04612
Antigen processing and presentation
ko04612
Antigen processing and presentation
ko04010 MAPK signaling pathway
ko04060 Cytokine-cytokine receptor interaction
ko04110 Cell cycle
ko04144 Endocytosis
ko04350 TGF-beta signaling pathway
ko04380 Osteoclast differentiation
ko04672 Intestinal immune network for IgA
production
ko05140 Leishmaniasis
ko05142 Chagas disease (American trypanosomiasis)
ko05144 Malaria
ko05145 Toxoplasmosis
ko05146 Amoebiasis
ko05152 Tuberculosis
ko05166 HTLV-I infection
ko05200 Pathways in cancer
ko05210 Colorectal cancer
ko05211 Renal cell carcinoma
ko05212 Pancreatic cancer
ko05220 Chronic myeloid leukemia
ko05323 Rheumatoid arthritis
ko05410 Hypertrophic cardiomyopathy (HCM)
ko05414 Dilated cardiomyopathy
ko04142 Lysosome
ko04145 Phagosome
ko04612 Antigen processing and presentation
ko05152 Tuberculosis
ko04114 Oocyte meiosis
ko04115 p53 signaling pathway
ko04150 mTOR signaling pathway
ko04510 Focal adhesion
ko04730 Long-term depression
ko04914 Progesterone-mediated oocyte maturation
ko04960 Aldosterone-regulated sodium reabsorption
ko05200 Pathways in cancer
ko05202 Transcriptional misregulation in cancer
ko05214 Glioma
ko05215 Prostate cancer
ko05218 Melanoma
ko05410 Hypertrophic cardiomyopathy (HCM)
ko05414 Dilated cardiomyopathy
85
Fig. 2. Gene expression. Expression data for hsp70, b2m, tgf, catS in A: distal intestine,
B: head kidney, C: liver. In liver also expression of IGF1 is shown.
86
4. Conclusion
The gradual substitution of sh meal by a plant mixture showed
signicant changes of the expression levels of four immune related
genes. The present results revealed that only a lower substitution
percentage (RH30) is tolerated and liver was proved to be the most
sensitive tissue in terms of variance in gene expression. However
gene expression was altered depending of the selected biological
triplicate. This study showed that only for two genes in liver (hsp70
and tgf) signicant differential expression was assured independently of the triplicates used and thus can be applied as trustable
marker for immune response using only three biological replicates
as representatives. Concerning the genes studied, 2-M was the
gene mostly affected by dietary treatment, and was found to be
signicantly up-regulated in head kidney and liver. In the distal
intestine only hsp70 was signicantly up-regulated by PP inclusion, which was also observed in cod when sh meal was totally
replaced by PP. Although the present results are robust due to the
high number of biological replicates (24), it should be noted that
they are still to be considered as rst indications on the effect of PP
inclusion in the diets for gilthead sea bream.
Conicts of interest
The authors declare no conict of interest.
Author contributions
E.S. conceived and designed the molecular experiments; F.K., J.K.
and A.S. performed the sh nutrition experiments; F.K. carried out
the molecular experiments, E.S. and F.K analyzed the data; A.S. and
J.K. contributed reagents/materials/analysis tools; E.S, F.K., A.S and
J.K. wrote the paper.
Acknowledgment
Authors would like to thank Ms. Eleni Karamichali, Researcher
at ICS-FORTH for writing scripts of sample size analysis.
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