Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare

Accepted Manuscript Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare Yu-Wen Dong, Lin Feng, Wei-Dan Jiang, Yang Liu, Pei Wu, Jun Jiang, Sheng-Yao Kuang, Ling Tang, Wu-Neng Tang, Yong-An Zhang, Xiao-Qiu Zhou PII:

S1050-4648(17)30655-1

DOI:

10.1016/j.fsi.2017.10.048

Reference:

YFSIM 4919

To appear in:

Fish and Shellfish Immunology

Received Date: 19 July 2017 Revised Date:

23 October 2017

Accepted Date: 26 October 2017

Please cite this article as: Dong Y-W, Feng L, Jiang W-D, Liu Y, Wu P, Jiang J, Kuang S-Y, Tang L, Tang W-N, Zhang Y-A, Zhou X-Q, Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare, Fish and Shellfish Immunology (2017), doi: 10.1016/ j.fsi.2017.10.048. 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.

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Dietary threonine deficiency ACCEPTED depressed the disease resistance, immune and physical MANUSCRIPT

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barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of

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Flavobacterium columnare

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Yu-Wen Dong

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Sheng-Yao Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c,*

, Lin Feng

a, b, c,1

, Wei-Dan Jiang

a, b, c

, Yang Liu

a, b, c

, Pei Wu

a, b, c

, Jun Jiang

a, b, c

,

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Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China

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b

Fish Nutrition and safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural

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University, Chengdu 611130, China

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c

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Agricultural University, Chengdu 611130, China

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d

Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China

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Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

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Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan

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*Corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,

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Sichuan, China. Tel.: +86 835 2885157; fax: + 86 8352 885968

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E-mail addresses: [email protected], [email protected] (X.-Q. Zhou);

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These two authors contributed to this work equally

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ACCEPTED MANUSCRIPT

Abstract

This study was conducted to investigate the effects of dietary threonine on the disease resistance, gill

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immune and physical barriers function of juvenile grass carp (Ctenopharyngodon idella). A total of 1080

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juveniles were fed six iso-nitrogenous diets containing graded levels of threonine (3.99-21.66 g kg-1 diet) for

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8 weeks, and then challenged with Flavobacterium columnare. Results showed that threonine deficiency

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(3.99 g kg-1 diet): (1) increased the gill rot morbidity after exposure to F. columnare; (2) attenuated the gill

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immune barrier function by decreasing antimicrobial substances production, up-regulating the mRNA levels

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of pro-inflammatory cytokines (except IL-12p40), and down-regulating the anti-inflammatory cytokines

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partly due to the modulation of NF-κB and TOR signaling. (3) disrupt the gill tight junction complexes by

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down-regulating TJs (claudin-3, -b, -c, 12, occludin, ZO-1 and ZO-2) and up-regulating TJs (claudin-7a, -7b)

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as well as related signaling molecule myosin light chain kinase mRNA levels (P < 0.05). (4) exacerbated the

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gill apoptosis by up-regulating cysteinyl aspartic acid-protease-3, 8, 9, c-Jun N-terminal kinases and

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mediating apoptosis related factors mRNA levels (P < 0.05); (5) exacerbated oxidative injury with increased

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reactive oxygen species, malondialdehyde and protein carbonyl contents (P < 0.05), decreased the

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antioxidant related enzymes activities and corresponding mRNA levels (except glutathione peroxidase-1b

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and glutathione-S-transferase-omega 2) as well as glutathione contents (P < 0.05) partly ascribe to the

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abridgement of NF-E2-related factor 2 signaling [Nrf2 / Keap1a (not Keap1b)] in fish gill. Overall,

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threonine deficiency depressed the disease resistance, and impaired immune and physical barriers in fish gill.

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Finally, based on the gill rot morbidity and biochemical indices (immune indices LA activity and

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antioxidant indices MDA content), threonine requirements for juvenile grass carp (9.53-53.43 g) were

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estimated to be 15.32 g kg-1 diet (4.73 g 100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1 protein ), 15.46 g

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kg-1 diet (4.77 g 100 g-1 protein ), respectively.

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Keywords: Threonine; Disease resistance; Immune barrier; Physical barrier; Gill; Juvenile grass carp

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(Ctenopharyngodon idella)

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ACCEPTED MANUSCRIPT

1. Introduction

Gill, a basic zone of oxygen entrance and excretion of nitrogenous in fish [1], have developed immune

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barriers and physical barriers to strengthen the resistance to pathogen invasion [2]. It was confirmed that the

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disruption of gill immune and physical barriers function leads to the depressed growth [3] and even high

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morbidity in fish [4]. Hence, it is of great importance to maintain gill immune and physical barriers function

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in fish. Our previous studies demonstrated that gill immune and physical barriers could be improved by

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nutrients like vitamin C [5], valine [6] and so on. Threonine is an essential amino acid limited for fish.

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Previous study of our lab found that threonine deficiency decreased the growth performance of Jian carp

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(Cyprinus carpio var.Jian) and juvenile grass carp (Ctenopharyngodon idella) [7, 8]. However, no reports

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focused on the relationship between threonine and gill immune and physical barriers function in fish. In

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liver, it was reported that threonine addition could increase the vitamin C levels in rat [9] and the valine

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levels in pigs [10]. Thus, there may be a possibility that threonine could influence the gill immune and

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physical barriers function, which awaits investigation.

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Fish immune barrier function was partly executed by antimicrobial compounds [e.g., lysozyme (LA),

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acid phosphatase (ACP)] and inflammatory cytokines [11-14]. To our knowledge, multiple cytokines could

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be mediated by nuclear factor κB (NF-κB) and target of rapamycin (TOR) signaling pathways [15].

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However, no direct evidence has been afforded about the effects of threonine on the antimicrobial

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compounds, cytokines and possible involved regulating mechanisms in fish gill. In the plasma of weaned

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pigs, threonine could increase the isoleucine level [16]. Previous study of our lab demonstrated that

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isoleucine could enhance the lysozyme and acid phosphatase activities in the serum of juvenile Jian carp

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(Cyprinus carpio var. Jian) [17]. Besides, threonine deficiency decreased the S-Adenosylmethionine (SAMe)

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synthesis in the mouse embryonic stem cells (mESCs) [18]. Li et al. reported that SAMe could promote the

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mTOR and inhibit NF-κBp65 signals activation, thus decreasing the lipopolysaccharide-induced TNF-α and

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IL-6 expression in the colon of mice [19]. Above observations remind us that threonine might modulate gill ACCEPTED MANUSCRIPT

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immune barrier and related signaling molecules, which is worthy of investigation. Apart from immune barrier, gill health is also depend on the physical barrier function [20]. In animals,

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damaged physical barrier function could be reflected in oxidative injury [21-25], cell apoptosis [26] and

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tight junction complexes (TJs) disruption [27, 28], which could be regulated by NF-E2-related factor 2

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(Nrf2) [29], c-Jun N-terminal protein kinase (JNK) [30] and myosin light chain kinase (MLCK) [31],

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respectively. However, no evidence has been given to show the effects of threonine on the physical barrier

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function and possible potential mechanisms in fish gill. Dai et al. [32] confirmed that threonine could be

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degraded into propionate in the small intestine of pigs. It was of note that sodium propionate could make a

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notable up-regulation of GPx in the liver of common carp [33]. In rat, threonine deficiency could make the

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GABAA receptor loss in the anterior piriform cortex [34]. Studies showed that GABAA receptor could

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decrease the Nrf2 expression in the astrocytes of mice [35] and the GSH content in rat hippocampus [36].

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Meanwhile, Hamard et al. [37] reported that threonine deficiency down-regulated the relative expression of

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insulin-like growth factor-2 (IGF-2) in the ileum of pigs. It was reported that IGF-2 stimulated the JNK

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activation and protected chondrosarcoma cells from apoptosis in mice [38]. Additionally, threonine

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deficiency decreased the cholesterol level in the liver of rat [39]. Studies confirmed that cholesterol could

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promote the expression of MLCK in the aorta [40] and occludin in the blood-testis barrier (BTB) of rabbit

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[41]. Therefore, there is a possibility that threonine might affect the physical barriers associated with

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oxidative status, apoptosis and TJs in fish gill, which is of great value to explore.

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Currently, dietary threonine requirement of juvenile grass carp for the optimal growth was estimated to

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be 14.53 g kg-1 diet (4.48 g threonine 100 g-1 protein) in our previous study [8]. However, nutrient

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requirements of fish may vary with different sensitive indices [42-44] and its metabolism utilization to make

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up for some loss during the acute-phase immune response [45]. Therefore, it is valuable to determine the

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threonine requirements based on different immune indices.

This study used the growth trial from our previous study [8], which is a part of a larger study ACCEPTED MANUSCRIPT

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conducted to investigate the effects of threonine on fish growth and health status. Based on the vital role that

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gill plays in fish growth and health, systematical study was intended to investigate the effects of threonine

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on antimicrobial substances, cytokines, antioxidants, apoptosis and intercellular TJs in the gills of juvenile

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grass carp after challenge with Flavobacterium columnare. Furthermore, we also investigated the effects of

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dietary threonine on related signaling molecules NF-κB, TOR, MLCK, JNK and Nrf2 in the gills of juvenile

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grass carp to reveal the underling mechanisms for threonine regulating gill immune and physical barriers

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function in fish. Additionally, threonine requirements of juvenile grass carp were also determined in

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different indices, which may provide a reference for formulating the commercial feed production of juvenile

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grass carp.

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2. Materials and methods

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2.1. Experimental design and diets

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The experiment diets were the same as our previous study (Table 1& Supplemental Table S1) [8].

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Dietary protein sources were supplied with fish meal. Casein and gelatin, and dietary lipid sources were

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supplied with fish oil and soybean oil. The overall composition of essential amino acids in the diets

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stimulated the amino acid pattern similar to that of found in 320.0 g kg-1 crude protein from grass carp

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whole-body protein, excluding threonine according to Wang et al. [46]. Threonine was added to the test

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diets at the determined concentrations of 3.99 (un-supplemented control), 7.70, 10.72, 14.10, 17.96 and

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21.66 g threonine kg-1 diet, respectively. Diets were made iso-nitrogenous with graded glycine instead of

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incremental threonine according to Tang et al. [47]. Pellets were produced and stored at -20 °C as refer to

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Hong et al.[48].

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(Table 1 inserted here) 2.2. Feeding trial

The procedures took place in theACCEPTED University of Sichuan Agricultural Animal Care Advisory Committee. MANUSCRIPT

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Juveniles were obtained from fisheries (Sichuan, China) and then acclimated to the experimental

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environment for 4 weeks as described by Hong et al. [48]. Then, 1080 fish (mean weight 9.53 ± 0.02 g)

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were randomly assigned to 18 experimental cages (1.4 L × 1.4 W × 1.4 H m), resulting in 60 fish per cage.

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Each cage was equipped with a disc of 100 cm diameter in the bottom to collect the uneaten feed according

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to our laboratory study [49]. Each cage was randomly assigned to one of three replicates of the six dietary

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treatments, and fish were fed with the respective diet four times daily for 8 weeks as refer to Wen et al.[50].

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Thirty minutes after feeding, uneaten feed was collected, dried, and weighed to calculate the feed intake, as

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described by Hong et al. [48]. During the experiment, water temperature was 28 ± 2 °C. The pH and

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dissolved oxygen were maintained at 7.0 ± 0.5 and not less than 6.0 mg/L, respectively. And the

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experimental units were under natural light and dark cycle, according to Yue et al. [51].

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2.3. Challenge test

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After the growth trial, we employed the successful model established by challenging with F. columnare

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to investigate the effects of threonine on gill immune and physical function of juvenile grass carp [5, 52].

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The F. columnare was kindly provided by College of Veterinary Medicine, Sichuan Agricultural University

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and cultured similar to the method described by Shoemaker et al.[53]. After feeding trial, thirty fish with

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similar body weight obtained from each experimental group were allowed to acclimate for 5 days before the

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challenge, as described by Arias et al.[54]. Then, fish were immersed into water containing 1.0×108

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colony-forming units (cfu) ml-1 F. columnare for 3 h, after which the fish were returned to corresponding

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cages to be observed for 3 days. The infection dose was sufficient to active the immune system and

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consequently enabled the investigation of effluent on reactivity against a threatening disease according to

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the results of our preliminary study data (unpubl. obs.). During the infectious trial, the experimental

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conditions were the same as those in the feeding trial.

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2.4. Sample collection after the challenge test

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After the challenge test, all fishesACCEPTED from each treatment were anaesthetized in a benzocaine bath as refer MANUSCRIPT

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to Geraylou et al. [55]. To examine the effects of threonine on the resistance of fish against gill rot, a score

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system was designed to evaluate the severity of gill rot as described by Taylor et al.[56]. Then fish gills were

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quickly removed, frozen in liquid nitrogen, and then stored at -80°C for later analysis as described by Chen

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et al. [42].

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2.5. Biochemical analysis

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The gill samples were homogenized in 10 volumes (w/v) of ice-cold physiological saline and

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centrifuged at 6,000 g at 4°C for 20 min, and the supernatants were stored until used for the determination

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of gill immune and antioxidant parameters as described by Luo et al. [57]. The acid phosphatase (ACP)

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activity, complement component 3 (C3) and C4 contents of gills were measured according to Zhao et al.

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[58]. The lysozyme (LA) activity of gills were assayed according to Li et al. [59]. The contents of reactive

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oxygen species (ROS), malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH) in the gills

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were determined by the method of Feng et al. [60]. The anti-superoxide anion (ASA) and antihydroxyl

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radical (AHR) activities were measured according to Li et al. [50]. The total superoxide dismutase (SOD)

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and copper, zinc superoxide dismutase (CuZnSOD) activities were determined as refer to Gavrilović et al.

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[61]. The manganese superoxide dismutase (MnSOD) activity was calculated by deducting CuZnSOD

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from total SOD. The catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and

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glutathione reductase (GR) activities were assayed as described by Elia et al. [62].

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2.6. Real-time polymerase chain reaction (PCR) analysis

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The procedures of RNA isolation, reverse transcription and quantitative real-time PCR were similar to

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another study conducted in our laboratory [50]. The total RNA was extracted from the gills using RNAiso

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Plus kit (TaKaRa, Dalian, Liaoning, China) according to the manufacturer's instructions followed by

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DNAse I treatment. RNA quality and quantity were assessed using agarose gel (1%) electrophoresis and

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spectrophotometric (A260:280 nm ratio) analysis, respectively. Subsequently, RNA was reverse transcribed

into cDNA using the PrimeScript™ RT reagent Kit MANUSCRIPT (TaKaRa) according to the manufacturer's instructions. ACCEPTED

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For quantitative real-time PCR, specific primers were designed according to the sequences cloned in our

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laboratory and the published sequences of grass carp (Provided in the Supplemental Table S2). According to

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the results of our preliminary experiment concerning the evaluation of internal control genes (data not

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shown), β-actin was used as a reference gene to normalize cDNA loading. The target and housekeeping

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gene amplification efficiency were calculated according to the specific gene standard curves generated from

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10-fold serial dilutions. The 2−∆∆CT method was used to calculate the expression results after verifying that

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the primers amplified with an efficiency of approximately 100% as described by Livak and Schmittgen [63].

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2.7. Western blotting analysis

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Protein homogenates from gills were prepared similar to the method described by Yao et al.[64].

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Protein concentrations were determined using the BCA assay kit (Betoyotime Biotechnology Inc., China).

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Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane for the western

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analysis. The membrane was blocked for 1h at room temperature and then incubated with the primary

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antibody overnight at 4 °C. The anti-total TOR, phosphorylation of TOR on residue Ser2448 (p-TOR Ser

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2448), Nrf2, β-actin and lamin B1 antibodies were used as the same as those in our previous studies [65, 66].

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The β-Actin and lamin B1 were used as control proteins for total and nuclear protein, respectively. The blots

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were washed three times, followed by a 2 h incubation with HRP-conjugated secondary antibody in TBST.

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The immune complexes were visualized using ECL reagents (Beyotime Biotechnology Inc., China), and

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then quantified using NIH Image 1.63 software. Different treatments were expressed relative to the level of

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control group. This experiment was repeated at least three times, and similar results were obtained each

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time.

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2.8. Calculations and statistical analysis

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The results were presented as the mean ± standard deviation (SD). All data were subjected to a

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one-way analysis of variance (ANOVA) to determine whether significant differences occurred among

treatments. If a significant difference ACCEPTED was identified, MANUSCRIPT differences among means were compared by Duncan’s

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multiple range tests at P < 0.05 with SPSS 18.0 (SPSS Inc., Chicago, IL, USA), according to Jiang et al.

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[67].

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3. Result

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3.1 Gill rot morbidity of grass carp after challenge with F. columnare

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Gill rot morbidity after challenge with F. columnare was decreased with threonine level added up to

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14.10 g threonine kg-1 diet, then increased gradually. Obviously, compared with optimal threonine

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supplementation, threonine deficiency accentuated the symptom of gill rot in fish (Fig. 1 & 2). Fig. 1 inserted here

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Fig. 2 inserted here

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3.2 Gill immune parameters

The activities of LA, ACP, and the contents of C3, C4 in the gills of juvenile grass carp are presented in

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Table 2. With threonine level added up to 14.10 g threonine kg-1 diet, increased activities or contents were

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obtained significantly in LA, ACP, C3 and C4 in the gills of juvenile grass carp (P < 0.05), then decreased

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gradually in LA, ACP and C3 but plateaued in C4 (P > 0.05), respectively.

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(Table 2 inserted here)

3.3 Relative mRNA levels of antimicrobial peptides, cytokines and related signaling molecules in the gill

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The effects of dietary threonine on antimicrobial peptides, cytokines and related signaling molecules in

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the gills of juvenile grass carp are displayed in Fig. 3-4. With dietary threonine rising up to 14.10, 17.96,

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14.10, 17.96 and 14.10 g threonine kg-1 diet, up-regulated mRNA levels were found in the hepcidin,

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β-defensin-1, TGF-β1, IL-4/13A and IL-10, and then all down-regulated gradually. Threonine addition

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up-regulated the liver expressed antimicrobial peptide 2A (LEAP-2A) mRNA abundances with its level up

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to 10.72 g threonine kg-1 diet, then plateaued (P > 0.05). Compared with the optimal threonine

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supplementation, threonine deficiency down-regulated mRNA levels of LEAP-2B, TGF-β2 and IL-4/13B in

fish gill (P < 0.05). On the contrary, with dietary threonine levels added up to 10.72, 14.10, 14.10 and 10.72 ACCEPTED MANUSCRIPT

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g threonine kg-1 diet, the mRNA levels of TNF-α, IL-1β, IFN-γ2, IL-8 and IL-17D were down-regulated,

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and then all increased gradually. Fish fed threonine deficiency diet showed the maximum mRNA levels of

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IL-6 and IL-12p35 in the gill (P < 0.05), respectively. However, there were no notable difference in the

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IL-12p40 mRNA level between fish fed graded levels of threonine (P > 0.05).

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The gill mRNA levels of NF-κB p65, c-Rel, IKKβ and 4E-BP1 were down-regulated with threonine

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level rising up to 14.10, 10.72, 10.72 and 14.10 g threonine kg-1 diet, and then up-regulated, gradually. Fish

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fed threonine-deficient diet showed the maximum NF-κB p52 mRNA level in the gill (P < 0.05). Inversely,

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the iκBα, TOR and S6K1 mRNA levels were up-regulated with threonine level rising up to 14.10 g

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threonine kg-1 diet, and then down-regulated gradually. However, no significant difference were detected in

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the mRNA levels of IKKα and IKKγ between fish fed graded levels of threonine (P > 0.05). Fig. 3 inserted here

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Fig. 4 inserted here

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3.4 Gill antioxidant-related parameters, Nrf2, Keap1a and Keap1b mRNA levels The effects of dietary threonine on gill antioxidant-related parameters of juvenile grass carp are

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displayed in Table 3. The contents of ROS, MDA and PC reduced significantly with rising threonine level

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up to 14.10 g threonine kg-1 diet (P < 0.05), and then increased significantly (P < 0.05). The activity of AHR,

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ASA, CuZnSOD, CAT and the reduced GSH contents increased significantly (P < 0.05) with threonine

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level rising up to 14.10 g threonine kg-1 diet, and then slipped gradually. With dietary threonine level added

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up to 10.72 and 14.10 g threonine kg-1 diet, GPx and GR activities were increased, and then decreased

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gradually. Additionally, the activities of MnSOD and GST were elevated significantly (P < 0.05) with

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dietary threonine added up to 10.72 g threonine kg-1 diet, and plateaued thereafter (P > 0.05). As shown in

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Fig. 5 (A&B)，gill CuZnSOD, MnSOD, CAT, GPx1a, GPx4a, GSTR and Nrf2 mRNA levels were

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up-regulated with threonine level added up to 14.10, 14.10, 14.10, 10.72, 14.10, 14.10 and 14.10 g threonine

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kg-1 diet, and then all down-regulated, gradually.MANUSCRIPT Compared with dietary threonine supplementation, ACCEPTED

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threonine deficiency down-regulated the mRNA levels of GPx4b, GSTO1 and GR significantly in fish gill

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(P < 0.05). The Keap1a mRNA level was down regulated with threonine level up to 14.10 g threonine kg-1

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diet, and then up regulated gradually. However, the gill mRNA levels of GPx1b, GSTO2 and Keep1b didn’t

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alter with graded levels of threonine supplemented in fish (P > 0.05). (Table 3 inserted here)

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Fig. 5A inserted here

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Fig. 5B inserted here 3.5 Relative mRNA levels of apoptosis related proteins in the gill

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As shown in Fig. 6, dietary threonine influenced the mRNA levels of apoptosis-related proteins and

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signaling molecules in the gills of juvenile grass carp. With threonine levels up to 14.10, 14.10, 14.10, 10.72,

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14.10 and 10.72 g threonine kg-1 diet, the mRNA levels of fas ligand (FasL), cysteinyl aspartic acid-protease

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3 (caspase3), apoptotic protease activating factor-1 (Apaf-1), Bax and JNK were down-regulated, and then

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up-regulated gradually. Meanwhile, compared with the optimal threonine supplementation, threonine

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deficiency up-regulated the caspase-8 and caspase-9 mRNA levels in the gills of juvenile grass carp,

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significantly (P < 0.05). The B-cell lymphoma protein-2 (Bc-2) and inhibitor of apoptosis proteins (IAP)

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mRNA levels in the gill of juvenile grass carp were up-regulated with threonine level added up to 14.10 g

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threonine kg-1 diet, and then all down-regulated gradually.

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Fig. 6 inserted here

3.6 Relative mRNA levels of TJ proteins and MLCK mRNA levels in the gill

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The effects of dietary threonine on mRNA levels of gills TJ proteins and MLCK are showed in Fig.7.

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Compared with the dietary threonine supplementation, threonine deficiency down-regulated the mRNA

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levels of occludin and ZO-2 in fish gill, significantly (P < 0.05). Each of the ZO-1, claudin-3, -b, -c and

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claudin-12 mRNA levels was up-regulated with dietary threonine level added up to 14.10 g threonine kg-1

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diet, and then all down-regulated, gradually. Conversely, the claudin-7a, -7b and MLCK mRNA levels in ACCEPTED MANUSCRIPT

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fish gill were down-regulated with threonine levels up to 14.10 g threonine kg-1 diet, and then all

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up-regulated, gradually. Interestingly, dietary threonine deficiency had no effects on the claudin-15a mRNA

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levels in the gills of juvenile grass carp (P > 0.05).

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Fig. 7 inserted here

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3.7 Protein levels of TOR and Nrf2 in the gill

The effects of dietary threonine on the protein levels of TOR and Nrf2 in the gills of fish are displayed

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in Fig.8. With the similar quantities of proteins were loaded into the gels, compared with the dietary

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threonine deficiency, fish fed 14.10 g threonine kg-1 diet showed the maximum total-TOR (T-TOR) and

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p-TOR Ser2448 protein levels in the gills of juvenile grass carp (P＜0.05), respectively. With dietary

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threonine level added up to 10.72 g threonine kg-1 diet, protein levels of nuclear Nrf2 in the gills of fish

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were enhanced gradually, and then plateaued (P＞0.05). Fig. 8A inserted here

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Fig. 8B inserted here

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4. Discussion

This study used the identical growth trial from our previous study, which is a part of a larger study

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conducted to explore the effects of threonine on fish growth and health status. Previous study demonstrated

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that optimal threonine level improved the growth performance of juvenile grass carp [8]. Generally, fish

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growth is often related to its gill health [3, 68]. Thus, for the first time, we next investigated the effect of

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threonine on gill health in fish.

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4.1. Threonine deficiency decreased gill disease resistance in fish.

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It was of note that gill health was partly reflected in the disease resistance [62]. In fish, F. columnare is

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a worldwide important pathogen, infection of which could make gills rot in a high morbidity [69]. In this

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study, threonine-deficiency caused the highest gill rot morbidity (17.0%) after exposure to F. columnare

while optimal threonine supplementation decreased the gill rot morbidity (3.5%), indicating that ACCEPTED MANUSCRIPT

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threonine-deficiency weakened the fish resistance against gill rot. Based on the quadratic regression analysis,

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the optimal threonine level for protecting juvenile grass carp against the gill rot morbidity was estimated to

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be 15.32 g threonine kg-1 diet (4.73 g threonine 100 g-1 protein). In addition, fish gill health is closely related

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to gill immune barrier function [2]. Thus, we investigated the effects of dietary threonine on gill immune

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barrier function in the gills of fish.

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4.2. Threonine deficiency impaired the gill immune barrier function

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4.2.1. Threonine deficiency decreased the antimicrobial compounds in fish gill

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In fish, improvement of the gill immune function mainly depends on the enhancement of antimicrobial

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compounds such as LA, ACP, complements and antimicrobial peptides [70, 71]. In the study, compared with

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threonine supplementation, threonine deficiency decreased the LA and ACP activities, C3 and C4 contents,

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and the mRNA levels of LEAP-2A, LEAP-2B, Hepcidin and β-defensin1 in the gills of juvenile grass carp

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after exposure to F. columnare, indicating that threonine deficiency attenuated the gill immune function in

297

fish. Moreover, gill immune function also relies on the inflammation response mediated by cytokines [72].

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Then we next examined the effects of dietary threonine on inflammation in fish gill.

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4.2.2. Threonine deficiency aggravated the inflammation response partly relate to NF-κB and TOR

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signaling pathways in the gills of fish

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In fish, it was the up-regulation of pro-inflammatory cytokines (like TNF-α, IL-β, IL-8, IFN-γ2) [73]

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and the down-regulation of anti-inflammatory cytokines (like IL-10 and TGF-β1) that sponsored the

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inflammatory response [72]. In this study, compared with the threonine supplementation, dietary threonine

304

deficiency up-regulated the mRNA levels of the pro-inflammatory cytokines (except IL-12p40) and

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down-regulated the anti-inflammatory cytokines (e.g., TGF-β1 and IL-10) mRNA levels in the gills of

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juvenile grass carp, indicating that threonine deficiency aggravated the inflammation in fish gill.

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Surprisingly, dietary threonine deficiency up-regulated the IL-12p35 (rather than IL-12p40) mRNA levels in

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the gills of juvenile grass carp, which might be relevant to the IL-1β. Lee et al. [74] reported that IL-1β ACCEPTED MANUSCRIPT

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could up-regulate the IL-12p35 but not IL-12p40 mRNA levels in human mature DCs. Our study showed

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that threonine deficiency up-regulated the IL-1β mRNA levels in fish gill, supporting our hypothesis. As we know, pro-inflammatory cytokines (like TNF-α and IL-1β) could be activated by NF-κB, which

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required a sequestering-protein named IκBα that could be catalyzed by the IKK complex (IKKα, IKKβ and

313

IKKγ) [75, 76]. In this study, dietary threonine deficiency up-regulated the NF-κB p65, NF-κB p52, c-Rel

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and IKKβ (not IKKα and IKKγ) mRNA levels in the gills of juvenile grass carp. Correlation analysis (Table

315

4) displayed that the mRNA levels of pro-inflammatory cytokines (except IL-12p40) were positively related

316

to NF-κB p65, NF-κB p52 and c-Rel, which were negatively correlated with IκBα. Moreover, the mRNA

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level of IκBα was negatively related to IKKβ in the gills of juvenile grass carp. These results suggested that

318

dietary threonine deficiency up-regulated the pro-inflammatory cytokines mRNA levels (except IL-12p40)

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partly ascribe to launching the signaling [IKKβ (not IKKα and IKKγ) / IκBα / (NF-κBp65 / p52 / c-Rel)] in

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the gills of fish. Intriguingly, threonine deficiency up-regulated IKKβ (not IKKα and IKKγ) mRNA

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transcript levels in the gills of juvenile grass carp, which might be relate to IFN-γ2. Eickhoff et al. [77]

322

reported that IFN-γ2 could induce the mRNA level of N-myc downstream-regulated gene 1 (NDRG1)

323

decreased in Hela cells. It was demonstrated that the repression of NDRG1 could up-regulate IKKβ (not

324

IKKα and IKKγ) expression in mice [78]. Our study displayed that threonine deficiency up-regulated the

325

IFN-γ mRNA level in the gills of juvenile grass carp. Thus, we hypothesized that threonine deficiency might

326

up-regulate the IFN-γ mRNA level to down-regulate the NDRG1 thereby resulting in the up-regulation of

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IKKβ (not IKKα and IKKγ) in the gills of juvenile grass carp. However, the hypothesis warrants further

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verification.

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Besides, evidences showed that the down-regulation of anti-inflammatory cytokines (like IL-10) could

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aggravate inflammation process, which could be modulated by the mTOR / (S6K1, 4EBP-1) signaling

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cascades with suppression of S6K1 and activation of 4E-BP1 in human [79]. Our study showed that,

compared with the dietary threonine ACCEPTED supplementation, threonine deficiency down regulated the TOR and MANUSCRIPT

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S6K1 mRNA levels, protein levels of T-TOR and p-TOR Ser2448, and up-regulated 4E-BP1 mRNA levels

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in the gill of juvenile grass carp. Correlation analysis (Table 4) indicated that the mRNA levels of

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anti-inflammatory cytokines (e.g., TGF-β1 and IL-10) and signaling molecule S6K1 were positively

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correlated to TOR, which was in inverse correlation with 4E-BP1 in the gills of juvenile grass carp,

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suggesting that threonine deficiency down-regulated the anti-inflammatory cytokines mRNA levels partly

338

due to diminishing the TOR / (S6K1, 4E-BP1) signaling cascades in fish gill.

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4.3. Threonine deficiency impaired the gill physical barrier function in the gill of fish

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4.3.1. Threonine deficiency exacerbated the gill oxidative damage and decreased the gill antioxidant

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capacity partly through the Nrf2 signaling pathway

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In fish gill, large amounts of ROS could be accumulated after infected with F. columnare, thus leading

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to oxidative damage which could be reflected in the increased MDA and PC [80]. For protecting gills from

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oxidative injury, it was invoked with non-enzymatic antioxidants and antioxidant enzymes in fish, such as

345

GSH, GPx and so on [81, 82]. In this study, compared with the optimal threonine supplementation,

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threonine deficiency increased the ROS, MDA and PC contents and decreased the activities of CuZnSOD,

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MnSOD, CAT, GPx, GST and GR, and GSH content in the gills of juvenile grass carp, suggesting that

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threonine deficiency decreased the antioxidant capacity in fish gill. Moreover, antioxidant enzyme activities

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are closely related to their mRNA levels, which could be regulated by Nrf2 signaling pathway in rat [29].

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Thus, we next investigated the effect of threonine on the antioxidant enzymes mRNA transcriptions and

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Nrf2 signaling pathway in fish gill.

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In present study, compared with the optimal threonine supplementation, threonine deficiency

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down-regulated the mRNA levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx4a, GPx4b, GSTR, GSTO1 and

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GR in the gills of juvenile grass carp. Correlation analysis (Table 4) indicated that CuZnSOD, MnSOD,

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CAT, GPx, GST and GR activities were positively correlated to their corresponding mRNA levels in the

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gills of grass carp, respectively. Above results manifested that threonine deficiency decreased the activities ACCEPTED MANUSCRIPT

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of CuZnSOD, MnSOD, CAT, GPx, GST and GR might be partly ascribed to the down-regulation of their

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corresponding mRNA levels in the gills of fish. Interestingly, threonine deficiency had no effects on the mRNA levels of GPx1b and GSTO2 in the gill

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of juvenile grass carp. Firstly, the reason that threonine deficiency down-regulated GPx1a (rather than

361

GPx1b) mRNA levels might be relate to the threonine efforts for the utilization of SeMet. Sarwar et al. [83]

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reported that threonine addition enhanced Serine contents in the plasma of rat. In HepG2 cells, serine could

363

promote the utilization of SeMet for the synthesis of GPx [84]. It was found that SeMet was more sensitive

364

and efficient to up-regulate the GPx1a gene expression than GPx1b in zebrafish [85]. Thus, we speculated

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that threonine deficiency might decreased the Serine level to hinder the utilization of SeMet, thus leading to

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a disposition of down-regulated GPx1a (rather than GPx1b) in fish. However, the specific mechanism needs

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further investigation. Secondly, no significant differences were observed on GSTO2 gene expression in the

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gills of fish fed graded levels of threonine, which might be concerned with ascorbic acid. In zebrafish,

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GSTO2 primarily mediated the reduction of dehydroascorbic acid into ascorbic acid, and it was critical for

370

maintaining ascorbic acid level [86]. Datta & Ghosh [9] found that threonine could enhance the ascorbic

371

acid level in rat liver. Thus, we speculated that threonine deficiency didn’t alter the GSTO2 mRNA level

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partly due to improving the ascorbic acid level as a result of substituting the role of GSTO2 in fish gill.

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However, it remains to be elucidated by further investigation.

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Furthermore, Nrf2 could regulate the transcriptions of antioxidant enzymes for being a key regulator,

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which could be prevented by blinding to Keap1 from translocation into the nucleus [87]. In this study,

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compared with the optimal threonine supplementation, threonine deficiency down-regulated the nuclear

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Nrf2 protein levels, Nrf2 mRNA levels and up-regulated the Keap1a (not Keap1b) mRNA levels in the gills

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of juvenile grass carp. Correlation analysis (Table 4) displayed that the mRNA levels of CuZnSOD,

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MnSOD, CAT, three isoforms of GPx (1a, 4a, 4b) and GR were positively correlated to Nrf2, which was in

inverse correlation with Keap1a in the gills of juvenile grass carp. These results indicated that threonine ACCEPTED MANUSCRIPT

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deficiency decreased the antioxidant related enzymes genes expression partly attribute to the Nrf2 / [Keap1a

382

(not Keap1b)] signaling in fish gill. Unexpectedly, threonine deficiency up-regulated the Keap1a (rather

383

than Keap1b) mRNA level in the gills of juvenile grass carp, which might be involved with fatty acid

384

binding protein (FABP). In the ileum of piglets, threonine deficiency down-regulated the FABP mRNA

385

levels [88]. Jefferson et al. [89] found that suppression of FABP could decline the phospholipid content in

386

mouse liver cells. Previous study of our lab confirmed that phospholipid deficiency could up-regulate the

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Keap1a (rather than Keap1b) genes expression in the intestines of juvenile grass carp [90]. Thus, we

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hypothesized the difference that threonine deficiency up-regulated the Keap1a (rather than Keap1b) mRNA

389

levels might be partly due to down-regulating the FABP mRNA level to decrease the phospholipid content

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in fish gill. However, further investigation should be conducted to support our hypothesis.

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4.3.2. Threonine deficiency aggravated apoptosis partly relate to signaling molecules JNK in fish gill.

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Apoptosis is programmed cell death that can be characterized in the extrinsic pathway triggered by

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extracellular cues delivered in the form of ligands binding to death receptors (DRs), such as FasL, which

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can induce the activation of caspase-8 and caspase-3 [91]. In this study, dietary threonine deficiency

395

up-regulated the FasL, caspase-8 and caspase-3 mRNA levels in the gills of juvenile grass carp. Correlation

396

analysis (Table 4) showed that the mRNA levels of caspase-3 was positively related to caspase-8, which was

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positively correlated with FasL in the gills of juvenile grass carp, indicating that threonine deficiency

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exacerbated the gill apoptosis partly relate to amplification of the extrinsic cascades FasL / caspase-8 /

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caspase-3 in fish.

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Apart from the extrinsic Pathway, intrinsic pathway also played a vital role in apoptosis characterized

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as mitochondrial apoptosis related factors released, such as Bcl-2, Bax, pro-apoptotic factors Apaf-1 and so

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on [30]. Moreover, study showed that JNK could increase the expression of anti-apoptotic associated protein

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IAP and Bcl-2 and decrease expression of Apaf-1 and Bax, thus inducing the subsequent enhancement of

caspase-9 [92]. In this study, dietaryACCEPTED threonine deficiency up-regulated the caspase-9, JNK, pro-apoptotic MANUSCRIPT

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factors Apaf-1 and Bax, and down regulated the anti-apoptotic factors Bcl-2 and IAP mRNA levels in the

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gills of juvenile grass carp. Correlation analysis (Table 4) displayed that mRNA levels of caspase-9 were

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negatively correlated with the Bcl-2 and IAP, and positively related to the Apaf-1 and Bax. Meanwhile, the

408

JNK mRNA level was positively correlated with Apaf-1 and Bax but negatively related to Bcl-2 and IAP in

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the gills of juvenile grass carp. Above observation demonstrated that threonine deficiency induced the gill

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pro-apoptosis partly ascribe to magnifying the signaling cascades JNK / (Apaf-1, Bax, Bcl-2 and IAP) /

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caspase-9 in fish.

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4.3.3. Threonine deficiency impaired the gill TJ complexes integrity partly via MLCK signaling pathway

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In fish, gill physical barrier function is partly depended on its integrity of the intercellular junctions

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involved with tight junction proteins (TJs) such as claudins, occludin, and ZO-1[93], which could be

415

regulated by MLCK [31]. Chen et al. [94] proved that up-regulating MLCK activity induced intestinal

416

epithelial barrier dysfunction with the disassembling of ZO-1 and occludin in Caco-2 cells. In this study,

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threonine deficiency down-regulated the TJs (claudin-3, -b, -c, -12, occludin and ZO-1) mRNA levels and

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up-regulated the MLCK in the gills of juvenile grass carp. Correlation analysis (Table 4) showed that the

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genes expression of TJs (claudin-3, -b, -c, -12, occludin and ZO-1) were negatively correlated with MLCK

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mRNA level, suggesting that threonine deficiency evoked the gill physical barrier dysfunction partly due to

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up-regulating the MLCK mRNA level in fish gills.

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Surprisingly, threonine deficiency up-regulated the claudin-7a and claudin-7b mRNA levels but didn’t

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influence the claudin-15a mRNA level in the gills of juvenile grass carp. Firstly, the reason that threonine

424

deficiency up-regulated mRNA levels of claudin-7a and claudin-7b might be relevant to plasma ammonia.

425

Alexandre et al. [95] proved that the up-regulation of claudin-7 made an increase in the paracellular

426

conductance to Na+ in LLC-PK1 cells. Evidence confirmed that the incremental Na+ transports contributed

427

to ammonia emission out of the gill in fish [96]. Our previous study found that threonine deficiency

increased the plasma ammonia content in juvenile grass carp [8]. Thus, the up-regulation of claudin-7a and ACCEPTED MANUSCRIPT

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claudin-7b caused by threonine deficiency might be a demand for the emission of ammonia in fish gill.

430

However, the speculated possibility requires more investigation to support. Secondly, threonine deficiency

431

didn’t alter the claudin-15a mRNA transcript abundances in the gills of juvenile grass carp, which might be

432

relate to claudin-7. In mouse intestine, claudin-15 was indispensable and induced efficiently for the

433

paracellular Na+ permeability [97], which is of the same function as claudin-7 [95]. In the study, threonine

434

deficiency up-regulated the claudin-7a and claudin-7b mRNA levels in the gills of juvenile grass carp,

435

which indicated that a substitute role of claudin-15a might be played by claudin-7. However, the speculated

436

reason needs further investigation.

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(Table 4 inserted here)

4.4. Comparison of the optimal threonine levels for juvenile grass carp based on different indices In this study, different indices were investigated for estimating the threonine requirements of juvenile

440

grass carp. As shown in Table 5, based on the gill rot morbidity, LA activity and MDA content, threonine

441

requirement was determined to be 15.32 g kg-1 diet (4.73 g 100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1

442

protein) and 15.46 g kg-1 diet (4.77 g 100 g-1 protein), respectively. Comparatively, based on the disease

443

resistance, immune and antioxidant-related indicators, the optimal threonine levels for juvenile grass carp

444

were slightly higher than that on the growth requirement with 14.53 g kg-1 diet (4.48 g 100 g-1 protein),

445

suggesting that a little more threonine supplementation in diet is essential to maintain a better status of gill

446

health in fish. Those phenomena might be due to the contribution of threonine to affording the requirements

447

for normal immune system and protecting tissues from collateral damage, rather than just for meeting the

448

growth performance [98].

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Additionally, for the data of genes expression in this study, similar folded changed in the genes

450

expression were also found in the other study addressed on the threonine improving health status in fish [99,

451

100]. For the different nutrients, the improvement for the immune modulation maybe different in fish [50]

452

[67]. However, it is worthy to be affirmed that theMANUSCRIPT accuracy and authenticity of the results in this study ACCEPTED

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benefits from our lots of preliminary experiments, thus advancing the experiment skills to diminish the

454

personal error as far as possible.

455

(Table 5 inserted here) 5. Conclusions

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In summary (Fig. 9), our study demonstrated that dietary threonine deficiency depressed the status of

458

gill health. For the first time, we found that threonine deficiency attenuated the immune barrier function

459

with decreasing the gill disease resistance, antimicrobial compounds production, up-regulating

460

pro-inflammatory cytokines (except IL-12p40) and down-regulating anti-inflammatory cytokines genes

461

expression, and that threonine deficiency impaired the physical barrier function with decreasing the

462

antioxidant capacity, induced apoptosis and disrupted the tight junction barriers in the gills of juvenile grass

463

carp after infection with F. columnare. Furthermore, the deteriorated fish immune and physical barriers

464

function by threonine deficiency might be partly associated with the modulated signaling molecules NF-κB,

465

TOR, Nrf2, JNK and MLCK. In addition, based on the gill rot morbidity, LA activity and MDA content,

466

threonine requirements for juvenile grass carp (9.55-53.43 g) were determined to be 15.32 g kg-1 diet (4.73 g

467

100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1 protein) and 15.46 g kg-1 diet (4.77 g 100 g-1 protein),

468

respectively.

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Fig. 9 inserted here Acknowledgements

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This research was financially supported by National Natural Science Foundation of China (31572632;

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31672662), the National Basic Research Program of China (973 Program) (2014CB138600), Outstanding

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Talents and Innovative Team of Agricultural Scientific Research (Ministry of Agriculture), Science and

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Technology Support Programme of Sichuan Province of China (2014NZ0003), Supported by the Earmarked

475

Fund for China Agriculture Research System (CARS-45), Major Scientific and Technological Achievement

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Transformation Project of Sichuan Province of ChinaMANUSCRIPT (2013NC0045), the Demonstration of Major Scientific ACCEPTED

477

and Technological Achievement Transformation Project of Sichuan Province of China (2015CC0011) and

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Foundation of Sichuan Youth Science and Technology Innovation Research Team (2017TD0002). The

479

authors would like to thank the personnel of these teams for their kind assistance.

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Table 1 Composition and nutrients content of basal diet.

ACCEPTED-1MANUSCRIPT

Nutrient contents1

g kg-1

Fish meal

50.4

Crude protein

324.0

Casein

30.0

Crude lipid

32.4

Crystal amino acid mix2

203.1

ω-3

5.0

Threonine premix3

50.0

ω-6

10.0

Gelatin

78.6

Available phosphorus

8.4

Fish oil

9.3

Soya bean oil

19.4

α-starch

290.0

Corn starch

150.5

Ca (H2PO4)2

33.2

Mineral premix4

20.0

Vitamin premix5

10.0

Choline chloride (60%)

5.0

RI PT

g kg

M AN U

Ingredients

SC

728

50.0

Cellulose

0.5

Ethoxyquin (30%) 729

1

730

according to NRC (2011).

731

2

732

18.64; isoleucine, 10.30; phenylalanine, 10.22; tyrosine, 6.44; valine, 11.25; Cysteine, 2.87; glutamic acid, 57.50; glycine,

733

46.35, respectively.

734

3

735

to compensate. Per kilogram of threonine premix composition from diet 1 to 6 was as follows (g kg-1): L-threonine 0.00,

736

71.40, 142.80, 214.20, 285.80, 357.20; glycine 222.80, 178.40, 133.80, 89.20, 44.60, 0.00 g and corn starch 777.20, 750.20,

737

723.40, 696.60, 669.60, 642.80g, respectively.

738

4

739

FeSO4.H2O (30.0% Fe), 24.5700; ZnSO4.H2O (34.5% Zn), 8.2500; CuSO4.5H2O (25.0% Cu), 0.9600; KI (76.9% I),

740

0.0668g; Na2SeO3 (44.7% Se), 0.0168. All ingredients were diluted with corn starch to 1 kg.

741

5

742

L-α-tocopherol acetate (50%), 12.58; menadione (22.9%), 0.83; cyanocobalamin (1%), 0.94; D-biotin (2%), 0.75; folic acid

743

(95%), 0.42; thiamine nitrate (98%), 0.11; ascorhyl acetate (95%), 4.31; niacin (99%), 2.58; meso-inositol (98%), 19.39;

744

calcium-D-pantothenate (98%), 2.56; riboflavin (80%), 0.63; pyridoxine hydrochloride (98%), 0.62. All ingredients were

745

diluted with corn starch to 1 kg.

746

Crude protein and crude lipid contents were measured value. Available phosphorus, ω-3 and ω-6 contents were calculated

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Crystal amino acid mix (g kg-1): lysine, 11.42; methionine, 7.81; tryptophan, 2.88; arginine, 9.56; histidine, 7.86; leucine,

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Threonine premix was added to obtain graded levels of threonine, and the amount of glycine and corn starch was reduced

Per kilogram of mineral premix (g kg-1): MnSO4.H2O (31.8% Mn), 1.8900; MgSO4⋅H2O (15.0% Mg), 200.0000;

Per kilogram of vitamin premix (g kg-1): retinyl acetate (500,000IU/g), 2.10; cholecalciferol (500,000IU/g), 0.40; D,

747

Table 2 Lysozyme (LA, U mg-1 protein), acid phosphatase (ACP, U mg-1 protein) activities, complement 3

ACCEPTED MANUSCRIPT

-1

748

(C3, mg g protein) and complement 4 (C4, mg g-1 protein) contents in the gills of juvenile grass carp

749

(Ctenopharyngodon idella) fed diets containing graded levels of threonine for 8 weeks1. Dietary threonine levels (g kg-1) 3.99

7.70

10.72

21.66

146.54±10.55b

179.90±13.74c

228.03±18.79d

191.84±15.37c

177.09±17.15c

ACP

174.10±13.44a

279.66±22.3b

335.62±22.37d

374.11±16.32e

313.13±12.39c

271.26±17.67b

C3

11.83±0.34a

15.92±0.37b

18.79±1.55c

21.23±0.74d

18.26±1.43c

16.59±0.92b

C4

1.05±0.06a

1.57±0.09b

1.74±0.11c

1.93±0.10d

1.88±0.09d

1.86±0.13d

R² = 0.966

P < 0.01

R² = 0.945

P < 0.05

R² = 0.939

P < 0.05

SC

Y C3 = -0.0752x2 + 2.1964x + 4.0310

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97.23±6.84a

Y ACP = -1.7329x2 + 49.4891x + 4.3730

Y C4 = 0.085x + 0.790; Y max = 1.888

Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).

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1

17.96

LA

Regression

750

14.10

AC C

EP

TE D

751

752

Table 3 MDA (nmol mg-1 protein), PC (nmol mg-1 protein) and ROS (% DCF florescence) contents, and

ACCEPTED MANUSCRIPT

-1

753

activities of AHR(U mg protein), ASA(U mg-1 protein), CuZnSOD (U mg-1 protein), MnSOD (U mg-1

754

protein), CAT (U mg-1 protein), GPx (U mg-1 protein), GST (U mg-1 protein) and GR (U mg-1 protein), and

755

GSH (mg g-1 protein) content in the gills of juvenile grass carp (Ctenopharyngodon idella) fed diets

756

containing graded levels of threonine for 8 weeks1. Dietary threonine levels (g kg-1) 7.70

10.72

14.10

ROS

100.00±5.66e

89.87±3.04d

73.17±4.54b

59.42±4.77a

70.22±4.18b

81.33±1.72c

MDA

17.19±1.16e

10.02±0.45d

7.66±0.50c

6.68±0.41a

7.64±0.67c

8.65±0.27b

PC

8.36±0.37e

7.18±0.38d

5.14±0.27b

3.91±0.27a

4.89±0.22b

5.51±0.17c

AHR

38.72±1.46a

44.20±2.30b

47.30±2.28bc

50.04±2.55c

46.50±2.77b

44.22±3.49b

ASA

71.32±4.86a

95.94±5.71b

108.34±6.15c

125.11±6.35d

111.52±5.52c

108.24±8.44c

CuZnSOD

4.82±0.51a

7.26±0.29c

7.96±0.37d

9.30±0.10e

6.19±0.28b

5.81±0.37b

MnSOD

4.77±0.29a

5.88±0.30b

7.23±0.59c

7.35±0.45c

7.43±0.50c

6.92±0.66c

CAT

1.95±0.08a

2.22±0.11b

2.50±0.13c

2.81±0.16d

2.61±0.16c

2.45±0.18c

GPx

107.26±8.09a

145.04±10.86b

168.78±6.34c

166.74±11.36c

163.59±2.98c

150.47±10.23b

GST

43.25±1.29a

60.72±3.58b

68.86±3.19c

70.36±5.56c

66.95±4.04c

67.17±5.93c

GR

21.44±1.93a

30.63±2.00c

33.51±1.24d

34.00±2.55d

29.54±1.15c

26.59±2.01b

GSH

4.77±0.29a

6.09±0.15b

6.68±0.20c

7.42±0.41d

6.55±0.21c

6.48±0.51c

Y ROS = 0.3129x2 – 9.3570x + 136.1482

SC

M AN U

TE D

Regression

17.96

21.66

RI PT

3.99

R² = 0.886

P < 0.05

Y MDA = 0.0788x – 2.4378x + 25.0555

R² = 0.964

P < 0.01

Y PC = 0.0307x2 - 0.9664x + 12.0618

R² = 0.914

P < 0.05

Y AHR = -0.0942x2 +2.7154x + 29.2422

R² = 0.957

P < 0.01

Y ASA = -0.3556x2 +11.1324x + 32.1404

R² = 0.947

P < 0.05

Y CuZnSOD = -0.0417x2 + 1.0994x +1.2185

R² = 0.794

P = 0.092

AC C

EP

2

R² = 0.986

P = 0.073

2

R² = 0.919

P < 0.05

2

R² = 0.969

P < 0.01

Y GR = -0.1285x + 3.4830x +10.3806

R² = 0.937

P < 0.05

Y GSH = -0.0186x2 + 0.5623x +2.8484

R² = 0.919

P < 0.05

Y MnSOD = 0.3619x +3.2555; Y max = 7.2306 Y CAT = -0.0059x +0.1833x + 1.2601 Y GPx = -0.5216x + 15.5722x +55.3629 2

757

1

758

reactive oxygen species; MDA, malondialdehyde; PC, protein carbonyl; AHR, antihydroxyl radical; ASA, anti-superoxide

759

anion; CuZnSOD, copper, zinc superoxide dismutase; MnSOD, manganese superoxide dismutase; CAT, catalase; GPx,

760

glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; GSH, glutathione.

761 762

Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). ROS,

Table 4 Correlation coefficient of parameters in the gill.

ACCEPTED MANUSCRIPT P

Independent parameters

Correlation coefficients

NF-κB p65

TNF-α

+ 0.973

< 0.01

IL-1β

+ 0.944

< 0.01

IFN-γ2

+ 0.978

< 0.01

IL-6

+ 0.753

= 0.084

IL-8

+ 0.868

< 0.05

IL-12p35

+ 0.794

= 0.059

IL-17D

+0.841

TNF-α

+ 0.932

IL-1β

+ 0.877

IFN-γ2

+ 0.944

IL-6

+ 0.944

IL-8

+ 0.960

IL-12p35

+ 0.922

IL-17D

+ 0.893

c-Rel

TNF-α

SC

< 0.05 < 0.01

< 0.01 < 0.05

< 0.05

+ 0.894

< 0.05

+ 0.902

< 0.05

+ 0.979

< 0.01

+ 0.968

< 0.01

IL-17D

+ 0.925

< 0.01

IKKβ

- 0.983

< 0.01

NF-κB p65

- 0.939

< 0.01

NF-κB p52

- 0.903

< 0.05

c-Rel

- 0.805

= 0.053

TGFβ1

+ 0.960

< 0.01

TGF-β2

+ 0.848

< 0.05

IL-4/13A

+ 0.913

< 0.05

IL-10

+ 0.906

< 0.05

S6K1

+ 0.872

< 0.05

4E-BP1

- 0.840

< 0.05

Claudin-3

- 0.875

< 0.05

Claudin-b

- 0.828

< 0.05

Claudin-c

- 0.873

< 0.05

Claudin-12

- 0.828

< 0.05

Claudin-7a

+ 0.931

< 0.01

Claudin-7b

+ 0.903

< 0.05

Occuldin

- 0.822

< 0.05

ZO-1

- 0.961

< 0.01

Caspase-8

+ 0.812

= 0.051

IL-8

EP

TE D

IL-12p35

Caspase-3

< 0.01

+ 0.859

IL-6

MLCK

< 0.05

< 0.05

IFN-γ2

TOR

< 0.01

+ 0.856

IL-1β

IκBα

< 0.05

M AN U

NF-κB p52

RI PT

Dependent parameters

AC C

763

Caspase-9

+ 0.950

< 0.01

Caspase-8

FasL

+ 0.850

< 0.05

Caspase-9

Apaf-1

+ 0.986

< 0.01

Bax

+ 0.980

< 0.01

IAP

- 0.948

< 0.01

Bcl-2

- 0.974

< 0.01

Apaf-1

+ 0.977

< 0.01

Bax

+ 0.995

< 0.01

IAP

- 0.943

< 0.01

Bcl-2

- 0.954

CuZnSOD activity

CuZnSOD mRNA

+ 0.784

MnSOD activity

MnSOD mRNA

+ 0.876

CAT activity

CAT mRNA

+ 0.965

GPx activity

GPx1a mRNA

+ 0.984

GPx4a mRNA

+ 0.914

GPx4b mRNA

+ 0.926

GST activity

GSTO1 mRNA

GR activity

GR mRNA

Nrf2

CuZnSOD

RI PT = 0.065

< 0.05

SC

< 0.01

< 0.01 < 0.05 < 0.01

+ 0.928

< 0.01

+ 0.896

< 0.05

+ 0.960

< 0.01

+ 0.989

< 0.01

+ 0.940

< 0.01

+ 0.932

< 0.01

GPx4a

+ 0.982

< 0.01

GPx4b

+ 0.885

< 0.05

GSTR

+ 0.758

= 0.081

GSTO1

+ 0.770

= 0.073

GR

+ 0.902

< 0.05

- 0.920

< 0.01

MnSOD CAT

EP

TE D

GPx1a

AC C

Keap1a

764

< 0.01

M AN U

JNK

ACCEPTED MANUSCRIPT

765

Table 5 Results from the quadratic regression analysis on gill rot morbidity, LA activity and MDA content

766

in the gills of juvenile grass carp (Ctenopharyngodon idella) fed diets containing graded levels of threonine

767

(g kg-1 diet) for 8 weeks.

ACCEPTED MANUSCRIPT

Indices

R2

Regressive equation

P

Threonine requirement

0.949

< 0.05

15.32 g kg-1 diet (4.73 g 100 g-1 protein )

LA

Y LA = -0.8740x2 + 27.1265x – 2.1121

0.923

< 0.05

15.52 g kg-1 diet (4.79 g 100 g-1 protein )

MDA

Y MDA = 0.0788x2 -2.4378x +25.0555

0.964

< 0.01

15.46 g kg-1 diet (4.77 g 100 g-1 protein )

768

AC C

EP

TE D

M AN U

SC

769

RI PT

Y gill rot morbidity = 0.1004x2 – 3.0772x + 27.459

Gill rot morbidity

Fig. 1

ACCEPTED MANUSCRIPT

RI PT

770

771

Fig. 1 Effect of dietary threonine on the gill rot morbidity of juvenile grass carp (Ctenopharyngodon

773

idella) after infected with Flavobacterium columnare.

Fig. 2

AC C

776

EP

TE D

775

M AN U

774

SC

772

777

Fig. 2 Deficiency of threonine led to obviously gill rot after infection with F. columnare, compared

778

with optimal threonine supplementation in juvenile grass carp (Ctenopharyngodon idella).

779

780

Fig. 3

ACCEPTED MANUSCRIPT

SC

RI PT

781

EP

783

TE D

M AN U

782

Fig. 3 Relative expression of antimicrobial peptides (Hepcidin, LEAP-2A, LEAP-2B and β-defensin1)

785

and anti-inflammatory cytokines (TGF-β1, TGF-β2, IL-4/13A, IL-4/13B and IL-10) (A), and

786

pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ2, IL-6, IL-8, IL-12p35, IL-12p40 and IL-17D) (B)

787

in the gills of juvenile grass carp (Ctenopharyngodon idella) fed diets containing graded levels of

788

threonine for 8 weeks. Data represent means of six fish in each group, error bars indicate S.D. Values

789

having different letters are significantly different (P < 0.05). TNF-α, tumor necrosis factor α; IFN-γ2, interferon γ2;

790

IL, interleukin; LEAP, liver expressed antimicrobial peptide; TGF, transforming growth factor.

791 792

AC C

784

Fig.4

ACCEPTED MANUSCRIPT

SC

RI PT

793

794

Fig. 4. Relative expression of NF-κB p65, NF-κB p52, c-Rel, IκBα, IKKα, IKKβ, IKKγ, TOR, S6K1

796

and 4E-BP1 in the gills of juvenile grass carp (Ctenopharyngodon idella) fed diets containing graded

797

levels of threonine for 8 weeks. Data represent means of six fish in each group, error bars indicate S.D.

798

Values having different letters are significantly different (P < 0.05). NF-κB, nuclear factor kappa B; IκBα,

799

inhibitor of κBα; IKK, IκB kinase; TOR, target of rapamycin; S6K1, ribosomal protein S6 kinases 1; 4E-BP,

800

eIF4E-binding proteins.

TE D EP

802

AC C

801

M AN U

795

Fig. 5

ACCEPTED MANUSCRIPT

RI PT

803

TE D

M AN U

SC

804

805

Fig. 5. Relative expression of antioxidant enzymes (CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a,

807

GPx4b, GSTR, GSTO1, GSTO2 and GR) (A) and related signaling molecules (Nrf2, Keap1a and

808

Keap1b) (B) in the gills of grass carp (Ctenopharyngodon idella) fed diets containing graded levels of

809

threonine for 8 weeks. Data represent means of six fish in each group, error bars indicate S.D. Values

810

having different letters are significantly different (P < 0.05). CuZnSOD: copper, zinc superoxide dismutase;

811

MnSOD: manganese superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GST: glutathione

812

S-transferase; GR: glutathione reductase; Nrf2: NF-E2-related factor 2; Keap1: Kelch-like-ECH-associated

813

protein 1.

814

AC C

EP

806

Fig. 6

ACCEPTED MANUSCRIPT

SC

RI PT

815

816

Fig. 6 Relative expression of apoptotic parameters (FasL, caspase-3, caspase-8, caspase-9, Apaf-1, Bax

818

Bcl-2, IAP and JNK) in the gills of juvenile grass carp (Ctenopharyngodon idella) fed diets containing

819

graded levels of threonine for 8 weeks. Data represent means of six fish in each group, error bars indicate

820

S.D. Values having different letters are significantly different (P < 0.05). Caspase, cysteinyl aspartic

821

acid-protease; Apaf-1, apoptotic protease activating factor-1; Bax, Bcl-2 associated X protein; FasL, Fas

822

ligand; Bcl-2, B-cell lymphoma protein-2; IAP, inhibitor of apoptosis proteins; JNK, c-Jun N-terminal

823

protein kinase.

TE D EP

AC C

824

M AN U

817

Fig. 7

ACCEPTED MANUSCRIPT

SC

RI PT

825

826

Fig. 7 Relative expression of tight junction complexes and MLCK in the gills of juvenile grass carp

828

(Ctenopharyngodon idella) fed diets containing graded levels of threonine for 8 weeks. Data represent

829

means of six fish in each group, error bars indicate S.D. Values having different letters are significantly

830

different (P < 0.05). ZO, zonula occludens; MLCK, myosin light chain kinase.

M AN U

827

AC C

EP

TE D

831

832

Fig. 8

ACCEPTED MANUSCRIPT

SC

RI PT

833

834

Fig. 8 Western blot analysis of TOR protein phosphorylation at Ser2448 (A) and nuclear Nrf2 (B) in

836

the gills of fish fed diets containing graded levels of threonine for 8 weeks. Data represent means of

837

three replicates in each group, error bars indicate SD, and different letters above bar denote the significant

838

difference between treatments (P < 0.05; ANOVA and Duncan’s multiple range tests).

M AN U

835

AC C

EP

TE D

839

840

Fig. 9

SC

RI PT

841

ACCEPTED MANUSCRIPT

843

M AN U

842

Fig. 9 The potential action pathways of threonine on the gill immune and physical barriers of fish.

AC C

EP

TE D

844

ACCEPTED MANUSCRIPT Highlights Compared with optimal threonine supplementation: Threonine deficiency decreased gill disease resistance in fish. Threonine deficiency impaired gill immune barrier function in fish.

RI PT

Threonine deficiency disturbed gill physical barrier function in fish.

AC C

EP

TE D

M AN U

SC

Threonine modulated molecules NF-κB, TOR, Nrf2, JNK and MLCK.