Residual pomegranate affecting the nonspecific immunity of juvenile Darkbarbel catfish

Journal Pre-proof Residual pomegranate affecting the nonspecific immunity of juvenile Darkbarbel catfish Pan Wu, Yonghe Gu, Rou Zhao, Yaxin Liu, Yanling Wang, Guozhong Lv, Zhenghai Li, Yajing Bao PII:

S1050-4648(19)30979-9

DOI:

https://doi.org/10.1016/j.fsi.2019.10.020

Reference:

YFSIM 6514

To appear in:

Fish and Shellfish Immunology

Received Date: 5 July 2019 Revised Date:

30 September 2019

Accepted Date: 7 October 2019

Please cite this article as: Wu P, Gu Y, Zhao R, Liu Y, Wang Y, Lv G, Li Z, Bao Y, Residual pomegranate affecting the nonspecific immunity of juvenile Darkbarbel catfish, Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.10.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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Residual pomegranate affecting the nonspecific immunity

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of juvenile Darkbarbel catfish Pan Wua, b, Yonghe Gua, Rou Zhaoa, Yaxin Liua, Yanling Wangc, Guozhong Lva, b*,

3 4 5 6 7

Zhenghai Lia, b**, Yajing Baoa, b*** a b c

School of Environment and Resources, Dalian Minzu University, Dalian 116600, China.

School of Resources and Environment, Northeast Agricultural University, Harbin 150030.

Department of Anesthesiology, the third affiliated hospital of SunYat-Sen University, Guangzhou, 510630, China.

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Abstract

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The improvement of resistance diseases, antioxidant and nonspecific immunity

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responses for Darkbarbel catfish by dietary residual pomegranate was investigated.

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The number of pathogenic bacteria and cumulative mortality of Darkbarbel catfish

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under 15-45% groups were decreased compared with control group (CK). Bioactive

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substances (polyphenols and vitamin) in residual pomegranate enhanced AKP, ACP,

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phagocytic, SOD, CAT activities through up-regulating AKP, ACP, SOD, CAT genes

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expression levels. Theoretical analysis showed bioactive substances regulated these

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genes expressions and enzyme activities as stimulus signal, component, active center.

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Moreover, residual pomegranate improved mTOR and NF-kB signaling pathway

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responses. Thus, residual pomegranate inhibited Aeromonas hydrophila that increased

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resistance to diseases. This technology completed the solid waste recovery and the

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Darkbarbel catfish growth performance simultaneously.

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Keyword: Darkbarbel catfish; Aeromonas hydrophila; antioxidant; resistance

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diseases; mTOR and NF-kB signaling pathway

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Introduction Pelteobagrus vachelli, namely Darkbarbel catfish meat is fine and tender, and is

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rich in the protein, fat, carbohydrate, vitamin, nicotinic acid and inorganic

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components such as calcium, phosphorus, iron [1]. Therefore, it is one of the most

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important and popular aquaculture species in China. But, the large-scale aquaculture

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for Darkbarbel catfish cause the pollution of aquaculture water and the frequent

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occurrence of disease by viruses, bacteria, fungi, parasites, and other pathogens *

Corresponding author. Tel.: +86 17866567878. E-mail address: [email protected].

**

Corresponding author. Tel.: +86 13904576826. E-mail address: [email protected]

***

Corresponding author. Tel.: +86 18567856666. E-mail address: [email protected].

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(Hemorrhagic edema, Saprolegniasis) [2]. Among, Aeromonas hydrophila (A.

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hydrophila) is the main pathogen for Darkbarbel catfish. These directly reduce the

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production performance of aquatic animals [3, 4]. Thus, the rapid growth of

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aquaculture and intensive production requires to adopt new strategies in breeding to

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improve the product quality. In recent years, non-synthetic dietary supplements

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(natural substances) have attracted much attention in aquaculture to replace chemical

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substances in order to improve the growth performance and disease resistance of fish

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[5, 6].

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Pomegranate is rich in anthocyanins, polyphenols, 1,1-diphenyl-2-picrylhydrazyl,

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flavonoids, polysaccharides, tannins, alkaloids, organic acids, with anti-aging and

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reducing blood lipid and blood pressure functions [7, 8]. As a result, pomegranate is

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widely grown and eaten. With the increase of demand, a large number of residual

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pomegranate (peel, core, even flesh) are produced as solid waste, which cause the

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environmental problems of solid waste pollution. Moreover, this also lead to the

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resource waste because pomegranate residue as nature resource has the potential of

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recycling to replace fish dietary.

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Therefore, a novel strategy of the improvement of disease resistance for using

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residual pomegranate is proposed in this work. The residual pomegranate is directly

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re-utilized. The new strategy is conducive to solid waste reduction, chemical feed

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reduction, resource recovery, aquaculture at the same time.

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To our best knowledge, the enhancement of disease resistance for Darkbarbel

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catfish by residual pomegranate was not researched. Moreover, the mechanism also is

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not clear that residual pomegranate regulate the immunity, antioxidant and disease

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resistance of Darkbarbel catfish. Therefore, the purpose of the work is to investigate

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the feasibility of the residual pomegranate enhancing disease resistance; to explain the

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mechanism of the residual pomegranate affecting disease resistance in terms of

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nonspecific immunity, antioxidant and mTOR and NF-kB signaling pathways

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responses.

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Materials and methods

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Fish rearing by the residual pomegranate and challenge test

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The experiments were carried out at May to June, 2018. Darkbarbel catfish (30 ±

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5 g and 11 ± 3 cm) were bought from the local fish farming plant. Pomegranate

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purchased from local market. Residual pomegranate (peel and core) was crushed to

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puree. A total of 150 fish were acclimated in tank at least 7 days. During the

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acclimatization, the fish were fed every day with commercial fish feed.

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After acclimatization, 120 fish was selected from 150 Darkbarbel catfish and

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assigned to triplicate four groups with 12 tanks (10 fish per 80 L tank containing 60 L

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water) randomly. Full-ration formula feed was partially replaced by residual

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pomegranate. The ingredients and inclusion levels of each feedstuff was presented in

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Table 1. These inclusion (protein and lipid) levels were chose based on the

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requirements of Darkbarbel catfish as per the method [1-2] and the ingredients and

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inclusion levels of pomegranate. These experimental diets was isonitrogenous and

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isolipidic (Table 1). Four processing groups were set and as follows: control group

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without substitution supplement; 15% substitution supplement group; 35%

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substitution supplement group; 45% substitution supplement group. Each processing

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group (CK, 15%, 35%, 45%) was repeated three times. The original water was

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renewed daily and the feces was removed. Darkbarbel catfish were fed once daily at a

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rate of 10% -15% of body weight during the experiment based on Lin et al. 2017 [9].

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Fish feces was removed with a siphon once daily during culturing. Water temperature

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(25.0 ± 1.0 °C), dissolved oxygen (6.0 ± 1.0 mg/L), and pH (7.0 ± 1.0) were

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respectively determined daily using a thermometer, DO meter, and pH meter.

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Afterward, in this work, all fish were administered in strict accordance with the

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National Institutes of Health Guide for the Care and Use of Laboratory Animals

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(China).

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After 30 days, the challenge test was carried out for all fishes according to Lin et

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al. 2017; Kong et al. 2017; Chiu et al. 2015; Yuan et al. 2019 [9-12]. Aeromonas

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hydrophila (A. hydrophila, strain number: ATCC7966, presentation from Dalian

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Ocean University), selected as the pathogen, was cultured on tryptic soy agar for 24h

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at 28 °C and transferred to 50mL of tryptic soy broth for 24h at 28 °C as the stock test

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culture. Broth cultures were centrifuged at 7000g for 10min at 4 °C. The supernatant

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was discarded, and bacterial pellets were re-suspended in sterilized saline solution

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(0.85% NaCl) as the stock bacterial solution. The stock bacterial solution was not

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washed before injection. The challenge test was carried out in triplicate by an

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intraperitoneal injection of 60µL of the stock bacterial solution, resulting in 4 × 106

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cfu/g body weight.

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Analysis and measurement

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After 45 days feeding (after 15 days of challenge), three individuals per tank

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were collected in each processing group. Darkbarbel catfish were anesthetized in ice

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water prior to euthanasia. The fish were then decontaminated with 70% ethanol and

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dissected immediately with sterile scissors. Small pieces of liver and head kidney

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were immersed in TRIzol reagent and stored in -80 °C until RNA extraction. Freshly

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dissected intestine were placed into filter-sterilized PBS and frozen at -20 °C until

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DNA extraction.

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Cumulative mortality and the populations of A. hydrophila

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The mortality was observed after 15 days of challenge. The cumulative

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mortality was calculated. The concentration of Aeromonas hydrophila (A. hydrophila)

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was also measured using selective media. Each dissected intestine sample (5 g) was

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put into 50 ml of sterile distilled water and incubated in a rotary shaker (160 rpm) at

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28 °C for 30 min. To assess the populations of A. hydrophila, the suspensions (200 µl)

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were smeared on Rimler-shotts and AHM culture mediums according to the National

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standard law of China GBT18652-2002 (Methods for detection of pathogenic A.

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hydrophila).

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Determination of various enzyme activities

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The superoxide dismutase (SOD) and catalase (CAT) activities in liver; the

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alkaline phosphatase (AKP), acid phosphatase (ACP) in head kidney were measured

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at 550nm, 240nm, 510nm, 440nm, 700nm using the assay kit (Nanjing Jiancheng

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Bioengineering Institute, China) by a UV/Vis spectrophotometer (Pharmacia Biotech

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Ultrospec 2000) respectively and according to Lin et al. 2017; Kong et al. 2017; Chiu

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et al. 2015; Yuan et al. 2019 [9-12]. The head kidney macrophages were isolated and

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prepared according to Secombes 1990 [13]. The phagocytic activity of macrophages

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was determined by the following Sakai et al. 1995 and Houwen, 2002 [14, 15].

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Immune enzymes-related genes and antioxidant enzymes-related genes expression

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According to Kong et al. 2017; Qi et al. 2017 [10, 16], total RNA was extracted

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from tissue samples by TRIzol Reagent (Cwbio, Beijing, China) and treated with

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4×gDNA wiper Mix to minimize the contamination of genomic DNA. The quality and

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purity of RNA were verified by electrophoresis on ethidium bromide staining 1.0%

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agarose gels and by A260 nm/A280 nm ratio. Complementary DNA was then

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synthesized using the HiScript® Reverse Transcriptase Kit (Vazyme, Jiangsu, China)

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following the instructions. The real-time quantitative PCR (RT-qPCR) was performed

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using AceQ™ qPCR SYBR ® Green Master Mix kit and CFX96 Real-Time PCR

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Detection System (Bio-Rad, USA). The β-actin gene was used as a house keeping

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gene. The PCR primer sequences and the reaction conditions used for real-time

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quantitative PCR are listed in Table S1, and the cycleindex was 30. The PCR

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efficiency of each primer was between 95.6% and 99.2%. RNA extracted from the

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head kidney was performed to detect the expression of immune enzymes-related

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genes (AKP, ACP) genes and TOR gene, 4E-BP gene in mTOR and NF-kB p65, IkB in

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NF-kB signaling pathway genes. RNA extracted from the livers was performed to

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detect the expression of antioxidant enzymes-related (SOD, CAT) genes. Each

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individual sample was run in triplicate wells. The RT-qPCR data were analyzed by the

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2-△△Ct method [17].

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Statistical analyses

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Data were subjected to one-way analysis of variance (ANOVA) in SPSS 18.0;

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mean differences among treatments were compared using Duncan's multiple-range

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test. The significant level was set at P < 0.05. Before statistical analyses, data were

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checked for normality of distribution and homogeneity of variance with the

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KolmogorovSmirnov test and Levene's test, respectively. If the data did not conform

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to normality of distribution and homogeneity of variance, the data were transformed

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by square root transformation or logarithmic transformation or arcsine transformation.

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Data were expressed as means ± SE.

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Results

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The feasibility of increasing resistance diseases ability for Darkbarbel catfish

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with residual pomegranate

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To research the effect of dietary residual pomegranate supplement on resistance

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diseases for Darkbarbel catfish, the Aeromonas hydrophila (A. hydrophila) challenge

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test was conducted. The mortality was observed after 15 days of Aeromonas

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hydrophila (A. hydrophila) challenge. The cumulative mortality was calculated (Table.

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2). Table 2 indicated that residual pomegranate inhibited A. hydrophila. The number

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of pathogenic bacteria and cumulative mortality in 15%, 35%, 45% groups were the

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lower than CK group, and presented significant difference (P<0.05). Among, 35%,

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45% groups was the best for the number of pathogenic bacteria and cumulative

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mortality. Meanwhile, the cumulative mortality (4-6%) was showed in non-challenged

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control group (Table S2). Further, figures S1-S2 showed the pathological changes of

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spleen and intestine were observed. These indicated that the cumulative mortality of

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challenge were mainly caused by A. hydrophila in CK, 15%, 35%, 45% groups.

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Darkbarbel catfish was infected by A. hydrophila. In addition, the growth of

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Darkbarbel catfish after feeding CK, 15%, 35% and 45% substitution supplement

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group were very well and its yield was significantly increased (Table S2). Table 2

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indicated that it had very good feasibility to increase the resistance diseases ability for

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Darkbarbel catfish using residual pomegranate.

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Improving non-specific immune and antioxidant response to fish diseases

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Table 2 showed that the resistance diseases were improved by residual

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pomegranate. These findings might be related to the nonspecific immunity and

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antioxidant responses. Therefore, superoxide dismutase (SOD) and catalase (CAT)

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activities in liver; the alkaline phosphatase (AKP), acid phosphatase (ACP) in head

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kidney were measured (Fig. 1). To investigate the mechanism by which the residual

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pomegranate affected resistance diseases, the alkaline phosphatase (AKP), acid

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phosphatase (ACP), phagocytic, superoxide dismutase (SOD) and catalase (CAT)

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activities of Darkbarbel catfish were determined. The results were showed in Fig. 1.

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The AKP, ACP, phagocytic, SOD, CAT activities of Darkbarbel catfish in

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15-45% groups were the better than the control group, and presented significant

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difference for CK group (P<0.05). Among, 35%, 45% groups was the best and

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showed significant difference for other groups (P<0.05).

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Meanwhile, to further investigate the molecular biological mechanism of the

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residual pomegranate regulating nonspecific immunity and antioxidant, AKP, ACP,

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SOD, CAT gene expression levels were determined. These results were showed in Fig.

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2. The AKP, ACP, SOD, CAT gene expression levels in other three given groups were

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the better than the control group, and 15%, 35%, 45% groups presented significant

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difference for the control group (P<0.05). Among, 35%, 45% groups had the best

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AKP, ACP, SOD, CAT gene expression levels, and showed significant difference for

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other groups (P<0.05). Figs. 1-2 indicated that residual pomegranate improved AKP,

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ACP, SOD, CAT activities through up-regulating AKP, ACP, SOD, CAT gene

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expression levels.

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Regulating mTOR and NF-kB signal transduction pathways response to fish

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diseases

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Table 2 showed that the resistance diseases were improved by residual

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pomegranate. These findings might be related to the mTOR and NF-kB signaling

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pathway responses. RNA extracted from the head kidney was performed to detect the

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expression of TOR gene, 4E-BP gene in mTOR and NF-kB p65, IkB in NF-kB

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signaling pathway genes (Fig. 3). To investigate the mechanism by which the residual

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pomegranate affected Darkbarbel catfish resistance diseases, the relative expression

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levels of mTOR and NF-kB signaling pathway genes in head kidney were exhibited in

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Fig. 3.

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Compared with the control group, the relative expression level of TOR gene,

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4E-BP gene in mTOR and NF-kB p65, IkBa in NF-kB was increased in 15%, 35%,

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45% groups, and 35%, 45% groups presented significant difference for CK group

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(P<0.05). Among, 35%, 45% groups was the best, and showed significant difference

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for other groups (P<0.05). Fig. 3 indicated that residual pomegranate promoted

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mTOR and NF-kB signaling pathway through up-regulating TOR gene, 4E-BP gene in

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mTOR and NF-kB p65, IkBa in NF-kB gene expression levels.

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Discussion

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Current research showed that 15-45% groups had better promoting effect on the

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resistance diseases than control group (CK) (Table 2). The main reason was the

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replacement of dietary residual pomegranate. It could be found form Table 1 that

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these diets are isonitrogenous and isolipidic. But, compared with CK group, the

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supplement of dietary residual pomegranate provided more rich and multiplex

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nutrients such as (flavonoids and polyphenols) for Darkbarbel catfish (Table 1). Costa

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et al. 2019; Khodabakhshian, 2017 [18,19] showed that residual pomegranate

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contained bioactive substances (flavonoids, vitamin, polyphenols) and metal ions.

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Further, Hoskin et al. 2018; Yi et al. 2017 [20, 21] observed the effects of polyphenol

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on enzyme activities of antioxidant and immune systems in vitro. Li et al. 2019 [22]

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used flavonoids to improve antioxidant and immune-related enzyme activities in

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juvenile northern snakehead fish. Ruiz et al. 2019; Wu et al. 2016 [23, 24] observed that

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vitamin C, E, K increased the antioxidant and non-specific immune enzyme activities

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in various aquatics. Table 2 indicated that it was feasible to promote resistance

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diseases by residual pomegranate.

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In this work, these findings in Figs. 1-2 indicated that the residual pomegranate

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improved the nonspecific immunity and antioxidant responses by regulating the

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expression level of related genes. For nonspecific immunity systems, both AKP and

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ACP were the marker enzyme of macrophage lysosome in organism, and were also

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the important hydrolytic enzyme in nonspecific immunity [25, 26]. They could kill

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invading pathogens, and also accelerate the phagocytosis of phagocytes and the

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degradation rate of foreign bodies. Moreover, AKP was closely related to the growth

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of aquatic animals, and played an important role in the absorption and utilization of

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nutrition, even the synthesis of protein. Thus, higher AKP and ACP activities had a

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positive effect on defense against external pathogens and microbial invasions.

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Meanwhile, it was also observed from Fig. 1 that phagocytic activity was significantly

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increased. Leukocyte had the function of the phagocytosis for pathogenic bacteria and

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bactericidal, which was an important aspect of non-specific immunization [9, 10]. As

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Fig. 1 shown, 15-45% groups significantly increased the AKP, ACP, phagocytic

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activities of Darkbarbel catfish, which improved the nonspecific immunity response

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ability, disease resistance (Table 2).

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As for antioxidant systems, the SOD, CAT were the vital enzymes in

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antioxidant defense system [10, 16]. They were able to scavenge reactive oxygen

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species (Ros) and alleviate its damage to cells. Moreover, SOD could enhance the

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defense function of macrophages, and was closely related to the immune system. As

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Fig. 2 shown, 15-45% groups significantly increased SOD, CAT activities. This

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finding indicated that residual pomegranate could enhance the antioxidant ability of

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Darkbarbel catfish, protected cells from damage, improved the survival rate and

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promoted the growth of Darkbarbel catfish (Table 2). Thus, Table 2 indicated that

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residual pomegranate inhibited A. hydrophila because that it enhanced the antioxidant

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and non-specific immune ability of Darkbarbel catfish (Figs. 1-2).

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Meanwhile, these findings of Figs. 1 showed the residual pomegranate enhanced

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AKP, ACP, SOD, CAT activities simultaneously. Prado et al. 2019; Nugroho et al.

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2017 [18, 19] showed that residual pomegranate contained bioactive substances

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(flavonoids, vitamin, polyphenols) and metal ions. Further, Hoskin et al. 2018; Yi et al.

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2017 [20, 21] observed the effects of polyphenol on enzyme activities of antioxidant

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and immune systems in vitro. Li et al. 2019 [22] used flavonoids to improve

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antioxidant and immune-related enzyme activities in juvenile northern snakehead fish.

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Ruiz et al. 2019; Wu et al. 2016 [23, 24] observed that vitamin C, E, K increased the

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antioxidant and non-specific immune enzyme activities in various aquatics. To sum up,

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residual pomegranate regulated AKP, ACP, SOD, CAT activities by containing

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bioactive substances and metal ions.

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Concerning the mechanism of bioactive substances and metal ions regulating

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these enzymes activities, there might be two reasons. Firstly, the bioactive substances

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and metal ions constituted enzymes or regulated enzyme synthesis pathway. Residual

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pomegranate contained a variety of amino acids, which were the basic components of

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enzymatic proteins [27]. At the same time, they contained a large number of vitamins,

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which constituted a variety of co-enzymes (flavin mononucleotide, Coenzyme A,

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transmethylase) [27]. Iron, magnesium and zinc were also the active center of the AKP,

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SOD, CAT in this work.

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Secondly, residual pomegranate also might induce or stimulate the expression of

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AKP, ACP, SOD, CAT gene as stimulation signal or activation factor (Fig. 2). The

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supplement of dietary residual pomegranate provided flavonoids and polyphenols for

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Darkbarbel catfish (Table 1). The substitution of residual pomegranate in diet affected

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the expression of AKP, ACP, SOD, CAT gene. This view was explained by some

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researches. Residual pomegranate were rich in vitamin, polyphenol, flavonoids,

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organic acid [18, 19]. Zhao et al. 2019 [28] observed that polyphenol modulated the

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gene expression in NRF2/HO-1 MAPK signaling. Rahman et al. 2019 [29] found that

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effect of vitamin C on the gene expression of the Nile tilapia. He et al. 2017 [30]

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observed that the organic acids increased the gene expression levels of TNF-α, LITAF

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and RAB6A in shrimp. Tian et al. 2019 [31] observed that flavonoids increased the

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gene expression in NF-κB and MAPK signalling pathways.

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As a signal transduction pathway, the mTOR signaling pathway, which plays a

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vital role in nutrition regulation and has complex impact on cell growth [32], widely

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exists in eukaryotes [33], food intake and environmental stresses [34]. TOR pathway

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is a key regulator of the balance between protein synthesis and degradation in

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response to nutrition quality and quantity [35, 36], and the protein synthesis is

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essential for cell growth, proliferation, apoptosis, and autophagy [37]. Moreover,

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immune protein synthesis and nutrient transport are also each related to mTOR [38].

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In this study, higher genes expression in mTOR signaling pathway was induced in

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resistance diseases, immune-related enzymes were enhanced in 15-45% groups.

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As for NF-kB signaling pathway, it regulated the congenital and acquired

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immunity, inflammation, stress response and the formation of B cell and lymphoid

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organ [39, 40]. It was closely related to the differentiation of immune cells [41]. In

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this study, higher genes expression in NF-kB signaling pathway was induced in

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15-45% groups, and then immune-related enzymes activities were enhanced in

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15-45% groups. Therefore, phagocytic activity was enhanced under 15-45% groups in

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this work. These results suggested that residual pomegranate promoted the activities

298

of immune-related enzymes by regulating NF-kB signaling pathway.Thus, the number

299

of pathogenic bacteria and cumulative mortality of Darkbarbel catfish was decreased

300

under 15-45% groups, and thus resistance diseases was increased (Table 2).

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To the best of researchers’ knowledge, the present study is the first one

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addressing to enhancing resistance diseases for Darkbarbel catfish with the residual

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pomegranate. Table 2 indicated that residual pomegranate could be reused directly to

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enhance resistance diseases. Residual pomegranate improved the technology reduced

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the use of chemical feeds in aquaculture, and completed the recycle and reuse of

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residual pomegranate.

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Conclusion

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Improvement of disease resistance for Darkbarbel catfish by residual pomegranate

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was feasible. Bioactive substances in residual pomegranate improved nonspecific

310

immunity, antioxidant, mTOR and NF-kB signaling pathway responses through

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up-regulating related genes expression levels. Theoretical analysis showed residual

312

pomegranate including bioactive substances regulated these genes expressions and

313

enzyme activities as stimulus signal, component, active center. Thus, residual

314

pomegranate inhibited the number of pathogenic bacteria and cumulative mortality of

315

Darkbarbel catfish. This technology increased the disease resistance of Darkbarbel

316

catfish.

317

Acknowledgments

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The authors gratefully acknowledge the National Nature Science Fund for

319

Distinguished Young Scholars of China (Grant No. 41625002), the National Natural

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Science Foundation Young of China (Grant No. 31700432), the National Natural

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Science Foundation of China (Grant No. 31700432; 31470550; 81500493; 31400386;

322

51778114), Post-doctoral Science Foundation Project of China and Heilongjiang

323

Province (Grant No. 2018M641797; 2018HLJ6578), Natural Science Foundation of

324

Guangdong Province (Grant No. 2015A030313098), Application Technology

325

Research and Development Projects of Harbin (Grant No. 2016RAXXJ103), the

326

MOA Modern Agricultural Talents Support Project for valuable financial support.

327

Supplementary material

328

The primers and anneal temperatures of AKP, ACP, SOD, CAT, TOR, 4E-BP, NF-kB

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p65, IkBa genes (Table S1); The cumulative mortality in non-challenged group and

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the growth of Darkbarbel catfish after feeding CK, 15%, 35% and 45% groups (Table

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S2); Pathological changes of intestine in Darkbarbel catfish infected with Aeromonas

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hydrophila (Fig. S1); Pathological changes of spleen in Darkbarbel catfish infected

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with Aeromonas hydrophila (Fig. S2).

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References

335

[1]

K.K. Zheng, X.M. Zhu, D. Han, Y.X. Yang, W. Lei, S.Q. Xie, Effects of dietary lipid levels on growth,

336

survival and lipid metabolism during early ontogeny of Pelteobagrus vachelli larvae, Aquaculture. 299 (2010)

337

121-127.

338

[2]

J. Zhang, N.N. Zhao, Z. Sharawyd, Y. Li, J. Ma, Y.N. Lou, Effects of dietary lipid and protein levels on

339

growth and physiological metabolism of Pelteobagrus fulvidraco larvae under recirculating aquaculture

340

system (RAS), Aquaculture. 495 (2018) 458-464.

341

[3]

H.K. Miandare, S. Farvardin, A. Shabani, S.H. Hoseinifar, S.S. Ramezanpour, The effects of

342

galactooligosaccharide on systemic and mucosal immune response, growth performance and appetite related

343

gene transcript in goldfish (Carassius auratus gibelio), Fish & Shellfish Immunology. 55 (2016) 479-483.

344

[4]

345 346

P. Samanta, H. Im, J. Na, J.H. Jung, Ecological risk assessment of a contaminated stream using multi-level integrated biomarker response in Carassius auratus, Environmental Pollution. 233 (2018) 429-438.

[5]

H. Dadras, M.R. Hayatbakhsh, W.L. Shelton, A. Golpour, Effects of dietary administration of Rose hip and

347

Safflower on growth performance, haematological, biochemical parameters and innate immune response of

348

Beluga, Huso huso (Linnaeus, 1758), Fish & Shellfish Immunology. 59 (2016) 109-114.

349

[6]

M. Yousefi, S.M. Hoseini, Y.A. Vatnikov, E.V. Kulikov, S.G. Drukovsky, Rosemary leaf powder improved

350

growth performance, immune and antioxidant parameters, and crowding stress responses in common carp

351

(Cyprinus carpio) fingerlings, Aquaculture. 505 (2019) 473-480.

352

[7]

A. Maity, J. Sharma, R.K. Pal, Novel potassium solubilizing bio-formulation improves nutrient availability,

353

fruit yield and quality of pomegranate (Punica granatum L.) in semi-arid ecosystem, Scientia Horticulturae.

354

255 (2019) 14-20.

355

[8]

J.M. Chater and L.C. Garner, Foliar nutrient applications to 'Wonderful' pomegranate (Punica granatum L.).

356

I. Effects on fruit mineral nutrient concentrations and internal quality, Scientia Horticulturae. 244 (2019)

357

421-427.

358

[9]

H.L. Lin, Y.L. Shiu, C.S. Chiu, SL. Huang, CH. Liu, Screening probiotic candidates for a mixture of

359

probiotics to enhance the growth performance, immunity, and disease resistance of Asian seabass, Lates

360

calcarifer (Bloch), against Aeromonas hydrophila, Fish & Shellfish Immunology. 60 (2017) 474-482.

361

[10] X.H. Kong, D. Qiao, X.L. Zhao, L. Wang, H.X. Zhang, The molecular characterizations of Cu/ZnSOD and

362

MnSOD and its responses of mRNA expression and enzyme activity to Aeromonas hydrophila or

363

lipopolysaccharide challenge in Qihe crucian carp Carassius auratus, Fish & Shellfish Immunology. 67

364

(2017) 429-440.

365

[11] S.T. Chiu, Y.L. Shiu, Wu Tsung-Meng, Y.S. Lin, C.H. Liu, Improvement in non-specific immunity and

366

disease resistance of barramundi, Lates calcarifer (Bloch), by diets containing Daphnia similis meal, Fish.

367

Shellfish Immunol. 44 (2015) 172-179.

368

[12] Y. Yuan, M. Jin, J. Xiong, Q.C. Zhou, Effects of dietary dosage forms of copper supplementation on growth,

369

antioxidant capacity, innate immunity enzyme activities and gene expressions for juvenile Litopenaeus

370

vannamei, Fish & Shellfish Immunology. 84 (2019) 1059-1067.

371

[13] C.J. Secombes, Isolation of salmonid macrophage and analysis of their killing activity, in: T.C. Fletcher, D.P.

372

Anderson, B.S. Roberson, W.B. Van Muiswinkel (Eds.), Techniques of Fish Immunology, SOS Publications,

373

Fair Haven. NJ. 1 (1990) 137-154.

374 375

[14] M. Sakai, M. Kobayashi, T. Yoshida, Activation of rainbow trout, Oncorhynchusmykiss, phagocytic cells by administration of bovine lactoferrin, Comp. Biochem. Physiology. 110B (1995) 755-759.

376

[15] B. Houwen, Blood film preparation and staining procedures, Clin. Lab. Med. 22 (2002) 1-14.

377

[16] X.Z. Qi, M.Y. Xue, S.B. Yang, J.W. Zha, F. Ling, Ammonia exposure alters the expression of immune-related

378

and antioxidant enzymes-related genes and the gut microbial community of crucian carp (Carassius auratus),

379

Fish & Shellfish Immunology. 70 (2017) 485-492.

380 381

[17] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method, Methods. 25 (2001) 402-408.

382

[18] A.M.M. Costa, L.O. Silva, A.G. Torres, Chemical composition of commercial cold-pressed pomegranate

383

(Punica granatum) seed oil from Turkey and Israel, and the use of bioactive compounds for samples’ origin

384

preliminary discrimination, Journal of Food Composition and Analysis. 75 (2019) 8-16.

385

[19] R. Khodabakhshian, Feasibility of using Raman spectroscopy for detection of tannin changes in pomegranate

386 387

fruits during maturity, Scientia Horticulturae. 257 (2019) 108670. [20]

R.T. Hoskin, J. Xiong, D.A. Esposito, M.A. Lila, Blueberry polyphenol-protein food ingredients: The

388

impact of spray drying on the in vitro antioxidant activity, anti-inflammatory markers, glucose metabolism

389

and fibroblast migration, Food Chemistry. 280 (2019) 187-194.

390

[21] J.J. Yi, H. Qu, Y.Z. Wu, Z.Y. Wang, L. Wang, Study on antitumor, antioxidant and immunoregulatory

391

activities of the purified polyphenols from pinecone of Pinus koraiensis on tumor-bearing S180 mice in vivo,

392

International Journal of Biological Macromolecules. 94 (2017) 735-744.

393

[22] M.Y. Li, X.M. Zhu, J.X. Tian, M. Liu, G.Q. Wang, Dietary flavonoids from Allium mongolicum Regel

394

promotes growth, improves immune, antioxidant status, immune-related signaling molecules and disease

395

resistance in juvenile northern snakehead fish (Channa argus), Aquaculture. 501 (2019) 473-481.

396

[23] M.A. Ruiz, M. B. Betancor, L. Robaina, D. Montero, M. J. Caballero, Dietary combination of vitamin E, C

397

and K affects growth, antioxidant activity, and the incidence of systemic granulomatosis in meagre

398

(Argyrosomus regius), Aquaculture. 498 (2019) 606-620.

399

[24] Y.S. Wu, S.Y. Liau, C.T. Huang, F.H. Nan, Beta 1,3/1,6-glucan and vitamin C immunostimulate the

400

non-specific immune response of white shrimp (Litopenaeus vannamei), Fish & Shellfish Immunology. 57

401

(2016) 269-277.

402

[25] E. Gisbert, H. Nolasco, M. Solovyev, Towards the standardization of brush border purification and intestinal

403

alkaline phosphatase quantification in fish with notes on other digestive enzymes, Aquaculture. 487 (2018)

404

102-108.

405 406 407 408

[26] Z.Q. Xia and S.J. Wu, Effects of glutathione on the survival, growth performance and non-specific immunity of white shrimps (Litopenaeus vannamei), Fish & Shellfish Immunology. 73 (2018) 141-144. [27] M. Kobayashi and Y.T. Tchan, Treatment of industrial waste solutions and production of useful by-products using a photosynthetic bacterial method, Water Res 7 (1973) 1219-1224.

409

[28] J. Zhao, J. Zang, Y. Lin, Y.H. Wang, X.J. Meng, Polyphenol-rich blue honeysuckle extract alleviates

410

silica-induced lung fibrosis by modulating Th immune response and NRF2/HO-1 MAPK signaling, Journal

411

of Functional Foods. 53 (2019) 176-186.

412

[29] A. Ramada, S.F. Sallam, M.S. Elsheikh, S.R. Ishak, M.G.R. Abdelsayed, S. Marwa, N. Rasha, K. Rabab,

413

O.M. Ibrahim, VDR gene expression in asthmatic children patients in relation to vitamin D status and

414

supplementation, Gene Reports. 15 (2019) 100387.

415

[30] W.Q. He, S. Rahimnejad, L. Wang, K. Song, C.X. Zhang, Effects of organic acids and essential oils blend on

416

growth, gut microbiota, immune response and disease resistance of Pacific white shrimp (Litopenaeus

417

vannamei) against Vibrio parahaemolyticus, Fish & Shellfish Immunology. 70 (2017) 164-173.

418

[31] C.L. Tian, P. Zhang, J. Yang, Z.H. Zhang, M.C. Liu, The protective effect of the flavonoid fraction of

419

Abutilon theophrasti Medic. leaves on LPS-induced acute lung injury in mice via the NF-κB and MAPK

420

signalling pathways, Biomedicine & Pharmacotherapy. 109 (2019) 1024-1031.

421 422

[32] N. Takei, K. Furukawa, O. Hanyu, H. Sone, H. Nawa, A possible link between BDNF and mTOR in control of food intake, Front. Psychol. 5 (2014) 1093-1095.

423

[33] A.M. Abuhagr, K.S. Maclea, E.S. Chang, D.L. Mykles, Mechanistic target of rapamycin (mTOR) signaling

424

genes in decapod crustaceans: cloning and tissue expression of mTOR, Akt, Rheb, and p70 S6 kinase in the

425

green crab, Carcinus maenas, and blackback land crab, Gecarcinus lateralis, Comp. Biochem. Physiol. A,

426

Mol. Integr. Physiol. 168 (2014) 25–39.

427

[34] H.D. Li, X.L. Tian, K. Zhao, W.W. Jiang, S.L. Dong, Effect of Clostridium butyricum in different forms on

428

growth performance, disease resistance, expression of genes involved in immune responses and mTOR

429

signaling pathway of Litopenaeus vannamai, Fish & Shellfish Immunology. 87 (2019) 13-21.

430 431

[35] S. Wullschleger, R. Loewith, M.N. Hall, TOR signaling in growth and metabolism, Cell. 124 (2006) 471-484.

432

[36] M. Wu, S. Lu, X. Wu, S. Jiang, Y. Luo, W. Yao, et al., Effects of dietary amino acid patterns on growth, feed

433

utilization and hepatic IGF-I, TOR gene expression levels of hybrid grouper (Epinephelus fuscoguttatus ♀ ×

434

Epinephelus lanceolatus ♂) juveniles, Aquaculture. 468 (2017) 508-514.

435 436 437 438

[37] J. Rohde, J. Heitman, M.E. Cardenas, The TOR kinases link nutrient sensing to cell growth, J. Biol. Chem. 276 (2001) 9583-9586. [38] M.E. Cardenas, N.S. Cutler, M.C. Lorenz, C.J.D. Como, J. Heitman, The TOR signaling cascade regulates gene expression in response to nutrients, Genes. 13 (1999) 3271-3279.

439

[39] J.H. Pan, L. Feng, W.D. Jiang, P. Wu, Y. Liu, Vitamin E deficiency depressed fish growth, disease resistance,

440

and the immunity and structural integrity of immune organs in grass carp (Ctenopharyngodon idella):

441

Referring to NF-κB, TOR and Nrf2 signaling, Fish & Shellfish Immunology. 60 (2017) 219-236.

442

[40] L. Li, F. Lin, W.D. Jiang, J. Jiang, Y. Liu, Dietary pantothenic acid deficiency and excess depress the growth,

443

intestinal mucosal immune and physical functions by regulating NF-κB, TOR, Nrf2 and MLCK signaling

444

pathways in grass carp (Ctenopharyngodon idella), Fish & Shellfish Immunology. 45 (2015) 399-413.

445

[41] G. Muthusamy, S. Gunaseelan, N.R. Prasad, Ferulic acid reverses P-glycoprotein-mediated multidrug

446

resistance via inhibition of PI3K/Akt/NF-κB signaling pathway, The Journal of Nutritional Biochemistry. 63

447

(2019) 62-71.

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

475

Figure and Table

476

Fig. 1. The nonspecific immune related enzyme and antioxidant related enzyme

477

activities of Darkbarbel catfish under CK, 15%, 35%, 45% groups Values (mean ±

478

S.E.) in the same column with different superscript letters significantly differ from

479

each other (P< 0.05).

480 481 482

Fig. 2. The relative expression levels of AKP, ACP, SOD, CAT genes under CK, 15%,

483

35%, 45% groups Values (mean ± S.E.) in the same column with different superscript

484

letters significantly differ from each other (P< 0.05).

485 486 487

Fig. 3. The relative expression levels of TOR, 4E-BP, NF-kB p65, IkBa genes in

488

mTOR and NF-kB signaling pathway under CK, 15%, 35%, 45% groups Values

489

(mean ± S.E.) in the same column with different superscript letters significantly differ

490

from each other (P< 0.05).

491 492 493

Table 1. The ingredients and inclusion levels under CK, 15%, 35%, 45% groups

494 495 496

Table 2. The number of pathogenic bacteria and cumulative mortality of Darkbarbel

497

catfish under CK, 15%, 35%, 45% groups Values (mean ± S.E.) in the same column

498

with different superscript letters significantly differ from each other (P< 0.05).

Antioxidant and non-speciafic immune enzymes activities (U/mg protein)

Fig. 1.

100

80

40 CK 15% 35% 45% c

60

SOD c c

b

CAT

c c

b

AKP

c

a b

a

a

20 c a b c

0 ACP

Fig. 2.

40

CK 15% 35% 45%

The relative expression levels

35 30

c b

25 a

20 c

15

c a

b b c

10

a

c a b

c c

5 0 SOD

CAT

AKP

ACP

c

Fig. 3.

70

The relative expression levels

60

CK 15% 35% 45%

c c

50 40 b

30 a

20 10 0

b c c a b c

4E-BP

c

a

NF-kB p65

a

b c c

IkBa

TOR

Table 1.

Composition

Control

15%

35%

45%

(g/kg)

group

group

group

group

424

424

424

424

Crude fat

80

80

80

80

Vitamin

5

7

11

14

Mineral

20

23

25

27

Flavonoids

0

0.05

0.08

0.1

Polyphenols

0

75

170

240

Crude protein

Table 2.

Group

Number of A.hydrophila (CFU/g)

Cumulative mortality (%)

CK

9.8×1011a

60.43±4.41a

15%

9.4×108b

50.87±3.49b

35%

4.4×104c

25.41±0.57c

45%

4.3×104c

25.65±0.53c

Highlights ▲ To culturing pelteobagrus vachelli by dietary residual pomegranate ▲ The residual pomegranate enhanced nonspecific immunity, antioxidant, disease resistance capacities ▲

The residual pomegranate improved mTOR, NF-kB signaling

ransduction pathway