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The effects of Natucin C-Natucin P mixture on blood biochemical parameters, antioxidant activity and non-specific immune responses in tilapia (Oreochromis niloticus)
Accepted Manuscript The effects of Natucin C-Natucin P mixture on blood biochemical parameters, antioxidant activity and non-specific immune responses in tilapia (Oreochromis niloticus) Yi-Bin Chen, Juan Hu, Qing-Ji Lv, Li-Jie Liu, Liu-Fa Wen, Xian-Kuan Yang, Hui-Hong Zhao PII:
S1050-4648(16)30376-X
DOI:
10.1016/j.fsi.2016.06.016
Reference:
YFSIM 4016
To appear in:
Fish and Shellfish Immunology
Received Date: 29 April 2016 Revised Date:
3 June 2016
Accepted Date: 8 June 2016
Please cite this article as: Chen Y-B, Hu J, Lv Q-J, Liu L-J, Wen L-F, Yang X-K, Zhao H-H, The effects of Natucin C-Natucin P mixture on blood biochemical parameters, antioxidant activity and non-specific immune responses in tilapia (Oreochromis niloticus), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.06.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The effects of Natucin C- Natucin P mixture on blood biochemical
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parameters, antioxidant activity and non-specific immune responses
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in tilapia (Oreochromis niloticus)
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Yi-Bin Chena, Juan Hua, Qing-Ji Lva, Li-Jie Liub, Liu-Fa Wenc, Xian-Kuan Yanga,
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Hui-Hong Zhaoa, ∗
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a
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510642, Guangdong Province, PR China
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b
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Biotechnology, Shantou University, Shantou 515063, Guangdong Province, PR China
College of Marine Sciences, South China Agricultural University, Guangzhou
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Marine Biology Institute & Guangdong Provincial Key Laboratory of Marine
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c
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510642, Guangdong Province, PR China
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Abstract: Natucin C (NC) and Natucin P (NP) are two kinds of antimicrobial
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peptides (AMPs). In the present study, the effects of NC-NP mixture on a tilapia
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species (Oreochromis niloticus) were examined. Animals were fed with either a
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control diet or one of five AMP-supplemented diets for eight weeks.
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AMP-supplemented diets contained five increasing levels of NP from G1 to G5 and
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one level of NC (200 mg/kg). Results showed that fish in the G3, G4 and G5 groups
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had significantly higher levels of total protein (TP), albumin (ALB) and globulin
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(GLO) in serum than fish in the control group. Fish fed with G4 and G5 diets
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exhibited significantly higher high-density lipoprotein cholesterol (HDL-C) levels
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compared
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AMP-supplemented groups were significantly lower than the control. In addition, the
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total antioxidant capacity (TAOC) and lysozyme (LZM) activities were significantly
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increased in fish fed with the G3 and G4 diets, respectively compared to the control.
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The serum malondialdehyde (MDA) levels in fish fed with AMP-supplemented diets
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were significantly decreased compared to those not supplemented with AMPs.
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College of Animal Science, South China Agricultural University, Guangzhou
the
control
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Lipopolysaccharide
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levels
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∗ Corresponding author: Hui-hong Zhao, College of Marine Sciences, South China Agricultural University, No.483 Wushan Street, Tianhe District, Guangzhou 510642, PR China. Tel: +86-20-85283529. Fax: +86-20-85280547 E-mail: [email protected] (H.-H. Zhao).
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ACCEPTED MANUSCRIPT Furthermore, the mRNA expressions of tumor necrosis factor alpha (TNF-α),
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interleukin-1-beta (IL-1β), gamma interferon (IFN-γ) and heat shock protein 70
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(HSP70) in the hepatopancreas, spleen, kidney and gill were measured. Overall, the
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expression levels were enhanced in an NP dose-dependent and tissue-specific manner.
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The expressions of four genes in four organs (except IL-1β in spleen, and TNF-α and
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HSP70 in gill) were significantly upregulated in fish fed with the G5 diet. Fish fed
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with the G4 diet had increased expression levels of IL-1β in spleen and IFN-γ in
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kidney. The relative expression levels of TNF-α, IL-1β and HSP70 in the
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hepatopancreas in fish fed with the G3 diet were significantly upregulated compared
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to the control. Transcriptional levels of IL-1β and HSP70 in the hepatopancreas, IFN-γ
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and HSP70 in the kidney and IL-1β in the gills of fish fed with the G2 diet were
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upregulated. Taken together, our results indicated that the NC-NP mixture can
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enhance the antioxidant capacity and innate immune ability of O. niloticus, indicating
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that this mixture might be a potential alternative to antibiotics when used as a feed
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additive.
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Keywords:
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biochemical parameters, Antioxidant, Immunity, Immune-related genes.
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Introduction
Oreochromis
niloticus,
Antimicrobial
peptides,
Blood
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Tilapia,
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Antibiotics are widely used as important medicines in cultivation and animal
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production to expedite growth and control diseases [1, 2]. However, the overuse of
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antibiotics has resulted in a number of serious problems, such as the enhancement of
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drug resistance, antibiotic-residues and environmental pollution [3, 4]. Therefore,
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investigations into antibiotic substitutes are of considerable value [5].
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Antimicrobial peptides (AMPs)，also called antibacterial peptides (ABPs), are
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vital components of an organism’s innate immune system and consist of dozens of
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amino acid residues [6, 7]. AMPs have a broad spectrum of antimicrobial activity
55
against pathogenic bacteria, fungi and viruses, and can eliminate mutant cells within
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organisms. To date, there have been more than 2000 AMPs reported in the database of
ACCEPTED MANUSCRIPT AMPs (http://aps.unmc.edu/AP/main.php). These have been isolated from a wide
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range of prokaryotes and eukaryotes including various viruses, bacteria, fungi,
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animals and plants [8, 9]. In spite of their diverse structures, these AMPs have
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common properties such as possessing a net positive charge of over +2 (usually +4,
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+5 or +6) [10] and adopting an α-helical linear and a β-sheet circular amphipathic
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second structure, which is tightly linked to antibacterial activities by interrelations
63
with bacterial cell-membranes [9, 11]. In contrast to antibiotics, AMPs have a broader
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spectrum, no drug residues, more rapid killing action, low resistance potential and
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highly selective toxicity [12–14]. Therefore, AMPs may be potential alternatives to
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antibiotics in livestock, poultry and fish farming. In addition, the major functions of
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AMPs used as additives in animal husbandry have been widely studied. It has been
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reported that AMPs could improve the growth performance and modulate the
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immunity of animals. Dietary supplementation of 5–10 mg/kg recombinant
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antimicrobial peptide (RAP) derived from Fenneropenaeus chinensis significantly
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increased weight gain, red blood cell numbers and catalase (CAT) activity and
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decreased the death rates of tilapia (GIFT, Oreochromis niloticus) after injection with
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Aeromonas hydrophila (P<0.05) [15]. Another study into koi (Cyprinus carpio koi)
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showed that the final body weight, special growth rate (SGR), CAT, superoxide
75
dismutase (SOD) and alkaline phosphatase (AKP) activities were significantly higher
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while cumulative mortality was lower after animals have been challenged with
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Aeromonas veronii compared to the control (P<0.05) at the level of 225 mg/kg of
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cecropin added into diets [16]. Furthermore, there were similar results reported in
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triploid crucian carp [17] and common carp (Cyprinus carpio) [18]. While the use of
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AMPs in aquaculture for enhancing growth, antioxidant capacity, immunity and
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resistance to infections has been studied in various animals, only a few studies have
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been conducted to investigate the effects of AMPs on the immune-related gene
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expressions of fish [19, 20].
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Several tilapia species are globally important in freshwater aquaculture [21].
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However, the conditions of high density culture and excessive use of antibiotics have
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led to many infectious diseases and antibiotic resistance of pathogens, which have
ACCEPTED MANUSCRIPT impacted tilapia production and increased costs [22, 23]. Therefore, the main
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objective of our study was to evaluate the effects of various levels of dietary AMPs on
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biochemical parameters, antioxidant activities, and immune responses in tilapia
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(Oreochromis niloticus) in order to evaluate their effectiveness as a substitute for
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antibiotics.
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2 Material and methods
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2.1 Peptides
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NC and NP (Guangzhou Bestide Bio-Science and Technology Co., Ltd,
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Guangzhou, China) were the two AMPs used in this study. Their values of
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antibacterial potency (Arbitrary Units, AU/g) and the method of measuring according
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to the instructions “Determination of Antimicrobial Activity of Peptide C/P in Natucin
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C/P —— Agar Diffusion Method” were provided by the company. The symbiotic
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bacteria were extracted from the intestines of free-range chickens in the countryside in
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Hainan Province, China. A mixture of symbiotic bacteria and Escherichia coli
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K12D31 was then cultured and fermented, and the fermentation broth was
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concentrated and purified into NC premix after sterilization. The purified NC
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displayed broad-spectrum antibacterial potency against pathogens, especially E. coli,
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with a minimum inhibitory concentration (MIC) of less than 155 µg/ml and a
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molecular mass of about 6 kDa. The activity of this peptide was ≥20,000 AU/g which
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meant that it had an inhibition zone of ≥9.8 mm in diameter when the indicative
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bacterium E. coli K12D31 with a concentration of 500µl/100ml medium and
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OD600nm=0.5 was used and the dry NC was prepared into solution in a mass fraction
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of 1/16.
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NP was secreted by Bacillus amyloliquefaciens obtained from pig intestine, and
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had
a
narrow-spectrum
mainly
against
Gram-positive
bacteria,
especially
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Streptococcus spp. It had a molecular mass of about 5 kDa and an activity of more
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than 20,000 AU/g in which the diameter of inhibition zone was ≥9.4 mm, the
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indicative bacterium was Streptococcus suis with the same concentration and OD600nm
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as E. coli K12D31 and the dry NP had a mass fraction of 1/8 in solution. Tilapia
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suffered a series of pathogenic infections in the process of culture including E. coli,
ACCEPTED MANUSCRIPT Streptococcus agalactiae and S. iniae. In consideration of the property of
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broad-spectrum antibacterial activity, especially against E. coli of NC and the
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anti-Streptococcus activity of NP, the two peptides were tested in the current study to
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explore their regulative and protective effects on tilapia. The nucleotide and amino
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acid sequences of the two peptides have been not published due to a patent
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application.
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2.2 Diet preparation and experimental design
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The formulation and chemical composition of the basal diet (control) is
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presented in Table 1. In the experiment groups, the AMPs were added to the basal diet
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as additives at different concentrations: 200 mg/kg of NC in all treatment groups (a
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previous preliminary test was conducted to find diets which promoted growth of
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tilapia best) and 25 mg/kg (G1), 100 mg/kg (G2), 400 mg/kg (G3), 800 mg/kg (G4) or
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1600 mg/kg (G5) of NP. All ingredients were ground (<0.375mm) and blended
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thoroughly with an additional 100 ml of water per 1 kg of diet in a food mixer for 30
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mins. Feed was extruded into pellets, 1.5 mm in diameter. The moist pellets were
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air-dried at ambient temperature until the content of moisture was less than 10 %.
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Finally, the dry pellets were stored in plastic bags and frozen at –20 °C until needed.
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2.3 Experimental fish and feeding
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Four hundred and fifty healthy tilapia (GIFT) fingerlings were obtained from
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Guangdong Tilapia Breeding Farm (Guangzhou, Guangdong, China). GIFT, called
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Genetic Improvement of Farmed Tilapias, is a genetically improved strain of Nile
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tilapia (O. niloticus), which was obtained by crossbreeding of eight different strains of
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O. niloticus. The stains were well documented germplasm from eight countries or
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areas which were Egypt, Kenya, Ghana, Senegal in Africa and Israel, Singapore,
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Thailand and Chinese Taiwan in Asia respectively[24]. The fish were transported in
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plastic bags filled with oxygenated water and acclimatized for two weeks prior to the
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feeding trial in a recirculating aquaculture system, which consisted of 22 circular
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glass aquaria (50 × 60 × 60 cm) with continuous aeration in the College of Animal
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Science, South China Agricultural University. After the acclimation, the fingerlings
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(2.12 ± 0.02 g) were randomly distributed into 18 tanks (all six groups in triplicate)
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ACCEPTED MANUSCRIPT with 25 ones in each tank for an 8-week feeding trial. During the period of feeding,
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25 % of water in fish tank was exchanged and feces were removed with a siphon daily.
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The temperature and pH of water were maintained in the range of 23-30 °C and
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7.0-8.0 respectively. The dissolved oxygen was higher than 5 mg/L, NH4 + -N lower
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than 0.5 mg/L. The fish were fed twice daily (at 9:00 and 16:00) at a rate of 4–5 %
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body weight, which was regulated according to the feed intake. The feed intake and
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the weight of dead fish were recorded daily.
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2.4 Sample collection and analytical methods
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At the end of the feeding trial, six fish from each tank were randomly sampled
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and anesthetized with MS-222 at a concentration of 200 mg/L after 24 h starvation.
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Blood samples were taken from the caudal vein using a 1 ml syringe and allowed to
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clot at 4 °C for 4 h. Serum was obtained by centrifugation at 1,520 g for 10 min and
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then stored at –20 °C until use. After the blood was collected, the fish were placed on
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ice for dissection to obtain hepatopancreas, spleen, kidney and gill samples, which
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were then frozen in liquid nitrogen for 2 h and stored at –80 °C until analyses.
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The serum samples were sent to Guangzhou Daan Clinical Laboratory Centre to
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determine the contents of total protein (TP), albumin (ALB), globulin (GLB),
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high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol
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(LDL-C) and blood glucose (GLU) by a BS-800 automatic biochemical analyzer
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(Mindray, Shenzhen, Guangdong, China). The serum lipopolysaccharide (LPS)
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contents were measured using the method of enzyme linked immunosorbent assay
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(ELISA).
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Total antioxidant capacity (TAOC), superoxide dismutase (SOD), catalase (CAT),
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malondialdehyde (MDA), lysozyme (LZM) and LPS were analyzed by kits (Nanjing
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Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the
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manufacturer’s instructions.
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The relative expression levels of four immune-related genes including tumor
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necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), gamma interferon (IFN-γ)
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and heat shock protein 70 (HSP70) in hepatopancreas, spleen, kidney and gill were
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examined with real-time Quantitative PCR (RT-qPCR). Total RNA was isolated using
ACCEPTED MANUSCRIPT a Hipure Universal RNA Kit (Magen, Guangzhou, Guangdong, China) according to
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the manufacturer's instructions and the nucleic acid concentration was quantified by
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measuring the absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer
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(Quawell Technology Inc., San Jose, CA, USA). Approximately 1 µg of total RNA
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from each sample was used to synthesize the first cDNA strand using an EasyScript
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One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech,
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Beijing, China). The β-actin gene was chosen as the reference gene for sample
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normalization. All qPCR primers were designed using the software of primer 5.0
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based on the gene sequences in GenBank and are presented in Table 2. The qPCR was
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performed using a TransStart Tip Green qPCR SuperMix Kit (TransGen Biotech) in a
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MX3005P real-time Quantitative PCR system (Stratagene, Santa Clara, CA, USA).
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The total mixture had a total volume of 20 µl and consisted of 0.4 µl of 10 µM
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forward primer, 0.4 µl of 10 µM reverse primer, 10 µl of 2× TransStart Tip Green
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qPCR SuperMix, 0.4 µl of Passive Reference Dye (50×), 1 µl of cDNA and 7.8 µl of
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double distilled H2O. The reaction conditions of qPCR amplification were as follows:
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94 °C for 30 s, 40 cycles of 94 °C for 5 s, 58 °C for 15 s, 72 °C for 10s, and the
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dissociation stage was as follows: dissociation-curve analysis was performed and
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showed one single peak in all cases. All samples were analyzed three times by qPCR.
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The products of qPCR and the primer sequences were detected by electrophoresis
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using 1.5 % agarose gel. The formula 2-
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transcript quantities.
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2.5 Statistical analysis
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All data were analyzed by one-way ANOVA using SPSS for windows version
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17.0 (SPSS, inc., USA) and expressed as mean ± standard error. LSD and Duncan’s
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multiple range tests were applied to determine significant differences between all
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groups. Significance level was considered at a value of P<0.05. The same letter
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between groups indicates no significant difference, while different letters indicate
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significant differences.
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3 Results
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3.1 Biochemical parameters of serum The fish fed with G3, G4 and G5 diets had significantly higher TP and ALB
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contents than the control fish (P<0.05), and the levels of GLB in fish fed with G2, G3,
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G4 and G5 diets were also markedly increased (P<0.05). The addition of AMPs
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increased serum HDL-C contents; these were markedly increased in the G4 and G5
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diet groups (P<0.05). The LDL-C level in fish fed with the G4 diet was higher than
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that in the control group (P<0.05) and was the highest value among treatment groups.
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LPS contents in serum significantly dropped in response to dietary AMP
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supplementation (P<0.05) and reached a minimum at the dose of 200 mg/kg NC with
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25 mg/kg of NP (G1), followed by the G5 diet.
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3.2 Antioxidant activity and non-specific immunity
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As shown in Table 4, the AMP-supplemented diets increased the antioxidant
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activity and non–specific immunity of tilapia. SOD and CAT activities of fish fed with
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AMPs-diets were higher than those fed with a control diet, although no significant
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difference was observed. TAOC of fish fed with the G3 diet was significantly higher
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compared to the control group (P<0.05); however, there was no significant difference
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among the AMP treatment groups (P>0.05). MDA levels in fish fed with
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AMP-supplemented diets appeared, while variable, to decrease with increasing doses
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of NP. All tested groups showed significantly lower MDA contents than the control
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group (P<0.05). As a non-specific immune parameter, the activity of LZM was
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investigated in this experiment. The G4 diet significantly improved the LZM level of
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fish (P<0.05), and other doses of AMPs also resulted in increasing LZM values
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without significant differences (P>0.05).
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3.3 Expression of immune-related genes
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To assess the effect of AMP-supplemented diets on further immune responses,
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the relative expression levels of four cytokines including TNF-α, IL-1β, IFN-γ and
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HSP70 were measured in hepatopancreas, spleen, kidney and gill of tilapia at the end
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of the 8-week feeding trial (Fig. 1). As shown in Fig. 1, the AMP-supplemented diets
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generally enhanced the mRNA expressions of cytokines in the four organs. At a
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concentration of 200 mg/kg NC with 1600 mg/kg NP (G5), the transcriptional levels
ACCEPTED MANUSCRIPT of the three pro-inflammatory immune factors (TNF-α, IL-1β, IFN-γ) and HSP70 in
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all organs (except IL-1β in spleen, and TNF-α and HSP70 in gill) were significantly
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upregulated compared to the control group (P<0.05). The G4 diet also lead to
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increased expression levels of IL-1β in spleen and IFN-γ in kidney (P<0.05). The
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relative expressions of TNF-α, IL-1β and HSP70 in G3 hepatopancreas samples were
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significantly higher than those in the control group (P<0.05). Significantly increased
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transcriptional levels of IL-1β and HSP70 in hepatopancreas, IFN-γ and HSP70 in
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kidney and IL-1β in gill tissue of fish fed with the G2 diet were seen (P<0.05). In
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contrast, the G1 diet resulted in no modulatory effect on expressions of the
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immune-related genes in all organs.
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4. Discussion
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In this study, we demonstrate that the use of NC in combination with NP could
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improve antioxidant activity and immunity of tilapia. Similar results have been
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reported in other studies, in which other AMPs were administered to different taxa [18,
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25]. It has been shown that a series of AMPs are likely to be new feed additives in the
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breeding industry, mainly due to their positive influence on growth and beneficial
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immunoregulation, which assists resistance to pathogens. In addition, there is
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considerable potential for their use in aquaculture.
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4.1 Effect of AMPs on serum biochemical parameters
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Serum TP content is used as an index to evaluate the nutritional and metabolic
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status of an organism, and reflects the level of health and immunity indirectly. TP
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measurements are based on dietary protein content, liver metabolism and even protein
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loss caused by some lesions [26, 27]. Our results indicated that AMP-supplemented
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diets could enhance protein synthesis and immunity of tilapia, especially when the
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concentration of NP was over 400 mg/kg. Different results have been reported in
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piglets fed with antimicrobial peptide (obtained from the intestine of Rongchang pig)
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diets [26] and common carp fed with cecropin diets [18], which may be in response to
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different tested animals or AMPs.
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The levels of serum HDL-C and LDL-C are vital indices that reflect
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lipometabolic status. HDL-C, which is usually considered the good cholesterol, can
ACCEPTED MANUSCRIPT prevent the occurrence of atherosclerosis, whereas LDL-C has the opposite influence
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on the body when present in high levels. In this study, the supplement of AMPs had an
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impact on anti-atherosclerosis of fish attributed to the increased HDL-C content in
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serum. LDL-C contents remained stable in all groups except in the fish fed with the
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G4 diet, where it was present at a higher level. This suggests that higher doses of NP
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supplementation may be capable of inducing atherosclerosis, but further studies are
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required due to the higher HDL-C content. Additionally, it was found that Glu content
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was not affected by dietary dosages of AMPs.
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LPS is a main component of cell walls of Gram-negative bacteria. LPS enters the
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bloodstream by way of intestinal absorption, bacteria that are released into the blood
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under the occurrence of sepsis, and exogenous injection. Detoxification is conducted
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by the liver and HDL, antibodies and phagocytes in blood [28]. The intestinal
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absorption results from intestinal inflammation and injury caused by proliferation of
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bacteria. The present study provides the first investigation into the effect of dietary
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AMP-supplementation on LPS content in serum. The results of this study indicated
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that the supplement of AMPs could significantly reduce the LPS level in tilapia, with
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the lowest value being found in fish fed with the G1 diet. This may partially be the
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result of the broad-spectrum antibacterial activity of NC to directly reduce the
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abundance of bacteria broad, partially due to the improvement of fish immunity.
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4.2 Effect of AMPs on antioxidant activity and non-specific immunity
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Like higher vertebrates, fish have the same susceptibility to the effects of
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reactive oxygen species (ROS) and innate and effective antioxidant defenses [29].
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Enzymatic and non-enzymatic antioxidant systems constitute “primary antioxidants”
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to protect organisms against oxidative stress and damage [29–31]. Non-enzymatic
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antioxidants are used as the first line of defense including substances such as certain
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vitamins, amino acids, metalloproteins and carotenoids. SOD, CAT, GR and GPx are
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important antioxidants that play a vital role in counteracting ROS when other
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antioxidant compounds are insufficient or depleted [31, 32]. Organisms can produce
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ROS such as the superoxide radical (O2-), hydrogen peroxide (H2O2) and the hydroxyl
295
radical (OH•), which are eliminated to maintain dynamic balance during normal
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ACCEPTED MANUSCRIPT aerobic metabolism. However, excessive ROS or a decrease in the capacity to
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eliminate ROS can result in oxidative stress when endogenous or exogenous
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stimulations exist [33]. TAOC is recognized as an index to assess the functional state
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and reflect the health status of an organism indirectly [34]. SOD is a key antioxidant,
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which can catalyze O2- into H2O2 and oxygen. Further, CAT and GPx convert H2O2 to
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water in order to thoroughly clear oxyradicals [18]. MDA, the breakdown product of
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lipid peroxides, has a strong cytotoxicity and exposes fish to oxidative stress [35].
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Furthermore, as the first line of defense against pathogen infection, the non-specific
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immune system protects fish from microbial damage [36]. LZM is an indispensable
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enzyme of the non-specific immune system that has the ability to disrupt cell walls by
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splitting peptidoglycans to resist bacteria, especially Gram-positive species [37].
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Previous studies have confirmed that AMPs can enhance antioxidant activity and
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non-specific immunity in various species, including one study in which SOD activity
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was significantly increased in serum of weaned piglets treated with 10 mg/kg
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Rongchang pig AMP [26]. Another study found TAOC and SOD activities were
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significantly improved in Litopenaeus vannamei treated with 300 mg/kg of AMPs of
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Musca domestica [38]. In addition, SOD and LZM activities were increased and MDA
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content was decreased in serum of Megalobrama amblycephala treated with 0.2 %
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AMP extracted from Bacillus subtilis fmbJ [39] and SOD, CAT, LZM activities were
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increased in serum of Cyprinus carpio treated with 100–400 mg/kg of cecropin [18].
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The present results were in agreement with these studies. Higher TAOC, SOD, CAT
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and LZM activities from tilapia fed with AMP-supplemented diets were observed, and
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MDA contents in all AMP-treated groups were significantly lower than the control
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group. The results clearly suggest that AMPs added to diets could improve antioxidant
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activity and non-specific immunity of tilapia.
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4.3 Effect of AMPs on expression of immune-related genes
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In this experiment, the expression levels of four immune-related genes were
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examined to evaluate immune responses to dietary levels of AMPs in tilapia. TNF-α,
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IL-1β and IFN-γ are three important pro-inflammatory cytokines that are considered
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to have regulatory roles in immune responses and can activate lymphocytes, natural
ACCEPTED MANUSCRIPT killer cells and macrophages, which lead to subsequent enhancements of nitric oxide
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production, respiratory burst activity and phagocytosis [40, 41]. As a multifunctional
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inflammatory factor, TNF-α is produced by a variety of immune cells [42, 43] and is
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found to be the strongest anti-tumor cytokine at present which directly inhibits
330
proliferation of tumor cells and resists bacteria and viruses by causing an
331
inflammatory response [44]. IL-1β induces growth of lymphocytes to enhance
332
immune responses and participates in resisting bacteria, viruses and tumors [45].
333
IFN-γ is acknowledged as a Type-II interferon that plays important anti-virus and
334
immune regulation roles [46]. The three are early-response markers of inflammation
335
and regulate the expression of many other cytokines [47]. Additionally, HSP70 is also
336
considered an important immune-related gene on account of its role in promoting
337
immune responses. It has been reported that HSP70 has a series of biological
338
functions including acting as an antioxidant and molecular chaperone, and in
339
anti-apoptosis and synergetic immunity [48, 49] in order to protect cells from
340
environmental stressors and pathogenic infections [50–52].
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Many studies have demonstrated that AMPs obtained from various sources could
342
induce expression of immune-related genes, in which TNF-α, IL-1β and IFN-γ were
343
involved, while no study has investigated HSP70. In in vitro models, BT
344
(Brevibacillus texasporus) cationic peptides upregulated the transcription of
345
pro-inflammatory cytokines IL-1β and IL-6, and inflammatory chemokines CXCLi1
346
and CXCLi2 in chicken heterophils and monocytes under phorbol myristate acetate
347
(PMA)-stimulation [53]. A study into weaned piglets challenged with E. coli found an
348
upregulation in expression levels of IL-1β and IL-6 in serum induced by cecropin AD,
349
which enhanced the immunity of the organisms [54]. It has also been reported that
350
diets supplemented with complex antibacterial peptides increased the contents of IL-2,
351
IL-4, IL-6, IFN-γ and TNF-α in serum of weaned piglets [55]. Transferring the genes
352
of tilapia hepcidin and shrimp chelonianin into zebrafish respectively also
353
significantly increased the expressions of immune-related genes IL-10, IL-21, IL-22,
354
Lyz, NF-κB, TNF-α, TLR-1 [56]. Another study on grouper (Epinephelus coioides)
355
indicated that the upregulation of IL-1β in muscle and of IL-8, TNF-α and IRF2 in
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ACCEPTED MANUSCRIPT liver was observed by electrotransfer of the epinecidin-1 gene into skeletal muscle.
357
However, cecropin used as an additive of common carp diets increased serum IL-1α,
358
IL-1β and IgM levels in lower doses and decreased IL-1α and IL-1β in higher doses
359
[18]. Additional studies using zebrafish [19], grouper [20] and half-smooth tongue
360
sole (Cynoglossus semilaevis) [57] have suggested that AMPs could modulate the
361
expression of immune-related genes including TNF-α, IL-1β and IFN-γ. Similar to the
362
reports above, it was observed that the supplement of NC-NP mixture enhanced the
363
expressions of TNF-α, IL-1β, IFN-γ and HSP70 in four organs (except in gill) in the
364
current study. The highest AMP dose also significantly upregulated the expressions of
365
four genes (except IL-1β in spleen, and TNF-α and HSP70 in gill). The expression
366
levels appeared, while variable, to increase with the increasing concentration of AMPs,
367
which indicates that the regulating capacity depends on the dosage of AMPs. This
368
study was initiated first to examine the effects of AMP additives on the expression of
369
the HSP70 gene. Our results have provided evidence that various antimicrobial
370
peptides can modulate immune-related gene expressions to play pivotal roles in
371
immune responses including inflammatory and antibacterial responses or antiviral
372
infection and stress resistance. Patterns will likely depend on the experimental
373
animals and peptides used. The mechanism has not been fully explored and needs
374
further investigation.
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In conclusion, the present study provided evidence that dietary NC-NP mixture
376
peptide supplementation can increase the contents of TP, ALB, GLB and HDL-C in
377
serum, improve antioxidant capacity and non-specific immunity and regulate
378
expression of immune-related genes to enhance positive immune responses for
379
disease resistance in tilapia (O. niloticus). This is the first record, to our knowledge, of
380
the application of NC and NP as additives in tilapia diets, which assessed
381
immune-regulation effects at the molecular level. Our results therefore provide an
382
important reference for further studies in this area. The present results indicated that
383
NC-NP mixture may be an attractive and potential candidate to enhance innate and
384
adaptive immunity to improve resistance to bodily diseases, and similar studies in the
385
near future are deemed necessary.
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ACCEPTED MANUSCRIPT 386 387
Acknowledgments The work was supported by the Ceeusro Project from Science and Technology
389
Program of Guangdong Province, China (grant No. 2015A090905022) awarded to Dr.
390
Hui-Hong Zhao.
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391 392
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ACCEPTED MANUSCRIPT Table 1 Formulation and approximate nutrient content of the basal diet (dry diet). Proportion (%)
Nutrients composition
Proportion (%)
Wheat middling
25.3
Moisture
9.02
Soybean meal
25
Crude protein
34.38
Rapeseed meal
15
Crude fat
Rice bran
10
Gross energy (KJ/g)
Peanut meal
10
Soybean oil
1
Corn gluten meal (60 %)
5.5
Fish meal
6
Betaine
0.3
Choline chloride (50 %)
0.2
Salt
0.1
Vitamin premix1
SC M AN U
0.1
EP
1
100
Vitamin premix: VA (IU/kg): 350,000, VD3 (IU/kg): 210,000, VE (g/kg): 6, VK3 (mg/kg): 500, VB1 (mg/kg): 450,
AC C
1
18.88
0.5
Mineral premixb2 Total
7.45
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Vit C Phosphate ester
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Ingredients
VB2 (mg/kg): 900, VB6 (mg/kg): 600, VB12 (mg/kg): 2, VC (mg/kg): 14000, nicotinamide (mg/kg): 3500, D-calcium pantothenate (mg/kg): 2000, folic acid (mg/kg): 160, biotin (mg/kg): 8. 2
Mineral premix: Mg (mg/kg): 5000, Fe (mg/kg): 5250, Mn (mg/kg): 1000, I (mg/kg): 168, Cu (mg/kg): 788, Zn
(mg/kg): 4200, Se (mg/kg): 25, Co (mg/kg): 131.
ACCEPTED MANUSCRIPT Table 2 Primers used in this study and their sequences and sizes of PCR amplicon. Size of PCR amplicon Primer name
Sequence (5'--3')
TNF-α-F
GAGGTCGGCGTGCCAAGA
TNF-α-R
TGGTTTCCGTCCACAGCGT
IL-1β-F
GTTCACCAGCAGGGATGAGATT
IL-1β-R
TGCGGTCTTCACTGCCTCC
HSP70-F
TGGAGTCCTACGCCTTCAACA
HSP70-R
CAGGTAGCACCAGTGGGCAT
IFN-γ-F
TGACCACATCGTTCAGAGCA
IFN-γ-R
GGCGACCTTTAGCCTTTGT
β-actin-F
TGGTGGGTATGGGTCAGAAAG
β-actin-R
GCTCCTCAGGGGCAACTCT
（bp）
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119
122
AC C
EP
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M AN U
SC
238
128
171
ACCEPTED MANUSCRIPT
Table 3
Control
G1
G2
G3
G4
G5
TP (g/L)
26.77±1.65c
29.30±0.61bc
30.70±1.23abc
32.33±1.27ab
32.20±0.59ab
34.23±1.60a
ALB (g/L)
13.23±1.31b
14.70±0.25ab
14.07±0.81ab
15.63±0.64a
15.87±0.34a
16.07±0.26a
GLB (g/L)
13.53±0.35c
14.60±0.61bc
16.63±0.89ab
16.70±0.65ab
16.33±0.34ab
18.17±1.63a
HDL-C (mmol/L)
1.60±0.07c
1.67±0.01bc
1.78±0.06abc
1.63±0.06bc
2.01±0.11a
1.87±0.11ab
LDL-C (mmol/L)
0.81±0.05b
0.78±0.02b
0.89±0.04ab
0.88±0.02ab
1.04±0.12a
0.90±0.05ab
Glu (mmol/L)
5.98±0.92ab
7.66±0.57a
7.34±0.37a
6.34±0.78ab
5.64±0.52ab
4.71±0.83b
LPS (pg/ml)
135.35±37.62a
69.02±6.35b
75.02±8.62b
80.85±6.26b
78.35±6.17b
72.68±11.74b
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Parameter
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Effect of AMP contents on serum biochemical parameters of tilapia (Oreochromis niloticus)
Values (mean ± S.E., n = 3) with different superscript letters (a, b, and c) in the same row are significantly different (P <0.05). TP: total protein; ALB: albumin, GLB: globulin; ALT: alkaline transaminase; HDL-C: high density lipoprotein
cholesterol;
LDL-C:
low
lipoprotein
AC C
EP
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lipopolysaccharide.
density
cholesterol;
GLU:
blood
glucose;
LPS:
ACCEPTED MANUSCRIPT Table 4 Effect of AMP contents on antioxidant activity and non-specific immunity of tilapia (Oreochromis
Control
G1
G2
G3
G4
G5
TAOC (U/ml)
9.85±0.87b
13.27±0.35ab
26.14±5.40ab
28.96±8.37a
18.40±6.64ab
24.34± 7.23ab
SOD (U/ml)
14.52±0.29
14.60±2.50
14.60±1.81
17.98±1.04
16.30±1.40
17.03±0.27
CAT (U/ml)
7.75±4.30
15.81±2.39
8.88±2.61
14.45±3.41
15.36±3.86
10.69±2.28
MDA (nmol/ml)
6.96±0.20a
4.71±0.23b
3.72±0.58b
4.12±0.98b
3.33±0.47b
3.34±0.43b
LZM (µg/ml)
10.82±0.43b
11.66±1.31b
12.94±0.78b
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Parameter
SC
niloticus)
11.35±0.46b
21.52±1.74a
12.72±0.73b
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Values (mean ± S.E., n = 3) with different superscript letters (a and b) in the same row are significantly different (P<0.05). TAOC: total antioxidant capacity; SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde;
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LZM: lysozyme.
ACCEPTED MANUSCRIPT Figure legends Fig.1. Effects of AMP-supplementation on the relative expression levels of TNF-α (A), IL-1β (B), IFN-γ (C) and HSP70 (D) in the hepatopancreas, spleen, kidney and gill of tilapia (Oreochromis niloticus). The different superscript letters (a, b, c and d) among diet treatments in the same organ
RI PT
indicate significance differences (P<0.05, one-way ANOVA). Bars represent the mean values ± S.E. (n=3). TNF-α: tumor necrosis factor α; IL-1β: interleukin 1β; IFN-γ: interferon γ; HSP70: heat shock protein 70.
G1
G2
G3
B
A a ab
bc
7
ab
6 5 a
4 3
b
bc c
c
b
b
1
bb
Spleen
Kidney
Gill
D
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EP
Hepatopancreas
Relative expression
bb
b
0
C
b
b
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2
Relative expression
a
10
Relative expression
15
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TNF-
G4
G5
SC
Control
ACCEPTED MANUSCRIPT Highlights
We assessed the effects of NC-NP mixture on antioxidant capacity, and immune responses in tilapia (GIFT), Oreochromis niloticus.
Diets supplemented with NC-NP mixture significantly decreased the LPS
Diets supplemented with NC-NP mixture increased the antioxidant capacity and improve the immunity of tilapia.
Diets supplemented with NC-NP mixture up-regulated the expressions of
EP
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SC
immune-related genes
AC C
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contents in serum of tilapia.
S1050-4648(16)30376-X
DOI:
10.1016/j.fsi.2016.06.016
Reference:
YFSIM 4016
To appear in:
Fish and Shellfish Immunology
Received Date: 29 April 2016 Revised Date:
3 June 2016
Accepted Date: 8 June 2016
Please cite this article as: Chen Y-B, Hu J, Lv Q-J, Liu L-J, Wen L-F, Yang X-K, Zhao H-H, The effects of Natucin C-Natucin P mixture on blood biochemical parameters, antioxidant activity and non-specific immune responses in tilapia (Oreochromis niloticus), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.06.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The effects of Natucin C- Natucin P mixture on blood biochemical
2
parameters, antioxidant activity and non-specific immune responses
3
in tilapia (Oreochromis niloticus)
4
Yi-Bin Chena, Juan Hua, Qing-Ji Lva, Li-Jie Liub, Liu-Fa Wenc, Xian-Kuan Yanga,
5
Hui-Hong Zhaoa, ∗
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a
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510642, Guangdong Province, PR China
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b
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Biotechnology, Shantou University, Shantou 515063, Guangdong Province, PR China
College of Marine Sciences, South China Agricultural University, Guangzhou
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Marine Biology Institute & Guangdong Provincial Key Laboratory of Marine
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c
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510642, Guangdong Province, PR China
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Abstract: Natucin C (NC) and Natucin P (NP) are two kinds of antimicrobial
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peptides (AMPs). In the present study, the effects of NC-NP mixture on a tilapia
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species (Oreochromis niloticus) were examined. Animals were fed with either a
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control diet or one of five AMP-supplemented diets for eight weeks.
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AMP-supplemented diets contained five increasing levels of NP from G1 to G5 and
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one level of NC (200 mg/kg). Results showed that fish in the G3, G4 and G5 groups
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had significantly higher levels of total protein (TP), albumin (ALB) and globulin
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(GLO) in serum than fish in the control group. Fish fed with G4 and G5 diets
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exhibited significantly higher high-density lipoprotein cholesterol (HDL-C) levels
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compared
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AMP-supplemented groups were significantly lower than the control. In addition, the
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total antioxidant capacity (TAOC) and lysozyme (LZM) activities were significantly
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increased in fish fed with the G3 and G4 diets, respectively compared to the control.
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The serum malondialdehyde (MDA) levels in fish fed with AMP-supplemented diets
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were significantly decreased compared to those not supplemented with AMPs.
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the
control
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fish.
Lipopolysaccharide
(LPS)
levels
in
∗ Corresponding author: Hui-hong Zhao, College of Marine Sciences, South China Agricultural University, No.483 Wushan Street, Tianhe District, Guangzhou 510642, PR China. Tel: +86-20-85283529. Fax: +86-20-85280547 E-mail: [email protected] (H.-H. Zhao).
all
ACCEPTED MANUSCRIPT Furthermore, the mRNA expressions of tumor necrosis factor alpha (TNF-α),
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interleukin-1-beta (IL-1β), gamma interferon (IFN-γ) and heat shock protein 70
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(HSP70) in the hepatopancreas, spleen, kidney and gill were measured. Overall, the
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expression levels were enhanced in an NP dose-dependent and tissue-specific manner.
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The expressions of four genes in four organs (except IL-1β in spleen, and TNF-α and
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HSP70 in gill) were significantly upregulated in fish fed with the G5 diet. Fish fed
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with the G4 diet had increased expression levels of IL-1β in spleen and IFN-γ in
34
kidney. The relative expression levels of TNF-α, IL-1β and HSP70 in the
35
hepatopancreas in fish fed with the G3 diet were significantly upregulated compared
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to the control. Transcriptional levels of IL-1β and HSP70 in the hepatopancreas, IFN-γ
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and HSP70 in the kidney and IL-1β in the gills of fish fed with the G2 diet were
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upregulated. Taken together, our results indicated that the NC-NP mixture can
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enhance the antioxidant capacity and innate immune ability of O. niloticus, indicating
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that this mixture might be a potential alternative to antibiotics when used as a feed
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additive.
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Keywords:
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biochemical parameters, Antioxidant, Immunity, Immune-related genes.
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Introduction
Oreochromis
niloticus,
Antimicrobial
peptides,
Blood
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Tilapia,
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Antibiotics are widely used as important medicines in cultivation and animal
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production to expedite growth and control diseases [1, 2]. However, the overuse of
49
antibiotics has resulted in a number of serious problems, such as the enhancement of
50
drug resistance, antibiotic-residues and environmental pollution [3, 4]. Therefore,
51
investigations into antibiotic substitutes are of considerable value [5].
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Antimicrobial peptides (AMPs)，also called antibacterial peptides (ABPs), are
53
vital components of an organism’s innate immune system and consist of dozens of
54
amino acid residues [6, 7]. AMPs have a broad spectrum of antimicrobial activity
55
against pathogenic bacteria, fungi and viruses, and can eliminate mutant cells within
56
organisms. To date, there have been more than 2000 AMPs reported in the database of
ACCEPTED MANUSCRIPT AMPs (http://aps.unmc.edu/AP/main.php). These have been isolated from a wide
58
range of prokaryotes and eukaryotes including various viruses, bacteria, fungi,
59
animals and plants [8, 9]. In spite of their diverse structures, these AMPs have
60
common properties such as possessing a net positive charge of over +2 (usually +4,
61
+5 or +6) [10] and adopting an α-helical linear and a β-sheet circular amphipathic
62
second structure, which is tightly linked to antibacterial activities by interrelations
63
with bacterial cell-membranes [9, 11]. In contrast to antibiotics, AMPs have a broader
64
spectrum, no drug residues, more rapid killing action, low resistance potential and
65
highly selective toxicity [12–14]. Therefore, AMPs may be potential alternatives to
66
antibiotics in livestock, poultry and fish farming. In addition, the major functions of
67
AMPs used as additives in animal husbandry have been widely studied. It has been
68
reported that AMPs could improve the growth performance and modulate the
69
immunity of animals. Dietary supplementation of 5–10 mg/kg recombinant
70
antimicrobial peptide (RAP) derived from Fenneropenaeus chinensis significantly
71
increased weight gain, red blood cell numbers and catalase (CAT) activity and
72
decreased the death rates of tilapia (GIFT, Oreochromis niloticus) after injection with
73
Aeromonas hydrophila (P<0.05) [15]. Another study into koi (Cyprinus carpio koi)
74
showed that the final body weight, special growth rate (SGR), CAT, superoxide
75
dismutase (SOD) and alkaline phosphatase (AKP) activities were significantly higher
76
while cumulative mortality was lower after animals have been challenged with
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Aeromonas veronii compared to the control (P<0.05) at the level of 225 mg/kg of
78
cecropin added into diets [16]. Furthermore, there were similar results reported in
79
triploid crucian carp [17] and common carp (Cyprinus carpio) [18]. While the use of
80
AMPs in aquaculture for enhancing growth, antioxidant capacity, immunity and
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resistance to infections has been studied in various animals, only a few studies have
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been conducted to investigate the effects of AMPs on the immune-related gene
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expressions of fish [19, 20].
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Several tilapia species are globally important in freshwater aquaculture [21].
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However, the conditions of high density culture and excessive use of antibiotics have
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led to many infectious diseases and antibiotic resistance of pathogens, which have
ACCEPTED MANUSCRIPT impacted tilapia production and increased costs [22, 23]. Therefore, the main
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objective of our study was to evaluate the effects of various levels of dietary AMPs on
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biochemical parameters, antioxidant activities, and immune responses in tilapia
90
(Oreochromis niloticus) in order to evaluate their effectiveness as a substitute for
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antibiotics.
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2 Material and methods
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2.1 Peptides
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NC and NP (Guangzhou Bestide Bio-Science and Technology Co., Ltd,
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Guangzhou, China) were the two AMPs used in this study. Their values of
96
antibacterial potency (Arbitrary Units, AU/g) and the method of measuring according
97
to the instructions “Determination of Antimicrobial Activity of Peptide C/P in Natucin
98
C/P —— Agar Diffusion Method” were provided by the company. The symbiotic
99
bacteria were extracted from the intestines of free-range chickens in the countryside in
100
Hainan Province, China. A mixture of symbiotic bacteria and Escherichia coli
101
K12D31 was then cultured and fermented, and the fermentation broth was
102
concentrated and purified into NC premix after sterilization. The purified NC
103
displayed broad-spectrum antibacterial potency against pathogens, especially E. coli,
104
with a minimum inhibitory concentration (MIC) of less than 155 µg/ml and a
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molecular mass of about 6 kDa. The activity of this peptide was ≥20,000 AU/g which
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meant that it had an inhibition zone of ≥9.8 mm in diameter when the indicative
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bacterium E. coli K12D31 with a concentration of 500µl/100ml medium and
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OD600nm=0.5 was used and the dry NC was prepared into solution in a mass fraction
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of 1/16.
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NP was secreted by Bacillus amyloliquefaciens obtained from pig intestine, and
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had
a
narrow-spectrum
mainly
against
Gram-positive
bacteria,
especially
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Streptococcus spp. It had a molecular mass of about 5 kDa and an activity of more
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than 20,000 AU/g in which the diameter of inhibition zone was ≥9.4 mm, the
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indicative bacterium was Streptococcus suis with the same concentration and OD600nm
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as E. coli K12D31 and the dry NP had a mass fraction of 1/8 in solution. Tilapia
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suffered a series of pathogenic infections in the process of culture including E. coli,
ACCEPTED MANUSCRIPT Streptococcus agalactiae and S. iniae. In consideration of the property of
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broad-spectrum antibacterial activity, especially against E. coli of NC and the
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anti-Streptococcus activity of NP, the two peptides were tested in the current study to
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explore their regulative and protective effects on tilapia. The nucleotide and amino
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acid sequences of the two peptides have been not published due to a patent
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application.
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2.2 Diet preparation and experimental design
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The formulation and chemical composition of the basal diet (control) is
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presented in Table 1. In the experiment groups, the AMPs were added to the basal diet
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as additives at different concentrations: 200 mg/kg of NC in all treatment groups (a
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previous preliminary test was conducted to find diets which promoted growth of
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tilapia best) and 25 mg/kg (G1), 100 mg/kg (G2), 400 mg/kg (G3), 800 mg/kg (G4) or
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1600 mg/kg (G5) of NP. All ingredients were ground (<0.375mm) and blended
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thoroughly with an additional 100 ml of water per 1 kg of diet in a food mixer for 30
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mins. Feed was extruded into pellets, 1.5 mm in diameter. The moist pellets were
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air-dried at ambient temperature until the content of moisture was less than 10 %.
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Finally, the dry pellets were stored in plastic bags and frozen at –20 °C until needed.
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2.3 Experimental fish and feeding
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Four hundred and fifty healthy tilapia (GIFT) fingerlings were obtained from
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Guangdong Tilapia Breeding Farm (Guangzhou, Guangdong, China). GIFT, called
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Genetic Improvement of Farmed Tilapias, is a genetically improved strain of Nile
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tilapia (O. niloticus), which was obtained by crossbreeding of eight different strains of
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O. niloticus. The stains were well documented germplasm from eight countries or
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areas which were Egypt, Kenya, Ghana, Senegal in Africa and Israel, Singapore,
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Thailand and Chinese Taiwan in Asia respectively[24]. The fish were transported in
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plastic bags filled with oxygenated water and acclimatized for two weeks prior to the
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feeding trial in a recirculating aquaculture system, which consisted of 22 circular
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glass aquaria (50 × 60 × 60 cm) with continuous aeration in the College of Animal
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Science, South China Agricultural University. After the acclimation, the fingerlings
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(2.12 ± 0.02 g) were randomly distributed into 18 tanks (all six groups in triplicate)
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ACCEPTED MANUSCRIPT with 25 ones in each tank for an 8-week feeding trial. During the period of feeding,
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25 % of water in fish tank was exchanged and feces were removed with a siphon daily.
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The temperature and pH of water were maintained in the range of 23-30 °C and
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7.0-8.0 respectively. The dissolved oxygen was higher than 5 mg/L, NH4 + -N lower
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than 0.5 mg/L. The fish were fed twice daily (at 9:00 and 16:00) at a rate of 4–5 %
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body weight, which was regulated according to the feed intake. The feed intake and
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the weight of dead fish were recorded daily.
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2.4 Sample collection and analytical methods
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At the end of the feeding trial, six fish from each tank were randomly sampled
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and anesthetized with MS-222 at a concentration of 200 mg/L after 24 h starvation.
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Blood samples were taken from the caudal vein using a 1 ml syringe and allowed to
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clot at 4 °C for 4 h. Serum was obtained by centrifugation at 1,520 g for 10 min and
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then stored at –20 °C until use. After the blood was collected, the fish were placed on
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ice for dissection to obtain hepatopancreas, spleen, kidney and gill samples, which
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were then frozen in liquid nitrogen for 2 h and stored at –80 °C until analyses.
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The serum samples were sent to Guangzhou Daan Clinical Laboratory Centre to
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determine the contents of total protein (TP), albumin (ALB), globulin (GLB),
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high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol
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(LDL-C) and blood glucose (GLU) by a BS-800 automatic biochemical analyzer
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(Mindray, Shenzhen, Guangdong, China). The serum lipopolysaccharide (LPS)
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contents were measured using the method of enzyme linked immunosorbent assay
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(ELISA).
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Total antioxidant capacity (TAOC), superoxide dismutase (SOD), catalase (CAT),
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malondialdehyde (MDA), lysozyme (LZM) and LPS were analyzed by kits (Nanjing
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Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the
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manufacturer’s instructions.
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The relative expression levels of four immune-related genes including tumor
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necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), gamma interferon (IFN-γ)
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and heat shock protein 70 (HSP70) in hepatopancreas, spleen, kidney and gill were
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examined with real-time Quantitative PCR (RT-qPCR). Total RNA was isolated using
ACCEPTED MANUSCRIPT a Hipure Universal RNA Kit (Magen, Guangzhou, Guangdong, China) according to
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the manufacturer's instructions and the nucleic acid concentration was quantified by
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measuring the absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer
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(Quawell Technology Inc., San Jose, CA, USA). Approximately 1 µg of total RNA
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from each sample was used to synthesize the first cDNA strand using an EasyScript
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One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech,
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Beijing, China). The β-actin gene was chosen as the reference gene for sample
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normalization. All qPCR primers were designed using the software of primer 5.0
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based on the gene sequences in GenBank and are presented in Table 2. The qPCR was
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performed using a TransStart Tip Green qPCR SuperMix Kit (TransGen Biotech) in a
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MX3005P real-time Quantitative PCR system (Stratagene, Santa Clara, CA, USA).
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The total mixture had a total volume of 20 µl and consisted of 0.4 µl of 10 µM
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forward primer, 0.4 µl of 10 µM reverse primer, 10 µl of 2× TransStart Tip Green
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qPCR SuperMix, 0.4 µl of Passive Reference Dye (50×), 1 µl of cDNA and 7.8 µl of
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double distilled H2O. The reaction conditions of qPCR amplification were as follows:
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94 °C for 30 s, 40 cycles of 94 °C for 5 s, 58 °C for 15 s, 72 °C for 10s, and the
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dissociation stage was as follows: dissociation-curve analysis was performed and
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showed one single peak in all cases. All samples were analyzed three times by qPCR.
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The products of qPCR and the primer sequences were detected by electrophoresis
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using 1.5 % agarose gel. The formula 2-
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transcript quantities.
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2.5 Statistical analysis
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All data were analyzed by one-way ANOVA using SPSS for windows version
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17.0 (SPSS, inc., USA) and expressed as mean ± standard error. LSD and Duncan’s
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multiple range tests were applied to determine significant differences between all
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groups. Significance level was considered at a value of P<0.05. The same letter
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between groups indicates no significant difference, while different letters indicate
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significant differences.
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3 Results
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3.1 Biochemical parameters of serum The fish fed with G3, G4 and G5 diets had significantly higher TP and ALB
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contents than the control fish (P<0.05), and the levels of GLB in fish fed with G2, G3,
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G4 and G5 diets were also markedly increased (P<0.05). The addition of AMPs
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increased serum HDL-C contents; these were markedly increased in the G4 and G5
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diet groups (P<0.05). The LDL-C level in fish fed with the G4 diet was higher than
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that in the control group (P<0.05) and was the highest value among treatment groups.
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LPS contents in serum significantly dropped in response to dietary AMP
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supplementation (P<0.05) and reached a minimum at the dose of 200 mg/kg NC with
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25 mg/kg of NP (G1), followed by the G5 diet.
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3.2 Antioxidant activity and non-specific immunity
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As shown in Table 4, the AMP-supplemented diets increased the antioxidant
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activity and non–specific immunity of tilapia. SOD and CAT activities of fish fed with
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AMPs-diets were higher than those fed with a control diet, although no significant
220
difference was observed. TAOC of fish fed with the G3 diet was significantly higher
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compared to the control group (P<0.05); however, there was no significant difference
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among the AMP treatment groups (P>0.05). MDA levels in fish fed with
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AMP-supplemented diets appeared, while variable, to decrease with increasing doses
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of NP. All tested groups showed significantly lower MDA contents than the control
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group (P<0.05). As a non-specific immune parameter, the activity of LZM was
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investigated in this experiment. The G4 diet significantly improved the LZM level of
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fish (P<0.05), and other doses of AMPs also resulted in increasing LZM values
228
without significant differences (P>0.05).
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3.3 Expression of immune-related genes
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To assess the effect of AMP-supplemented diets on further immune responses,
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the relative expression levels of four cytokines including TNF-α, IL-1β, IFN-γ and
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HSP70 were measured in hepatopancreas, spleen, kidney and gill of tilapia at the end
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of the 8-week feeding trial (Fig. 1). As shown in Fig. 1, the AMP-supplemented diets
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generally enhanced the mRNA expressions of cytokines in the four organs. At a
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concentration of 200 mg/kg NC with 1600 mg/kg NP (G5), the transcriptional levels
ACCEPTED MANUSCRIPT of the three pro-inflammatory immune factors (TNF-α, IL-1β, IFN-γ) and HSP70 in
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all organs (except IL-1β in spleen, and TNF-α and HSP70 in gill) were significantly
238
upregulated compared to the control group (P<0.05). The G4 diet also lead to
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increased expression levels of IL-1β in spleen and IFN-γ in kidney (P<0.05). The
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relative expressions of TNF-α, IL-1β and HSP70 in G3 hepatopancreas samples were
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significantly higher than those in the control group (P<0.05). Significantly increased
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transcriptional levels of IL-1β and HSP70 in hepatopancreas, IFN-γ and HSP70 in
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kidney and IL-1β in gill tissue of fish fed with the G2 diet were seen (P<0.05). In
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contrast, the G1 diet resulted in no modulatory effect on expressions of the
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immune-related genes in all organs.
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4. Discussion
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In this study, we demonstrate that the use of NC in combination with NP could
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improve antioxidant activity and immunity of tilapia. Similar results have been
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reported in other studies, in which other AMPs were administered to different taxa [18,
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25]. It has been shown that a series of AMPs are likely to be new feed additives in the
251
breeding industry, mainly due to their positive influence on growth and beneficial
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immunoregulation, which assists resistance to pathogens. In addition, there is
253
considerable potential for their use in aquaculture.
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4.1 Effect of AMPs on serum biochemical parameters
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Serum TP content is used as an index to evaluate the nutritional and metabolic
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status of an organism, and reflects the level of health and immunity indirectly. TP
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measurements are based on dietary protein content, liver metabolism and even protein
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loss caused by some lesions [26, 27]. Our results indicated that AMP-supplemented
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diets could enhance protein synthesis and immunity of tilapia, especially when the
260
concentration of NP was over 400 mg/kg. Different results have been reported in
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piglets fed with antimicrobial peptide (obtained from the intestine of Rongchang pig)
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diets [26] and common carp fed with cecropin diets [18], which may be in response to
263
different tested animals or AMPs.
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The levels of serum HDL-C and LDL-C are vital indices that reflect
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lipometabolic status. HDL-C, which is usually considered the good cholesterol, can
ACCEPTED MANUSCRIPT prevent the occurrence of atherosclerosis, whereas LDL-C has the opposite influence
267
on the body when present in high levels. In this study, the supplement of AMPs had an
268
impact on anti-atherosclerosis of fish attributed to the increased HDL-C content in
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serum. LDL-C contents remained stable in all groups except in the fish fed with the
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G4 diet, where it was present at a higher level. This suggests that higher doses of NP
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supplementation may be capable of inducing atherosclerosis, but further studies are
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required due to the higher HDL-C content. Additionally, it was found that Glu content
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was not affected by dietary dosages of AMPs.
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LPS is a main component of cell walls of Gram-negative bacteria. LPS enters the
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bloodstream by way of intestinal absorption, bacteria that are released into the blood
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under the occurrence of sepsis, and exogenous injection. Detoxification is conducted
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by the liver and HDL, antibodies and phagocytes in blood [28]. The intestinal
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absorption results from intestinal inflammation and injury caused by proliferation of
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bacteria. The present study provides the first investigation into the effect of dietary
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AMP-supplementation on LPS content in serum. The results of this study indicated
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that the supplement of AMPs could significantly reduce the LPS level in tilapia, with
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the lowest value being found in fish fed with the G1 diet. This may partially be the
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result of the broad-spectrum antibacterial activity of NC to directly reduce the
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abundance of bacteria broad, partially due to the improvement of fish immunity.
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4.2 Effect of AMPs on antioxidant activity and non-specific immunity
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Like higher vertebrates, fish have the same susceptibility to the effects of
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reactive oxygen species (ROS) and innate and effective antioxidant defenses [29].
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Enzymatic and non-enzymatic antioxidant systems constitute “primary antioxidants”
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to protect organisms against oxidative stress and damage [29–31]. Non-enzymatic
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antioxidants are used as the first line of defense including substances such as certain
291
vitamins, amino acids, metalloproteins and carotenoids. SOD, CAT, GR and GPx are
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important antioxidants that play a vital role in counteracting ROS when other
293
antioxidant compounds are insufficient or depleted [31, 32]. Organisms can produce
294
ROS such as the superoxide radical (O2-), hydrogen peroxide (H2O2) and the hydroxyl
295
radical (OH•), which are eliminated to maintain dynamic balance during normal
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ACCEPTED MANUSCRIPT aerobic metabolism. However, excessive ROS or a decrease in the capacity to
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eliminate ROS can result in oxidative stress when endogenous or exogenous
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stimulations exist [33]. TAOC is recognized as an index to assess the functional state
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and reflect the health status of an organism indirectly [34]. SOD is a key antioxidant,
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which can catalyze O2- into H2O2 and oxygen. Further, CAT and GPx convert H2O2 to
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water in order to thoroughly clear oxyradicals [18]. MDA, the breakdown product of
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lipid peroxides, has a strong cytotoxicity and exposes fish to oxidative stress [35].
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Furthermore, as the first line of defense against pathogen infection, the non-specific
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immune system protects fish from microbial damage [36]. LZM is an indispensable
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enzyme of the non-specific immune system that has the ability to disrupt cell walls by
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splitting peptidoglycans to resist bacteria, especially Gram-positive species [37].
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Previous studies have confirmed that AMPs can enhance antioxidant activity and
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non-specific immunity in various species, including one study in which SOD activity
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was significantly increased in serum of weaned piglets treated with 10 mg/kg
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Rongchang pig AMP [26]. Another study found TAOC and SOD activities were
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significantly improved in Litopenaeus vannamei treated with 300 mg/kg of AMPs of
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Musca domestica [38]. In addition, SOD and LZM activities were increased and MDA
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content was decreased in serum of Megalobrama amblycephala treated with 0.2 %
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AMP extracted from Bacillus subtilis fmbJ [39] and SOD, CAT, LZM activities were
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increased in serum of Cyprinus carpio treated with 100–400 mg/kg of cecropin [18].
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The present results were in agreement with these studies. Higher TAOC, SOD, CAT
317
and LZM activities from tilapia fed with AMP-supplemented diets were observed, and
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MDA contents in all AMP-treated groups were significantly lower than the control
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group. The results clearly suggest that AMPs added to diets could improve antioxidant
320
activity and non-specific immunity of tilapia.
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4.3 Effect of AMPs on expression of immune-related genes
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In this experiment, the expression levels of four immune-related genes were
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examined to evaluate immune responses to dietary levels of AMPs in tilapia. TNF-α,
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IL-1β and IFN-γ are three important pro-inflammatory cytokines that are considered
325
to have regulatory roles in immune responses and can activate lymphocytes, natural
ACCEPTED MANUSCRIPT killer cells and macrophages, which lead to subsequent enhancements of nitric oxide
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production, respiratory burst activity and phagocytosis [40, 41]. As a multifunctional
328
inflammatory factor, TNF-α is produced by a variety of immune cells [42, 43] and is
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found to be the strongest anti-tumor cytokine at present which directly inhibits
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proliferation of tumor cells and resists bacteria and viruses by causing an
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inflammatory response [44]. IL-1β induces growth of lymphocytes to enhance
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immune responses and participates in resisting bacteria, viruses and tumors [45].
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IFN-γ is acknowledged as a Type-II interferon that plays important anti-virus and
334
immune regulation roles [46]. The three are early-response markers of inflammation
335
and regulate the expression of many other cytokines [47]. Additionally, HSP70 is also
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considered an important immune-related gene on account of its role in promoting
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immune responses. It has been reported that HSP70 has a series of biological
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functions including acting as an antioxidant and molecular chaperone, and in
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anti-apoptosis and synergetic immunity [48, 49] in order to protect cells from
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environmental stressors and pathogenic infections [50–52].
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Many studies have demonstrated that AMPs obtained from various sources could
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induce expression of immune-related genes, in which TNF-α, IL-1β and IFN-γ were
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involved, while no study has investigated HSP70. In in vitro models, BT
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(Brevibacillus texasporus) cationic peptides upregulated the transcription of
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pro-inflammatory cytokines IL-1β and IL-6, and inflammatory chemokines CXCLi1
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and CXCLi2 in chicken heterophils and monocytes under phorbol myristate acetate
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(PMA)-stimulation [53]. A study into weaned piglets challenged with E. coli found an
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upregulation in expression levels of IL-1β and IL-6 in serum induced by cecropin AD,
349
which enhanced the immunity of the organisms [54]. It has also been reported that
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diets supplemented with complex antibacterial peptides increased the contents of IL-2,
351
IL-4, IL-6, IFN-γ and TNF-α in serum of weaned piglets [55]. Transferring the genes
352
of tilapia hepcidin and shrimp chelonianin into zebrafish respectively also
353
significantly increased the expressions of immune-related genes IL-10, IL-21, IL-22,
354
Lyz, NF-κB, TNF-α, TLR-1 [56]. Another study on grouper (Epinephelus coioides)
355
indicated that the upregulation of IL-1β in muscle and of IL-8, TNF-α and IRF2 in
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ACCEPTED MANUSCRIPT liver was observed by electrotransfer of the epinecidin-1 gene into skeletal muscle.
357
However, cecropin used as an additive of common carp diets increased serum IL-1α,
358
IL-1β and IgM levels in lower doses and decreased IL-1α and IL-1β in higher doses
359
[18]. Additional studies using zebrafish [19], grouper [20] and half-smooth tongue
360
sole (Cynoglossus semilaevis) [57] have suggested that AMPs could modulate the
361
expression of immune-related genes including TNF-α, IL-1β and IFN-γ. Similar to the
362
reports above, it was observed that the supplement of NC-NP mixture enhanced the
363
expressions of TNF-α, IL-1β, IFN-γ and HSP70 in four organs (except in gill) in the
364
current study. The highest AMP dose also significantly upregulated the expressions of
365
four genes (except IL-1β in spleen, and TNF-α and HSP70 in gill). The expression
366
levels appeared, while variable, to increase with the increasing concentration of AMPs,
367
which indicates that the regulating capacity depends on the dosage of AMPs. This
368
study was initiated first to examine the effects of AMP additives on the expression of
369
the HSP70 gene. Our results have provided evidence that various antimicrobial
370
peptides can modulate immune-related gene expressions to play pivotal roles in
371
immune responses including inflammatory and antibacterial responses or antiviral
372
infection and stress resistance. Patterns will likely depend on the experimental
373
animals and peptides used. The mechanism has not been fully explored and needs
374
further investigation.
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In conclusion, the present study provided evidence that dietary NC-NP mixture
376
peptide supplementation can increase the contents of TP, ALB, GLB and HDL-C in
377
serum, improve antioxidant capacity and non-specific immunity and regulate
378
expression of immune-related genes to enhance positive immune responses for
379
disease resistance in tilapia (O. niloticus). This is the first record, to our knowledge, of
380
the application of NC and NP as additives in tilapia diets, which assessed
381
immune-regulation effects at the molecular level. Our results therefore provide an
382
important reference for further studies in this area. The present results indicated that
383
NC-NP mixture may be an attractive and potential candidate to enhance innate and
384
adaptive immunity to improve resistance to bodily diseases, and similar studies in the
385
near future are deemed necessary.
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ACCEPTED MANUSCRIPT 386 387
Acknowledgments The work was supported by the Ceeusro Project from Science and Technology
389
Program of Guangdong Province, China (grant No. 2015A090905022) awarded to Dr.
390
Hui-Hong Zhao.
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391 392
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ACCEPTED MANUSCRIPT Table 1 Formulation and approximate nutrient content of the basal diet (dry diet). Proportion (%)
Nutrients composition
Proportion (%)
Wheat middling
25.3
Moisture
9.02
Soybean meal
25
Crude protein
34.38
Rapeseed meal
15
Crude fat
Rice bran
10
Gross energy (KJ/g)
Peanut meal
10
Soybean oil
1
Corn gluten meal (60 %)
5.5
Fish meal
6
Betaine
0.3
Choline chloride (50 %)
0.2
Salt
0.1
Vitamin premix1
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0.1
EP
1
100
Vitamin premix: VA (IU/kg): 350,000, VD3 (IU/kg): 210,000, VE (g/kg): 6, VK3 (mg/kg): 500, VB1 (mg/kg): 450,
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18.88
0.5
Mineral premixb2 Total
7.45
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Vit C Phosphate ester
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Ingredients
VB2 (mg/kg): 900, VB6 (mg/kg): 600, VB12 (mg/kg): 2, VC (mg/kg): 14000, nicotinamide (mg/kg): 3500, D-calcium pantothenate (mg/kg): 2000, folic acid (mg/kg): 160, biotin (mg/kg): 8. 2
Mineral premix: Mg (mg/kg): 5000, Fe (mg/kg): 5250, Mn (mg/kg): 1000, I (mg/kg): 168, Cu (mg/kg): 788, Zn
(mg/kg): 4200, Se (mg/kg): 25, Co (mg/kg): 131.
ACCEPTED MANUSCRIPT Table 2 Primers used in this study and their sequences and sizes of PCR amplicon. Size of PCR amplicon Primer name
Sequence (5'--3')
TNF-α-F
GAGGTCGGCGTGCCAAGA
TNF-α-R
TGGTTTCCGTCCACAGCGT
IL-1β-F
GTTCACCAGCAGGGATGAGATT
IL-1β-R
TGCGGTCTTCACTGCCTCC
HSP70-F
TGGAGTCCTACGCCTTCAACA
HSP70-R
CAGGTAGCACCAGTGGGCAT
IFN-γ-F
TGACCACATCGTTCAGAGCA
IFN-γ-R
GGCGACCTTTAGCCTTTGT
β-actin-F
TGGTGGGTATGGGTCAGAAAG
β-actin-R
GCTCCTCAGGGGCAACTCT
（bp）
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119
122
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238
128
171
ACCEPTED MANUSCRIPT
Table 3
Control
G1
G2
G3
G4
G5
TP (g/L)
26.77±1.65c
29.30±0.61bc
30.70±1.23abc
32.33±1.27ab
32.20±0.59ab
34.23±1.60a
ALB (g/L)
13.23±1.31b
14.70±0.25ab
14.07±0.81ab
15.63±0.64a
15.87±0.34a
16.07±0.26a
GLB (g/L)
13.53±0.35c
14.60±0.61bc
16.63±0.89ab
16.70±0.65ab
16.33±0.34ab
18.17±1.63a
HDL-C (mmol/L)
1.60±0.07c
1.67±0.01bc
1.78±0.06abc
1.63±0.06bc
2.01±0.11a
1.87±0.11ab
LDL-C (mmol/L)
0.81±0.05b
0.78±0.02b
0.89±0.04ab
0.88±0.02ab
1.04±0.12a
0.90±0.05ab
Glu (mmol/L)
5.98±0.92ab
7.66±0.57a
7.34±0.37a
6.34±0.78ab
5.64±0.52ab
4.71±0.83b
LPS (pg/ml)
135.35±37.62a
69.02±6.35b
75.02±8.62b
80.85±6.26b
78.35±6.17b
72.68±11.74b
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Parameter
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Effect of AMP contents on serum biochemical parameters of tilapia (Oreochromis niloticus)
Values (mean ± S.E., n = 3) with different superscript letters (a, b, and c) in the same row are significantly different (P <0.05). TP: total protein; ALB: albumin, GLB: globulin; ALT: alkaline transaminase; HDL-C: high density lipoprotein
cholesterol;
LDL-C:
low
lipoprotein
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EP
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lipopolysaccharide.
density
cholesterol;
GLU:
blood
glucose;
LPS:
ACCEPTED MANUSCRIPT Table 4 Effect of AMP contents on antioxidant activity and non-specific immunity of tilapia (Oreochromis
Control
G1
G2
G3
G4
G5
TAOC (U/ml)
9.85±0.87b
13.27±0.35ab
26.14±5.40ab
28.96±8.37a
18.40±6.64ab
24.34± 7.23ab
SOD (U/ml)
14.52±0.29
14.60±2.50
14.60±1.81
17.98±1.04
16.30±1.40
17.03±0.27
CAT (U/ml)
7.75±4.30
15.81±2.39
8.88±2.61
14.45±3.41
15.36±3.86
10.69±2.28
MDA (nmol/ml)
6.96±0.20a
4.71±0.23b
3.72±0.58b
4.12±0.98b
3.33±0.47b
3.34±0.43b
LZM (µg/ml)
10.82±0.43b
11.66±1.31b
12.94±0.78b
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Parameter
SC
niloticus)
11.35±0.46b
21.52±1.74a
12.72±0.73b
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Values (mean ± S.E., n = 3) with different superscript letters (a and b) in the same row are significantly different (P<0.05). TAOC: total antioxidant capacity; SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde;
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LZM: lysozyme.
ACCEPTED MANUSCRIPT Figure legends Fig.1. Effects of AMP-supplementation on the relative expression levels of TNF-α (A), IL-1β (B), IFN-γ (C) and HSP70 (D) in the hepatopancreas, spleen, kidney and gill of tilapia (Oreochromis niloticus). The different superscript letters (a, b, c and d) among diet treatments in the same organ
RI PT
indicate significance differences (P<0.05, one-way ANOVA). Bars represent the mean values ± S.E. (n=3). TNF-α: tumor necrosis factor α; IL-1β: interleukin 1β; IFN-γ: interferon γ; HSP70: heat shock protein 70.
G1
G2
G3
B
A a ab
bc
7
ab
6 5 a
4 3
b
bc c
c
b
b
1
bb
Spleen
Kidney
Gill
D
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Hepatopancreas
Relative expression
bb
b
0
C
b
b
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2
Relative expression
a
10
Relative expression
15
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TNF-
G4
G5
SC
Control
ACCEPTED MANUSCRIPT Highlights
We assessed the effects of NC-NP mixture on antioxidant capacity, and immune responses in tilapia (GIFT), Oreochromis niloticus.
Diets supplemented with NC-NP mixture significantly decreased the LPS
Diets supplemented with NC-NP mixture increased the antioxidant capacity and improve the immunity of tilapia.
Diets supplemented with NC-NP mixture up-regulated the expressions of
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immune-related genes
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contents in serum of tilapia.