- Email: [email protected]
Effect of dietary phosphorus deficiency on the growth, immune function and structural integrity of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella)
Accepted Manuscript Effect of dietary phosphorus deficiency on the growth, immune function and structural integrity of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) Kang Chen, Wei-Dan Jiang, Pei Wu, Yang Liu, Sheng-Yao Kuang, Ling Tang, WuNeng Tang, Yong-An Zhang, Xiao-Qiu Zhou, Lin Feng PII:
S1050-4648(17)30070-0
DOI:
10.1016/j.fsi.2017.02.007
Reference:
YFSIM 4429
To appear in:
Fish and Shellfish Immunology
Received Date: 2 October 2016 Revised Date:
7 February 2017
Accepted Date: 8 February 2017
Please cite this article as: Chen K, Jiang W-D, Wu P, Liu Y, Kuang S-Y, Tang L, Tang W-N, Zhang Y-A, Zhou X-Q, Feng L, Effect of dietary phosphorus deficiency on the growth, immune function and structural integrity of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella), Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.007. 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.
1
Effect of dietary phosphorus deficiency on the growth, immune function and structural
2
integrity of head kidney, spleenACCEPTED and skin inMANUSCRIPT young grass carp (Ctenopharyngodon idella)
3 a, 1
, Wei-Dan Jiang a, b, c, 1, Pei Wu
a, b, c
, Sheng-Yao Kuang d, Ling Tang d,
Kang Chen
5
Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, *, Lin Feng a, b, c, *
9 10 11 12
Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan
Agricultural University, Sichuan, Chengdu 611130, China c
RI PT
8
b
Animal Nutrition Institute, Sichuan Agricultural University, Sichuan, Chengdu 611130, China
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan
Agricultural University, Sichuan, Chengdu 611130, China
SC
7
a
d
Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China
e
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
M AN U
6
, Yang Liu
a, b, c
4
13
* Corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,
15
Sichuan, China. Tel.: +86 835 2885157; fax: +86 835 2885968.
16
E-mail addresses: [email protected], [email protected] (X.-Q. Zhou); [email protected] (L.
17
Feng).
20 21
1
EP
19
These two authors contributed to this work equally
AC C
18
TE D
14
22
Abstract
23
This study evaluates the effects of dietary phosphorus on the growth, immune function and structural
24
integrity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) that were fed graded
25
levels of available phosphorus (0.95-8.75 g/kg diet). Results indicated that phosphorus deficiency decreased
26
the growth performance of young grass carp. In addition, the results first demonstrated that compared with
27
the optimal phosphorus level, phosphorus deficiency depressed the lysozyme (LZ) and acid phosphatase
28
(ACP) activities and the complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and
29
down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα
30
(IκBα) and target of rapamycin (TOR), whereas it up-regulated pro-inflammatory cytokines, nuclear factor
31
kappa B (NF-κB) p65 and NF-κB p52 mRNA levels to decrease fish head kidney and spleen immune
32
functions. Moreover, phosphorus deficiency up-regulated the mRNA levels of Kelch-like-ECH-associated
33
protein 1a (Keap1a), Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X
34
protein (Bax), caspase -2, -3, -7, -8 and -9, p38 mitogen-activated protein kinase (MAPK) and myosin light
35
chain kinase (MLCK), whereas it depressed the glutathione (GSH) contents and antioxidant enzymes
36
activities, and down-regulated the mRNA levels of antioxidant enzymes, NF-E2-related factor 2 (Nrf2),
37
B-cell lymphoma protein-2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and tight junction complexes to
38
attenuate fish head kidney and spleen structural integrity. In addition, phosphorus deficiency increased skin
39
hemorrhage and lesions morbidity. Finally, based on the percent weight gain (PWG) and the ability to
40
combat skin hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp
41
(254.56-898.23 g) were estimated to be 4.10 and 4.13 g/kg diet, respectively. In summary, phosphorus
42
deficiency decreases the growth performance, and impairs immune function and structural integrity in the
43
head kidney, spleen and skin of young grass carp.
44
Keywords: Phosphorus deficiency; Grass carp (Ctenopharyngodon idella); Immune function; Structural
45
integrity; Head kidney; Spleen
46
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
47
1. Introduction
48
Currently, aquaculture is frequently faced with disease problems resulting from its intensification [1].
49
The disease problems of fish are closely associated with the depressed immune function of immune organs
50
in fish [2]. The head kidney and spleen of fish are the major immune organs [3]. Meanwhile, fish skin is
51
believed to be an important mucosal immune organ and the first line of defence [4]. It is well known that
52
providing enough nutrients is an important approach to improve the immune function of immune organs in
53
animals [2]. Phosphorus is an essential nutrient for the normal growth of fish [5]. Studies show that dietary
54
phosphorus deficiency induces poor bone mineralization, skeletal and operculum deformities, an increase in
55
body lipid content and a decline of growth performance in several fish species [6-9]. In fish, growth is partly
56
related to the immune function of immune organs [10, 11]. However, no study has investigated the effect of
57
dietary phosphorus on the immune function of the immune organs in fish. A low dietary level of phosphorus
58
increases the whole body lipid level in silver perch (Bidyanus bidyanus) [12]. A study from our lab showed
59
that excess dietary levels of lipids reduced the immune function of the immune organs in young grass carp
60
(Ctenopharyngodon idella) [10]. The above studies indicate that there might be a relationship between
61
dietary phosphorus deficiency and the immune function of the immune organs in fish, which is worth
62
investigation.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
The immune function of fish immune organs largely relies on antibacterial substances [such as
64
immunoglobulin M (IgM) and lysozyme (LZ)] and cytokines [13]. In fish, cytokines are regulated by the
65
transcription factors nuclear factor-κB (NF-κB) and target of rapamycin (TOR) [14, 15]. However, there is
66
no study demonstrating the effects of phosphorus on antibacterial substances and cytokines and its possible
67
mechanisms in fish. A low level of phosphorus decreased the serum antibody titer in channel catfish
68
(Ictalurus punctatus) [16] and the plasma IgM concentration, as well as LZ activity, in European whitefish
69
(Coregonus lavaretus L.) [17]. Sarasa et al. reported that phosphorus deficiency down-regulated peroxisome
70
proliferative activated receptor α (PPARα) expression in mice livers [18]. The inhibition of PPARα
71
increased the interleukin 8 (IL-8) content by activating NF-κB in human microvessel endothelial cells [19].
72
In addition, phosphorus deficiency depressed Akt phosphorylation in NHBE cells [20]. A study showed that
73
the inhibition of Akt phosphorylation inactivated mTOR in rat skeletal muscle cells [21]. According to these
74
studies, it follows that phosphorus deficiency may affect the production of antimicrobial substances,
75
cytokines and the relevant signalling molecules to decrease the immune function of the immune organs in
AC C
EP
63
76
fish, which deserves investigation.
77
The immune function of the immune organs is also correlated with the structural integrity of these
78
organs in fish [22], which is related to cellular integrity and intercellular integrity [23]. Cellular integrity is
79
impaired by oxidative damage and cell apoptosis [24]. The intercellular integrity is mainly maintained by
80
tight junction complexes (TJs) (such as occludin, zonula occludens 1 and claudins) in rats [25]. Studies have
81
indicated that the gene expression of antioxidant, apoptosis and TJs could be regulated by NF-E2-related
82
factor 2 (Nrf2) [26], p38 mitogen-activated protein kinase (MAPK) [27] and myosin light chain kinase
83
(MLCK) [28] in humans, respectively. However, no study has addressed the effects of phosphorus on
84
antioxidant, apoptosis, and tight junction and their possible signalling pathways in the immune organs of
85
terrestrial animals and fish. In human endothelial cells, phosphorus deficiency decreases nitric oxide
86
production [29]. Decreased nitric oxide inhibits the activation of Nrf2 in rat pheochromocytoma cells [30].
87
Glinn et al. reported that phosphorus deficiency increased the Ca2+ concentration in rat neuronal cells [31].
88
Ca2+ induces apoptosis through the activation of p38 MAPK in mice macrophage cells [32]. To our
89
knowledge, phosphorus is an important component of phospholipids [33]. It was reported that phospholipids
90
down-regulate the expression of MLCK, leading to the up-regulation of occludin, ZO-1 and claudins mRNA
91
levels in the gills of juvenile grass carp [34]. Based on these data, there may be a correlation between dietary
92
phosphorus deficiency and the structural integrity of immune organs associated with antioxidants, apoptosis
93
and TJs as well as their related signalling molecules in fish, which requires investigation.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Grass carp is an important farmed fish, accounting for ~16% of the global freshwater aquaculture [35].
95
Currently, phosphorus requirements have been confirmed in juvenile grass carp [9] and young grass carp
96
[36], which is mainly based on growth performance. However, the nutrient requirements of fish may vary
97
with different indices. A study from our lab showed that myo-inositol requirements for juvenile Jian carp
98
based on the head kidney acid phosphatase (ACP) activity (660.7 mg/kg diet) were higher than those based
99
on a percent weight gain (PWG) (518.0 mg/kg diet) [37]. However, no study has reported the phosphorus
100
requirements based on the immune function and structural integrity of the immune organs in fish, which
101
needs further investigation.
AC C
EP
94
102
Here, we hypothesized that phosphorus deficiency might impair immune function and structural
103
integrity in the head kidney, spleen and skin to decrease the growth performance of fish. To test this
104
hypothesis, the present study, for the first time, investigates the influence of phosphorus on antibacterial
105
substances, cytokines, antioxidants, apoptosis and TJs in the head kidney, spleen and skin of young grass
106
carp after challenge with Aeromonas hydrophila. In addition, we further investigated the influence of
107
phosphorus on the relevant signalling molecules, including NF-κB, TOR, Nrf2, p38 MAPK and MLCK,
108
which might imply partial theoretical evidence for the mechanisms of the phosphorus-enhanced fish immune
109
function and structural integrity. Meanwhile, the phosphorus requirements for the young grass carp based on
110
the immune function and structural integrity of the immune organs were also evaluated, which may provide
111
a reference for the commercial feed preparation and healthy breeding of grass carp.
112
2. Materials and methods
113
2.1. Experimental diet and procedures
RI PT
ACCEPTED MANUSCRIPT
Formulation of the basal diet and proximate analysis of the diets are presented in Table 1. Fish meal,
115
casein, gelatin and rice gluten meal were used as dietary protein sources. Fish oil and soybean oil were used
116
as dietary lipid sources. Six experimental diets were obtained by supplementing the basal diet with
117
monosodium phosphate (NaH2PO4•2H2O, AR) which was from Chengdu Kelong Chemicals Reagents
118
(Chengdu, China). The dietary total phosphorus were determined by the method of the AOAC (2000), and
119
final total phosphorus levels of the six experimental diets were 2.3 (un-supplemented), 4.0, 5.6, 7.6, 9.2 and
120
11.0 g/kg diet, respectively, which was based on the previous study from our lab [36]. The diets preparation
121
was conducted as described by our previous study [8]. Diets were prepared by thoroughly mixing all the
122
ingredients. Distilled water was added to produce a proper pelleting consistency, and then the mixture was
123
homogenized and extruded through a 2-mm die. The noodle-like diets were dried using an electrical fan at
124
room temperature. After being prepared completely, the diets were stored at -20 °C according to Xie et al.
125
[8].
127
M AN U
TE D
EP
AC C
126
SC
114
(Table 1 inserted here)
2.2. Growth trial and sample collection
128
All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan
129
Agricultural University. After obtainment from fishery (Sichuan, China), grass carp were acclimated to
130
experimental conditions for 4 weeks according to Xie et al. [8]. Subsequently, 540 fish with similar body
131
weights (mean weight 256.22 ± 0.60 g) were randomly assigned to 18 experimental cages (1.4 L × 1.4 W ×
132
1.4 H m), namely 30 fish per cage as described in our lab study [23, 36]. Every cage was equipped with a
133
disc of 100 cm diameter in the bottom to collect the uneaten feed according to Wu et al. [38]. Each
134
experimental diet was randomly fed to fish one of triplicate cages of the six dietary treatments to apparent
135
satiation, and fish were fed with respective diet four times daily according to Du et al. [39] for 60 days. The
136
uneaten feed was collected 30 min after each meal as described by our previous study [40]. During the
137
experiment, water temperature and pH were 27 ± 2 °C and 7.0 ± 0.4, respectively. The dissolved oxygen was
138
higher than 6.0 mg/L. The treatment groups were under natural light and dark cycle as described by Wen et
139
al. [15]. Before and during the experiment, daily water samples were collected and analyzed. Average
140
waterborne phosphorus concentration was below 0.054 mg/L.
RI PT
ACCEPTED MANUSCRIPT
Fish in each cage were weighed and counted at the initiation and termination of the feeding trial to
142
calculate the growth performance related indices. Twelve fish from each treatment were anaesthetized in a
143
benzocaine bath according to our lab previous study [8]. To analyze serum phosphorus content and alkaline
144
phosphatase (AKP) activity, the blood samples of six fish from each treatment were drawn from the caudal
145
vein then plasma was removed and stored -20 °C until analysis as described by Wen et al. [36]. To calculate
146
the index of the head kidney and spleen, the head kidney and spleen of twelve fish were quickly removed
147
and weighed.
148
2.3. Digestibility trial and sample collection
M AN U
SC
141
The digestibility trial was performed according to a previous study by our lab [8]. During the
150
digestibility trial, diet 1 (total phosphorus 2.3 g/kg) and diet 3 (total phosphorus 5.6 g/kg) were the same as
151
treatments 1 and 3 of the growth trial, respectively, which were added with 5 g/kg Cr2O3 according to Roy &
152
Lall [7]. One hundred and eighty fish with similar body weights (mean weight 255.67 ± 0.37 g) were
153
randomly assigned to six extra above-mentioned experimental cages (30 fish per cage) and were fed either
154
diet 1 or 3 in triplicate four times daily to satiation as described by Liang et al. [9]. After 10 days of feeding,
155
faecal collection was conducted 6 h after the first meal according to Mai et al. [41]. As Austreng described,
156
the faeces were stripped from all of the fish by applying gentle pressure in the anal area [42]. The faecal
157
samples were pooled and stored at -20 °C for subsequent analyses according to Zhang et al. [43].
158
2.4. Challenge trial and sample collection
AC C
EP
TE D
149
159
A. hydrophila was friendly provided by College of Veterinary Medicine, Sichuan Agricultural
160
University, China. The challenge test was conducted as described by our previous study [23]. After the
161
growth trial, fifteen fish of similar body weights were obtained from each treatment group as described by
162
Liu et al. [44] and moved to six new experimental cages. After 5 days acclimation according to Kuang et al.
163
[22], fish were intraperitoneally injected with 1.0 ml of 2.5×109 colony-forming units (cfu) ml-1
164
A. hydrophila for each individual. The challenge dose was selected as an appropriate dose which could
165
effectively induce inflammation and consequently enable the investigation on fish reactivity against a
166
threatening disease without causing death according to our preliminary test (data was not shown).
167
Managements during the challenge test were the same as the feeding trial for 14 days according to our lab
168
previous study [11]. To evaluate the severity of skin hemorrhage and lesions morbidity in grass carp, a
169
scoring system was designed which were similar to Song et al. [45]. After the challenge trial, all fish from
170
each treatment were anaesthetized in a benzocaine bath according to Xie et al. [8]. Skin samples were
171
collected from grass carp using the method of Caiping et al. [46]. Skin sections (approx. 2 cm × 2 cm) were
172
thereafter aseptically excised from both the dorsal and ventral regions (about 1 cm above the pectoral and
173
pelvic fin, respectively) along the left side of the fish. Then sacrificed, the head kidney, spleen and skin of
174
the fish were quickly removed and frozen in liquid nitrogen, then stored at -80 °C until analysis as described
175
by Ni et al. [10].
176
2.5. Biochemical analysis
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
The approximate compositions of the diets were analysed according to the standard methods of AOAC
178
[47]. Briefly, crude protein was determined by the micro-Kjeldahl method, crude lipid by the Soxhlet
179
method. Phosphorus contents of experimental diets and faeces were measured by method of AOAC [47].
180
Chromium contents of experimental diets and faeces were analysed according to Wen et al. [36].
TE D
177
Tissue homogenates of head kidney, spleen and skin were prepared in 10 volumes (w/v) of ice-cold
182
normal saline and centrifuged at 6000 g for 20 min at 4 °C. The supernatant was conserved and used to
183
determine the immune and antioxidant parameters, which was similar to Wu et al. [48]. The determination
184
methods of serum phosphorus and AKP are similar to Shao et al. [49]. LZ, ACP activities and complement 3
185
(C3), C4 and immunoglobulin M (IgM) contents were assayed according to Zhao et al. [50]. The reactive
186
oxygen species (ROS) and malondialdehyde (MDA) contents were determined according to Yang et al. [51]
187
and Ji et al [52], respectively. The contents of protein carbonyl (PC) and glutathione (GSH) were assayed as
188
described by Ni et al. [10]. The anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were
189
measured as previously study [22]. The total superoxide dismutase (SOD) and copper, zinc superoxide
190
dismutase (CuZnSOD) activities were determined as described by Lu et al. [53]. The manganese superoxide
191
dismutase (MnSOD) activity was calculated by deducting CuZnSOD from total SOD. The catalase (CAT),
AC C
EP
181
192
glutathione-S-transferases (GST) and glutathione reductase (GR) activities were assayed according to Wen et
193
al. [36]. The glutathione peroxidase (GPx) activity was measured according to Zhang et al. [54]. The choline
194
content was determined as described by Zhang et al. [55].
195
2.6. Real-time polymerase chain reaction (PCR) analysis
ACCEPTED MANUSCRIPT
Total RNA samples were isolated from the head kidney, spleen and skin using RNAiso Plus Kit (Takara,
197
Dalian, China) according to the manufacturer’s instructions, then quality and quantity were assessed using
198
agarose gel (1%) electrophoresis and spectrophotometric (A260: 280 nm ratio) analysis, respectively, as
199
described by Luo et al. [56]. Subsequently, RNA was reverse transcribed into cDNA using the
200
PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer’s instructions. For quantitative
201
real-time PCR, specific primers were designed according to the sequences cloned in our laboratory and the
202
published sequences of grass carp (Table 2). According to the results of our preliminary experiment
203
concerning the evaluation of internal control genes (data not shown), β-actin was used as a reference gene to
204
normalize cDNA loading. The target and housekeeping gene amplification efficiency were calculated
205
according to the specific gene standard curves generated from 10-fold serial dilutions. The 2−∆∆CT method
206
was used to calculate the expression results after verifying that the primers amplified with an efficiency of
207
approximately 100% according to Livak & Schmittgen et al. [57].
209
SC
M AN U
(Table 2 inserted here)
TE D
208
RI PT
196
2.7. Calculations and statistical analysis
The data of initial body weight (IBW), final body weight (FBW) and feed intake (FI) were used to
211
calculate the percent weight gain (PWG), feed efficiency (FE) and specific growth rate (SGR) according to
212
Xie et al. [8]. The weight of head kidney and spleen were used to calculate the index of head kidney and
213
spleen, respectively, as described by Kuang et al. [22].
AC C
EP
210
214
PWG =100 × [FBW (g/fish) – IBW (g/fish)]/IBW (g/fish);
215
FE = 100 × [FBW (g/fish) – IBW (g/fish)]/FI (g/fish);
216
SGR = 100 × [ln (mean final weight) – ln (mean initial weight)]/days;
217
Head kidney index = (g head kidney weight/g body weight) × 100;
218
Spleen index = (g spleen weight/g body weight) × 100;
219
The apparent phosphorus digestibility in the diet (D) calculation equation D (%) = 100 × [1 – (CrD ×
220
PF)/(CrF × PD)], where CrD and PD were the concentrations of Cr2O3 and phosphorus in the diet, respectively,
221
CrF and PF were the concentrations of Cr2O3 and phosphorus in the faeces, respectively [National Research
222
Council (NRC) 2011] [58]. According to this, apparent phosphorus digestibility in the diet 1 and diet 3
223
could be calculated.
ACCEPTED MANUSCRIPT
The apparent phosphorus digestibility in the NaH2PO4 (Di) calculation equation: Di (%) = 100 × (P2 ×
225
D2 – P1 × D1)/I, where P1 and P2 were the concentrations of phosphorus in diet 1 and diet 3, respectively; D1
226
and D2 were the apparent phosphorus digestibility in diet 1 and diet 3, respectively; I was concentration of
227
inorganic phosphorus in diet 3 [7]. According to this, apparent phosphorus digestibility in the NaH2PO4
228
could be calculated.
229
RI PT
224
All data were subjected to one-way analysis of variance followed by Duncan’s multiple range tests to determine significant differences among treatment groups using the software SPSS 18.0 (SPSS Inc., Chicago,
231
IL, USA) at a level of P < 0.05. The results are presented as the means ± SD. Broken-line analysis was used
232
to evaluate the dietary phosphorus requirement of young grass carp according to Xie et al. [8].
233
3. Results
234
3.1. Phosphorus availability
M AN U
SC
230
Apparent digestibility of phosphorus in diet 1 and diet 3 were 41.14% and 69.48%, respectively.
236
Apparent digestibility of phosphorus in NaH2PO4 was calculated to be 89.20%. According to these values,
237
available phosphorus contents in the six experimental diets were estimated to be 0.95 (un-supplemented
238
control), 2.46, 3.96, 5.68, 7.10 and 8.75 g/kg diet, respectively.
239
3.2. Growth performance and serum phosphorus concentration and alkaline phosphatase activity, as well
240
as skin hemorrhage and lesions morbidity of young grass carp after infection with A. hydrophila
EP
TE D
235
As shown in Table 3, the growth performance (FBW, PWG, SGR FI, and FE) and head kidney weight
242
and index all significantly increased as the dietary available phosphorus levels increased to 3.96 g/kg diet (P
243
< 0.05), and then plateaued. Fish fed 3.96 and 5.68 g/kg diet showed the highest spleen weight. As the
244
dietary available phosphorus levels increased to 3.96 g/kg diet, the spleen index was no significant changes
245
(P > 0.05), and then gradually decreased. The serum phosphorus concentration and AKP activity increased
246
as the dietary available phosphorus levels increased to 5.68 g/kg diet, and then plateaued. After infection
247
with A. hydrophila in young grass carp, the skin hemorrhage and lesions morbidity significantly decreased
248
as the dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and then plateaued.
249
Compared with optimal available phosphorus level, phosphorus deficiency led to an obvious skin
AC C
241
250
hemorrhage and lesions symptom (Fig. 1&2).
251
(Table 3 inserted here)
252
(Fig. 1 inserted here)
253
(Fig. 2 inserted here)
ACCEPTED MANUSCRIPT
254
3.3. Immunological parameters in immune organs of young grass carp after infection with A. hydrophila
255
3.3.1. Antimicrobial substances activities or contents in the immune organs The LZ and ACP activities, C3, C4 and IgM contents in the head kidney, spleen and skin are presented
257
in Table 4. In the head kidney, the LZ and ACP activities, C3, C4 and IgM contents gradually increased as
258
the dietary available phosphorus levels increased to 5.68, 3.96, 7.10, 7.10 and 2.46 g/kg diet, respectively,
259
and then plateaued. As the dietary available phosphorus levels increased to 2.46, 3.96, 5.68, 5.68 and 5.68
260
g/kg diet, the LZ and ACP activities and C3, C4 and IgM contents in the spleen gradually improved,
261
respectively, and then plateaued. The LZ and ACP activities, C3, C4 and IgM contents in the skin gradually
262
increased as the dietary available phosphorus levels increased to 3.96, 5.68, 5.68, 7.10 and 3.96 g/kg diet,
263
respectively, and then plateaued.
264
(Table 4 inserted here)
M AN U
SC
RI PT
256
3.3.2. Relative mRNA levels of antimicrobial peptides, cytokines, GATA-3 and related signalling
266
molecules in the immune organs
TE D
265
The effects of dietary available phosphorus on immune-related indices in the head kidney, spleen and
268
skin of young grass carp are presented in Fig. 3-6. In the head kidney, the liver-expressed antimicrobial
269
peptide 2A (LEAP-2A), LEAP-2B and hepcidin mRNA levels were gradually up-regulated as the dietary
270
available phosphorus levels increased to 3.96 g/kg diet and then plateaued. Fish fed 5.68 g/kg available
271
phosphorus diet showed the highest β-defensin-1 mRNA level. In the spleen, the LEAP-2A, LEAP-2B,
272
hepcidin and β-defensin-1 mRNA levels were gradually up-regulated as the dietary available phosphorus
273
levels increased to 3.96, 5.68, 3.96 and 3.96 g/kg diet, respectively, and then plateaued. In the skin, the
274
LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 mRNA levels were gradually up-regulated as the dietary
275
available phosphorus levels increased to 3.96, 2.46, 3.96 and 5.68 g/kg diet, respectively, and then plateaued.
276
In the head kidney, the mRNA levels of IL-6, IL-12p35, interferon γ2 (IFN-γ2), NF-κB p52, c-Rel,
277
IκB kinases γ (IKKγ) and eIF4E-binding proteins 1 (4E-BP1) were gradually down-regulated as the dietary
278
available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96, 3.96, 3.96 and 5.68 g/kg diet, respectively,
AC C
EP
267
279
simultaneously, IL-1β, IL-17D, tumor necrosis factor α (TNF-α), NF-κB p65, IKKβ and 4E-BP2 were
280
significantly down-regulated as the dietary available phosphorus levels increased to 5.68, 3.96, 5.68, 5.68,
281
3.96 and 2.46 g/kg diet (P < 0.05), respectively, and plateaued thereafter. Fish fed 5.68 g/kg available
282
phosphorus diet showed the lowest IL-15 mRNA level. The mRNA levels of IL-4/13A, transforming growth
283
factor β1 (TGF-β1), inhibitor protein κBα (IκBα), p70 S6 kinase (S6K1) and GATA-3 were gradually
284
up-regulated as the dietary available phosphorus levels increased to 3.96, 5.68, 3.96, 3.96 and 3.96 g/kg diet,
285
respectively, simultaneously, IL-10, IL-11, TGF-β2 and TOR were all significantly up-regulated as the
286
dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and plateaued after. In the spleen,
287
the mRNA levels of IL-1β, IL-6, IFN-γ2, c-Rel and IKKβ were gradually down-regulated as the dietary
288
available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively,
289
simultaneously, IL-12p35, IL-17D, TNF-α, NF-κB p52, NF-κB p65, IKKγ, 4E-BP1 and 4E-BP2 were
290
significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 3.96, 3.96, 2.46,
291
3.96, 2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then plateaued. The IL-10, IL-11, IL-4/13A,
292
TGF-β2, IκBα, TOR, and S6K1 mRNA levels were gradually up-regulated as the dietary available
293
phosphorus levels increased to 3.96, 2.46, 3.96, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively, and
294
remained constant thereafter. Fish fed 5.68 g/kg available phosphorus diet had the highest mRNA levels of
295
TGF-β1 and GATA-3, and the lowest mRNA level of IL-15. Interestingly, dietary available phosphorus had
296
no impact on the mRNA levels of IL-8, IL-12p40, IL-4/13B and IKKα in the head kidney and spleen of
297
young grass carp. In the skin, the mRNA levels of IL-12p35, NF-κB p65, IKKα and 4E-BP2 were gradually
298
down-regulated as the dietary available phosphorus levels increased to 3.96, 3.96, 5.68 and 3.96 g/kg diet,
299
respectively, simultaneously, IL-1β, IL-6, IL-8, IL-17D, TNF-α, IFN-γ2, NF-κB p52, c-Rel, IKKβ and IKKγ
300
were significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 2.46, 3.96,
301
3.96, 3.96, 3.96, 2.46, 2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then plateaued. The mRNA
302
levels of IL-10, IL-11, IL-4/13A, TGF-β1, IκBα and TOR were gradually up-regulated as the dietary
303
available phosphorus levels increased to 3.96, 3.96, 3.96, 3.96, 2.46 and 2.46 g/kg diet, respectively, and
304
plateaued thereafter. Fish fed 5.68 g/kg available phosphorus diet showed the highest mRNA levels of
305
TGF-β2 and GATA-3, and the lowest mRNA level of IL-15. Interestingly, dietary available phosphorus had
306
no impact on the mRNA levels of IL-12p40, IL-4/13B, S6K1 and 4E-BP1.
307
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
(Fig. 3 inserted here)
308
(Fig. 4 inserted here)
309
(Fig. 5 inserted here)
310
(Fig. 6 inserted here)
ACCEPTED MANUSCRIPT
311
3.4. Immune organs structural integrity of young grass carp after infection of A. hydrophila
312
3.4.1. Antioxidant-related parameters in the immune organs The head kidney, spleen and skin oxidative status and antioxidant capacity are displayed in Table 5. In
314
the head kidney, the ROS, MDA and PC contents significantly decreased as the dietary available
315
phosphorus levels increased to 5.68 g/kg diet (P < 0.05), and there were no significant changes in the MDA
316
and PC contents thereafter (P > 0.05), but an increase was observed in the ROS content. The AHR, ASA,
317
CuZnSOD, MnSOD, GPx and GST activities as well as GSH and choline contents gradually increased as the
318
dietary available phosphorus levels increased to 5.68, 5.68, 5.68, 3.96, 3.96, 5.68, 3.96 and 2.46 g/kg diet,
319
respectively, and then plateaued. The CAT activity was the highest for fish fed 3.96 and 5.68 g/kg available
320
phosphorus diet. In the spleen, the ROS, MDA and PC contents significantly decreased as the dietary
321
available phosphorus levels increased to 5.68, 5.68 and 3.96 g/kg diet (P < 0.05), respectively, and the MDA
322
and PC plateaued, but ROS increased thereafter. The AHR, ASA, CuZnSOD, MnSOD and GPx activities as
323
well as GSH and choline contents gradually increased as the dietary available phosphorus levels increased to
324
3.96, 7.10, 2.46, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively, and then plateaued. Fish fed 5.68 g/kg
325
available phosphorus diet had the highest GST activity. Interestingly, the GR activities significantly
326
decreased as the dietary available phosphorus levels increased to 2.46 and 3.96 g/kg diet in the head kidney
327
and spleen (P < 0.05), respectively, and then plateaued. However, dietary available phosphorus had no
328
impact on the CAT activity in the spleen of grass carp. In the skin, the ROS, MDA and PC contents
329
gradually decreased as the dietary available phosphorus levels increased to 5.68 g/kg diet, and ROS and
330
MDA increased thereafter, but PC plateaued. The AHR, ASA, CuZnSOD, MnSOD, GPx, GST and GR
331
activities as well as GSH and choline contents gradually increased as the dietary available phosphorus levels
332
increased to 3.96, 3.96, 7.10, 5.68, 3.96, 5.68, 3.96, 3.96 and 3.96 g/kg diet, respectively, and then plateaued.
333
The CAT activity was the highest for fish fed 3.96 and 5.68 g/kg diet available phosphorus.
334 335 336
AC C
EP
TE D
M AN U
SC
RI PT
313
(Table 5 inserted here) 3.4.2. Relative mRNA levels of antioxidant enzymes, Nrf2, Keap1a and Keap1b in the immune organs As shown in Fig. 7, in the head kidney, the mRNA levels of CuZnSOD, CAT, GPx1a, GPx4a and Nrf2
337
were gradually up-regulated as the dietary available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96
338
and 5.68 g/kg diet, respectively, simultaneously, the MnSOD, GPx1b, GSTR and GSTO1 mRNA levels were
339
all significantly up-regulated as the dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05),
340
and plateaued thereafter. Fish fed 5.68 g/kg available phosphorus diet showed the highest GPx4b and
341
GSTP1 mRNA levels. In the spleen, the mRNA levels of CuZnSOD, GPx1a, GPx1b, GPx4a, GPx4b,
342
GSTR and GSTP1 were gradually up-regulated as the dietary available phosphorus levels increased to 3.96
343
g/kg diet, and then plateaued. As dietary available phosphorus levels increased to 3.96, 2.46, 3.96 and 2.46
344
g/kg diet, MnSOD, CAT, GSTO1 and Nrf2 mRNA levels were significantly up-regulated (P < 0.05),
345
respectively, and remained constant thereafter. In the skin, the mRNA levels of CuZnSOD, CAT, GPx1a,
346
GPx1b, GPx4b, GSTR, GSTO1 and Nrf2 were gradually up-regulated as the dietary available phosphorus
347
levels increased to 2.46, 3.96, 3.96, 3.96, 3.96, 2.46, 3.96 and 3.96 g/kg diet, respectively, simultaneously,
348
the MnSOD, GPx4a, GSTP1 and GR mRNA levels were significantly up-regulated as the dietary available
349
phosphorus levels increased to 2.46, 3.96, 5.68 and 2.46 g/kg diet (P < 0.05), respectively, and plateaued
350
after. The mRNA levels of Keap1a were all down-regulated as the dietary available phosphorus level
351
increased to 5.68 g/kg diet in the head kidney, spleen and skin, and then plateaued. Interestingly, in the head
352
kidney and spleen, the GR expression was significantly down-regulated as the dietary available phosphorus
353
level increased to 5.68 and 3.96 g/kg diet (P < 0.05), respectively, and then kept unchanged. In the head
354
kidney, spleen and skin, dietary available phosphorus had no impact on the mRNA levels of Keap1b.
356
RI PT
SC
M AN U
TE D
(Fig. 7 inserted here)
EP
355
ACCEPTED MANUSCRIPT
3.4.3. Relative mRNA levels of apoptosis-related parameters in the immune organs The effects of dietary available phosphorus on apoptosis and related signalling molecules in the head
358
kidney, spleen and skin of young grass carp are presented in Fig. 8. In the head kidney, the mRNA levels of
359
Apaf-1, caspase-2 and -7 were gradually down-regulated as the dietary available phosphorus levels
360
increased to 5.68, 3.96 and 3.96 g/kg diet, respectively, simultaneously, FasL, Bax, caspase-3, -8, -9 and p38
361
MAPK were significantly down-regulated as the dietary available phosphorus levels increased to 5.68, 5.68,
362
3.96, 2.46, 3.96 and 3.96 g/kg diet (P < 0.05), respectively, and plateaued thereafter. The mRNA levels of
363
Bcl-2, Mcl-1 and IAP were gradually up-regulated as the dietary available phosphorus levels increased to
364
3.96, 5.68 and 5.68 g/kg diet, respectively, and plateaued thereafter. In the spleen, the mRNA levels of
365
Apaf-1, caspase-7 and -9 were gradually down-regulated as the dietary available phosphorus levels
AC C
357
366
increased to 5.68, 3.96 and 3.96 g/kg diet, respectively, simultaneously, FasL, Bax, caspase-2, -3 and p38
367
MAPK were significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 2.46,
368
2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then remained constant. Fish fed 5.68 g/kg
369
available phosphorus diet showed the lowest caspase-8 mRNA level. The mRNA levels of Bcl-2, Mcl-1 and
370
IAP were gradually up-regulated as the dietary available phosphorus levels increased to 3.96, 3.96 and 2.46
371
g/kg diet, respectively, and then plateaued. However, dietary available phosphorus had no influence on the
372
mRNA levels of JNK in the head kidney and spleen. In the skin, the mRNA levels of FasL, Bax, Apaf-1,
373
caspase-3, -7 and -8 were significantly down-regulated as the dietary available phosphorus levels increased
374
to 3.96, 3.96, 5.68, 3.96, 2.46 and 3.96 g/kg diet (P < 0.05), respectively, simultaneously, caspase-9 and JNK
375
were both gradually down-regulated as the dietary available phosphorus levels increased to 3.96 g/kg diet,
376
and plateaued thereafter. The mRNA levels of Bcl-2, Mcl-1 and IAP were significantly up-regulated as the
377
dietary available phosphorus levels increased to 2.46, 3.96 and 3.96 g/kg diet (P < 0.05), respectively, and
378
then plateaued.
380
RI PT
SC
M AN U
379
ACCEPTED MANUSCRIPT
(Fig. 8 inserted here)
3.4.4. Relative mRNA levels of TJs, transporter and MLCK in the immune organs The effects of dietary available phosphorus on TJs, type-IIb sodium-phosphate cotransporter (NaPi-IIb)
382
and MLCK relative mRNA levels in the head kidney, spleen and skin are presented in Fig 9. In the head
383
kidney, the mRNA levels of occludin, claudin-f, -7a, -11 and -15a were gradually up-regulated as the dietary
384
available phosphorus levels increased to 3.96, 3.96, 3.96, 2.46 and 3.96 g/kg diet, respectively,
385
simultaneously, ZO-1, ZO-2 and claudin-c were significantly up-regulated as the dietary available
386
phosphorus levels increased to 3.96, 2.46 and 3.96 g/kg diet (P < 0.05), respectively, and plateaued after.
387
The mRNA levels of claudin-12, NaPi-IIb and MLCK were all significantly down-regulated as the dietary
388
available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and then plateaued. The mRNA level of
389
claudin-7b showed the highest for fish fed 5.68g/kg available phosphorus diet. In the spleen, as dietary
390
available phosphorus levels increased to 3.96, 2.46, 3.96, 3.96, 2.46, 2.46, 2.46 and 3.96 g/kg diet, the
391
mRNA levels of occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -11 and -15a were gradually up-regulated,
392
respectively, and plateaued thereafter. The mRNA levels of claudin-12, NaPi-IIb and MLCK were
393
down-regulated as the dietary available phosphorus levels increased to 2.46, 3.96 and 2.46 g/kg diet,
394
respectively, and then plateaued. Fish fed 5.68 g/kg available phosphorus diet had the highest claudin-7b
AC C
EP
TE D
381
395
mRNA level. Interestingly, dietary available phosphorus had no impacts on the mRNA levels of claudin-b,
396
-3c and -15b in the head kidney and spleen. In the skin, the mRNA levels of occludin, ZO-1, ZO-2,
397
claudin-b, -c, -f, -7a, -11 and -15a were up-regulated as the dietary available phosphorus levels increased to
398
3.96, 2.46, 3.96, 3.96, 3.96, 3.96, 3.96, 5.68 and 3.96 g/kg diet, respectively, and plateaued after. As the
399
dietary available phosphorus levels increased to 3.96, 2.46 and 2.46 g/kg diet, the mRNA levels of
400
claudin-12, NaPi-IIb and MLCK were significantly down-regulated (P < 0.05), respectively, and then
401
plateaued. Fish fed 5.68 g/kg available phosphorus diet had highest mRNA level of claudin-7b. Interestingly,
402
dietary available phosphorus had no impacts on the mRNA levels of claudin-3c and -15b.
RI PT
403
ACCEPTED MANUSCRIPT
(Fig. 9 inserted here) 4. Discussion
405
4.1. Phosphorus deficiency decreased the growth performance of fish
SC
404
This study indicated that dietary phosphorus deficiency decreased the growth performance (PWG, FI,
407
FE and SGR) and immune organs size (head kidney and spleen weight) of young grass carp. The serum
408
phosphorus concentration and AKP activity were used as indicators of the phosphorus nutrition status in fish
409
[36]. In the present study, phosphorus deficiency decreased the serum phosphorus concentration and AKP
410
activity, indicating that phosphorus deficiency worsened the nutrition status. Based on the broken-line
411
analysis of the PWG, the available phosphorus requirement for young grass carp was estimated to be 4.10
412
g/kg diet, which was consistent with the result of our previous study [36]. Fish growth is closely related to
413
disease resistance [59], which is partly associated with the ability to combat skin hemorrhage and lesions,
414
immune function and structural integrity in the immune organs of fish [11, 23]. A. hydrophila is one of the
415
predominant pathogens in freshwater fish, which causes fish skin hemorrhage and lesions as well as kidney
416
and spleen immune function depression and integrity damage [10, 45]. Thus, in this study, after the feeding
417
trial, the fish were infected with A. hydrophila to investigate the effects of phosphorus on the ability to
418
combat skin hemorrhage and lesions, immune function and structural integrity in the immune organs of
419
young grass carp.
420
4.2. Phosphorus deficiency decreased the head kidney and spleen immune function partly relating to
421
NF-κB and TOR signalling in fish after infection with A. hydrophila
422
4.2.1. Phosphorus deficiency reduced the head kidney and spleen antibacterial substances of fish
423
AC C
EP
TE D
M AN U
406
The immune function of fish largely relies on antibacterial substances, which consist of LZ, ACP, IgM,
424
complement factors and antimicrobial peptides [60]. Thus, we first investigated the effect of phosphorus on
425
antibacterial substances in the head kidney and spleen of fish, and concluded that, compared with optimal
426
phosphorus level, phosphorus deficiency decreased the LZ and ACP activities, and the C3, C4 and IgM
427
contents and down-regulated antimicrobial peptides, including LEAP-2A, LEAP-2B, hepcidin and
428
β-defensin-1 mRNA levels, implying that phosphorus deficiency depressed the immune function in the head
429
kidney and spleen of fish. Fish immune function is closely associated with the inflammation response, which
430
are primarily mediated by cytokines [56]. Therefore, we next investigated the relationships between the
431
dietary available phosphorus and the cytokines in the head kidney and spleen of young grass carp.
432
4.2.2. Phosphorus deficiency aggravated inflammatory responses partly relating to NF-κB and TOR
433
signalling in the head kidney and spleen of fish
SC
RI PT
ACCEPTED MANUSCRIPT
The inflammatory response is often regulated by cytokines, which include pro-inflammatory cytokines,
435
such as IL-1β, IL-6, IFN-γ and TNF-α, in humans [61], and anti-inflammatory cytokines, such as IL-4,
436
IL-10, IL-11 and TGF-β, in rats [62]. In teleost, the up-regulation of pro-inflammatory cytokines and the
437
down-regulation of anti-inflammatory cytokines initiates and accelerates additional inflammatory processes
438
[63]. In this study, compared with optimal phosphorus level, phosphorus deficiency up-regulated the
439
pro-inflammatory cytokines IL-1β, IL-6, IL-12p35, IL-15, IL-17D, IFN-γ2 and TNF-α mRNA levels and
440
down-regulated the anti-inflammatory cytokines IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA levels
441
in the head kidney and spleen of young grass carp. These results implied that phosphorus deficiency might
442
induce inflammatory responses in the head kidney and spleen of fish. Interestingly, phosphorus deficiency
443
had no effects on the mRNA levels of IL-8, IL-12p40 and IL-4/13B in the head kidney and spleen of young
444
grass carp.
AC C
EP
TE D
M AN U
434
445
First, the IL-8 mRNA levels unaffected by phosphorus deficiency in the head kidney and spleen of
446
young grass carp may be related to the unaffected JNK. The inhibition of JNK reduces IL-8 mRNA level in
447
EAGS cells [64]. In this study, phosphorus deficiency had no impacts on the JNK mRNA levels in the head
448
kidney and spleen, supporting our hypothesis.
449
Second, phosphorus deficiency up-regulated IL-12p35 (rather than IL-12p40) mRNA levels in the head
450
kidney and spleen of young grass carp, which may be partially associated with IL-1β. IL-1β could
451
up-regulates IL-12p35 mRNA level but has no influence on the mRNA level of IL-12p40 in the mature DCs
452
[65]. Our study showed that phosphorus deficiency elevated the mRNA levels of IL-1β in the head kidney
453
and spleen. A further correlation analysis showed that IL-12p35 mRNA levels were positively related to
454
IL-1β mRNA levels in the head kidney and spleen of young grass carp (Table 6), supporting our hypothesis.
ACCEPTED MANUSCRIPT
Lastly, phosphorus deficiency down-regulated the mRNA levels of IL-4/13A (rather than IL-4/13B) in
456
the head kidney and spleen of young grass carp, which may be related to GATA-3. The inhibition of
457
GATA-3 down-regulates the gene transcription of IL-4/13A by reducing the binding with a TATA box [66]
458
which is not found in IL-4/13B in puffer (Tetraodon nigroviridis) [67, 68]. In the present study, phosphorus
459
deficiency down-regulated the GATA-3 mRNA levels in the head kidney and spleen of young grass carp.
460
According to the above studies, phosphorus deficiency down-regulating GATA-3 expression might lead to
461
the down-regulation of IL-4/13A (rather than IL-4/13B) mRNA levels in the head kidney and spleen of fish.
RI PT
455
In human mast cells, the expression of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8
463
was dependent on the activation of the transcription factor NF-κB [69]. NF-κB p65, NF-κB p52 and c-Rel
464
are important members in the NF-κB family [70]. In HeLa and 293 cells, IKKα, IKKβ and IKKγ form the
465
IKK complexes and phosphorylate IκBs which activate NF-κB [71]. In the present study, compared with
466
optimal phosphorus level, phosphorus deficiency up-regulated NF-κB p65, NF-κB p52, c-Rel, IKKβ and
467
IKKγ mRNA levels and down-regulated IκBα mRNA levels in the head kidney and spleen of young grass
468
carp. Correlation analysis indicated that the pro-inflammatory cytokines IL-1β, IL-6, IL-12p35 (rather than
469
IL-12p40), IL-15, IL-17D, IFN-γ2 and TNF-α mRNA levels were positively related to NF-κB p65, NF-κB
470
p52 and c-Rel mRNA levels (Table 6). These results implied that phosphorus deficiency up-regulated
471
pro-inflammatory cytokines (except IL-8 and IL-12p40) mRNA levels, which may be partly referred to
472
(IKKβ, IKKγ) /IκBα/ (NF-κB p65, NF-κB p52 and c-Rel) in the head kidney and spleen of fish.
EP
TE D
M AN U
SC
462
Interestingly, we found that phosphorus deficiency up-regulated IKKβ and IKKγ (but not IKKα) in the
474
head kidney and spleen of young grass carp, and this interesting phenomenon might be correlated with
475
phospholipids. It was reported that dietary phospholipids in an un-supplemented group up-regulate the
476
expression of IKKβ and IKKγ (but not IKKα) in the intestine of juvenile grass carp [72]. Phosphorus
477
deficiency reduces the synthesis of phospholipid [33]. Hence, we hypothesize that phosphorus deficiency
478
might decrease phospholipids content, leading to up-regulated IKKβ and IKKγ (but not IKKα) expression in
479
the head kidney and spleen of fish. However, this hypothesis requires further investigation.
AC C
473
480
mTOR enhances the anti-inflammatory cytokine IL-10 activity in human PBMCs [73]. In mammals,
481
mTOR directly phosphorylates S6K1 and inhibits the initiation factor 4E-BP to initiate the translation of
482
distinct mRNAs [74]. In the present study, compared with the optimal phosphorus level, phosphorus
483
deficiency down-regulated TOR and S6K1 mRNA levels, and up-regulated 4E-BP1 and 4E-BP2 mRNA
484
levels in the head kidney and spleen of young grass carp. Correlation analysis showed that the mRNA levels
485
of the anti-inflammatory cytokines IL-4/13A (rather than IL-4/13B), IL-10, IL-11, TGF-β1 and TGF-β2
486
were positively correlated with TOR gene expression (Table 6), suggesting that phosphorus deficiency
487
down-regulated anti-inflammatory cytokines (except IL-4/13B) gene expression, which might be partly
488
correlated to the TOR/ (S6K1, 4E-BP) signalling pathway in the head kidney and spleen of fish.
RI PT
ACCEPTED MANUSCRIPT
In summary, our data first implied that phosphorus deficiency decreased fish head kidney and spleen
490
immune function, which may be related to two aspects: (1) reduced LZ and ACP activities, C3, C4 and IgM
491
contents and the antimicrobial peptides LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 expression; (2)
492
up-regulated mRNA levels of the pro-inflammatory cytokines (except IL-8) IL-1β, IL-6, IL-12p35 (rather
493
than IL-12p40), IL-15, IL-17D, IFN-γ2 and TNF-α, and down-regulated mRNA levels of the
494
anti-inflammatory cytokines IL-4/13A (rather than IL-4/13B), IL-10, IL-11, TGF-β1 and TGF-β2, which
495
might be partly related to the IKKβ, IKKγ (rather than IKKα) /IκBα/NF-κB and TOR/ (S6K1, 4E-BP)
496
signalling pathways. In addition, the immune function of the immune organs is also correlated with the
497
structural integrity of these organs in fish [48]. Thus, we next investigated the effects of phosphorus on the
498
structural integrity in the head kidney and spleen of young grass carp.
M AN U
(Table 6 inserted here)
TE D
499
SC
489
4.3. Phosphorus deficiency attenuated the structural integrity relating to Nrf2, p38 MAPK and MLCK
501
signalling in the head kidney and spleen of fish after infection with A. hydrophila
502
4.3.1. Phosphorus deficiency induced oxidative damage and impaired the antioxidant system partly by
503
Nrf2 signalling in the head kidney and spleen of fish
AC C
EP
500
504
Infection with pathogens is often associated with an increase in the free radical content, which may lead
505
to oxidative damage [75] and then impair structural integrity in fish [37]. In the present study, compared
506
with the optimal phosphorus level, phosphorus deficiency increased ROS, MDA and PC contents and
507
decreased the ASA, AHR, CuZnSOD, MnSOD, GPx and GST activities and GSH contents in the head
508
kidney and spleen, and decreased the CAT activity only in the head kidney of young grass carp, indicating
509
that phosphorus deficiency induced oxidative damage by increasing the free radical production and
510
decreasing the antioxidant ability in fish. In addition, fish antioxidant enzyme activities were partly
511
dependent on their gene expression [53]. Thus, we next explored the effects of phosphorus on antioxidant
512
enzyme gene expression in the head kidney and spleen of young grass carp. In this study, compared with the
513
optimal phosphorus level, phosphorus deficiency down-regulated CuZnSOD, MnSOD, CAT, GPx1a, GPx1b,
514
GPx4a, GPx4b, GSTR, GSTO1 and GSTP1 expression in the head kidney and spleen of young grass carp.
515
These results indicate that phosphorus deficiency decreased those enzyme activities, which might partly
516
contribute to their down-regulated mRNA levels in the head kidney and spleen of fish. Surprisingly, we
517
found that phosphorus deficiency promoted GR activities and mRNA levels in the head kidney and spleen
518
and had no effect on the activity of CAT in the spleen of young grass carp.
RI PT
ACCEPTED MANUSCRIPT
First, phosphorus deficiency-enhanced GR activities and expression might be partially due to an
520
adaptive mechanism against the declined GSH contents. It was reported that GR catalyzes the reduction in
521
GSSG back to GSH in mammalian tissues [76]. Our data showed that phosphorus deficiency decreased the
522
GSH contents in the head kidney and spleen of young grass carp. According to the above data, the
523
phosphorus deficiency-decreased GSH contents might cause adaptive increases in the GR activities and
524
mRNA levels in the head kidney and spleen of fish.
M AN U
SC
519
Moreover, phosphorus deficiency did not influence the activity of CAT in the spleen (not the head
526
kidney) of young grass carp, which may be explained by the similar function of CAT and GPx. It was
527
reported that GPx and CAT both participated in the reduction of H2O2 in fish [77, 78]. In our study, dietary
528
phosphorus enhanced the GPx activities in the head kidney and spleen, where the GPx activity in the spleen
529
was higher than that in head kidney. Hence, the stable CAT activity in the spleen (not head kidney) as a
530
result of phosphorus deficiency might due to a higher GPx activity in the spleen than that in the head kidney
531
of fish.
AC C
EP
TE D
525
532
In addition, the antioxidant enzyme transcription factor Nrf2 is released by the repression of Keap1 in
533
zebrafish (Danio rerio) [79]. In the current study, compared with the optimal phosphorus level, phosphorus
534
deficiency down-regulated Nrf2 mRNA levels and up-regulated Keap1a mRNA levels in the head kidney
535
and spleen of young grass carp. Correlation analysis showed that the mRNA levels of CuZnSOD, MnSOD,
536
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTO1 and GSTP1 in the head kidney and spleen were
537
positively correlated with the Nrf2 mRNA levels which were negatively related to the mRNA levels of
538
Keap1a (Table 6). These results indicated that phosphorus deficiency down-regulated the antioxidant
539
enzyme mRNA levels partly by inhibiting Nrf2 gene transcription, which was regulated by Keap1a in the
540
head kidney and spleen of fish. Interestingly, phosphorus deficiency had no effect on the Keap1b mRNA
541
levels in the head kidney and spleen of young grass carp.
ACCEPTED MANUSCRIPT
Phosphorus deficiency up-regulated the mRNA levels of Keap1a (rather than Keap1b) in the head
543
kidney and spleen of young grass carp, which may be concerned with choline deficiency. A study from our
544
lab found that choline deficiency up-regulated Keap1a but had no impact on the Keap1b mRNA levels in the
545
gills of grass carp [80]. In our study, phosphorus deficiency decreased the choline content in the head kidney
546
and spleen of young grass carp, supporting our hypothesis.
RI PT
542
In summary, the results of current study first indicated that phosphorus deficiency induced oxidative
548
damage and impaired cellular integrity, which might be partly modulated by Keap1a (rather than Keap1b)
549
/Nrf2 pathway, leading to down-regulated mRNA levels of the antioxidant enzyme CuZnSOD, MnSOD,
550
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTO1 and GSTP1, and weaken the antioxidant enzyme
551
CuZnSOD, MnSOD, CAT (except spleen), GPx and GST activities in the head kidney and spleen of fish.
552
Moreover, a study showed that oxidative damage could induce cell apoptosis in SH-SY5Y cells, which was
553
partly regulated by p38 MAPK [27]. Hence, we further examined the effects of phosphorus on apoptosis in
554
the head kidney and spleen of young grass carp.
555
4.3.2. Phosphorus deficiency induced apoptosis partly relating to p38 MAPK signalling in the head
556
kidney and spleen of fish
TE D
M AN U
SC
547
Caspases, which are closely associated with apoptosis, based on their function, are divided into two
558
categories, the initiator caspases (including caspase-2, -8, -9) and the effector caspases (including caspase-3,
559
-7) [81]. Two major apoptosis pathways, the death receptor pathway (FasL/caspase-8) and the mitochondria
560
pathway [(Bcl-2, Mcl-1 and Bax)/Apaf-1/caspase-9], have been defined in mammalian cells [82]. The IAP
561
inhibits caspase in human cells [83]. In the present study, compared with the optimal phosphorus level,
562
phosphorus deficiency up-regulated caspase-2, -3, -7, -8 and -9, pro-apoptotic FasL, Apaf-1 and Bax mRNA
563
levels, and down-regulated anti-apoptotic Bcl-2, Mcl-1 and IAP mRNA levels in the head kidney and spleen
564
of young grass carp. Correlation analysis showed that the mRNA levels of FasL were positively correlated
565
with caspase-8; and caspase-9 mRNA levels were positively correlated with pro-apoptotic Apaf-1 and Bax
566
mRNA levels, while were negatively correlated with the anti-apoptotic Bcl-2, Mcl-1 and IAP mRNA levels;
567
and caspase-3, -7 were positively correlated with caspase-9 in the head kidney and spleen (Table 6),
568
suggesting that phosphorus deficiency induces apoptosis in the head kidney and spleen of fish. In addition,
AC C
EP
557
569
p38 MAPK and JNK mediates apoptosis in human hepatoma cells [84]. In the present study, compared with
570
the optimal phosphorus level, phosphorus deficiency up-regulated p38 MAPK mRNA levels in the head
571
kidney and spleen of young grass carp. Correlation analysis showed that p38 MAPK mRNA levels were
572
positively correlated with pro-apoptotic FasL, Apaf-1 and Bax, while they were negatively correlated with
573
the anti-apoptotic Bcl-2 and Mcl-1 mRNA levels in the head kidney and spleen of young grass carp (Table
574
6), suggesting that phosphorus deficiency-induced apoptosis is partly associated with p38 MAPK in the head
575
kidney and spleen of fish. Interestingly, phosphorus had no effect on the JNK expression in the head kidney
576
and spleen of young grass carp. This phenomenon might be partly correlated with the unchanged IKKα.
577
Yuan et al. reported that IKKα inhibited the activation of JNK in human epithelial cells [85]. Our study
578
showed that phosphorus deficiency had no impact on IKKα expression in the head kidney and spleen,
579
supporting our hypothesis.
SC
RI PT
ACCEPTED MANUSCRIPT
In summary, the above findings first implied that phosphorus deficiency induced apoptosis and
581
impaired cellular integrity, which was partly associated with p38 MAPK (rather than JNK), leading to the
582
activation of the death receptor pathway (FasL/caspase-8) and the mitochondria pathway [(Bcl-2, Mcl-1 and
583
Bax)/Apaf-1/caspase-9] in the head kidney and spleen of fish. In addition, the structural integrity of the
584
organs is also generally correlated with the intercellular tight junctions in epithelial cells [86]. Hence, we
585
further examined the effects of phosphorus on TJs in the head kidney and spleen of young grass carp.
586
4.3.3. Phosphorus deficiency weakened intercellular integrity partly relating to MLCK signalling in the
587
head kidney and spleen of fish
EP
TE D
M AN U
580
TJs (like occludin, ZO-1 and claudins) are responsible for intercellular integrity in mouse kidneys [87],
589
which includes barrier-forming and pore-forming TJs [88]. Ma et al. reported that TJ barrier function was
590
prevented by MLCK in Caco-2 cells [89]. Thus, we first investigated the effects of phosphorus on TJs in the
591
head kidney and spleen of fish and presented that phosphorus deficiency down-regulated the mRNA levels
592
of occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -7b and -15a and up-regulated the mRNA levels of the
593
pore-forming TJ claudin-12 and MLCK. Correlation analysis displayed that the mRNA levels of these TJs
594
were negatively (claudin-12 was positively) related to the MLCK mRNA levels in the head kidney and
595
spleen of young grass carp (Table 6), suggesting that phosphorus deficiency might up-regulate MLCK
596
mRNA levels to damage tight junction function and weaken the intercellular integrity in the head kidney and
597
spleen of fish. Interestingly, in this study, phosphorus deficiency had no impact on the mRNA levels of
AC C
588
598
claudin-b, -3c and -15b in the head kidney and spleen of young grass carp.
599
First, phosphorus deficiency had no effect on the mRNA levels of claudin-b and -3c in the head kidney
600
and spleen of young grass carp, which may be all correlated with cortisol influenced by IL-6. In humans,
601
IL-6 increases plasma cortisol levels [90]. Cortisol had no effect on the mRNA levels of claudin-b in the
602
goldfish (Carassius auratus) [91] and claudin-3c in puffer [92] gill epithelia cells. In this study, phosphorus
603
deficiency up-regulated the expression of IL-6 in the head kidney and spleen of young grass carp. Therefore,
604
we presume that phosphorus deficiency up-regulated IL-6 expression leading to the rise of cortisol levels,
605
while cortisol might have no impact on claudin-b and -3c in the head kidney and spleen of fish. However,
606
further investigation should be conducted to support this supposition.
RI PT
ACCEPTED MANUSCRIPT
In addition, phosphorus deficiency down-regulated claudin-15a mRNA levels but had no impact on
608
claudin-15b in the head kidney and spleen of young grass carp. This interesting phenomenon may be related
609
to the same function of type-IIb sodium-phosphate cotransporter (NaPi-IIb) and claudin-15a. In fish,
610
NaPi-IIb is a phosphorus transporter that mediates the transport of Na+ [93]. It was reported that claudin-15a
611
(rather than claudin-15b) plays an important role in Na+ transport in Japanese medaka (Oryziaslatipes) [94].
612
In the present study, phosphorus deficiency up-regulated NaPi-IIb mRNA levels in the head kidney and
613
spleen. According to these data, phosphorus deficiency up-regulated NaPi-II mRNA levels to enhance Na+
614
transport, which might weaken claudin-15a function leading to the down-regulation of claudin-15a (rather
615
than claudin-15b) in the head kidney and spleen of fish.
TE D
M AN U
SC
607
In summary, the above data indicated for the first time that phosphorus deficiency damaged tight
617
junction function and weakened intercellular integrity partly correlating with MLCK to down-regulate the
618
mRNA levels of the TJs (except claudin-b, -3c and -15b) occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -7b and
619
-15a and up-regulate the mRNA levels of pore-forming TJ claudin-12 in the head kidney and spleen of fish.
620
The underlying mechanism requires further investigation.
621
4.4. Phosphorus deficiency decreased the immune function and attenuated the structural integrity in the
622
skin of fish compared with the head kidney and spleen
AC C
EP
616
623
The skin is the structure that covers the body and protects it not only from the entry of pathogens or
624
allergens but also from the leakage of water, solutes, or nutrients, which are related to the immune function
625
and structural integrity of fish skin [4]. Grass carp infected with A. hydrophila might have a skin
626
hemorrhage and lesions [45]. In this study, compared with the optimal phosphorus level, phosphorus
627
deficiency increased the skin hemorrhage and lesions morbidity of young grass carp after infection with A.
628
hydrophila (Figs 1&2), suggesting that phosphorus deficiency decreases the defence against A. hydrophila
629
in the skin of fish. Based on the broken-line analysis of the ability to combat skin hemorrhage and lesions,
630
the dietary available phosphorus requirement of young grass carp was estimated to be 4.13 g/kg diet.
ACCEPTED MANUSCRIPT
In the skin of young grass carp, except for 4E-BP1 and S6K1, the effects of phosphorus on antibacterial
632
substances, cytokines, antioxidant, apoptosis and TJs related parameters models were similar to those in the
633
head kidney and spleen. Interestingly, phosphorus deficiency had no impact on the mRNA levels of 4E-BP1
634
and S6K1 in the skin. The mRNA level of 4E-BP1 unaffected by phosphorus deficiency in the skin may be
635
correlated with insulin. A study reported that insulin had no effect on the 4E-BP1 levels in the mouse skin
636
JB6 Cl 41 cells [95]. In humans, a low level of phosphorus, causing hypophosphatemia, increases the serum
637
insulin level [96]. Hence, in the condition of phosphorus deficiency, the unaffected mRNA levels of 4E-BP1
638
might due to insulin, which might have no effect on 4E-BP1 mRNA level in the skin of fish. In addition, the
639
stable mRNA level of S6K1 by phosphorus deficiency in the skin may be associated with Akt1. A study
640
reported that the knockout of Akt1, which is predominantly expressed in the skin, had no effect on S6K1
641
levels in mice embryo fibroblasts [97]. Meanwhile, phosphorus deficiency depressed Akt1 levels in NHBE
642
cells [20]. Hence, the stable mRNA level of S6K1 by phosphorus deficiency may be related to Akt1 which
643
was highly expressed in the skin and might have no effect on S6K1 levels in the skin of fish. However, these
644
further reasons need deeper investigation.
645
4.5. Interesting results: high levels of phosphorus impaired some indices that differed from the traditional
646
growth results
EP
TE D
M AN U
SC
RI PT
631
Based on the function of the kidney (metanephros in fish) in the excretion of phosphorus, no negative
648
effects on the growth performances were observed in this study and the previous studies in juvenile grass
649
carp and juvenile Jian carp when high doses were administered [8, 9]. Interestingly, in the present study,
650
compared with the optimal phosphorus level, high levels of phosphorus increased the ROS contents in the
651
head kidney and spleen, and down-regulated the mRNA levels of GPx4b and GSTP1 in the head kidney,
652
anti-inflammatory cytokine TGF-β1 in the spleen, and up-regulated the mRNA levels of caspase-8 in the
653
head kidney, the pro-inflammatory cytokine IL-15 in the head kidney and spleen after infection with A.
654
hydrophila. First, high levels of phosphorus increased the ROS contents in the head kidney and spleen,
655
which may be partly related to NADPH oxidase. A study displayed that NADPH oxidase induced ROS
AC C
647
656
production in BAECs [98]. High levels of phosphorus increased NADPH oxidase activity in bovine aortic
657
endothelial cells [99]. Second, high levels of phosphorus down-regulated the mRNA levels of GPx4b and
658
GSTP1 and up-regulated IL-15 and caspase-8, which may be partly due to the rise of ROS contents. It was
659
reported that ROS down-regulated the mRNA levels of GPx4b and GSTP1 in grass carp [10] and increased
660
IL-15 levels in human mononuclear cells [100] and caspase-8 levels in DU-145 cells [101]. As mentioned
661
above, our study displayed that a high level of phosphorus increased the ROS contents in the head kidney
662
and spleen. Furthermore, high levels of phosphorus down-regulated TGF-β1 in the spleen partly by IL-15. A
663
study displayed that IL-15 inhibits TGF-β1 expression in human T lymphocytes [102]. Meanwhile, in this
664
study, a high level of phosphorus up-regulated IL-15 expression in the spleen of young grass carp. However,
665
the detail reasons need a deeper investigation.
666
4.6. Comparisons of the optimal dietary available phosphorus levels for young grass carp based on the
667
different indices
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
In this study, after infection with A. hydrophila, phosphorus deficiency increased the skin hemorrhage
669
and lesions morbidity and impaired the immune function and structural integrity in the immune organs of
670
fish. Thus, an evaluation of phosphorus requirement is quite necessary in fish. As shown in Table 7, based
671
on the growth performance (PWG and FI), the available phosphorus requirements for young grass carp were
672
estimated to be 4.10 and 3.89 g/kg diet, respectively. Meanwhile, the available phosphorus requirement for
673
ability to combat skin hemorrhage and lesions was estimated to be 4.13 g/kg diet, suggesting that the
674
available phosphorus requirement for ability to combat skin hemorrhage and lesions was close to that based
675
on the growth performance. Our further investigation on fish immune function and structural integrity
676
indicated that the available phosphorus requirements for young grass carp (254.56-898.23 g), according to
677
the immune indices (head kidney LZ activity and spleen C4 content) and the antioxidant indices (head
678
kidney and spleen MDA contents), were estimated to be 5.71, 5.57, 5.54 and 5.40 g/kg diet, respectively.
679
The results implied that the available phosphorus requirements for young grass carp based on the immune
680
and antioxidant indices were higher than those based on growth performance, indicating that more
681
phosphorus is required for enhancing immune function and improving structural integrity in the immune
682
organs of fish. In addition, the available phosphorus requirements for young grass carp based on immune
683
and antioxidant indices higher than that based on the ability to combat skin hemorrhage and lesions, which
684
may be associated with the function of VD. In humans, the skin is an important place for VD to synthesize
AC C
EP
TE D
668
685
which could increase serum phosphorus concentrations [103], indicating that no more phosphorus is
686
required for skin of fish.
687 688
ACCEPTED MANUSCRIPT
(Table 7 inserted here) 5. Conclusions In summary (Fig. 10), on the basis of our previous study about phosphorus on the growth performance
690
of young grass carp, the present study first demonstrated that phosphorus deficiency decreased the immune
691
function in the head kidney and spleen of fish indicated by: (1) depressed LZ and ACP activities, C3, C4 and
692
IgM contents, and antimicrobial peptides LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 mRNA levels; (2)
693
and enhanced IKKβ, IKKγ (rather than IKKα)/IκBα/NF-κB signalling pathway to up-regulate the mRNA
694
levels of the pro-inflammatory cytokines (except IL-8 and IL-12p40); (3) and repressed the
695
TOR/(S6K1,4E-BP) signalling pathway to down-regulate the mRNA levels of the anti-inflammatory
696
cytokines (except IL-4/13B). Simultaneously, phosphorus deficiency attenuated the structural integrity in the
697
head kidney and spleen of fish displayed by the following aspects: (1) a modulated Keap1a (rather than
698
Keap1b)/Nrf2 signalling pathway to reduce the antioxidant enzyme gene expressions and activities (but
699
elevate GR, and except CAT activity in the spleen), and thus induced oxidative damage and impaired
700
cellular integrity; (2) up-regulated p38 MAPK mRNA levels to activate the death receptor pathway and the
701
mitochondria pathway, which induced apoptosis and impaired cellular integrity; (3) promoted MLCK
702
expression to down-regulate TJs gene expressions (not including claudin-b, -3c and -15b, and up-regulate
703
claudin-12), which damaged TJs function and weakened intercellular integrity. Meanwhile, phosphorus
704
deficiency increased skin hemorrhage and lesions morbidity, and impaired immune function and structural
705
integrity in the skin of young grass carp. Lastly, based on the PWG and the ability to combat skin
706
hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp (254.56-898.23
707
g) were estimated to be 4.10 and 4.13 g/kg diet, respectively.
708 709
AC C
EP
TE D
M AN U
SC
RI PT
689
(Fig. 10 inserted here) Acknowledgements
710
This research was financially supported by the National Basic Research Program of China (973
711
Program) (2014CB138600), National Department Public Benefit Research Foundation (Agriculture) of
712
China (201003020), Outstanding Talents and Innovative Team of Agricultural Scientific Research (Ministry
713
of Agriculture), Science and Technology Support Program of Sichuan Province of China (2014NZ0003),
714
Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China
715
(2012NC0007; 2013NC0045), The Demonstration of Major Scientific and Technological Achievement
716
Transformation Project of Sichuan Province of China (2015CC0011) and Natural Science Foundation for
717
Young Scientists of Sichuan Province (2014JQ0007). The authors would like to thank the personnel of these
718
teams for their kind assistance.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
719
720
References
721
[1] M.G. Bondad-Reantaso, R.P. Subasinghe, J.R. Arthur, K. Ogawa, S. Chinabut, R. Adlard, et al., Disease and health
722
MANUSCRIPT management in Asian aquaculture ☆, Vet.ACCEPTED Parasitol. 132 (2005) 249-272.
723
[2] P. Li, Y.-L. Yin, D. Li, S.W. Kim, G. Wu, Amino acids and immune function, Brit. J. Nutr 98 (2007) 237-252.
724
[3] C. Uribe, H. Folch, R. Enriquez, G. Moran, Innate and adaptive immunity in teleost fish: a review, Vet. Med-Czech 56
725
(2011) 486-503.
726
[4] M.Á. Esteban, An overview of the immunological defenses in fish skin, Isrn Immunol. 2012 (2012).
727
[5] G. Baeverfjord, T. Asgard, K. Shearer, Development and detection of phosphorus deficiency in Atlantic salmon, Salmo
729 730 731 732
salar L., parr and post-smolts, Aquac. Nutr. 4 (1998) 1-12. [6] P.G. Fjelldal, T. Hansen, S. Albrektsen, Inadequate phosphorus nutrition in juvenile Atlantic salmon has a negative effect on long-term bone health, Aquaculture 334 (2012) 117-123.
RI PT
728
[7] P.K. Roy, S.P. Lall, Dietary phosphorus requirement of juvenile haddock (Melanogrammus aeglefinus L.), Aquaculture 221 (2003) 451-468.
[8] N.B. Xie, L. Feng, Y. Liu, J. Jiang, W.D. Jiang, K. Hu, et al., Growth, body composition, intestinal enzyme activities
734
and microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary phosphorus, Aquac. Nutr. 17
735
(2011) 645-656.
737
[9] J.J. Liang, Y.J. Liu, L.X. Tian, H.J. Yang, G.Y. Liang, Dietary available phosphorus requirement of juvenile grass carp
M AN U
736
SC
733
(Ctenopharyngodon idella), Aquac. Nutr. 18 (2012) 181-188.
738
[10] P.J. Ni, W.D. Jiang, W. Pei, L. Yang, S.Y. Kuang, T. Ling, et al., Dietary low or excess levels of lipids reduced growth
739
performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp
740
(Ctenopharyngodon idella) under the infection of Aeromonas hydrophila, Fish Shellfish Immunol. 55 (2016) 28–47. [11] F.-Y. Pan, L. Feng, W.-D. Jiang, J. Jiang, P. Wu, S.-Y. Kuang, et al., Methionine hydroxy analogue enhanced fish
742
immunity via modulation of NF-κB, TOR, MLCK, MAPKs and Nrf2 signaling in young grass carp (Ctenopharyngodon
743
idella), Fish Shellfish Immunol. 56 (2016) 208-228.
745 746 747
[12] S.D. Yang, T.S. Lin, F.G. Liu, C.H. Liou, Influence of dietary phosphorus levels on growth, metabolic response and body composition of juvenile silver perch (Bidyanus bidyanus), Aquaculture 253 (2006) 592-601. [13] L. Tort, J. Balasch, S. Mackenzie, Fish immune system. A crossroads between innate and adaptive responses, Inmunología 22 (2003) 277-286.
EP
744
TE D
741
[14] V.H. Olavarría, M.P. Sepulcre, J.E. Figueroa, V. Mulero, Prolactin-induced production of reactive oxygen species and
749
IL-1β in leukocytes from the bony fish gilthead seabream involves Jak/Stat and NF-κB signaling pathways, J. Immunol.
750
185 (2010) 3873-3883.
AC C
748
751
[15] H. Wen, L. Feng, W. Jiang, Y. Liu, J. Jiang, S. Li, et al., Dietary tryptophan modulates intestinal immune response,
752
barrier function, antioxidant status and gene expression of TOR and Nrf2 in young grass carp (Ctenopharyngodon idella),
753
Fish Shellfish Immunol. 40 (2014) 275-287.
754 755 756 757 758 759 760 761 762 763
[16] J.C. Eya, R. Lovell, Effects of dietary phosphorus on resistance of channel catfish to Edwardsiella ictaluri challenge, J. Aquat. Anim. Health 10 (1998) 28-34. [17] E. Jokinen, J. Vielma, T. Aaltonen, J. Koskela, The effect of dietary phosphorus deficiency on the immune responses of European whitefish (Coregonus lavaretus L.), Fish Shellfish Immunol. 15 (2003) 159-168. [18] T. Sarasa, Y. Hironori, N. Otoki, K. Tomohiro, I. Mariko, M. Masashi, et al., Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice, Nutr. Res. 33 (2013) 586-593. [19] A. Mishra, A. Chaudhary, S. Sethi, Oxidized omega-3 fatty acids inhibit NF-kappaB activation via a PPARalpha-dependent pathway, Arterio. Thromb. Vasc. Biol. 24 (2004) 1621-1627. [20] S.-H. Chang, K.N. Yu, Y.-S. Lee, G.-H. An, G.R. Beck Jr, N.H. Colburn, et al., Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells, Am. J. Resp. Cell Mol. 35 (2006) 528-539.
764 765
[21] S. Bodine, T. Stitt, M. Gonzalez, W. Kline, G. Stover, R. Bauerlein, et al., Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo, Nat. Cell Biol. 3 (2001) 1014-1019.
766
[22] S.-Y. Kuang, W.-W. Xiao, L. Feng, Y.ACCEPTED Liu, J. Jiang, W.-D. Jiang, et al., Effects of graded levels of dietary methionine MANUSCRIPT
767
hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio
768
var. Jian), Fish Shellfish Immunol. 32 (2012) 629-636.
769
[23] H.-J. Xu, W.-D. Jiang, L. Feng, Y. Liu, P. Wu, J. Jiang, et al., Dietary vitamin C deficiency depresses the growth, head
770
kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in
771
young grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 52 (2016) 111-138.
773
[24] A. Sarmento, L. Guilhermino, A. Afonso, Mercury chloride effects on the function and cellular integrity of sea bass (Dicentrarchus labrax) head kidney macrophages, Fish Shellfish Immunol. 17 (2004) 489-498.
RI PT
772
774
[25] O.J. Baker, J.M. Camden, R.S. Redman, J.E. Jones, C.I. Seye, L. Erb, et al., Proinflammatory cytokines tumor necrosis
775
factor-α and interferon-γ alter tight junction structure and function in the rat parotid gland Par-C10 cell line, Am. J.
776
Physiol-Cell Ph. 295 (2008) C1191-C1201.
778 779 780
[26] S. Homma, Y. Ishii, Y. Morishima, T. Yamadori, Y. Matsuno, N. Haraguchi, et al., Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer, Clin. Cancer. Res. 15 (2009) 3423-3432.
SC
777
[27] Y.-W. Ki, J.H. Park, J.E. Lee, I.C. Shin, H.C. Koh, JNK and p38 MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis, Toxicol. Lett. 218 (2013) 235-245.
[28] T.Y. Ma, M.A. Boivin, D. Ye, A. Pedram, H.M. Said, Mechanism of TNF-{alpha} modulation of Caco-2 intestinal
782
epithelial tight junction barrier: role of myosin light-chain kinase protein expression, Am. J. Physiol-Gastr. L 288 (2005)
783
95-96.
785 786 787 788 789 790 791
[29] A. Peng, T. Wu, C. Zeng, D. Rakheja, J. Zhu, T. Ye, et al., Adverse effects of simulated hyper- and hypo-phosphatemia on endothelial cell function and viability, PloS One 6 (2011) 442-442.
[30] H.C. Um, J.H. Jang, D.H. Kim, L. Chan, Y.J. Surh, Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in PC12 cells, Nitric Oxide 25 (2011) 161-168.
TE D
784
M AN U
781
[31] M. Glinn, B. Ni, R.P. Irwin, S. Kelley, S.Z. Lin, S.M. Paul, Inorganic Pi increases neuronal survival in the acute early phase following excitotoxic/oxidative insults, J. Neurochem. 70 (1998) 1850-1858. [32] H.K. Sang, R.P. Sharma, Mercury-induced apoptosis and necrosis in murine macrophages: role of calcium-induced reactive oxygen species and p38 mitogen-activated protein kinase signaling, Toxicol. Appl. Pharm. 196 (2004) 47-57. [33] M.A. Pasek, Rethinking early Earth phosphorus geochemistry, P. Natl. Acad. Sci. 105 (2008) 853-858.
793
[34] L. Feng, Y.-P. Chen, W.-D. Jiang, Y. Liu, J. Jiang, P. Wu, et al., Modulation of immune response, physical barrier and
794
related signaling factors in the gills of juvenile grass carp (Ctenopharyngodon idella) fed supplemented diet with
795
phospholipids, Fish Shellfish Immunol. 48 (2016) 79-93.
797
AC C
796
EP
792
[35] Y. Wang, Y. Lu, Y. Zhang, Z. Ning, Y. Li, Q. Zhao, et al., The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation, Nat. Genet. 47 (2015) 625-631.
798
[36] J. Wen, W. Jiang, L. Feng, S. Kuang, J. Jiang, L. Tang, et al., The influence of graded levels of available phosphorus on
799
growth performance, muscle antioxidant and flesh quality of young grass carp (Ctenopharyngodon idella), Anim. Nutr. 1
800
(2015) 77-84.
801
[37] W.-D. Jiang, K. Hu, Y. Liu, J. Jiang, P. Wu, J. Zhao, et al., Dietary myo-inositol modulates immunity through
802
antioxidant activity and the Nrf2 and E2F4/cyclin signalling factors in the head kidney and spleen following infection of
803
juvenile fish with Aeromonas hydrophila, Fish Shellfish Immunol. 49 (2016) 374-386.
804
[38] Y.P. Wu, L. Feng, W.D. Jiang, Y. Liu, J. Jiang, S.H. Li, et al., Influence of dietary zinc on muscle composition, flesh
805
quality and muscle antioxidant status of young grass carp (Ctenopharyngodon idella Val.), Aquac. Res. 46 (2015)
806
2360-2373.
807 808
[39] Z.-Y. Du, Y.-J. Liu, L.-X. Tian, J.-G. He, J.-M. Cao, G.-Y. Liang, The influence of feeding rate on growth, feed efficiency and body composition of juvenile grass carp (Ctenopharyngodon idella), Aquacult. Int. 14 (2006) 247-257.
809
[40] Q. Tang, L. Feng, W. Jiang, Y. Liu, J. Jiang, S. Li, et al., Effects of dietary copper on growth, digestive, and brush
810
border enzyme activities and antioxidant defense of hepatopancreas and intestine for young grass carp
811
(Ctenopharyngodon idella), Biol. Trace Elem. Res. 155 (2013) 370-380. ACCEPTED MANUSCRIPT
812 813 814 815 816 817
[41] K. Mai, C. Zhang, Q. Ai, Q. Duan, W. Xu, L. Zhang, et al., Dietary phosphorus requirement of large yellow croaker, Pseudosciaena crocea R ☆, Aquaculture 251 (2006) 346-353. [42] E. Austreng, Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract, Aquaculture 13 (1978) 265-272. [43] C. Zhang, K. Mai, Q. Ai, W. Zhang, Q. Duan, B. Tan, et al., Dietary phosphorus requirement of juvenile Japanese seabass, Lateolabrax japonicus, Aquaculture 255 (2006) 201-209. [44] B. Liu, L. Xu, X. Ge, J. Xie, P. Xu, Q. Zhou, et al., Effects of mannan oligosaccharide on the physiological responses,
819
HSP70 gene expression and disease resistance of Allogynogenetic crucian carp (Carassius auratus gibelio) under
820
Aeromonas hydrophila infection, Fish Shellfish Immunol. 34 (2013) 1395-1403.
821 822
RI PT
818
[45] X. Song, J. Zhao, Y. Bo, Z. Liu, K. Wu, C. Gong, Aeromonas hydrophila induces intestinal inflammation in grass carp (Ctenopharyngodon idella): An experimental model, Aquaculture 434 (2014) 171-178.
[46] C.M.A. Caipang, C.C. Lazado, M.F. Brinchmann, J.H. Rombout, V. Kiron, Differential expression of immune and
824
stress genes in the skin of Atlantic cod (Gadus morhua), Comp. Biochem. Physiol. D: Genom. Proteom. 6 (2011)
825
158-162.
SC
823
[47] A. International. Official methods of analysis of AOAC International: AOAC International; 2005.
827
[48] P. Wu, W.D. Jiang, Y. Liu, G.F. Chen, J. Jiang, S.H. Li, et al., Effect of choline on antioxidant defenses and gene
828
expressions of Nrf2 signaling molecule in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian),
829
Fish Shellfish Immunol. 38 (2014) 374-382.
830 831
M AN U
826
[49] Q. Shao, J. Ma, Z. Xu, W. Hu, J. Xu, S. Xie, Dietary phosphorus requirement of juvenile black seabream, Sparus macrocephalus, Aquaculture 277 (2008) 92-100.
[50] J. Zhao, Y. Liu, J. Jiang, P. Wu, W. Jiang, S. Li, et al., Effects of dietary isoleucine on the immune response, antioxidant
833
status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian), Fish Shellfish Immunol.
834
35 (2013) 572-580.
TE D
832
[51] Q. Yang, Y. Rui, L. Ming, Q. Zhou, X. Liang, Z.C. Elmada, Effects of dietary fucoidan on the blood constituents,
836
anti-oxidation and innate immunity of juvenile yellow catfish (Pelteobagrus fulvidraco), Fish Shellfish Immunol. 41
837
(2014) 264-270.
839 840 841 842 843 844 845
[52] H. Ji, L. Jie, P. Liu, Regulation of growth performance and lipid metabolism by dietary n-3 highly unsaturated fatty acids in juvenile grass carp, Ctenopharyngodon idellus, Comp. Biochem. Physiol. 159 (2011) 49–56.
AC C
838
EP
835
[53] X. Lu, C. Wang, B. Liu, The role of Cu/Zn-SOD and Mn-SOD in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix, Fish Shellfish Immunol. 42 (2015) 58-65. [54] X.D. Zhang, Y.F. Zhu, L.S. Cai, T.X. Wu, Effects of fasting on the meat quality and antioxidant defenses of market-size farmed large yellow croaker (Pseudosciaena crocea), Aquaculture 280 (2008) 136-139. [55] Z. Zhang, R.P. Wilson, A modified enzymatic assay for quantifying choline in fish tissue and common feed ingredients, J. Agr. Food Chem. 46 (1998) 3673-3676.
846
[56] J.B. Luo, L. Feng, W.D. Jiang, Y. Liu, P. Wu, J. Jiang, et al., The impaired intestinal mucosal immune system by valine
847
deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating
848
tight junction proteins transcript abundance in the intestine, Fish Shellfish Immunol. 40 (2014) 197-207.
849 850
[57] K.J. Livak, T.D. Schmittgen, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 −∆∆ C T Method, Methods 25 (2001) 402-408.
851
[58] NRC. Nutrient requirements of fish and shrimp: National academies press; 2011.
852
[59] B.C. Peterson, E. Peatman, D.D. Ourth, G.C. Waldbieser, Effects of a phytogenic feed additive on growth performance,
853
susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin, Fish Shellfish Immunol.
854
44 (2015) 21-25.
855
[60] B. Magnadóttir, Innate immunity of fish (overview), Fish Shellfish Immunol. 20 (2006) 137-151.
856
[61] Y. Liu, R.C.-M. Ho, A. Mak, Interleukin (IL)-6, tumour necrosis factor alpha (TNF-α) and soluble interleukin-2 ACCEPTED MANUSCRIPT
857
receptors (sIL-2R) are elevated in patients with major depressive disorder: a meta-analysis and meta-regression, J. Affect.
858
Disorders 139 (2012) 230-239.
859
[62] R. Wei, G.M. Jonakait, Neurotrophins and the anti-inflammatory agents interleukin-4 (IL-4), IL-10, IL-11 and
860
transforming growth factor-β1 (TGF-β1) down-regulate T cell costimulatory molecules B7 and CD40 on cultured rat
861
microglia, J. Neuroimmunol. 95 (1999) 8-18.
863
[63] B.M.L.V.V. Kemenade, E.H. Stolte, J.R. Metz, M. Chadzinska, Chapter 7 Neuroendocrine–Immune Interactions in Teleost Fish, Fish Physiol. 28 (2009) 313-364.
RI PT
862
864
[64] X. Huang, Y. Huang, Z. Ouyang, J. Cai, Y. Yan, Q. Qin, Roles of stress-activated protein kinases in the replication of
865
Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells, J. Gen. Virol. 92 (2011)
866
1292-1301.
869 870 871 872 873 874
23 p19 and p40 in lesional skin of patients with psoriasis vulgaris, J. Exp. Med. 199 (2004) 125-130.
SC
868
[65] E. Lee, W.L. Trepicchio, J.L. Oestreicher, D. Pittman, F. Wang, F. Chamian, et al., Increased expression of interleukin
[66] M. Ohtani, N. Hayashi, K. Hashimoto, T. Nakanishi, J.M. Dijkstra, Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish, Immunogenetics 60 (2008) 383-397.
[67] O. Jaillon, J.M. Aury, J.L. Petit, N. Stangethomann, E. Mauceli, L. Bouneau, et al., Genome duplication in the teleost
M AN U
867
fish : Tetraodon nigroviridis: reveals the early vertebrate proto-karyotype : Article : Nature, Nat. (2004). [68] J.H. Li, J.Z. Shao, L.X. Xiang, Y. Wen, Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: The first evidence of Th2-type cytokine in fish, Mol. Cell. Biol. 44 (2007) 2078-2086. [69] Y.D. Min, C.H. Choi, H. Bark, H.Y. Son, H.H. Park, S. Lee, et al., Quercetin inhibits expression of inflammatory
876
cytokines through attenuation of NF-κB and p38 MAPK in HMC-1 human mast cell line, Inflamm. Res. 56 (2007)
877
210-215.
878 879 880 881
TE D
875
[70] P. Viatour, M.P. Merville, V. Bours, A. Chariot, Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation, Trends Biochem. Sci. 30 (2005) 43-52.
[71] S. Tegethoff, J. Behlke, C. Scheidereit, Tetrameric oligomerization of IκB kinase γ (IKKγ) is obligatory for IKK complex activity and NF-κB activation, Mol. Cell. Biol. 23 (2003) 2029-2041. [72] Y.-P. Chen, W.-D. Jiang, Y. Liu, J. Jiang, P. Wu, J. Zhao, et al., Exogenous phospholipids supplementation improves
883
growth and modulates immune response and physical barrier referring to NF-κB, TOR, MLCK and Nrf2 signaling factors
884
in the intestine of juvenile grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 47 (2015) 46-62.
886
AC C
885
EP
882
[73] T. Weichhart, G. Costantino, M. Poglitsch, M. Rosner, M. Zeyda, K.M. Stuhlmeier, et al., The TSC-mTOR signaling pathway regulates the innate inflammatory response, Immunity 29 (2008) 565-577.
887
[74] N. Hay, N. Sonenberg, Upstream and downstream of mTOR, Genes. Dev. 18 (2004) 1926-1945.
888
[75] J. Deng, B. Kang, L. Tao, H. Rong, X. Zhang, Effects of dietary cholesterol on antioxidant capacity, non-specific
889
immune response, and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean
890
meal-based diets, Fish Shellfish Immunol. 34 (2013) 324-331.
891
[76] S.C. Lu, Glutathione synthesis, Biochim. Biophys. Acta-Gen. Subjects 1830 (2013) 3143-3153.
892
[77] M. Li, W. Wu, P. Zhou, F. Xie, Q. Zhou, K. Mai, et al., Comparison effect of dietary astaxanthin and Haematococcus
893
pluvialis on growth performance, antioxidant status and immune response of large yellow croaker Pseudosciaena crocea,
894
Aquaculture 434 (2014) 227–232.
895
[78] H. Elabd, H.P. Wang, A. Shaheen, H. Yao, A. Abbass, Feeding Glycyrrhiza glabra (liquorice) and Astragalus
896
membranaceus (AM) alters innate immune and physiological responses in yellow perch (Perca flavescens), Fish Shellfish
897
Immunol. 54 (2016) 374-384.
898
[79] K. Mukaigasa, L.T. Nguyen, L. Li, H. Nakajima, M. Yamamoto, M. Kobayashi, Genetic evidence of an evolutionarily
899
conserved role for Nrf2 in the protection against oxidative stress, Mol. Cell. Biol. 32 (2012) 4455-4461.
900
[80] H.F. Zhao, W.D. Jiang, Y. Liu, J. Jiang, P. Wu, S.Y. Kuang, et al., Dietary choline regulates antibacterial activity,
901
inflammatory response and barrier function in the gills of MANUSCRIPT grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. ACCEPTED
902
52 (2016) 139–150.
903
[81] M.G. Grütter, Caspases: key players in programmed cell death, Curr. Opin. Struct. Biol. 10 (2000) 649-655.
904
[82] Y. Xiao-Ming, Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and
905
mitochondria apoptosis pathways, Cell Res. 10 (2000) 161-167. [83] Q.L. Deveraux, J.C. Reed, IAP family proteins—suppressors of apoptosis, Genes. Dev. 13 (1999) 239-252.
907
[84] B.J. Kim, S.W. Ryu, B.J. Song, JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and
908
mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells, J. Biol. Chem. 281 (2006) 21256-21265.
909
[85] Z.Q. Yuan, R.I. Feldman, M. Sun, N.E. Olashaw, D. Coppola, G.E. Sussman, et al., Inhibition of JNK by cellular stress-
910
and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells,
911
J. Biol. Chem. 277 (2002) 29973-29982.
RI PT
906
[86] K. Shin, V.C. Fogg, B. Margolis, Tight junctions and cell polarity, Annu. Rev. Cell Dev. Bi. 22 (2006) 207-235.
913
[87] Y. Kiuchi-Saishin, S. Gotoh, M. Furuse, A. Takasuga, Y. Tano, S. Tsukita, Differential expression patterns of claudins,
914
SC
912
tight junction membrane proteins, in mouse nephron segments, J. Am. Soc. Nephrol. 13 (2002) 875-886. [88] C.M. Van Itallie, J.M. Anderson, Claudins and epithelial paracellular transport, Annu. Rev. Physiol. 68 (2006) 403-429.
916
[89] T.Y. Ma, D. Tran, N. Hoa, D. Nguyen, M. Merryfield, A. Tarnawski, Mechanism of extracellular calcium regulation of
917
intestinal epithelial tight junction permeability: Role of cytoskeletal involvement, Microsc. Res. Techniq. 51 (2000)
918
156-168.
920 921 922 923 924
[90] A. Steensberg, C.P. Fischer, C. Keller, K. Møller, B.K. Pedersen, IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans, Am. J. Physiol-Endoc 285 (2003) E433-437.
[91] H. Chasiotis, S.P. Kelly, Effect of cortisol on permeability and tight junction protein transcript abundance in primary cultured gill epithelia from stenohaline goldfish and euryhaline trout, Gen. Comp. Endocr. 172 (2011) 494-504.
TE D
919
M AN U
915
[92] P. Bui, B.L.M. Sp, Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia, Mol. Cell. Endocrino. 317 (2010) 120-126.
[93] J. Lake, C. Gravel, G.K.D. Koko, C. Robert, G.W. Vandenberg, Combining suppressive subtractive hybridization and
926
cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney,
927
Comp. Biochem. Physiol. D: Genom. Proteom. 5 (2010) 24-35.
EP
925
[94] M.C. Bossus, S.S. Madsen, C.K. Tipsmark, Functional dynamics of claudin expression in Japanese medaka (Oryzias
929
latipes): Response to environmental salinity, Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol. 187 (2015) 74-85.
930
[95] G. Liu, Y. Zhang, A.M. Bode, W.Y. Ma, Z. Dong, Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in
931 932 933
AC C
928
response to UVB irradiation, J. Biol. Chem. 277 (2002) 8810-8816. [96] F.J. Paula, A.E. Plens, M.C. Foss, Effects of hypophosphatemia on glucose tolerance and insulin secretion, Horm. Metab. Res. 30 (1998) 281-284.
934
[97] B.X. Peng, P.Z. Xu, M.L. Chen, A. Hahnwindgassen, J. Skeen, Dwarfism, impaired skin development, skeletal muscle
935
atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev 17, (2010).
936
[98] E. Takeda, Y. Taketani, K, M. Nomoto, E. Shuto, N. Sawada, H. Yamamoto, et al., A novel function of
937 938 939
phosphate-mediated intracellular signal transduction pathways, Adv. Enzyme. Regul. 46 (2006) 154-161. [99] E. Shuto, Y. Taketani, R. Tanaka, N. Harada, M. Isshiki, M. Sato, et al., Dietary phosphorus acutely impairs endothelial function, J. Am. Soc. Nephrol. 20 (2009) 1504-1512.
940
[100] A. Liu, J.L. Arbiser, A. Holmgren, G. Klein, E. Klein, PSK and Trx80 inhibit B-cell growth in EBV-infected cord
941
blood mononuclear cells through T cells activated by the monocyte products IL-15 and IL-12, Blood 105 (2005)
942
1606-1613.
943
[101] I. Perez-Cruz, J.M. Cárcamo, D.W. Golde, Caspase-8 dependent TRAIL-induced apoptosis in cancer cell lines is
944
inhibited by vitamin C and catalase, Apoptosis 12 (2007) 225-234.
945
[102] M. Benahmed, B. Meresse, B. Arnulf, U. Barbe, J.J. Mention, V. Verkarre, et al., Inhibition of TGF-β signaling by
946
IL-15: a new role for IL-15 in the loss ofACCEPTED immune homeostasis in celiac disease, Gastroenterology 132 (2007) 994-1008. MANUSCRIPT
947
[103] M.F. Holick, Holick MF.Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and
948
cardiovascular disease. Am J Clin Nutr 80:1678S-1688SS.
949
[104] Y.Y. Zeng, W.D. Jiang, Y. Liu, P. Wu, J. Zhao, J. Jiang, et al., Optimal dietary alpha-linolenic acid/linoleic acid ratio
950
improved digestive and absorptive capacities and target of rapamycin gene expression of juvenile grass carp
951
(Ctenopharyngodon idellus), Aquac. Nutr. (2015). [105] L. Wang, N. Shang, H. Feng, Q. Guo, H. Dai, Molecular cloning of grass carp (Ctenopharyngodon idellus) T-bet and
953
GATA-3, and their expression profiles with IFN-γ in response to grass carp reovirus (GCRV) infection, Fish Physiol.
954
Biochem. 39 (2013) 793-805.
RI PT
952
955
AC C
EP
TE D
M AN U
SC
956
Table 1 Composition and nutrients content of basal diet.
958
ACCEPTED MANUSCRIPT
Ingredients
g/kg
Nutrients content
40.00
Fish meal
Crude protein
Casein
146.00
Crude lipid
Gelatin
66.00
n-3 5
g/kg
4
307.10
4
55.10 10.40
5
153.80
n-6
α-starch
240.00
Total phosphorus 6
2.30
Corn starch
162.20
Available phosphorus 6
0.95
Fish oil
29.00
Soybean oil
9.30
Cellulose
50.00
Vitamin premix 1
10.00
Mineral premix 2
20.00
Monosodium phosphate mixture 3
60.00
Choline chloride (50%)
10.00
DL-Met (99%)
2.60
L-Trp (99%)
0.60
Ethoxyquin (30%)
0.50
9.60
RI PT
Rice gluten meal
SC
957
959 960
1
961
L-α-tocopherol acetate (50%), 12.58; menadione (22.9%), 0.83; cyanocobalamin (1%), 0.94; D-biotin (2%), 0.75; folic acid
962
(95%), 0.42; thiamine nitrate (98%), 0.09; ascorhyl acetate (95%), 4.31; niacin (99%), 4.04; meso-inositol (98%), 19.39;
963
calcium-D-pantothenate (98%), 3.85; riboflavin (80%), 0.73; pyridoxine hydrochloride (98%), 0.62. All ingredients were
964
diluted with corn starch to 1 kg.
965
2
M AN U
Per kilogram of vitamin premix (g/kg): retinyl acetate (500,000 IU/g), 2.10; cholecalciferol (500,000 IU/g), 0.40; D,
Per kilogram of mineral premix (g/kg): MnSO4⋅H2O (31.8% Mn), 2.6590; MgSO4⋅H2O (15.0% Mg), 200.0000;
FeSO4⋅H2O (30.0% Fe), 12.2500; ZnSO4⋅H2O (34.5% Zn), 8.2460; CuSO4⋅5H2O (25.0% Cu), 0.9560; KI (76.9% I), 0.0650;
967
Na2SeO3 (44.7% Se), 0.0168. All ingredients were diluted with corn starch to 1 kg.
968
3
Monosodium phosphate mixture: premix was added to obtain graded level of phosphorus.
969
4
Crude protein and crude lipid contents were measured value.
970
5
n-3 and n-6 contents were calculated according to Zeng et al. [104].
971
6
Total phosphorus content were determined according to the method of the AOAC (2000) [47], the value of available
972
phosphorus was calculated based on the digestibility of basal diet as determined in the digestibility trial according to NRC
973
(2011) [58].
975
EP
AC C
974
TE D
966
Table 2 Real-time PCR primer sequences 1.
977
ACCEPTED MANUSCRIPT Target gene
Primer sequence Forward (5’→3’)
hepcidin
AGCAGGAGCAGGATGAGC
GCCAGGGGATTTGTTTGT
59.3
Accession number JQ246442.1
LEAP-2A
TGCCTACTGCCAGAACCA
AATCGGTTGGCTGTAGGA
59.3
FJ390414
LEAP-2B
TGTGCCATTAGCGACTTCTGAG
ATGATTCGCCACAAAGGGG
59.3
KT625603
β-defensin-1
TTGCTTGTCCTTGCCGTCT
AATCCTTTGCCACAGCCTAA
58.4
KT445868
IFN-γ2
TGTTTGATGACTTTGGGATG
TCAGGACCCGCAGGAAGAC
60.4
JX657682
TNF-α
CGCTGCTGTCTGCTTCAC
CCTGGTCCTGGTTCACTC
58.4
HQ696609
IL-1β
AGAGTTTGGTGAAGAAGAGG
TTATTGTGGTTACGCTGGA
57.1
JQ692172 KC535507.1
Primer sequence Reverse (5’→3’)
Temperature(°C)
CAGCAGAATGGGGGAGTTATC
CTCGCAGAGTCTTGACATCCTT
62.3
ATGAGTCTTAGAGGTCTGGGT
ACAGTGAGGGCTAGGAGGG
60.3
JN663841
IL-10
AATCCCTTTGATTTTGCC
GTGCCTTATCCTACAGTATGTG
61.4
HQ388294
IL-11
GGTTCAAGTCTCTTCCAGCGAT
TGCGTGTTATTTTGTTCAGCCA
57.0
KT445870
IL-12p35
TGGAAAAGGAGGGGAAGATG
AGACGGACGCTGTGTGAGTGTA
55.4
KF944667.1
IL-12p40
ACAAAGATGAAAAACTGGAGGC
GTGTGTGGTTTAGGTAGGAGCC
59.0
KF944668.1
IL-15
CCTTCCAACAATCTCGCTTC
AACACATCTTCCAGTTCTCCTT
61.4
KT445872
IL-17D
GTGTCCAGGAGAGCACCAAG
GCGAGAGGCTGAGGAAGTTT
62.3
KF245426.1
IL-4/13A
CTACTGCTCGCTTTCGCTGT
CCCAGTTTTCAGTTCTCTCAGG
55.9
KT445871
IL-4/13B
TGTGAACCAGACCCTACATAACC
TTCAGGACCTTTGCTGCTTG
55.9
KT625600
TGF-β1
TTGGGACTTGTGCTCTAT
AGTTCTGCTGGGATGTTT
55.9
EU099588
TGF-β2
TACATTGACAGCAAGGTGGTG
TCTTGTTGGGGATGATGTAGTT
55.9
KM279716
GATA-3
AGCCCACACCTCTTCACCTT
CACTCTTTGTCCTCCTGCCG
58.5
JX021295, [105]
NF-κB p52
TCAGTGTAACGACAACGGGAT
ATACTTCAGCCACACCTCTCTTAG
58.4
KM279720
NF-κB p65
GAAGAAGGATGTGGGAGATG
TGTTGTCGTAGATGGGCTGAG
62.3
KJ526214
c-Rel
GCGTCTATGCTTCCAGATTTACC
ACTGCCACTGTTCTTGTTCACC
59.3
KT445865
IκBα
TCTTGCCATTATTCACGAGG
TGTTACCACAGTCATCCACCA
62.3
KJ125069
IKKα
GGCTACGCCAAAGACCTG
CGGACCTCGCCATTCATA
60.3
KM279718
SC
RI PT
IL-6 IL-8
M AN U
976
GTGGCGGTGGATTATTGG
GCACGGGTTGCCAGTTTG
60.3
KP125491
AGAGGCTCGTCATAGTGG
CTGTGATTGGCTTGCTTT
58.4
KM079079
TOR
TCCCACTTTCCACCAACT
ACACCTCCACCTTCTCCA
61.4
JX854449
S6K1
TGGAGGAGGTAATGGACG
ACATAAAGCAGCCTGACG
54.0
EF373673
4E-BP1
GCTGGCTGAGTTTGTGGTTG
CGAGTCGTGCTAAAAAGGGTC
60.3
KT757305
4E-BP2
CACTTTATTCTCCACCACCCC
TTCATTGAGGATGTTCTTGCC
60.3
KT757306
occludin
TATCTGTATCACTACTGCGTCG
CATTCACCCAATCCTCCA
59.4
KF193855
ZO-1
CGGTGTCTTCGTAGTCGG
CAGTTGGTTTGGGTTTCAG
59.4
KJ000055
ZO-2
TACAGCGGGACTCTAAAATGG
TCACACGGTCGTTCTCAAAG
60.3
KM112095
claudin-b
GAGGGAATCTGGATGAGC
ATGGCAATGATGGTGAGA
57.0
KF193860
claudin-c
GAGGGAATCTGGATGAGC
claudin-f
GCTGGAGTTGCCTGTCTTATTC
claudin-3c claudin-7a
EP
TE D
IKKβ IKKγ
59.4
KF193859
57.1
KM112097
ATCACTCGGGACTTCTA
CAGCAAACCCAATGTAG
57.0
KF193858
ACTTACCAGGGACTGTGGATGT
CACTATCATCAAAGCACGGGT
59.3
KT625604
claudin-7b
CTAACTGTGGTGGTGATGAC
AACAATGCTACAAAGGGCTG
59.3
KT445866
claudin-11
TCTCAACTGCTCTGTATCACTGC
TTTCTGGTTCACTTCCGAGG
62.3
KT445867
claudin-12
CCCTGAAGTGCCCACAA
GCGTATGTCACGGGAGAA
55.4
KF998571
claudin-15a
TGCTTTATTTCTTGGCTTTC
CTCGTACAGGGTTGAGGTG
59.0
KF193857
claudin-15b
AGTGTTCTAAGATAGGAGGGGAG
AGCCCTTCTCCGATTTCAT
62.3
KT757304
NaPi-IIb
CTGGACTCAAGAGAACCAAACG
GCAATGACAGAGCCAACAAAAT
62.7
KX823957
MLCK
GAAGGTCAGGGCATCTCA
GGGTCGGGCTTATCTACT
53.0
KM279719
FasL
AGGAAATGCCCGCACAAATG
AACCGCTTTCATTGACCTGGAG
61.4
KT445873
p38 MAPK
TGGGAGCAGACCTCAACAAT
TACCATCGGGTGGCAACATA
60.4
KM112098
JNK
ACAGCGTAGATGTGGGTGATT
GCTCAAGGTTGTGGTCATACG
62.3
KT757312
Bcl-2
AGGAAAATGGAGGTTGGGAT
CTGAGCAAAAAAGGCGATG
60.3
JQ713862.1
Mcl-1
TGGAAAGTCTCGTGGTAAAGCA
ATCGCTGAAGATTTCTGTTGCC
58.4
KT757307
Bax
CATCTATGAGCGGGTTCGTC
TTTATGGCTGGGGTCACACA
60.3
JQ793788.1
Apaf-1
AAGTTCTGGAGCCTGGACAC
AACTCAAGACCCCACAGCAC
61.4
KM279717
IAP
CACAATCCTGGTATGCGTCG
GGGTAATGCCTCTGGTGCTC
58.4
FJ593503.1
caspase-2
CGCTGTTGTGTGTTTACTGTCTCA
ACGCCATTATCCATCTCCTCTC
60.3
KT757313
AC C
CTGTTATGAAAGCGGCAC ACCAATCTCCCTCTTTTGTGTC
caspase-3
GCTGTGCTTCATTTGTTTG
TCTGAGATGTTATGGCTGTC
55.9
JQ793789
caspase-7
GCCATTACAGGATTGTTTCACC
CCTTATCTGTGCCATTGCGT
57.1
KT625601
caspase-8
ATCTGGTTGAAATCCGTGAA
TCCATCTGATGCCCATACAC
59.0
KM016991
caspase-9
CTGTGGCGGAGGTGAGAA
59.0
JQ793787
CuZnSOD
CGCACTTCAACCCTTACA
ACTTTCCTCATTGCCTCC
61.5
GU901214
MnSOD
ACGACCCAAGTCTCCCTA
ACCCTGTGGTTCTCCTCC
60.4
GU218534
CAT
GAAGTTCTACACCGATGAGG
CCAGAAATCCCAAACCAT
58.7
FJ560431
GPx1a
GGGCTGGTTATTCTGGGC
AGGCGATGTCATTCCTGTTC
61.5
EU828796
GPx1b
TTTTGTCCTTGAAGTATGTCCGTC
GGGTCGTTCATAAAGGGCATT
60.3
KT757315
GPx4a
TACGCTGAGAGAGGTTTACACAT
CTTTTCCATTGGGTTGTTCC
60.4
KU255598
GPx4b
CTGGAGAAATACAGGGGTTACG
CTCCTGCTTTCCGAACTGGT
60.3
KU255599
ACCEPTED MANUSCRIPT GTGCTGGAGGACATGGGAAT
TCTCAAGGAACCCGTCTG
CCAAGTATCCGTCCCACA
58.4
EU107283
GSTP1
ACAGTTGCCCAAGTTCCAG
CCTCACAGTCGTTTTTTCCA
59.3
KM112099
GSTO1
GGTGCTCAATGCCAAGGGAA
CTCAAACGGGTCGGATGGAA
GR
GTGTCCAACTTCTCCTGTG
ACTCTGGGGTCCAAAACG
Nrf2
CTGGACGAGGAGACTGGA
ATCTGTGGTAGGTGGAAC
Keap1a
TTCCACGCCCTCCTCAA
TGTACCCTCCCGCTATG
Keap1b
TCTGCTGTATGCGGTGGGC
CTCCTCCATTCATCTTTCTCG
β-actin
GGCTGTGCTGTCCCTGTA
GGGCATAACCCTCGTAGAT
58.4
KT757314
59.4
JX854448
62.5
KF733814
63.0
KF811013
57.9
KJ729125
61.4
M25013
SC
978
RI PT
GSTR
979
1
980
TGF-β, transforming growth factor β; NF-κB, nuclear factor kappa B; IκBα, inhibitor of κBα; IKK, IκB kinase; TOR, target
981
of rapamycin; S6K1, ribosomal protein S6 kinases 1; 4E-BP, eIF4E-binding proteins; ZO, zonula occludens; NaPi-IIb,
982
type-IIb sodium-phosphate cotransporter; MLCK, myosin light chain kinase; FasL, fatty acid synthetase ligand; p38 MAPK,
983
p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal protein kinase; Bcl-2, B-cell lymphoma protein-2; Mcl-1,
984
myeloid cell leukemia-1; Bax, Bcl-2 associated X protein; Apaf-1, apoptotic protease activating factor-1; IAP, inhibitor of
985
apoptosis proteins; caspase, cysteinyl aspartic acid-protease; CuZnSOD, copper, zinc superoxide dismutase; MnSOD,
986
manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR,
987
glutathione reductase; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like-ECH-associated protein 1.
M AN U
TE D
EP
989
AC C
988
LEAP-2, liver expressed antimicrobial peptide 2; IFN-γ2, interferon γ2; TNF-α, tumor necrosis factor α; IL, interleukin;
990
Table 3 Growth performance, head kidney and spleen weight (g), index (%) and serum phosphorus
991
concentration (mmol/L) and serum alkaline phosphatase activity (AKP, King’s unit/100mL) of young grass
992
carp (Ctenopharyngodon idellus) fed diets containing graded levels of available phosphorus for 60 days.
ACCEPTED MANUSCRIPT
993 Available P in the diet（ （g/kg diet） ） 2.46
3.96
5.68
7.10
8.75
IBW1
256.44±0.38a
256.00±0.67a
256.67±0.67a
256.22±0.38a
255.78±1.02a
254.56±0.51a
FBW1
610.87±12.09a
734.33±21.83b
880.63±16.57c
884.63±15.50c
898.23±18.11c
889.10±9.30c
PWG1
138.20±4.39a
186.86±9.04b
243.11±6.87c
245.26±6.39c
251.20±8.44c
247.00±4.09c
SGR1
1.45±0.03a
1.76±0.05b
2.05±0.03c
2.07±0.03c
2.09±0.04c
2.07±0.02c
FI1
688.48±0.99a
780.57±0.26b
851.03±0.69c
850.72±0.63c
850.52±0.81c
851.02±0.38c
FE1
51.48±1.67a
61.28±2.87b
73.32±1.92c
73.87±1.86c
75.51±2.31c
74.37±1.15c
Weight2
0.688±0.067a
1.087±0.092b
1.542±0.138c
1.578±0.086c
1.583±0.141c
1.559±0.159c
Index2
0.140±0.008a
0.161±0.012b
0.178±0.012c
0.174±0.013c
0.177±0.010c
0.176±0.015c
Weight2
0.842±0.067a
1.117±0.103c
1.417±0.103e
1.358±0.124de
1.292±0.138d
0.936±0.103b
Index2
0.173±0.018c
0.165±0.016c
1.49±0.13a
1.79±0.09b
13.20±0.90a
15.46±1.10b
Serum phosphorus3 Serum AKP
3
Regression YFBW = 89.6147x + 521.7909
0.150±0.015b
0.145±0.014b
0.105±0.008a
2.19±0.14c
2.48±0.14d
2.42±0.19d
2.51±0.14d
16.79±1.15b
20.14±1.51c
19.55±1.74c
19.60±1.43c
R2 = 0.9974
P < 0.05
Yhead kidney weight = 0.2838x + 0.4082 Yhead kidney index = 0.0125x + 0.1288 Yspleen weight = -0.0342x2 + 0.3477x + 0.5227
EP
Y Serum phosphorus = 0.2127x + 1.2948
TE D
Ymax = 2.07
YFE = 7.2550x + 44.2025
994
0.164±0.013c
Ymax = 888.15
YSGR = 0.2021x + 1.2559
Y Serum AKP = 1.4189x + 11.7693
M AN U
Spleen
SC
Head kidney
RI PT
0.95
2
P < 0.05
2
R = 0.9999
Ymax = 74.27
R = 0.9963
P < 0.05
Ymax = 1.565
R2 = 0.9984
P < 0.05
Ymax = 0.176
Ymax = 2.47 Ymax = 19.76
2
R = 0.9968
P < 0.05
R2 = 0.9704
P < 0.01
R2 = 0.9923
P < 0.01
2
R = 0.9802
P = 0.01
995
1
996
rate (%/day); FI: feed intake (g/fish); FE: feed efficiency (%).
997
Values are means ± SD for three replicate groups, with 30 fish in each group, and different superscripts in the same row are
998
significantly different (P < 0.05).
999
2
Values are means ± SD (n = 12), and different superscripts in the same row are significantly different (P < 0.05).
1000
3
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1001
1002
AC C
IBW: Initial body weight (g/fish); FBW: final body weight (g/fish); PWG: percent weight gain (%); SGR: specific growth
1003
Table 4 Immune parameters in the head kidney, spleen and skin of young grass carp (Ctenopharyngodon
1004
idella) fed diets containing graded levels of available phosphorus for 60 days, and then challenge with
1005
Aeromonas hydrophila for 14 days1
ACCEPTED MANUSCRIPT
1006 Available P in the diet（ （g/kg diet） ） 0.95
2.46
3.96
5.68
7.10
8.75
Head kidney 192.88±11.50a
213.24±20.70a
242.65±13.78b
266.16±23.39c
265.25±17.12c
271.09±19.62c
ACP
432.01±41.72a
552.73±52.40b
611.05±31.51c
656.39±30.62c
663.46±65.80c
664.11±44.34c
C3
14.96±1.44a
17.77±1.64b
20.06±1.09c
23.32±2.10d
26.25±1.73e
26.68±2.03e
C4
9.50±0.81a
10.31±0.45a
11.84±1.11b
13.50±1.13c
15.73±1.49d
15.82±1.45d
IgM
70.06±6.54a
79.88±7.10b
82.23±4.34b
82.28±7.02b
80.89±4.39b
79.74±7.70b
Spleen LZ
143.81±12.11a
165.89±11.45b
a
b
443.83±43.04
168.35±5.92b 532.75±51.50
177.59±11.89b c
548.63±38.33
c
RI PT
LZ
179.28±14.23b
173.35±11.75b
c
556.85±17.38c
553.14±53.41
380.01±29.75
C3
28.85±2.42a
32.19±2.85a
38.43±3.38b
40.77±2.11bc
42.43±3.96c
43.34±3.31c
C4
4.77±0.47a
5.95±0.38b
7.64±0.72c
8.51±0.63d
8.73±0.71d
8.58±0.66d
IgM
31.79±2.83a
44.80±3.89b
51.98±4.74c
52.67±4.78cd
57.76±5.61d
55.90±4.31cd
LZ
53.61±2.00a
103.36±7.33b
170.37±17.45c
159.47±15.52c
174.36±17.52c
162.33±15.80c
ACP
182.00±14.68a
251.22±22.56b
273.36±21.94b
316.40±13.24c
307.62±20.18c
313.29±26.68c
C3
25.46±2.16a
30.23±2.98b
35.96±2.75c
42.24±3.02d
43.34±3.94d
41.52±3.40d
C4
5.75±0.54a
8.43±0.83b
11.94±1.13c
12.59±1.26c
14.09±1.06d
15.24±1.08d
IgM
49.87±4.96a
90.36±5.94b
119.19±9.55c
117.89±9.75c
124.01±11.39c
125.78±12.31c
Ymax = 648.75
R2 = 0.9618
P = 0.125
YACP in head kidney = 59.5039x + 385.7503 YC3 in head kidney = 1.8124x + 13.1685 YC4 in head kidney = 1.0073x + 8.1159 YACP in spleen = 50.7319x + 327.5648 YC3 in spleen = 2.6641x + 26.3691 2
TE D
Regression
M AN U
Skin
SC
ACP
Ymax = 26.46 Ymax = 15.77 Ymax = 547.84 Ymax = 42.88
YLZ in skin = 38.7853x + 13.8321 YACP in skin = 27.0312x + 167.5545
AC C
YC3 in skin = 3.5755x + 21.8066 YC4 in skin = 1.3383x + 5.1679
YIgM in skin = 23.0345x + 29.8872
1007
EP
YIgM in spleen = -0.6184x + 8.8951x + 25.0505
2
P < 0.01
2
P < 0.01
2
P = 0.061
2
P < 0.05
2
P < 0.01
2
P = 0.055
2
P < 0.05
2
P < 0.01
2
P < 0.01
2
P = 0.060
R = 0.9978 R = 0.9740 R = 0.9907 R = 0.9632 R = 0.9668
Ymax = 166.63 Ymax = 313.43 Ymax = 42.36 Ymax = 14.67 Ymax = 121.72
R = 0.9924 R = 0.9529 R = 0.9986 R = 0.9347 R = 0.9910
1008
1
1009
Lysozyme (U/mg protein); ACP: acid phosphatase (U/mg protein); C3: complement component 3 (mg/g protein); C4:
1010
complement component 4 (mg/g protein); IgM: immunoglobulin M (mg/g protein).
1011
1012
Values are means ± SD (n = 6), and superscripted different letters in the same row are significantly different (P < 0.05). LZ:
1013
Table 5 Antioxidant related parameters in the head kidney, spleen and skin of young grass carp
1014
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus for 60 days, and then
1015
challenge with Aeromonas hydrophila for 14 days1
ACCEPTED MANUSCRIPT
1016 Available P in the diet（ （g/kg diet） ） 0.95
2.46
3.96
5.68
7.10
8.75
Head kidney 100.00±6.13d d
69.84±2.94c
60.52±1.68b
43.09±3.70a
65.79±2.56c
65.95±2.37c
c
b
a
a
34.35±1.64a
MDA
53.12±3.29
PC
6.87±0.57d
5.47±0.32c
4.43±0.42b
3.50±0.26a
AHR
60.91±3.76a
63.30±2.10a
68.03±1.90b
73.33±3.34c
ASA
40.71±3.61a
54.98±5.39b
62.56±5.34c
72.78±5.09d
a
46.45±4.19
d
3.98±0.35
MnSOD
3.80±0.36a
4.36±0.29a
5.47±0.51b
5.68±0.48b
CAT
0.75±0.06a
0.92±0.09bc
1.08±0.08d
1.11±0.09d
GPx
172.60±16.35a
186.34±14.03ab
193.41±13.57bc
210.04±18.98c
GST
82.94±7.61a
101.60±10.49b
129.87±9.30c
155.11±11.37d
74.98±6.08c
72.00±4.72d
74.33±4.86d
d
7.48±0.46d
5.72±0.51b
5.64±0.50b
0.98±0.09c
0.85±0.04b
207.80±13.43c
208.57±14.78c
157.08±13.62d
157.04±9.90d 22.77±1.56a
GSH
1.37±0.12a
1.76±0.14b
2.57±0.23c
2.51±0.19c
2.58±0.25c
2.62±0.20c
Choline
88.40±8.34a
111.24±8.17b
124.51±11.28b
128.77±10.18b
114.49±8.06b
119.45±7.43b
ROS
100.00±7.91e
70.00±4.61c
52.03±2.73b
39.92±2.68a
68.10±4.10c
76.86±5.08d
MDA
87.55±7.97d
79.99±3.90c
70.93±5.27b
63.63±5.66a
64.18±5.46ab
65.81±3.47ab
PC
5.44±0.50c
3.80±0.33b
2.46±0.20a
2.51±0.25a
2.72±0.14a
2.66±0.19a
AHR
39.74±2.50a
42.90±3.33ab
45.24±2.48bc
46.97±1.92c
49.07±2.97c
49.05±4.48c
ASA
47.85±4.18a
60.17±5.48b
73.64±5.35c
83.28±7.11d
95.53±6.48e
94.72±5.99e
CuZnSOD
4.87±0.45a
5.87±0.29b
5.54±0.32b
5.51±0.40b
5.49±0.50b
5.70±0.46b
MnSOD
4.78±0.41a
6.02±0.17b
7.48±0.56c
7.43±0.38c
7.49±0.72c
7.31±0.69c
CAT
0.50±0.04a
0.51±0.04a
0.52±0.05a
0.52±0.04a
0.53±0.04a
0.53±0.05a
GPx
213.54±19.80a
244.72±15.39b
284.86±19.13c
288.08±19.03c
282.21±24.96c
289.47±25.70c
GST
106.96±7.57a
116.92±5.75a
128.67±5.81b
150.84±8.86d
140.47±9.60c
129.81±12.08b
GR
24.96±1.93c
22.53±1.44b
20.35±1.75a
19.62±1.17a
19.53±1.74a
19.83±1.18a
GSH
1.02±0.08a
1.83±0.13b
3.05±0.27c
3.04±0.18c
3.12±0.21c
3.16±0.24c
Choline
93.60±4.99a
96.12±4.05a
102.29±7.51ab
112.07±7.24b
106.49±9.31ab
105.21±5.91ab
TE D
EP
AC C
23.25±0.93
a
29.20±1.66
Skin
21.57±2.15
a
GR
Spleen
22.56±1.15
a
3.67±0.27a
74.25±3.48c
7.60±0.30
M AN U
23.19±1.47
a
7.51±0.24
35.11±1.67 3.40±0.32a
CuZnSOD
b
6.21±0.41
c
34.47±0.75
SC
5.05±0.39
b
40.48±2.00
RI PT
ROS
ROS
100.00±3.27d
82.79±4.57c
78.40±7.35bc
66.30±6.52a
62.67±3.59a
74.82±7.32b
MDA
23.53±1.85c
20.17±1.96b
15.24±1.42a
16.22±0.93a
18.66±1.83b
19.90±1.97b
PC
6.15±0.50d
5.50±0.39c
4.43±0.35b
3.88±0.38a
3.71±0.37a
3.74±0.31a
AHR
27.80±1.80a
30.72±1.52a
47.09±1.41b
48.46±3.93b
45.71±3.63b
47.22±2.46b
ASA
36.36±3.52a
58.09±2.53b
97.29±9.17c
99.19±8.64c
96.62±6.00c
97.59±9.03c
CuZnSOD
4.89±0.48a
5.97±0.50b
7.39±0.37c
8.15±0.40d
9.62±0.35e
9.57±0.16e
MnSOD
4.61±0.44a
5.86±0.52b
6.38±0.59b
7.59±0.61c
7.72±0.71c
7.70±0.75c
CAT
0.72±0.07a
0.82±0.08b
0.92±0.09c
0.92±0.07c
0.84±0.07bc
0.77±0.07ab
GPx
99.69±6.82a
126.22±7.21b
148.66±13.75c
141.26±10.14c
143.52±10.83c
144.84±8.88c
GST
71.65±1.87a
82.60±5.31b
95.82±5.99c
118.42±11.14d
116.41±9.81d
116.70±6.39d
GR
18.63±1.94a
29.49±2.18b
42.17±2.28c
41.26±3.59c
39.68±2.05c
39.41±1.58c
a
GSH
4.85±0.47
Choline
54.42±4.70a
6.30±0.50
b
59.96±4.03ab
9.67±0.55
c
64.49±4.52bc
9.31±0.68
c
71.78±6.07c
9.44±0.68
c
9.41±0.60c
65.81±5.54bc
64.74±3.35bc
R2 = 0.8716
P < 0.05
Regression YROS in head kidney = 2.0516x2 - 23.5161x + 118.6128
YPC in head kidney = -0.7069x + 7.3761
R2 = 0.9845
Ymin = 3.53
YAHR in head kidney = 2.6843x + 57.6346
Ymax = 74.19
YCuZnSOD in head kidney = 0.7478x + 3.2482
Ymax = 5.63
P < 0.01
2
P < 0.01
2
P < 0.01
2
P = 0.120
2
P < 0.05
2
P < 0.05
2
P < 0.05
2
P < 0.01
2
P < 0.05
2
P < 0.05
2
P = 0.053
2
P < 0.05
R = 0.9642
YGpx in head kidney = 7.6283x + 165.7090
Ymax = 208.80
YGST in head kidney = 15.6009x + 66.4829
R = 0.9850
Ymax = 156.41
YGSH in head kidney = 0.3986x + 0.9198
R = 0.9947
Ymax = 2.57
R = 0.9624
2
YROS in spleen = 2.7772x - 29.3119x + 124.8207
R = 0.9016
YPC in spleen = -0.9880x + 6.3272
Ymax = 48.36
YASA in spleen = 7.6202x + 41.3830
Ymax = 95.13
YMnSOD in spleen = 0.8946x + 3.8958
Ymax = 7.43
YGpx in spleen = 23.6900x + 189.5043
Ymax = 286.16
2
YGST in spleen = -1.4327x + 17.6660x + 87.2845
R = 0.9966
RI PT
Ymin = 2.59
YAHR in spleen = 1.5229x + 38.7456
R = 0.9737
R = 0.9951 R = 0.9977
R = 0.9945
R = 0.8582
Ymin = 19.84
R = 0.9991
SC
YGR in spleen = -1.5319x + 26.3782 YGSH in spleen = 0.6718x + 0.3157
Ymax = 3.09
2
YROS in skin = 1.1271x - 14.5238x + 113.2893
2
P = 0.076
2
P < 0.05
2
P < 0.05
2
P = 0.010
2
P < 0.05
2
P < 0.01
2
P = 0.010
2
R = 0.9470
P < 0.05
R2 = 0.9979
P < 0.05
R = 0.9860 R = 0.9414
2
M AN U
YMDA in skin = 0.3858x - 4.1251x + 27.1300 YPC in skin = -0.4997x + 6.6210
Ymin = 3.78
YASA in skin = 20.2353x + 14.2006
Ymax = 97.67
YCuZnSOD in skin = 0.7465x + 4.1952
Ymax = 9.59
YMnSOD in skin = 0.6022x + 4.1449
Ymax = 7.67
2
YCAT in skin = -0.0117x + 0.1188x + 0.6135 YGpx in skin = 16.2702x + 84.8885
Ymax = 144.57
YGST in skin = 9.8306x + 60.0501
Ymax = 117.18
TE D
1017
P = 0.120
2
R = 0.9653
YCAT in head kidney = -0.0196x + 0.2025x + 0.5670
YGSH in skin = 1.6017x + 3.0042
P < 0.01
2
R = 0.9996
2
YGR in skin = 7.8204x + 10.8856
P = 0.010
2
R = 0.9809
Ymax = 7.53
YMnSOD in head kidney = 0.5563x + 3.1772
P < 0.01
2
R = 0.9817
Ymax = 73.04 ACCEPTED MANUSCRIPT
YASA in head kidney = 6.5996x + 36.2278
Ymax = 40.63 Ymax = 9.46
P < 0.01
2
R = 0.8740
R = 0.9794 R = 0.9727 R = 0.9884 R = 0.9801
2
P = 0.010
2
P < 0.05
2
P < 0.05
R = 0.9801 R = 0.9978 R = 0.9485
1018
1
1019
ROS, reactive oxygen species (% DCF florescence); MDA, malondialdehyde (nmol/g tissue); PC, protein carbonyl
1020
(nmol/mg protein); ASA, anti-superoxide anion (U/g protein); AHR, anti-hydroxyl radical (U/mg protein); CuZnSOD,
1021
copper/zinc superoxide dismutase (U/mg protein); MnSOD, manganese superoxide dismutase (U/mg protein); CAT,
1022
catalase (U/mg protein); GPx, glutathione peroxidase (U/mg protein); GST, glutathione-S-transferase (U/mg protein); GR,
1023
glutathione reductase (U/g protein); GSH, glutathione (mg/g protein); Choline (ug/g tissue).
1025
EP
AC C
1024
Values are means ± SD (n = 6), and superscripted different letters in the same row are significantly different (P < 0.05).
1027
1028
Table 6 Correlation coefficient of parameters in the head kidney and spleen.
c-Rel
IL-1β IκBα
TOR
Nrf2
FasL caspase-9
caspase-3 caspase-7 p38 MAPK
MLCK
< 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 = 0.072 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.05
RI PT
NF-κB p52
+0.991 +0.990 +0.951 +0.844 +0.934 +0.936 +0.944 +0.953 +0.948 +0.973 +0.928 +0.974 +0.961 +0.959 +0.962 +0.979 +0.942 +0.906 +0.944 +0.984 +0.972 +0.944 -0.978 -0.999 -0.989 -0.954 -0.994 +0.998 +0.986 +0.995 +0.959 +0.993 +0.993 +0.969 +0.987 +0.992 +0.957 +0.991 +0.926 +0.965 +0.964 +0.866 -0.983 -0.993 +0.772 +0.958 +0.919 -0.978 -0.917 -0.941 +0.972 +0.947 +0.988 +0.945 +0.982 -0.961 -0.979 -0.949 -0.986 -0.918 -0.993 -0.964 -0.948 -0.829 -0.984 +0.982 -0.933
Correlation coefficients +0.922 +0.966 +0.986 +0.919 +0.989 +0.999 +0.992 +0.935 +0.961 +0.961 +0.891 +0.988 +0.985 +0.999 +0.928 +0.968 +0.979 +0.820 +0.981 +0.974 +0.962 +0.907 -0.991 -0.974 -0.975 -0.976 -0.919 +0.979 +0.957 +0.953 +0.919 +0.956 +0.933 +0.996 +0.996 +0.966 +0.914 +0.990 +0.942 +0.951 +0.987 +0.997 -0.998 -0.948 +0.879 +0.946 +0.955 -0.962 -0.942 -0.772 +0.972 +0.993 +0.925 +0.885 +0.983 -0.895 -0.879 -0.905 -0.881 -0.879 -0.888 -0.976 -0.910 -0.802 -0.982 +0.971 -0.851
SC
IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-12p35 NF-κB p65 NF-κB p52 c-Rel IKKβ IKKγ IL-4/13A IL-10 IL-11 TGF-β1 TGF-β2 CuZnSOD MnSOD CAT GPx1a GPx1b GPx4a GPx4b GSTR GSTO1 GSTP1 GR Keap1a caspase-8 Apaf-1 Bax Bcl-2 Mcl-1 IAP caspase-9 caspase-9 FasL Apaf-1 Bax Bcl-2 Mcl-1 occludin ZO-1 ZO-2 claudin-c claudin-f claudin-7a claudin-7b claudin-11 claudin-12 claudin-15a
Spleen
M AN U
NF-κB p65
Head kidney
Correlation MANUSCRIPT ACCEPTED P coefficients
TE D
Dependent parameters
EP
Independent parameters
AC C
1026
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.01 < 0.05 = 0.055 < 0.01 < 0.01 < 0.05
1029
Table 7 The available phosphorus (AP) requirements based on PWG, FI, skin hemorrhage and lesions
1030
morbidity, head kidney LZ activity and MDA content, spleen C4 and MDA contents of young grass carp
1031
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus.
ACCEPTED MANUSCRIPT
1032 Indices
Regressive equation
R2
P
AP requirement
Y = 34.8502x + 103.7754
Ymax = 246.65
0.9805
< 0.05
4.10 g/kg
FI
Y = 54.0100x + 640.6770
Ymax = 850.82
0.9943
< 0.05
3.89 g/kg
Y = -5.3136x + 27.2760
Ymin = 5.33
0.8498
< 0.01
4.13 g/kg
Y = 15.8684x + 176.9600
Ymax = 267.50
0.7385
< 0.01
5.71 g/kg
Y = -3.9404x + 56.4860
Ymin = 34.64
0.8722
Y = 0.8200x + 4.0421
Ymax = 8.61
0.8704
Y = -5.1413x + 92.2952
Ymin = 64.54
0.7358
Morbidity LZ in head kidney MDA in head kidney C4 in spleen MDA in spleen
< 0.01
5.54 g/kg
= 0.01
5.57 g/kg
< 0.01
5.40 g/kg
SC
1033
RI PT
PWG
AC C
EP
TE D
M AN U
1034
Fig. 1.
ACCEPTED MANUSCRIPT
RI PT
1035
1036
Fig. 1. Effects of available phosphorus levels on skin hemorrhage and lesions morbidity in young grass
1038
carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila. Values having different
1039
letters are significantly different (P < 0.05).
SC
1037
1040
1042
EP
TE D
M AN U
Fig. 2.
AC C
1041
1043
Fig. 2. Phosphorus deficiency led to obviously skin hemorrhage and lesions, compared to available
1044
phosphorus 3.96 g/kg diet, in young grass carp (Ctenopharyngodon idella) after infection with
1045
Aeromonas hydrophila.
1046
Fig. 3.
ACCEPTED MANUSCRIPT
RI PT
1047
M AN U
SC
1048
AC C
EP
TE D
1049
1050
1051
Fig. 3. Relative expression of LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 in the head kidney (A),
1052
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1053
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1054
Values having different letters are significantly different (P < 0.05). LEAP-2, liver-expressed antimicrobial
1055
peptide 2.
1056
Fig. 4.
ACCEPTED MANUSCRIPT
RI PT
1057
M AN U
SC
1058
AC C
EP
TE D
1059
1060
1061
Fig. 4. Relative expression of pro-inflammatory cytokines in the head kidney (A), spleen (B) and skin
1062
(C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of available
1063
phosphorus. Data represent means of six fish in each group, error bars indicate S.D. Values having different
1064
letters are significantly different (P < 0.05). IL, interleukin; IFN-γ2, interferon γ2 and TNF-α, tumor necrosis
1065
factor α.
1066
Fig. 5.
ACCEPTED MANUSCRIPT
RI PT
1067
M AN U
SC
1068
EP AC C
1070
TE D
1069
1071
Fig. 5. Relative expression of anti-inflammatory cytokines and GATA-3 in the head kidney (A), spleen
1072
(B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1073
available phosphorus. Data represent means of six fish in each group, error bars indicate S.D. Values
1074
having different letters are significantly different (P < 0.05). TGF-β, transforming growth factor β.
1075
1076
Fig. 6.
ACCEPTED MANUSCRIPT
RI PT
1077
M AN U TE D EP AC C
1079
SC
1078
1080
1081
Fig. 6. Relative expression of NF-κB p52, NF-κB p65, c-Rel, IκBα, IKKα, IKKβ, IKKγ, TOR, S6K1,
1082
4E-BP1 and 4E-BP2 in the head kidney (A), spleen (B) and skin (C) of young grass carp
1083
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus. Data represent
1084
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1085
different (P < 0.05). NF-κB, nuclear factor kappa B; IκBα, inhibitor of κBα; IKK, IκB kinase; TOR, target of
1086
rapamycin; S6K1, ribosomal protein S6 kinases 1; 4E-BP, eIF4E-binding proteins.
1087
1088
Fig. 7.
ACCEPTED MANUSCRIPT
RI PT
1089
M AN U
SC
1090
EP AC C
1092
TE D
1091
1093
Fig. 7. Relative expression of antioxidant enzymes, Nrf2, Keap1a and Keap1b in the head kidney (A),
1094
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1095
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1096
Values having different letters are significantly different (P < 0.05). CuZnSOD, copper, zinc superoxide
1097
dismutase; MnSOD, manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST,
1098
glutathione-S-transferase;
1099
Kelch-like-ECH-associated protein 1.
1100
1101
GR,
glutathione
reductase;
Nrf2,
NF-E2-related
factor
2;
Keap1,
Fig. 8.
ACCEPTED MANUSCRIPT
RI PT
1102
M AN U TE D EP AC C
1104
SC
1103
1105
1106
Fig. 8. Relative expression of apoptotic parameters in the head kidney (A), spleen (B) and skin (C) of
1107
young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus.
1108
Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1109
significantly different (P < 0.05). caspase, cysteinyl aspartic acid-protease; Apaf-1, apoptotic protease
1110
activating factor-1; Bax, Bcl-2 associated X protein; FasL, Fas ligand; Bcl-2, B-cell lymphoma protein-2;
1111
IAP, inhibitor of apoptosis proteins; Mcl-1, myeloid cell leukemia-1; p38 MAPK, p38 mitogen-activated
1112
protein kinase; JNK, c-Jun N-terminal protein kinase.
1113
Fig. 9.
ACCEPTED MANUSCRIPT
RI PT
1114
M AN U
SC
1115
EP AC C
1117
TE D
1116
1118
Fig. 9. Relative expression of tight junction complexes, transporter and MLCK in the head kidney (A),
1119
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1120
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1121
Values having different letters are significantly different (P < 0.05). ZO, zonula occludens; NaPi-IIb:
1122
type-IIb sodium-phosphate cotransporter; MLCK, myosin light chain kinase.
1123
Fig. 10.
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
1124
1125
Fig. 10. The potential action pathways of phosphorus in the head kidney and spleen immune function
1127
and structural integrity of fish.
EP AC C
1128
TE D
1126
ACCEPTED MANUSCRIPT Highlights 1、 Phosphorus deficiency decreased immune function of fish immune organs. 2、 Phosphorus deficiency induced oxidative damage and apoptosis, and thus impaired cellular integrity of fish immune organs.
RI PT
3、 Phosphorus deficiency damaged tight junction, and thus weakened intercellular integrity of fish immune organs.
4、 Dietary phosphorus regulated NF-κB, TOR, Nrf2, p38 MAPK and MLCK signaling of fish
AC C
EP
TE D
M AN U
SC
immune oregans.
S1050-4648(17)30070-0
DOI:
10.1016/j.fsi.2017.02.007
Reference:
YFSIM 4429
To appear in:
Fish and Shellfish Immunology
Received Date: 2 October 2016 Revised Date:
7 February 2017
Accepted Date: 8 February 2017
Please cite this article as: Chen K, Jiang W-D, Wu P, Liu Y, Kuang S-Y, Tang L, Tang W-N, Zhang Y-A, Zhou X-Q, Feng L, Effect of dietary phosphorus deficiency on the growth, immune function and structural integrity of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella), Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.007. 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.
1
Effect of dietary phosphorus deficiency on the growth, immune function and structural
2
integrity of head kidney, spleenACCEPTED and skin inMANUSCRIPT young grass carp (Ctenopharyngodon idella)
3 a, 1
, Wei-Dan Jiang a, b, c, 1, Pei Wu
a, b, c
, Sheng-Yao Kuang d, Ling Tang d,
Kang Chen
5
Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, *, Lin Feng a, b, c, *
9 10 11 12
Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan
Agricultural University, Sichuan, Chengdu 611130, China c
RI PT
8
b
Animal Nutrition Institute, Sichuan Agricultural University, Sichuan, Chengdu 611130, China
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan
Agricultural University, Sichuan, Chengdu 611130, China
SC
7
a
d
Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China
e
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
M AN U
6
, Yang Liu
a, b, c
4
13
* Corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,
15
Sichuan, China. Tel.: +86 835 2885157; fax: +86 835 2885968.
16
E-mail addresses: [email protected], [email protected] (X.-Q. Zhou); [email protected] (L.
17
Feng).
20 21
1
EP
19
These two authors contributed to this work equally
AC C
18
TE D
14
22
Abstract
23
This study evaluates the effects of dietary phosphorus on the growth, immune function and structural
24
integrity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) that were fed graded
25
levels of available phosphorus (0.95-8.75 g/kg diet). Results indicated that phosphorus deficiency decreased
26
the growth performance of young grass carp. In addition, the results first demonstrated that compared with
27
the optimal phosphorus level, phosphorus deficiency depressed the lysozyme (LZ) and acid phosphatase
28
(ACP) activities and the complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and
29
down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα
30
(IκBα) and target of rapamycin (TOR), whereas it up-regulated pro-inflammatory cytokines, nuclear factor
31
kappa B (NF-κB) p65 and NF-κB p52 mRNA levels to decrease fish head kidney and spleen immune
32
functions. Moreover, phosphorus deficiency up-regulated the mRNA levels of Kelch-like-ECH-associated
33
protein 1a (Keap1a), Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X
34
protein (Bax), caspase -2, -3, -7, -8 and -9, p38 mitogen-activated protein kinase (MAPK) and myosin light
35
chain kinase (MLCK), whereas it depressed the glutathione (GSH) contents and antioxidant enzymes
36
activities, and down-regulated the mRNA levels of antioxidant enzymes, NF-E2-related factor 2 (Nrf2),
37
B-cell lymphoma protein-2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and tight junction complexes to
38
attenuate fish head kidney and spleen structural integrity. In addition, phosphorus deficiency increased skin
39
hemorrhage and lesions morbidity. Finally, based on the percent weight gain (PWG) and the ability to
40
combat skin hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp
41
(254.56-898.23 g) were estimated to be 4.10 and 4.13 g/kg diet, respectively. In summary, phosphorus
42
deficiency decreases the growth performance, and impairs immune function and structural integrity in the
43
head kidney, spleen and skin of young grass carp.
44
Keywords: Phosphorus deficiency; Grass carp (Ctenopharyngodon idella); Immune function; Structural
45
integrity; Head kidney; Spleen
46
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
47
1. Introduction
48
Currently, aquaculture is frequently faced with disease problems resulting from its intensification [1].
49
The disease problems of fish are closely associated with the depressed immune function of immune organs
50
in fish [2]. The head kidney and spleen of fish are the major immune organs [3]. Meanwhile, fish skin is
51
believed to be an important mucosal immune organ and the first line of defence [4]. It is well known that
52
providing enough nutrients is an important approach to improve the immune function of immune organs in
53
animals [2]. Phosphorus is an essential nutrient for the normal growth of fish [5]. Studies show that dietary
54
phosphorus deficiency induces poor bone mineralization, skeletal and operculum deformities, an increase in
55
body lipid content and a decline of growth performance in several fish species [6-9]. In fish, growth is partly
56
related to the immune function of immune organs [10, 11]. However, no study has investigated the effect of
57
dietary phosphorus on the immune function of the immune organs in fish. A low dietary level of phosphorus
58
increases the whole body lipid level in silver perch (Bidyanus bidyanus) [12]. A study from our lab showed
59
that excess dietary levels of lipids reduced the immune function of the immune organs in young grass carp
60
(Ctenopharyngodon idella) [10]. The above studies indicate that there might be a relationship between
61
dietary phosphorus deficiency and the immune function of the immune organs in fish, which is worth
62
investigation.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
The immune function of fish immune organs largely relies on antibacterial substances [such as
64
immunoglobulin M (IgM) and lysozyme (LZ)] and cytokines [13]. In fish, cytokines are regulated by the
65
transcription factors nuclear factor-κB (NF-κB) and target of rapamycin (TOR) [14, 15]. However, there is
66
no study demonstrating the effects of phosphorus on antibacterial substances and cytokines and its possible
67
mechanisms in fish. A low level of phosphorus decreased the serum antibody titer in channel catfish
68
(Ictalurus punctatus) [16] and the plasma IgM concentration, as well as LZ activity, in European whitefish
69
(Coregonus lavaretus L.) [17]. Sarasa et al. reported that phosphorus deficiency down-regulated peroxisome
70
proliferative activated receptor α (PPARα) expression in mice livers [18]. The inhibition of PPARα
71
increased the interleukin 8 (IL-8) content by activating NF-κB in human microvessel endothelial cells [19].
72
In addition, phosphorus deficiency depressed Akt phosphorylation in NHBE cells [20]. A study showed that
73
the inhibition of Akt phosphorylation inactivated mTOR in rat skeletal muscle cells [21]. According to these
74
studies, it follows that phosphorus deficiency may affect the production of antimicrobial substances,
75
cytokines and the relevant signalling molecules to decrease the immune function of the immune organs in
AC C
EP
63
76
fish, which deserves investigation.
77
The immune function of the immune organs is also correlated with the structural integrity of these
78
organs in fish [22], which is related to cellular integrity and intercellular integrity [23]. Cellular integrity is
79
impaired by oxidative damage and cell apoptosis [24]. The intercellular integrity is mainly maintained by
80
tight junction complexes (TJs) (such as occludin, zonula occludens 1 and claudins) in rats [25]. Studies have
81
indicated that the gene expression of antioxidant, apoptosis and TJs could be regulated by NF-E2-related
82
factor 2 (Nrf2) [26], p38 mitogen-activated protein kinase (MAPK) [27] and myosin light chain kinase
83
(MLCK) [28] in humans, respectively. However, no study has addressed the effects of phosphorus on
84
antioxidant, apoptosis, and tight junction and their possible signalling pathways in the immune organs of
85
terrestrial animals and fish. In human endothelial cells, phosphorus deficiency decreases nitric oxide
86
production [29]. Decreased nitric oxide inhibits the activation of Nrf2 in rat pheochromocytoma cells [30].
87
Glinn et al. reported that phosphorus deficiency increased the Ca2+ concentration in rat neuronal cells [31].
88
Ca2+ induces apoptosis through the activation of p38 MAPK in mice macrophage cells [32]. To our
89
knowledge, phosphorus is an important component of phospholipids [33]. It was reported that phospholipids
90
down-regulate the expression of MLCK, leading to the up-regulation of occludin, ZO-1 and claudins mRNA
91
levels in the gills of juvenile grass carp [34]. Based on these data, there may be a correlation between dietary
92
phosphorus deficiency and the structural integrity of immune organs associated with antioxidants, apoptosis
93
and TJs as well as their related signalling molecules in fish, which requires investigation.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Grass carp is an important farmed fish, accounting for ~16% of the global freshwater aquaculture [35].
95
Currently, phosphorus requirements have been confirmed in juvenile grass carp [9] and young grass carp
96
[36], which is mainly based on growth performance. However, the nutrient requirements of fish may vary
97
with different indices. A study from our lab showed that myo-inositol requirements for juvenile Jian carp
98
based on the head kidney acid phosphatase (ACP) activity (660.7 mg/kg diet) were higher than those based
99
on a percent weight gain (PWG) (518.0 mg/kg diet) [37]. However, no study has reported the phosphorus
100
requirements based on the immune function and structural integrity of the immune organs in fish, which
101
needs further investigation.
AC C
EP
94
102
Here, we hypothesized that phosphorus deficiency might impair immune function and structural
103
integrity in the head kidney, spleen and skin to decrease the growth performance of fish. To test this
104
hypothesis, the present study, for the first time, investigates the influence of phosphorus on antibacterial
105
substances, cytokines, antioxidants, apoptosis and TJs in the head kidney, spleen and skin of young grass
106
carp after challenge with Aeromonas hydrophila. In addition, we further investigated the influence of
107
phosphorus on the relevant signalling molecules, including NF-κB, TOR, Nrf2, p38 MAPK and MLCK,
108
which might imply partial theoretical evidence for the mechanisms of the phosphorus-enhanced fish immune
109
function and structural integrity. Meanwhile, the phosphorus requirements for the young grass carp based on
110
the immune function and structural integrity of the immune organs were also evaluated, which may provide
111
a reference for the commercial feed preparation and healthy breeding of grass carp.
112
2. Materials and methods
113
2.1. Experimental diet and procedures
RI PT
ACCEPTED MANUSCRIPT
Formulation of the basal diet and proximate analysis of the diets are presented in Table 1. Fish meal,
115
casein, gelatin and rice gluten meal were used as dietary protein sources. Fish oil and soybean oil were used
116
as dietary lipid sources. Six experimental diets were obtained by supplementing the basal diet with
117
monosodium phosphate (NaH2PO4•2H2O, AR) which was from Chengdu Kelong Chemicals Reagents
118
(Chengdu, China). The dietary total phosphorus were determined by the method of the AOAC (2000), and
119
final total phosphorus levels of the six experimental diets were 2.3 (un-supplemented), 4.0, 5.6, 7.6, 9.2 and
120
11.0 g/kg diet, respectively, which was based on the previous study from our lab [36]. The diets preparation
121
was conducted as described by our previous study [8]. Diets were prepared by thoroughly mixing all the
122
ingredients. Distilled water was added to produce a proper pelleting consistency, and then the mixture was
123
homogenized and extruded through a 2-mm die. The noodle-like diets were dried using an electrical fan at
124
room temperature. After being prepared completely, the diets were stored at -20 °C according to Xie et al.
125
[8].
127
M AN U
TE D
EP
AC C
126
SC
114
(Table 1 inserted here)
2.2. Growth trial and sample collection
128
All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan
129
Agricultural University. After obtainment from fishery (Sichuan, China), grass carp were acclimated to
130
experimental conditions for 4 weeks according to Xie et al. [8]. Subsequently, 540 fish with similar body
131
weights (mean weight 256.22 ± 0.60 g) were randomly assigned to 18 experimental cages (1.4 L × 1.4 W ×
132
1.4 H m), namely 30 fish per cage as described in our lab study [23, 36]. Every cage was equipped with a
133
disc of 100 cm diameter in the bottom to collect the uneaten feed according to Wu et al. [38]. Each
134
experimental diet was randomly fed to fish one of triplicate cages of the six dietary treatments to apparent
135
satiation, and fish were fed with respective diet four times daily according to Du et al. [39] for 60 days. The
136
uneaten feed was collected 30 min after each meal as described by our previous study [40]. During the
137
experiment, water temperature and pH were 27 ± 2 °C and 7.0 ± 0.4, respectively. The dissolved oxygen was
138
higher than 6.0 mg/L. The treatment groups were under natural light and dark cycle as described by Wen et
139
al. [15]. Before and during the experiment, daily water samples were collected and analyzed. Average
140
waterborne phosphorus concentration was below 0.054 mg/L.
RI PT
ACCEPTED MANUSCRIPT
Fish in each cage were weighed and counted at the initiation and termination of the feeding trial to
142
calculate the growth performance related indices. Twelve fish from each treatment were anaesthetized in a
143
benzocaine bath according to our lab previous study [8]. To analyze serum phosphorus content and alkaline
144
phosphatase (AKP) activity, the blood samples of six fish from each treatment were drawn from the caudal
145
vein then plasma was removed and stored -20 °C until analysis as described by Wen et al. [36]. To calculate
146
the index of the head kidney and spleen, the head kidney and spleen of twelve fish were quickly removed
147
and weighed.
148
2.3. Digestibility trial and sample collection
M AN U
SC
141
The digestibility trial was performed according to a previous study by our lab [8]. During the
150
digestibility trial, diet 1 (total phosphorus 2.3 g/kg) and diet 3 (total phosphorus 5.6 g/kg) were the same as
151
treatments 1 and 3 of the growth trial, respectively, which were added with 5 g/kg Cr2O3 according to Roy &
152
Lall [7]. One hundred and eighty fish with similar body weights (mean weight 255.67 ± 0.37 g) were
153
randomly assigned to six extra above-mentioned experimental cages (30 fish per cage) and were fed either
154
diet 1 or 3 in triplicate four times daily to satiation as described by Liang et al. [9]. After 10 days of feeding,
155
faecal collection was conducted 6 h after the first meal according to Mai et al. [41]. As Austreng described,
156
the faeces were stripped from all of the fish by applying gentle pressure in the anal area [42]. The faecal
157
samples were pooled and stored at -20 °C for subsequent analyses according to Zhang et al. [43].
158
2.4. Challenge trial and sample collection
AC C
EP
TE D
149
159
A. hydrophila was friendly provided by College of Veterinary Medicine, Sichuan Agricultural
160
University, China. The challenge test was conducted as described by our previous study [23]. After the
161
growth trial, fifteen fish of similar body weights were obtained from each treatment group as described by
162
Liu et al. [44] and moved to six new experimental cages. After 5 days acclimation according to Kuang et al.
163
[22], fish were intraperitoneally injected with 1.0 ml of 2.5×109 colony-forming units (cfu) ml-1
164
A. hydrophila for each individual. The challenge dose was selected as an appropriate dose which could
165
effectively induce inflammation and consequently enable the investigation on fish reactivity against a
166
threatening disease without causing death according to our preliminary test (data was not shown).
167
Managements during the challenge test were the same as the feeding trial for 14 days according to our lab
168
previous study [11]. To evaluate the severity of skin hemorrhage and lesions morbidity in grass carp, a
169
scoring system was designed which were similar to Song et al. [45]. After the challenge trial, all fish from
170
each treatment were anaesthetized in a benzocaine bath according to Xie et al. [8]. Skin samples were
171
collected from grass carp using the method of Caiping et al. [46]. Skin sections (approx. 2 cm × 2 cm) were
172
thereafter aseptically excised from both the dorsal and ventral regions (about 1 cm above the pectoral and
173
pelvic fin, respectively) along the left side of the fish. Then sacrificed, the head kidney, spleen and skin of
174
the fish were quickly removed and frozen in liquid nitrogen, then stored at -80 °C until analysis as described
175
by Ni et al. [10].
176
2.5. Biochemical analysis
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
The approximate compositions of the diets were analysed according to the standard methods of AOAC
178
[47]. Briefly, crude protein was determined by the micro-Kjeldahl method, crude lipid by the Soxhlet
179
method. Phosphorus contents of experimental diets and faeces were measured by method of AOAC [47].
180
Chromium contents of experimental diets and faeces were analysed according to Wen et al. [36].
TE D
177
Tissue homogenates of head kidney, spleen and skin were prepared in 10 volumes (w/v) of ice-cold
182
normal saline and centrifuged at 6000 g for 20 min at 4 °C. The supernatant was conserved and used to
183
determine the immune and antioxidant parameters, which was similar to Wu et al. [48]. The determination
184
methods of serum phosphorus and AKP are similar to Shao et al. [49]. LZ, ACP activities and complement 3
185
(C3), C4 and immunoglobulin M (IgM) contents were assayed according to Zhao et al. [50]. The reactive
186
oxygen species (ROS) and malondialdehyde (MDA) contents were determined according to Yang et al. [51]
187
and Ji et al [52], respectively. The contents of protein carbonyl (PC) and glutathione (GSH) were assayed as
188
described by Ni et al. [10]. The anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were
189
measured as previously study [22]. The total superoxide dismutase (SOD) and copper, zinc superoxide
190
dismutase (CuZnSOD) activities were determined as described by Lu et al. [53]. The manganese superoxide
191
dismutase (MnSOD) activity was calculated by deducting CuZnSOD from total SOD. The catalase (CAT),
AC C
EP
181
192
glutathione-S-transferases (GST) and glutathione reductase (GR) activities were assayed according to Wen et
193
al. [36]. The glutathione peroxidase (GPx) activity was measured according to Zhang et al. [54]. The choline
194
content was determined as described by Zhang et al. [55].
195
2.6. Real-time polymerase chain reaction (PCR) analysis
ACCEPTED MANUSCRIPT
Total RNA samples were isolated from the head kidney, spleen and skin using RNAiso Plus Kit (Takara,
197
Dalian, China) according to the manufacturer’s instructions, then quality and quantity were assessed using
198
agarose gel (1%) electrophoresis and spectrophotometric (A260: 280 nm ratio) analysis, respectively, as
199
described by Luo et al. [56]. Subsequently, RNA was reverse transcribed into cDNA using the
200
PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer’s instructions. For quantitative
201
real-time PCR, specific primers were designed according to the sequences cloned in our laboratory and the
202
published sequences of grass carp (Table 2). According to the results of our preliminary experiment
203
concerning the evaluation of internal control genes (data not shown), β-actin was used as a reference gene to
204
normalize cDNA loading. The target and housekeeping gene amplification efficiency were calculated
205
according to the specific gene standard curves generated from 10-fold serial dilutions. The 2−∆∆CT method
206
was used to calculate the expression results after verifying that the primers amplified with an efficiency of
207
approximately 100% according to Livak & Schmittgen et al. [57].
209
SC
M AN U
(Table 2 inserted here)
TE D
208
RI PT
196
2.7. Calculations and statistical analysis
The data of initial body weight (IBW), final body weight (FBW) and feed intake (FI) were used to
211
calculate the percent weight gain (PWG), feed efficiency (FE) and specific growth rate (SGR) according to
212
Xie et al. [8]. The weight of head kidney and spleen were used to calculate the index of head kidney and
213
spleen, respectively, as described by Kuang et al. [22].
AC C
EP
210
214
PWG =100 × [FBW (g/fish) – IBW (g/fish)]/IBW (g/fish);
215
FE = 100 × [FBW (g/fish) – IBW (g/fish)]/FI (g/fish);
216
SGR = 100 × [ln (mean final weight) – ln (mean initial weight)]/days;
217
Head kidney index = (g head kidney weight/g body weight) × 100;
218
Spleen index = (g spleen weight/g body weight) × 100;
219
The apparent phosphorus digestibility in the diet (D) calculation equation D (%) = 100 × [1 – (CrD ×
220
PF)/(CrF × PD)], where CrD and PD were the concentrations of Cr2O3 and phosphorus in the diet, respectively,
221
CrF and PF were the concentrations of Cr2O3 and phosphorus in the faeces, respectively [National Research
222
Council (NRC) 2011] [58]. According to this, apparent phosphorus digestibility in the diet 1 and diet 3
223
could be calculated.
ACCEPTED MANUSCRIPT
The apparent phosphorus digestibility in the NaH2PO4 (Di) calculation equation: Di (%) = 100 × (P2 ×
225
D2 – P1 × D1)/I, where P1 and P2 were the concentrations of phosphorus in diet 1 and diet 3, respectively; D1
226
and D2 were the apparent phosphorus digestibility in diet 1 and diet 3, respectively; I was concentration of
227
inorganic phosphorus in diet 3 [7]. According to this, apparent phosphorus digestibility in the NaH2PO4
228
could be calculated.
229
RI PT
224
All data were subjected to one-way analysis of variance followed by Duncan’s multiple range tests to determine significant differences among treatment groups using the software SPSS 18.0 (SPSS Inc., Chicago,
231
IL, USA) at a level of P < 0.05. The results are presented as the means ± SD. Broken-line analysis was used
232
to evaluate the dietary phosphorus requirement of young grass carp according to Xie et al. [8].
233
3. Results
234
3.1. Phosphorus availability
M AN U
SC
230
Apparent digestibility of phosphorus in diet 1 and diet 3 were 41.14% and 69.48%, respectively.
236
Apparent digestibility of phosphorus in NaH2PO4 was calculated to be 89.20%. According to these values,
237
available phosphorus contents in the six experimental diets were estimated to be 0.95 (un-supplemented
238
control), 2.46, 3.96, 5.68, 7.10 and 8.75 g/kg diet, respectively.
239
3.2. Growth performance and serum phosphorus concentration and alkaline phosphatase activity, as well
240
as skin hemorrhage and lesions morbidity of young grass carp after infection with A. hydrophila
EP
TE D
235
As shown in Table 3, the growth performance (FBW, PWG, SGR FI, and FE) and head kidney weight
242
and index all significantly increased as the dietary available phosphorus levels increased to 3.96 g/kg diet (P
243
< 0.05), and then plateaued. Fish fed 3.96 and 5.68 g/kg diet showed the highest spleen weight. As the
244
dietary available phosphorus levels increased to 3.96 g/kg diet, the spleen index was no significant changes
245
(P > 0.05), and then gradually decreased. The serum phosphorus concentration and AKP activity increased
246
as the dietary available phosphorus levels increased to 5.68 g/kg diet, and then plateaued. After infection
247
with A. hydrophila in young grass carp, the skin hemorrhage and lesions morbidity significantly decreased
248
as the dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and then plateaued.
249
Compared with optimal available phosphorus level, phosphorus deficiency led to an obvious skin
AC C
241
250
hemorrhage and lesions symptom (Fig. 1&2).
251
(Table 3 inserted here)
252
(Fig. 1 inserted here)
253
(Fig. 2 inserted here)
ACCEPTED MANUSCRIPT
254
3.3. Immunological parameters in immune organs of young grass carp after infection with A. hydrophila
255
3.3.1. Antimicrobial substances activities or contents in the immune organs The LZ and ACP activities, C3, C4 and IgM contents in the head kidney, spleen and skin are presented
257
in Table 4. In the head kidney, the LZ and ACP activities, C3, C4 and IgM contents gradually increased as
258
the dietary available phosphorus levels increased to 5.68, 3.96, 7.10, 7.10 and 2.46 g/kg diet, respectively,
259
and then plateaued. As the dietary available phosphorus levels increased to 2.46, 3.96, 5.68, 5.68 and 5.68
260
g/kg diet, the LZ and ACP activities and C3, C4 and IgM contents in the spleen gradually improved,
261
respectively, and then plateaued. The LZ and ACP activities, C3, C4 and IgM contents in the skin gradually
262
increased as the dietary available phosphorus levels increased to 3.96, 5.68, 5.68, 7.10 and 3.96 g/kg diet,
263
respectively, and then plateaued.
264
(Table 4 inserted here)
M AN U
SC
RI PT
256
3.3.2. Relative mRNA levels of antimicrobial peptides, cytokines, GATA-3 and related signalling
266
molecules in the immune organs
TE D
265
The effects of dietary available phosphorus on immune-related indices in the head kidney, spleen and
268
skin of young grass carp are presented in Fig. 3-6. In the head kidney, the liver-expressed antimicrobial
269
peptide 2A (LEAP-2A), LEAP-2B and hepcidin mRNA levels were gradually up-regulated as the dietary
270
available phosphorus levels increased to 3.96 g/kg diet and then plateaued. Fish fed 5.68 g/kg available
271
phosphorus diet showed the highest β-defensin-1 mRNA level. In the spleen, the LEAP-2A, LEAP-2B,
272
hepcidin and β-defensin-1 mRNA levels were gradually up-regulated as the dietary available phosphorus
273
levels increased to 3.96, 5.68, 3.96 and 3.96 g/kg diet, respectively, and then plateaued. In the skin, the
274
LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 mRNA levels were gradually up-regulated as the dietary
275
available phosphorus levels increased to 3.96, 2.46, 3.96 and 5.68 g/kg diet, respectively, and then plateaued.
276
In the head kidney, the mRNA levels of IL-6, IL-12p35, interferon γ2 (IFN-γ2), NF-κB p52, c-Rel,
277
IκB kinases γ (IKKγ) and eIF4E-binding proteins 1 (4E-BP1) were gradually down-regulated as the dietary
278
available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96, 3.96, 3.96 and 5.68 g/kg diet, respectively,
AC C
EP
267
279
simultaneously, IL-1β, IL-17D, tumor necrosis factor α (TNF-α), NF-κB p65, IKKβ and 4E-BP2 were
280
significantly down-regulated as the dietary available phosphorus levels increased to 5.68, 3.96, 5.68, 5.68,
281
3.96 and 2.46 g/kg diet (P < 0.05), respectively, and plateaued thereafter. Fish fed 5.68 g/kg available
282
phosphorus diet showed the lowest IL-15 mRNA level. The mRNA levels of IL-4/13A, transforming growth
283
factor β1 (TGF-β1), inhibitor protein κBα (IκBα), p70 S6 kinase (S6K1) and GATA-3 were gradually
284
up-regulated as the dietary available phosphorus levels increased to 3.96, 5.68, 3.96, 3.96 and 3.96 g/kg diet,
285
respectively, simultaneously, IL-10, IL-11, TGF-β2 and TOR were all significantly up-regulated as the
286
dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and plateaued after. In the spleen,
287
the mRNA levels of IL-1β, IL-6, IFN-γ2, c-Rel and IKKβ were gradually down-regulated as the dietary
288
available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively,
289
simultaneously, IL-12p35, IL-17D, TNF-α, NF-κB p52, NF-κB p65, IKKγ, 4E-BP1 and 4E-BP2 were
290
significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 3.96, 3.96, 2.46,
291
3.96, 2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then plateaued. The IL-10, IL-11, IL-4/13A,
292
TGF-β2, IκBα, TOR, and S6K1 mRNA levels were gradually up-regulated as the dietary available
293
phosphorus levels increased to 3.96, 2.46, 3.96, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively, and
294
remained constant thereafter. Fish fed 5.68 g/kg available phosphorus diet had the highest mRNA levels of
295
TGF-β1 and GATA-3, and the lowest mRNA level of IL-15. Interestingly, dietary available phosphorus had
296
no impact on the mRNA levels of IL-8, IL-12p40, IL-4/13B and IKKα in the head kidney and spleen of
297
young grass carp. In the skin, the mRNA levels of IL-12p35, NF-κB p65, IKKα and 4E-BP2 were gradually
298
down-regulated as the dietary available phosphorus levels increased to 3.96, 3.96, 5.68 and 3.96 g/kg diet,
299
respectively, simultaneously, IL-1β, IL-6, IL-8, IL-17D, TNF-α, IFN-γ2, NF-κB p52, c-Rel, IKKβ and IKKγ
300
were significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 2.46, 3.96,
301
3.96, 3.96, 3.96, 2.46, 2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then plateaued. The mRNA
302
levels of IL-10, IL-11, IL-4/13A, TGF-β1, IκBα and TOR were gradually up-regulated as the dietary
303
available phosphorus levels increased to 3.96, 3.96, 3.96, 3.96, 2.46 and 2.46 g/kg diet, respectively, and
304
plateaued thereafter. Fish fed 5.68 g/kg available phosphorus diet showed the highest mRNA levels of
305
TGF-β2 and GATA-3, and the lowest mRNA level of IL-15. Interestingly, dietary available phosphorus had
306
no impact on the mRNA levels of IL-12p40, IL-4/13B, S6K1 and 4E-BP1.
307
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
(Fig. 3 inserted here)
308
(Fig. 4 inserted here)
309
(Fig. 5 inserted here)
310
(Fig. 6 inserted here)
ACCEPTED MANUSCRIPT
311
3.4. Immune organs structural integrity of young grass carp after infection of A. hydrophila
312
3.4.1. Antioxidant-related parameters in the immune organs The head kidney, spleen and skin oxidative status and antioxidant capacity are displayed in Table 5. In
314
the head kidney, the ROS, MDA and PC contents significantly decreased as the dietary available
315
phosphorus levels increased to 5.68 g/kg diet (P < 0.05), and there were no significant changes in the MDA
316
and PC contents thereafter (P > 0.05), but an increase was observed in the ROS content. The AHR, ASA,
317
CuZnSOD, MnSOD, GPx and GST activities as well as GSH and choline contents gradually increased as the
318
dietary available phosphorus levels increased to 5.68, 5.68, 5.68, 3.96, 3.96, 5.68, 3.96 and 2.46 g/kg diet,
319
respectively, and then plateaued. The CAT activity was the highest for fish fed 3.96 and 5.68 g/kg available
320
phosphorus diet. In the spleen, the ROS, MDA and PC contents significantly decreased as the dietary
321
available phosphorus levels increased to 5.68, 5.68 and 3.96 g/kg diet (P < 0.05), respectively, and the MDA
322
and PC plateaued, but ROS increased thereafter. The AHR, ASA, CuZnSOD, MnSOD and GPx activities as
323
well as GSH and choline contents gradually increased as the dietary available phosphorus levels increased to
324
3.96, 7.10, 2.46, 3.96, 3.96, 3.96 and 3.96 g/kg diet, respectively, and then plateaued. Fish fed 5.68 g/kg
325
available phosphorus diet had the highest GST activity. Interestingly, the GR activities significantly
326
decreased as the dietary available phosphorus levels increased to 2.46 and 3.96 g/kg diet in the head kidney
327
and spleen (P < 0.05), respectively, and then plateaued. However, dietary available phosphorus had no
328
impact on the CAT activity in the spleen of grass carp. In the skin, the ROS, MDA and PC contents
329
gradually decreased as the dietary available phosphorus levels increased to 5.68 g/kg diet, and ROS and
330
MDA increased thereafter, but PC plateaued. The AHR, ASA, CuZnSOD, MnSOD, GPx, GST and GR
331
activities as well as GSH and choline contents gradually increased as the dietary available phosphorus levels
332
increased to 3.96, 3.96, 7.10, 5.68, 3.96, 5.68, 3.96, 3.96 and 3.96 g/kg diet, respectively, and then plateaued.
333
The CAT activity was the highest for fish fed 3.96 and 5.68 g/kg diet available phosphorus.
334 335 336
AC C
EP
TE D
M AN U
SC
RI PT
313
(Table 5 inserted here) 3.4.2. Relative mRNA levels of antioxidant enzymes, Nrf2, Keap1a and Keap1b in the immune organs As shown in Fig. 7, in the head kidney, the mRNA levels of CuZnSOD, CAT, GPx1a, GPx4a and Nrf2
337
were gradually up-regulated as the dietary available phosphorus levels increased to 5.68, 3.96, 3.96, 3.96
338
and 5.68 g/kg diet, respectively, simultaneously, the MnSOD, GPx1b, GSTR and GSTO1 mRNA levels were
339
all significantly up-regulated as the dietary available phosphorus levels increased to 3.96 g/kg diet (P < 0.05),
340
and plateaued thereafter. Fish fed 5.68 g/kg available phosphorus diet showed the highest GPx4b and
341
GSTP1 mRNA levels. In the spleen, the mRNA levels of CuZnSOD, GPx1a, GPx1b, GPx4a, GPx4b,
342
GSTR and GSTP1 were gradually up-regulated as the dietary available phosphorus levels increased to 3.96
343
g/kg diet, and then plateaued. As dietary available phosphorus levels increased to 3.96, 2.46, 3.96 and 2.46
344
g/kg diet, MnSOD, CAT, GSTO1 and Nrf2 mRNA levels were significantly up-regulated (P < 0.05),
345
respectively, and remained constant thereafter. In the skin, the mRNA levels of CuZnSOD, CAT, GPx1a,
346
GPx1b, GPx4b, GSTR, GSTO1 and Nrf2 were gradually up-regulated as the dietary available phosphorus
347
levels increased to 2.46, 3.96, 3.96, 3.96, 3.96, 2.46, 3.96 and 3.96 g/kg diet, respectively, simultaneously,
348
the MnSOD, GPx4a, GSTP1 and GR mRNA levels were significantly up-regulated as the dietary available
349
phosphorus levels increased to 2.46, 3.96, 5.68 and 2.46 g/kg diet (P < 0.05), respectively, and plateaued
350
after. The mRNA levels of Keap1a were all down-regulated as the dietary available phosphorus level
351
increased to 5.68 g/kg diet in the head kidney, spleen and skin, and then plateaued. Interestingly, in the head
352
kidney and spleen, the GR expression was significantly down-regulated as the dietary available phosphorus
353
level increased to 5.68 and 3.96 g/kg diet (P < 0.05), respectively, and then kept unchanged. In the head
354
kidney, spleen and skin, dietary available phosphorus had no impact on the mRNA levels of Keap1b.
356
RI PT
SC
M AN U
TE D
(Fig. 7 inserted here)
EP
355
ACCEPTED MANUSCRIPT
3.4.3. Relative mRNA levels of apoptosis-related parameters in the immune organs The effects of dietary available phosphorus on apoptosis and related signalling molecules in the head
358
kidney, spleen and skin of young grass carp are presented in Fig. 8. In the head kidney, the mRNA levels of
359
Apaf-1, caspase-2 and -7 were gradually down-regulated as the dietary available phosphorus levels
360
increased to 5.68, 3.96 and 3.96 g/kg diet, respectively, simultaneously, FasL, Bax, caspase-3, -8, -9 and p38
361
MAPK were significantly down-regulated as the dietary available phosphorus levels increased to 5.68, 5.68,
362
3.96, 2.46, 3.96 and 3.96 g/kg diet (P < 0.05), respectively, and plateaued thereafter. The mRNA levels of
363
Bcl-2, Mcl-1 and IAP were gradually up-regulated as the dietary available phosphorus levels increased to
364
3.96, 5.68 and 5.68 g/kg diet, respectively, and plateaued thereafter. In the spleen, the mRNA levels of
365
Apaf-1, caspase-7 and -9 were gradually down-regulated as the dietary available phosphorus levels
AC C
357
366
increased to 5.68, 3.96 and 3.96 g/kg diet, respectively, simultaneously, FasL, Bax, caspase-2, -3 and p38
367
MAPK were significantly down-regulated as the dietary available phosphorus levels increased to 3.96, 2.46,
368
2.46, 3.96 and 2.46 g/kg diet (P < 0.05), respectively, and then remained constant. Fish fed 5.68 g/kg
369
available phosphorus diet showed the lowest caspase-8 mRNA level. The mRNA levels of Bcl-2, Mcl-1 and
370
IAP were gradually up-regulated as the dietary available phosphorus levels increased to 3.96, 3.96 and 2.46
371
g/kg diet, respectively, and then plateaued. However, dietary available phosphorus had no influence on the
372
mRNA levels of JNK in the head kidney and spleen. In the skin, the mRNA levels of FasL, Bax, Apaf-1,
373
caspase-3, -7 and -8 were significantly down-regulated as the dietary available phosphorus levels increased
374
to 3.96, 3.96, 5.68, 3.96, 2.46 and 3.96 g/kg diet (P < 0.05), respectively, simultaneously, caspase-9 and JNK
375
were both gradually down-regulated as the dietary available phosphorus levels increased to 3.96 g/kg diet,
376
and plateaued thereafter. The mRNA levels of Bcl-2, Mcl-1 and IAP were significantly up-regulated as the
377
dietary available phosphorus levels increased to 2.46, 3.96 and 3.96 g/kg diet (P < 0.05), respectively, and
378
then plateaued.
380
RI PT
SC
M AN U
379
ACCEPTED MANUSCRIPT
(Fig. 8 inserted here)
3.4.4. Relative mRNA levels of TJs, transporter and MLCK in the immune organs The effects of dietary available phosphorus on TJs, type-IIb sodium-phosphate cotransporter (NaPi-IIb)
382
and MLCK relative mRNA levels in the head kidney, spleen and skin are presented in Fig 9. In the head
383
kidney, the mRNA levels of occludin, claudin-f, -7a, -11 and -15a were gradually up-regulated as the dietary
384
available phosphorus levels increased to 3.96, 3.96, 3.96, 2.46 and 3.96 g/kg diet, respectively,
385
simultaneously, ZO-1, ZO-2 and claudin-c were significantly up-regulated as the dietary available
386
phosphorus levels increased to 3.96, 2.46 and 3.96 g/kg diet (P < 0.05), respectively, and plateaued after.
387
The mRNA levels of claudin-12, NaPi-IIb and MLCK were all significantly down-regulated as the dietary
388
available phosphorus levels increased to 3.96 g/kg diet (P < 0.05), and then plateaued. The mRNA level of
389
claudin-7b showed the highest for fish fed 5.68g/kg available phosphorus diet. In the spleen, as dietary
390
available phosphorus levels increased to 3.96, 2.46, 3.96, 3.96, 2.46, 2.46, 2.46 and 3.96 g/kg diet, the
391
mRNA levels of occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -11 and -15a were gradually up-regulated,
392
respectively, and plateaued thereafter. The mRNA levels of claudin-12, NaPi-IIb and MLCK were
393
down-regulated as the dietary available phosphorus levels increased to 2.46, 3.96 and 2.46 g/kg diet,
394
respectively, and then plateaued. Fish fed 5.68 g/kg available phosphorus diet had the highest claudin-7b
AC C
EP
TE D
381
395
mRNA level. Interestingly, dietary available phosphorus had no impacts on the mRNA levels of claudin-b,
396
-3c and -15b in the head kidney and spleen. In the skin, the mRNA levels of occludin, ZO-1, ZO-2,
397
claudin-b, -c, -f, -7a, -11 and -15a were up-regulated as the dietary available phosphorus levels increased to
398
3.96, 2.46, 3.96, 3.96, 3.96, 3.96, 3.96, 5.68 and 3.96 g/kg diet, respectively, and plateaued after. As the
399
dietary available phosphorus levels increased to 3.96, 2.46 and 2.46 g/kg diet, the mRNA levels of
400
claudin-12, NaPi-IIb and MLCK were significantly down-regulated (P < 0.05), respectively, and then
401
plateaued. Fish fed 5.68 g/kg available phosphorus diet had highest mRNA level of claudin-7b. Interestingly,
402
dietary available phosphorus had no impacts on the mRNA levels of claudin-3c and -15b.
RI PT
403
ACCEPTED MANUSCRIPT
(Fig. 9 inserted here) 4. Discussion
405
4.1. Phosphorus deficiency decreased the growth performance of fish
SC
404
This study indicated that dietary phosphorus deficiency decreased the growth performance (PWG, FI,
407
FE and SGR) and immune organs size (head kidney and spleen weight) of young grass carp. The serum
408
phosphorus concentration and AKP activity were used as indicators of the phosphorus nutrition status in fish
409
[36]. In the present study, phosphorus deficiency decreased the serum phosphorus concentration and AKP
410
activity, indicating that phosphorus deficiency worsened the nutrition status. Based on the broken-line
411
analysis of the PWG, the available phosphorus requirement for young grass carp was estimated to be 4.10
412
g/kg diet, which was consistent with the result of our previous study [36]. Fish growth is closely related to
413
disease resistance [59], which is partly associated with the ability to combat skin hemorrhage and lesions,
414
immune function and structural integrity in the immune organs of fish [11, 23]. A. hydrophila is one of the
415
predominant pathogens in freshwater fish, which causes fish skin hemorrhage and lesions as well as kidney
416
and spleen immune function depression and integrity damage [10, 45]. Thus, in this study, after the feeding
417
trial, the fish were infected with A. hydrophila to investigate the effects of phosphorus on the ability to
418
combat skin hemorrhage and lesions, immune function and structural integrity in the immune organs of
419
young grass carp.
420
4.2. Phosphorus deficiency decreased the head kidney and spleen immune function partly relating to
421
NF-κB and TOR signalling in fish after infection with A. hydrophila
422
4.2.1. Phosphorus deficiency reduced the head kidney and spleen antibacterial substances of fish
423
AC C
EP
TE D
M AN U
406
The immune function of fish largely relies on antibacterial substances, which consist of LZ, ACP, IgM,
424
complement factors and antimicrobial peptides [60]. Thus, we first investigated the effect of phosphorus on
425
antibacterial substances in the head kidney and spleen of fish, and concluded that, compared with optimal
426
phosphorus level, phosphorus deficiency decreased the LZ and ACP activities, and the C3, C4 and IgM
427
contents and down-regulated antimicrobial peptides, including LEAP-2A, LEAP-2B, hepcidin and
428
β-defensin-1 mRNA levels, implying that phosphorus deficiency depressed the immune function in the head
429
kidney and spleen of fish. Fish immune function is closely associated with the inflammation response, which
430
are primarily mediated by cytokines [56]. Therefore, we next investigated the relationships between the
431
dietary available phosphorus and the cytokines in the head kidney and spleen of young grass carp.
432
4.2.2. Phosphorus deficiency aggravated inflammatory responses partly relating to NF-κB and TOR
433
signalling in the head kidney and spleen of fish
SC
RI PT
ACCEPTED MANUSCRIPT
The inflammatory response is often regulated by cytokines, which include pro-inflammatory cytokines,
435
such as IL-1β, IL-6, IFN-γ and TNF-α, in humans [61], and anti-inflammatory cytokines, such as IL-4,
436
IL-10, IL-11 and TGF-β, in rats [62]. In teleost, the up-regulation of pro-inflammatory cytokines and the
437
down-regulation of anti-inflammatory cytokines initiates and accelerates additional inflammatory processes
438
[63]. In this study, compared with optimal phosphorus level, phosphorus deficiency up-regulated the
439
pro-inflammatory cytokines IL-1β, IL-6, IL-12p35, IL-15, IL-17D, IFN-γ2 and TNF-α mRNA levels and
440
down-regulated the anti-inflammatory cytokines IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA levels
441
in the head kidney and spleen of young grass carp. These results implied that phosphorus deficiency might
442
induce inflammatory responses in the head kidney and spleen of fish. Interestingly, phosphorus deficiency
443
had no effects on the mRNA levels of IL-8, IL-12p40 and IL-4/13B in the head kidney and spleen of young
444
grass carp.
AC C
EP
TE D
M AN U
434
445
First, the IL-8 mRNA levels unaffected by phosphorus deficiency in the head kidney and spleen of
446
young grass carp may be related to the unaffected JNK. The inhibition of JNK reduces IL-8 mRNA level in
447
EAGS cells [64]. In this study, phosphorus deficiency had no impacts on the JNK mRNA levels in the head
448
kidney and spleen, supporting our hypothesis.
449
Second, phosphorus deficiency up-regulated IL-12p35 (rather than IL-12p40) mRNA levels in the head
450
kidney and spleen of young grass carp, which may be partially associated with IL-1β. IL-1β could
451
up-regulates IL-12p35 mRNA level but has no influence on the mRNA level of IL-12p40 in the mature DCs
452
[65]. Our study showed that phosphorus deficiency elevated the mRNA levels of IL-1β in the head kidney
453
and spleen. A further correlation analysis showed that IL-12p35 mRNA levels were positively related to
454
IL-1β mRNA levels in the head kidney and spleen of young grass carp (Table 6), supporting our hypothesis.
ACCEPTED MANUSCRIPT
Lastly, phosphorus deficiency down-regulated the mRNA levels of IL-4/13A (rather than IL-4/13B) in
456
the head kidney and spleen of young grass carp, which may be related to GATA-3. The inhibition of
457
GATA-3 down-regulates the gene transcription of IL-4/13A by reducing the binding with a TATA box [66]
458
which is not found in IL-4/13B in puffer (Tetraodon nigroviridis) [67, 68]. In the present study, phosphorus
459
deficiency down-regulated the GATA-3 mRNA levels in the head kidney and spleen of young grass carp.
460
According to the above studies, phosphorus deficiency down-regulating GATA-3 expression might lead to
461
the down-regulation of IL-4/13A (rather than IL-4/13B) mRNA levels in the head kidney and spleen of fish.
RI PT
455
In human mast cells, the expression of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8
463
was dependent on the activation of the transcription factor NF-κB [69]. NF-κB p65, NF-κB p52 and c-Rel
464
are important members in the NF-κB family [70]. In HeLa and 293 cells, IKKα, IKKβ and IKKγ form the
465
IKK complexes and phosphorylate IκBs which activate NF-κB [71]. In the present study, compared with
466
optimal phosphorus level, phosphorus deficiency up-regulated NF-κB p65, NF-κB p52, c-Rel, IKKβ and
467
IKKγ mRNA levels and down-regulated IκBα mRNA levels in the head kidney and spleen of young grass
468
carp. Correlation analysis indicated that the pro-inflammatory cytokines IL-1β, IL-6, IL-12p35 (rather than
469
IL-12p40), IL-15, IL-17D, IFN-γ2 and TNF-α mRNA levels were positively related to NF-κB p65, NF-κB
470
p52 and c-Rel mRNA levels (Table 6). These results implied that phosphorus deficiency up-regulated
471
pro-inflammatory cytokines (except IL-8 and IL-12p40) mRNA levels, which may be partly referred to
472
(IKKβ, IKKγ) /IκBα/ (NF-κB p65, NF-κB p52 and c-Rel) in the head kidney and spleen of fish.
EP
TE D
M AN U
SC
462
Interestingly, we found that phosphorus deficiency up-regulated IKKβ and IKKγ (but not IKKα) in the
474
head kidney and spleen of young grass carp, and this interesting phenomenon might be correlated with
475
phospholipids. It was reported that dietary phospholipids in an un-supplemented group up-regulate the
476
expression of IKKβ and IKKγ (but not IKKα) in the intestine of juvenile grass carp [72]. Phosphorus
477
deficiency reduces the synthesis of phospholipid [33]. Hence, we hypothesize that phosphorus deficiency
478
might decrease phospholipids content, leading to up-regulated IKKβ and IKKγ (but not IKKα) expression in
479
the head kidney and spleen of fish. However, this hypothesis requires further investigation.
AC C
473
480
mTOR enhances the anti-inflammatory cytokine IL-10 activity in human PBMCs [73]. In mammals,
481
mTOR directly phosphorylates S6K1 and inhibits the initiation factor 4E-BP to initiate the translation of
482
distinct mRNAs [74]. In the present study, compared with the optimal phosphorus level, phosphorus
483
deficiency down-regulated TOR and S6K1 mRNA levels, and up-regulated 4E-BP1 and 4E-BP2 mRNA
484
levels in the head kidney and spleen of young grass carp. Correlation analysis showed that the mRNA levels
485
of the anti-inflammatory cytokines IL-4/13A (rather than IL-4/13B), IL-10, IL-11, TGF-β1 and TGF-β2
486
were positively correlated with TOR gene expression (Table 6), suggesting that phosphorus deficiency
487
down-regulated anti-inflammatory cytokines (except IL-4/13B) gene expression, which might be partly
488
correlated to the TOR/ (S6K1, 4E-BP) signalling pathway in the head kidney and spleen of fish.
RI PT
ACCEPTED MANUSCRIPT
In summary, our data first implied that phosphorus deficiency decreased fish head kidney and spleen
490
immune function, which may be related to two aspects: (1) reduced LZ and ACP activities, C3, C4 and IgM
491
contents and the antimicrobial peptides LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 expression; (2)
492
up-regulated mRNA levels of the pro-inflammatory cytokines (except IL-8) IL-1β, IL-6, IL-12p35 (rather
493
than IL-12p40), IL-15, IL-17D, IFN-γ2 and TNF-α, and down-regulated mRNA levels of the
494
anti-inflammatory cytokines IL-4/13A (rather than IL-4/13B), IL-10, IL-11, TGF-β1 and TGF-β2, which
495
might be partly related to the IKKβ, IKKγ (rather than IKKα) /IκBα/NF-κB and TOR/ (S6K1, 4E-BP)
496
signalling pathways. In addition, the immune function of the immune organs is also correlated with the
497
structural integrity of these organs in fish [48]. Thus, we next investigated the effects of phosphorus on the
498
structural integrity in the head kidney and spleen of young grass carp.
M AN U
(Table 6 inserted here)
TE D
499
SC
489
4.3. Phosphorus deficiency attenuated the structural integrity relating to Nrf2, p38 MAPK and MLCK
501
signalling in the head kidney and spleen of fish after infection with A. hydrophila
502
4.3.1. Phosphorus deficiency induced oxidative damage and impaired the antioxidant system partly by
503
Nrf2 signalling in the head kidney and spleen of fish
AC C
EP
500
504
Infection with pathogens is often associated with an increase in the free radical content, which may lead
505
to oxidative damage [75] and then impair structural integrity in fish [37]. In the present study, compared
506
with the optimal phosphorus level, phosphorus deficiency increased ROS, MDA and PC contents and
507
decreased the ASA, AHR, CuZnSOD, MnSOD, GPx and GST activities and GSH contents in the head
508
kidney and spleen, and decreased the CAT activity only in the head kidney of young grass carp, indicating
509
that phosphorus deficiency induced oxidative damage by increasing the free radical production and
510
decreasing the antioxidant ability in fish. In addition, fish antioxidant enzyme activities were partly
511
dependent on their gene expression [53]. Thus, we next explored the effects of phosphorus on antioxidant
512
enzyme gene expression in the head kidney and spleen of young grass carp. In this study, compared with the
513
optimal phosphorus level, phosphorus deficiency down-regulated CuZnSOD, MnSOD, CAT, GPx1a, GPx1b,
514
GPx4a, GPx4b, GSTR, GSTO1 and GSTP1 expression in the head kidney and spleen of young grass carp.
515
These results indicate that phosphorus deficiency decreased those enzyme activities, which might partly
516
contribute to their down-regulated mRNA levels in the head kidney and spleen of fish. Surprisingly, we
517
found that phosphorus deficiency promoted GR activities and mRNA levels in the head kidney and spleen
518
and had no effect on the activity of CAT in the spleen of young grass carp.
RI PT
ACCEPTED MANUSCRIPT
First, phosphorus deficiency-enhanced GR activities and expression might be partially due to an
520
adaptive mechanism against the declined GSH contents. It was reported that GR catalyzes the reduction in
521
GSSG back to GSH in mammalian tissues [76]. Our data showed that phosphorus deficiency decreased the
522
GSH contents in the head kidney and spleen of young grass carp. According to the above data, the
523
phosphorus deficiency-decreased GSH contents might cause adaptive increases in the GR activities and
524
mRNA levels in the head kidney and spleen of fish.
M AN U
SC
519
Moreover, phosphorus deficiency did not influence the activity of CAT in the spleen (not the head
526
kidney) of young grass carp, which may be explained by the similar function of CAT and GPx. It was
527
reported that GPx and CAT both participated in the reduction of H2O2 in fish [77, 78]. In our study, dietary
528
phosphorus enhanced the GPx activities in the head kidney and spleen, where the GPx activity in the spleen
529
was higher than that in head kidney. Hence, the stable CAT activity in the spleen (not head kidney) as a
530
result of phosphorus deficiency might due to a higher GPx activity in the spleen than that in the head kidney
531
of fish.
AC C
EP
TE D
525
532
In addition, the antioxidant enzyme transcription factor Nrf2 is released by the repression of Keap1 in
533
zebrafish (Danio rerio) [79]. In the current study, compared with the optimal phosphorus level, phosphorus
534
deficiency down-regulated Nrf2 mRNA levels and up-regulated Keap1a mRNA levels in the head kidney
535
and spleen of young grass carp. Correlation analysis showed that the mRNA levels of CuZnSOD, MnSOD,
536
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTO1 and GSTP1 in the head kidney and spleen were
537
positively correlated with the Nrf2 mRNA levels which were negatively related to the mRNA levels of
538
Keap1a (Table 6). These results indicated that phosphorus deficiency down-regulated the antioxidant
539
enzyme mRNA levels partly by inhibiting Nrf2 gene transcription, which was regulated by Keap1a in the
540
head kidney and spleen of fish. Interestingly, phosphorus deficiency had no effect on the Keap1b mRNA
541
levels in the head kidney and spleen of young grass carp.
ACCEPTED MANUSCRIPT
Phosphorus deficiency up-regulated the mRNA levels of Keap1a (rather than Keap1b) in the head
543
kidney and spleen of young grass carp, which may be concerned with choline deficiency. A study from our
544
lab found that choline deficiency up-regulated Keap1a but had no impact on the Keap1b mRNA levels in the
545
gills of grass carp [80]. In our study, phosphorus deficiency decreased the choline content in the head kidney
546
and spleen of young grass carp, supporting our hypothesis.
RI PT
542
In summary, the results of current study first indicated that phosphorus deficiency induced oxidative
548
damage and impaired cellular integrity, which might be partly modulated by Keap1a (rather than Keap1b)
549
/Nrf2 pathway, leading to down-regulated mRNA levels of the antioxidant enzyme CuZnSOD, MnSOD,
550
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTO1 and GSTP1, and weaken the antioxidant enzyme
551
CuZnSOD, MnSOD, CAT (except spleen), GPx and GST activities in the head kidney and spleen of fish.
552
Moreover, a study showed that oxidative damage could induce cell apoptosis in SH-SY5Y cells, which was
553
partly regulated by p38 MAPK [27]. Hence, we further examined the effects of phosphorus on apoptosis in
554
the head kidney and spleen of young grass carp.
555
4.3.2. Phosphorus deficiency induced apoptosis partly relating to p38 MAPK signalling in the head
556
kidney and spleen of fish
TE D
M AN U
SC
547
Caspases, which are closely associated with apoptosis, based on their function, are divided into two
558
categories, the initiator caspases (including caspase-2, -8, -9) and the effector caspases (including caspase-3,
559
-7) [81]. Two major apoptosis pathways, the death receptor pathway (FasL/caspase-8) and the mitochondria
560
pathway [(Bcl-2, Mcl-1 and Bax)/Apaf-1/caspase-9], have been defined in mammalian cells [82]. The IAP
561
inhibits caspase in human cells [83]. In the present study, compared with the optimal phosphorus level,
562
phosphorus deficiency up-regulated caspase-2, -3, -7, -8 and -9, pro-apoptotic FasL, Apaf-1 and Bax mRNA
563
levels, and down-regulated anti-apoptotic Bcl-2, Mcl-1 and IAP mRNA levels in the head kidney and spleen
564
of young grass carp. Correlation analysis showed that the mRNA levels of FasL were positively correlated
565
with caspase-8; and caspase-9 mRNA levels were positively correlated with pro-apoptotic Apaf-1 and Bax
566
mRNA levels, while were negatively correlated with the anti-apoptotic Bcl-2, Mcl-1 and IAP mRNA levels;
567
and caspase-3, -7 were positively correlated with caspase-9 in the head kidney and spleen (Table 6),
568
suggesting that phosphorus deficiency induces apoptosis in the head kidney and spleen of fish. In addition,
AC C
EP
557
569
p38 MAPK and JNK mediates apoptosis in human hepatoma cells [84]. In the present study, compared with
570
the optimal phosphorus level, phosphorus deficiency up-regulated p38 MAPK mRNA levels in the head
571
kidney and spleen of young grass carp. Correlation analysis showed that p38 MAPK mRNA levels were
572
positively correlated with pro-apoptotic FasL, Apaf-1 and Bax, while they were negatively correlated with
573
the anti-apoptotic Bcl-2 and Mcl-1 mRNA levels in the head kidney and spleen of young grass carp (Table
574
6), suggesting that phosphorus deficiency-induced apoptosis is partly associated with p38 MAPK in the head
575
kidney and spleen of fish. Interestingly, phosphorus had no effect on the JNK expression in the head kidney
576
and spleen of young grass carp. This phenomenon might be partly correlated with the unchanged IKKα.
577
Yuan et al. reported that IKKα inhibited the activation of JNK in human epithelial cells [85]. Our study
578
showed that phosphorus deficiency had no impact on IKKα expression in the head kidney and spleen,
579
supporting our hypothesis.
SC
RI PT
ACCEPTED MANUSCRIPT
In summary, the above findings first implied that phosphorus deficiency induced apoptosis and
581
impaired cellular integrity, which was partly associated with p38 MAPK (rather than JNK), leading to the
582
activation of the death receptor pathway (FasL/caspase-8) and the mitochondria pathway [(Bcl-2, Mcl-1 and
583
Bax)/Apaf-1/caspase-9] in the head kidney and spleen of fish. In addition, the structural integrity of the
584
organs is also generally correlated with the intercellular tight junctions in epithelial cells [86]. Hence, we
585
further examined the effects of phosphorus on TJs in the head kidney and spleen of young grass carp.
586
4.3.3. Phosphorus deficiency weakened intercellular integrity partly relating to MLCK signalling in the
587
head kidney and spleen of fish
EP
TE D
M AN U
580
TJs (like occludin, ZO-1 and claudins) are responsible for intercellular integrity in mouse kidneys [87],
589
which includes barrier-forming and pore-forming TJs [88]. Ma et al. reported that TJ barrier function was
590
prevented by MLCK in Caco-2 cells [89]. Thus, we first investigated the effects of phosphorus on TJs in the
591
head kidney and spleen of fish and presented that phosphorus deficiency down-regulated the mRNA levels
592
of occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -7b and -15a and up-regulated the mRNA levels of the
593
pore-forming TJ claudin-12 and MLCK. Correlation analysis displayed that the mRNA levels of these TJs
594
were negatively (claudin-12 was positively) related to the MLCK mRNA levels in the head kidney and
595
spleen of young grass carp (Table 6), suggesting that phosphorus deficiency might up-regulate MLCK
596
mRNA levels to damage tight junction function and weaken the intercellular integrity in the head kidney and
597
spleen of fish. Interestingly, in this study, phosphorus deficiency had no impact on the mRNA levels of
AC C
588
598
claudin-b, -3c and -15b in the head kidney and spleen of young grass carp.
599
First, phosphorus deficiency had no effect on the mRNA levels of claudin-b and -3c in the head kidney
600
and spleen of young grass carp, which may be all correlated with cortisol influenced by IL-6. In humans,
601
IL-6 increases plasma cortisol levels [90]. Cortisol had no effect on the mRNA levels of claudin-b in the
602
goldfish (Carassius auratus) [91] and claudin-3c in puffer [92] gill epithelia cells. In this study, phosphorus
603
deficiency up-regulated the expression of IL-6 in the head kidney and spleen of young grass carp. Therefore,
604
we presume that phosphorus deficiency up-regulated IL-6 expression leading to the rise of cortisol levels,
605
while cortisol might have no impact on claudin-b and -3c in the head kidney and spleen of fish. However,
606
further investigation should be conducted to support this supposition.
RI PT
ACCEPTED MANUSCRIPT
In addition, phosphorus deficiency down-regulated claudin-15a mRNA levels but had no impact on
608
claudin-15b in the head kidney and spleen of young grass carp. This interesting phenomenon may be related
609
to the same function of type-IIb sodium-phosphate cotransporter (NaPi-IIb) and claudin-15a. In fish,
610
NaPi-IIb is a phosphorus transporter that mediates the transport of Na+ [93]. It was reported that claudin-15a
611
(rather than claudin-15b) plays an important role in Na+ transport in Japanese medaka (Oryziaslatipes) [94].
612
In the present study, phosphorus deficiency up-regulated NaPi-IIb mRNA levels in the head kidney and
613
spleen. According to these data, phosphorus deficiency up-regulated NaPi-II mRNA levels to enhance Na+
614
transport, which might weaken claudin-15a function leading to the down-regulation of claudin-15a (rather
615
than claudin-15b) in the head kidney and spleen of fish.
TE D
M AN U
SC
607
In summary, the above data indicated for the first time that phosphorus deficiency damaged tight
617
junction function and weakened intercellular integrity partly correlating with MLCK to down-regulate the
618
mRNA levels of the TJs (except claudin-b, -3c and -15b) occludin, ZO-1, ZO-2, claudin-c, -f, -7a, -7b and
619
-15a and up-regulate the mRNA levels of pore-forming TJ claudin-12 in the head kidney and spleen of fish.
620
The underlying mechanism requires further investigation.
621
4.4. Phosphorus deficiency decreased the immune function and attenuated the structural integrity in the
622
skin of fish compared with the head kidney and spleen
AC C
EP
616
623
The skin is the structure that covers the body and protects it not only from the entry of pathogens or
624
allergens but also from the leakage of water, solutes, or nutrients, which are related to the immune function
625
and structural integrity of fish skin [4]. Grass carp infected with A. hydrophila might have a skin
626
hemorrhage and lesions [45]. In this study, compared with the optimal phosphorus level, phosphorus
627
deficiency increased the skin hemorrhage and lesions morbidity of young grass carp after infection with A.
628
hydrophila (Figs 1&2), suggesting that phosphorus deficiency decreases the defence against A. hydrophila
629
in the skin of fish. Based on the broken-line analysis of the ability to combat skin hemorrhage and lesions,
630
the dietary available phosphorus requirement of young grass carp was estimated to be 4.13 g/kg diet.
ACCEPTED MANUSCRIPT
In the skin of young grass carp, except for 4E-BP1 and S6K1, the effects of phosphorus on antibacterial
632
substances, cytokines, antioxidant, apoptosis and TJs related parameters models were similar to those in the
633
head kidney and spleen. Interestingly, phosphorus deficiency had no impact on the mRNA levels of 4E-BP1
634
and S6K1 in the skin. The mRNA level of 4E-BP1 unaffected by phosphorus deficiency in the skin may be
635
correlated with insulin. A study reported that insulin had no effect on the 4E-BP1 levels in the mouse skin
636
JB6 Cl 41 cells [95]. In humans, a low level of phosphorus, causing hypophosphatemia, increases the serum
637
insulin level [96]. Hence, in the condition of phosphorus deficiency, the unaffected mRNA levels of 4E-BP1
638
might due to insulin, which might have no effect on 4E-BP1 mRNA level in the skin of fish. In addition, the
639
stable mRNA level of S6K1 by phosphorus deficiency in the skin may be associated with Akt1. A study
640
reported that the knockout of Akt1, which is predominantly expressed in the skin, had no effect on S6K1
641
levels in mice embryo fibroblasts [97]. Meanwhile, phosphorus deficiency depressed Akt1 levels in NHBE
642
cells [20]. Hence, the stable mRNA level of S6K1 by phosphorus deficiency may be related to Akt1 which
643
was highly expressed in the skin and might have no effect on S6K1 levels in the skin of fish. However, these
644
further reasons need deeper investigation.
645
4.5. Interesting results: high levels of phosphorus impaired some indices that differed from the traditional
646
growth results
EP
TE D
M AN U
SC
RI PT
631
Based on the function of the kidney (metanephros in fish) in the excretion of phosphorus, no negative
648
effects on the growth performances were observed in this study and the previous studies in juvenile grass
649
carp and juvenile Jian carp when high doses were administered [8, 9]. Interestingly, in the present study,
650
compared with the optimal phosphorus level, high levels of phosphorus increased the ROS contents in the
651
head kidney and spleen, and down-regulated the mRNA levels of GPx4b and GSTP1 in the head kidney,
652
anti-inflammatory cytokine TGF-β1 in the spleen, and up-regulated the mRNA levels of caspase-8 in the
653
head kidney, the pro-inflammatory cytokine IL-15 in the head kidney and spleen after infection with A.
654
hydrophila. First, high levels of phosphorus increased the ROS contents in the head kidney and spleen,
655
which may be partly related to NADPH oxidase. A study displayed that NADPH oxidase induced ROS
AC C
647
656
production in BAECs [98]. High levels of phosphorus increased NADPH oxidase activity in bovine aortic
657
endothelial cells [99]. Second, high levels of phosphorus down-regulated the mRNA levels of GPx4b and
658
GSTP1 and up-regulated IL-15 and caspase-8, which may be partly due to the rise of ROS contents. It was
659
reported that ROS down-regulated the mRNA levels of GPx4b and GSTP1 in grass carp [10] and increased
660
IL-15 levels in human mononuclear cells [100] and caspase-8 levels in DU-145 cells [101]. As mentioned
661
above, our study displayed that a high level of phosphorus increased the ROS contents in the head kidney
662
and spleen. Furthermore, high levels of phosphorus down-regulated TGF-β1 in the spleen partly by IL-15. A
663
study displayed that IL-15 inhibits TGF-β1 expression in human T lymphocytes [102]. Meanwhile, in this
664
study, a high level of phosphorus up-regulated IL-15 expression in the spleen of young grass carp. However,
665
the detail reasons need a deeper investigation.
666
4.6. Comparisons of the optimal dietary available phosphorus levels for young grass carp based on the
667
different indices
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
In this study, after infection with A. hydrophila, phosphorus deficiency increased the skin hemorrhage
669
and lesions morbidity and impaired the immune function and structural integrity in the immune organs of
670
fish. Thus, an evaluation of phosphorus requirement is quite necessary in fish. As shown in Table 7, based
671
on the growth performance (PWG and FI), the available phosphorus requirements for young grass carp were
672
estimated to be 4.10 and 3.89 g/kg diet, respectively. Meanwhile, the available phosphorus requirement for
673
ability to combat skin hemorrhage and lesions was estimated to be 4.13 g/kg diet, suggesting that the
674
available phosphorus requirement for ability to combat skin hemorrhage and lesions was close to that based
675
on the growth performance. Our further investigation on fish immune function and structural integrity
676
indicated that the available phosphorus requirements for young grass carp (254.56-898.23 g), according to
677
the immune indices (head kidney LZ activity and spleen C4 content) and the antioxidant indices (head
678
kidney and spleen MDA contents), were estimated to be 5.71, 5.57, 5.54 and 5.40 g/kg diet, respectively.
679
The results implied that the available phosphorus requirements for young grass carp based on the immune
680
and antioxidant indices were higher than those based on growth performance, indicating that more
681
phosphorus is required for enhancing immune function and improving structural integrity in the immune
682
organs of fish. In addition, the available phosphorus requirements for young grass carp based on immune
683
and antioxidant indices higher than that based on the ability to combat skin hemorrhage and lesions, which
684
may be associated with the function of VD. In humans, the skin is an important place for VD to synthesize
AC C
EP
TE D
668
685
which could increase serum phosphorus concentrations [103], indicating that no more phosphorus is
686
required for skin of fish.
687 688
ACCEPTED MANUSCRIPT
(Table 7 inserted here) 5. Conclusions In summary (Fig. 10), on the basis of our previous study about phosphorus on the growth performance
690
of young grass carp, the present study first demonstrated that phosphorus deficiency decreased the immune
691
function in the head kidney and spleen of fish indicated by: (1) depressed LZ and ACP activities, C3, C4 and
692
IgM contents, and antimicrobial peptides LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 mRNA levels; (2)
693
and enhanced IKKβ, IKKγ (rather than IKKα)/IκBα/NF-κB signalling pathway to up-regulate the mRNA
694
levels of the pro-inflammatory cytokines (except IL-8 and IL-12p40); (3) and repressed the
695
TOR/(S6K1,4E-BP) signalling pathway to down-regulate the mRNA levels of the anti-inflammatory
696
cytokines (except IL-4/13B). Simultaneously, phosphorus deficiency attenuated the structural integrity in the
697
head kidney and spleen of fish displayed by the following aspects: (1) a modulated Keap1a (rather than
698
Keap1b)/Nrf2 signalling pathway to reduce the antioxidant enzyme gene expressions and activities (but
699
elevate GR, and except CAT activity in the spleen), and thus induced oxidative damage and impaired
700
cellular integrity; (2) up-regulated p38 MAPK mRNA levels to activate the death receptor pathway and the
701
mitochondria pathway, which induced apoptosis and impaired cellular integrity; (3) promoted MLCK
702
expression to down-regulate TJs gene expressions (not including claudin-b, -3c and -15b, and up-regulate
703
claudin-12), which damaged TJs function and weakened intercellular integrity. Meanwhile, phosphorus
704
deficiency increased skin hemorrhage and lesions morbidity, and impaired immune function and structural
705
integrity in the skin of young grass carp. Lastly, based on the PWG and the ability to combat skin
706
hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp (254.56-898.23
707
g) were estimated to be 4.10 and 4.13 g/kg diet, respectively.
708 709
AC C
EP
TE D
M AN U
SC
RI PT
689
(Fig. 10 inserted here) Acknowledgements
710
This research was financially supported by the National Basic Research Program of China (973
711
Program) (2014CB138600), National Department Public Benefit Research Foundation (Agriculture) of
712
China (201003020), Outstanding Talents and Innovative Team of Agricultural Scientific Research (Ministry
713
of Agriculture), Science and Technology Support Program of Sichuan Province of China (2014NZ0003),
714
Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China
715
(2012NC0007; 2013NC0045), The Demonstration of Major Scientific and Technological Achievement
716
Transformation Project of Sichuan Province of China (2015CC0011) and Natural Science Foundation for
717
Young Scientists of Sichuan Province (2014JQ0007). The authors would like to thank the personnel of these
718
teams for their kind assistance.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
719
720
References
721
[1] M.G. Bondad-Reantaso, R.P. Subasinghe, J.R. Arthur, K. Ogawa, S. Chinabut, R. Adlard, et al., Disease and health
722
MANUSCRIPT management in Asian aquaculture ☆, Vet.ACCEPTED Parasitol. 132 (2005) 249-272.
723
[2] P. Li, Y.-L. Yin, D. Li, S.W. Kim, G. Wu, Amino acids and immune function, Brit. J. Nutr 98 (2007) 237-252.
724
[3] C. Uribe, H. Folch, R. Enriquez, G. Moran, Innate and adaptive immunity in teleost fish: a review, Vet. Med-Czech 56
725
(2011) 486-503.
726
[4] M.Á. Esteban, An overview of the immunological defenses in fish skin, Isrn Immunol. 2012 (2012).
727
[5] G. Baeverfjord, T. Asgard, K. Shearer, Development and detection of phosphorus deficiency in Atlantic salmon, Salmo
729 730 731 732
salar L., parr and post-smolts, Aquac. Nutr. 4 (1998) 1-12. [6] P.G. Fjelldal, T. Hansen, S. Albrektsen, Inadequate phosphorus nutrition in juvenile Atlantic salmon has a negative effect on long-term bone health, Aquaculture 334 (2012) 117-123.
RI PT
728
[7] P.K. Roy, S.P. Lall, Dietary phosphorus requirement of juvenile haddock (Melanogrammus aeglefinus L.), Aquaculture 221 (2003) 451-468.
[8] N.B. Xie, L. Feng, Y. Liu, J. Jiang, W.D. Jiang, K. Hu, et al., Growth, body composition, intestinal enzyme activities
734
and microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary phosphorus, Aquac. Nutr. 17
735
(2011) 645-656.
737
[9] J.J. Liang, Y.J. Liu, L.X. Tian, H.J. Yang, G.Y. Liang, Dietary available phosphorus requirement of juvenile grass carp
M AN U
736
SC
733
(Ctenopharyngodon idella), Aquac. Nutr. 18 (2012) 181-188.
738
[10] P.J. Ni, W.D. Jiang, W. Pei, L. Yang, S.Y. Kuang, T. Ling, et al., Dietary low or excess levels of lipids reduced growth
739
performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp
740
(Ctenopharyngodon idella) under the infection of Aeromonas hydrophila, Fish Shellfish Immunol. 55 (2016) 28–47. [11] F.-Y. Pan, L. Feng, W.-D. Jiang, J. Jiang, P. Wu, S.-Y. Kuang, et al., Methionine hydroxy analogue enhanced fish
742
immunity via modulation of NF-κB, TOR, MLCK, MAPKs and Nrf2 signaling in young grass carp (Ctenopharyngodon
743
idella), Fish Shellfish Immunol. 56 (2016) 208-228.
745 746 747
[12] S.D. Yang, T.S. Lin, F.G. Liu, C.H. Liou, Influence of dietary phosphorus levels on growth, metabolic response and body composition of juvenile silver perch (Bidyanus bidyanus), Aquaculture 253 (2006) 592-601. [13] L. Tort, J. Balasch, S. Mackenzie, Fish immune system. A crossroads between innate and adaptive responses, Inmunología 22 (2003) 277-286.
EP
744
TE D
741
[14] V.H. Olavarría, M.P. Sepulcre, J.E. Figueroa, V. Mulero, Prolactin-induced production of reactive oxygen species and
749
IL-1β in leukocytes from the bony fish gilthead seabream involves Jak/Stat and NF-κB signaling pathways, J. Immunol.
750
185 (2010) 3873-3883.
AC C
748
751
[15] H. Wen, L. Feng, W. Jiang, Y. Liu, J. Jiang, S. Li, et al., Dietary tryptophan modulates intestinal immune response,
752
barrier function, antioxidant status and gene expression of TOR and Nrf2 in young grass carp (Ctenopharyngodon idella),
753
Fish Shellfish Immunol. 40 (2014) 275-287.
754 755 756 757 758 759 760 761 762 763
[16] J.C. Eya, R. Lovell, Effects of dietary phosphorus on resistance of channel catfish to Edwardsiella ictaluri challenge, J. Aquat. Anim. Health 10 (1998) 28-34. [17] E. Jokinen, J. Vielma, T. Aaltonen, J. Koskela, The effect of dietary phosphorus deficiency on the immune responses of European whitefish (Coregonus lavaretus L.), Fish Shellfish Immunol. 15 (2003) 159-168. [18] T. Sarasa, Y. Hironori, N. Otoki, K. Tomohiro, I. Mariko, M. Masashi, et al., Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice, Nutr. Res. 33 (2013) 586-593. [19] A. Mishra, A. Chaudhary, S. Sethi, Oxidized omega-3 fatty acids inhibit NF-kappaB activation via a PPARalpha-dependent pathway, Arterio. Thromb. Vasc. Biol. 24 (2004) 1621-1627. [20] S.-H. Chang, K.N. Yu, Y.-S. Lee, G.-H. An, G.R. Beck Jr, N.H. Colburn, et al., Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells, Am. J. Resp. Cell Mol. 35 (2006) 528-539.
764 765
[21] S. Bodine, T. Stitt, M. Gonzalez, W. Kline, G. Stover, R. Bauerlein, et al., Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo, Nat. Cell Biol. 3 (2001) 1014-1019.
766
[22] S.-Y. Kuang, W.-W. Xiao, L. Feng, Y.ACCEPTED Liu, J. Jiang, W.-D. Jiang, et al., Effects of graded levels of dietary methionine MANUSCRIPT
767
hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio
768
var. Jian), Fish Shellfish Immunol. 32 (2012) 629-636.
769
[23] H.-J. Xu, W.-D. Jiang, L. Feng, Y. Liu, P. Wu, J. Jiang, et al., Dietary vitamin C deficiency depresses the growth, head
770
kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in
771
young grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 52 (2016) 111-138.
773
[24] A. Sarmento, L. Guilhermino, A. Afonso, Mercury chloride effects on the function and cellular integrity of sea bass (Dicentrarchus labrax) head kidney macrophages, Fish Shellfish Immunol. 17 (2004) 489-498.
RI PT
772
774
[25] O.J. Baker, J.M. Camden, R.S. Redman, J.E. Jones, C.I. Seye, L. Erb, et al., Proinflammatory cytokines tumor necrosis
775
factor-α and interferon-γ alter tight junction structure and function in the rat parotid gland Par-C10 cell line, Am. J.
776
Physiol-Cell Ph. 295 (2008) C1191-C1201.
778 779 780
[26] S. Homma, Y. Ishii, Y. Morishima, T. Yamadori, Y. Matsuno, N. Haraguchi, et al., Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer, Clin. Cancer. Res. 15 (2009) 3423-3432.
SC
777
[27] Y.-W. Ki, J.H. Park, J.E. Lee, I.C. Shin, H.C. Koh, JNK and p38 MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis, Toxicol. Lett. 218 (2013) 235-245.
[28] T.Y. Ma, M.A. Boivin, D. Ye, A. Pedram, H.M. Said, Mechanism of TNF-{alpha} modulation of Caco-2 intestinal
782
epithelial tight junction barrier: role of myosin light-chain kinase protein expression, Am. J. Physiol-Gastr. L 288 (2005)
783
95-96.
785 786 787 788 789 790 791
[29] A. Peng, T. Wu, C. Zeng, D. Rakheja, J. Zhu, T. Ye, et al., Adverse effects of simulated hyper- and hypo-phosphatemia on endothelial cell function and viability, PloS One 6 (2011) 442-442.
[30] H.C. Um, J.H. Jang, D.H. Kim, L. Chan, Y.J. Surh, Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in PC12 cells, Nitric Oxide 25 (2011) 161-168.
TE D
784
M AN U
781
[31] M. Glinn, B. Ni, R.P. Irwin, S. Kelley, S.Z. Lin, S.M. Paul, Inorganic Pi increases neuronal survival in the acute early phase following excitotoxic/oxidative insults, J. Neurochem. 70 (1998) 1850-1858. [32] H.K. Sang, R.P. Sharma, Mercury-induced apoptosis and necrosis in murine macrophages: role of calcium-induced reactive oxygen species and p38 mitogen-activated protein kinase signaling, Toxicol. Appl. Pharm. 196 (2004) 47-57. [33] M.A. Pasek, Rethinking early Earth phosphorus geochemistry, P. Natl. Acad. Sci. 105 (2008) 853-858.
793
[34] L. Feng, Y.-P. Chen, W.-D. Jiang, Y. Liu, J. Jiang, P. Wu, et al., Modulation of immune response, physical barrier and
794
related signaling factors in the gills of juvenile grass carp (Ctenopharyngodon idella) fed supplemented diet with
795
phospholipids, Fish Shellfish Immunol. 48 (2016) 79-93.
797
AC C
796
EP
792
[35] Y. Wang, Y. Lu, Y. Zhang, Z. Ning, Y. Li, Q. Zhao, et al., The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation, Nat. Genet. 47 (2015) 625-631.
798
[36] J. Wen, W. Jiang, L. Feng, S. Kuang, J. Jiang, L. Tang, et al., The influence of graded levels of available phosphorus on
799
growth performance, muscle antioxidant and flesh quality of young grass carp (Ctenopharyngodon idella), Anim. Nutr. 1
800
(2015) 77-84.
801
[37] W.-D. Jiang, K. Hu, Y. Liu, J. Jiang, P. Wu, J. Zhao, et al., Dietary myo-inositol modulates immunity through
802
antioxidant activity and the Nrf2 and E2F4/cyclin signalling factors in the head kidney and spleen following infection of
803
juvenile fish with Aeromonas hydrophila, Fish Shellfish Immunol. 49 (2016) 374-386.
804
[38] Y.P. Wu, L. Feng, W.D. Jiang, Y. Liu, J. Jiang, S.H. Li, et al., Influence of dietary zinc on muscle composition, flesh
805
quality and muscle antioxidant status of young grass carp (Ctenopharyngodon idella Val.), Aquac. Res. 46 (2015)
806
2360-2373.
807 808
[39] Z.-Y. Du, Y.-J. Liu, L.-X. Tian, J.-G. He, J.-M. Cao, G.-Y. Liang, The influence of feeding rate on growth, feed efficiency and body composition of juvenile grass carp (Ctenopharyngodon idella), Aquacult. Int. 14 (2006) 247-257.
809
[40] Q. Tang, L. Feng, W. Jiang, Y. Liu, J. Jiang, S. Li, et al., Effects of dietary copper on growth, digestive, and brush
810
border enzyme activities and antioxidant defense of hepatopancreas and intestine for young grass carp
811
(Ctenopharyngodon idella), Biol. Trace Elem. Res. 155 (2013) 370-380. ACCEPTED MANUSCRIPT
812 813 814 815 816 817
[41] K. Mai, C. Zhang, Q. Ai, Q. Duan, W. Xu, L. Zhang, et al., Dietary phosphorus requirement of large yellow croaker, Pseudosciaena crocea R ☆, Aquaculture 251 (2006) 346-353. [42] E. Austreng, Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract, Aquaculture 13 (1978) 265-272. [43] C. Zhang, K. Mai, Q. Ai, W. Zhang, Q. Duan, B. Tan, et al., Dietary phosphorus requirement of juvenile Japanese seabass, Lateolabrax japonicus, Aquaculture 255 (2006) 201-209. [44] B. Liu, L. Xu, X. Ge, J. Xie, P. Xu, Q. Zhou, et al., Effects of mannan oligosaccharide on the physiological responses,
819
HSP70 gene expression and disease resistance of Allogynogenetic crucian carp (Carassius auratus gibelio) under
820
Aeromonas hydrophila infection, Fish Shellfish Immunol. 34 (2013) 1395-1403.
821 822
RI PT
818
[45] X. Song, J. Zhao, Y. Bo, Z. Liu, K. Wu, C. Gong, Aeromonas hydrophila induces intestinal inflammation in grass carp (Ctenopharyngodon idella): An experimental model, Aquaculture 434 (2014) 171-178.
[46] C.M.A. Caipang, C.C. Lazado, M.F. Brinchmann, J.H. Rombout, V. Kiron, Differential expression of immune and
824
stress genes in the skin of Atlantic cod (Gadus morhua), Comp. Biochem. Physiol. D: Genom. Proteom. 6 (2011)
825
158-162.
SC
823
[47] A. International. Official methods of analysis of AOAC International: AOAC International; 2005.
827
[48] P. Wu, W.D. Jiang, Y. Liu, G.F. Chen, J. Jiang, S.H. Li, et al., Effect of choline on antioxidant defenses and gene
828
expressions of Nrf2 signaling molecule in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian),
829
Fish Shellfish Immunol. 38 (2014) 374-382.
830 831
M AN U
826
[49] Q. Shao, J. Ma, Z. Xu, W. Hu, J. Xu, S. Xie, Dietary phosphorus requirement of juvenile black seabream, Sparus macrocephalus, Aquaculture 277 (2008) 92-100.
[50] J. Zhao, Y. Liu, J. Jiang, P. Wu, W. Jiang, S. Li, et al., Effects of dietary isoleucine on the immune response, antioxidant
833
status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian), Fish Shellfish Immunol.
834
35 (2013) 572-580.
TE D
832
[51] Q. Yang, Y. Rui, L. Ming, Q. Zhou, X. Liang, Z.C. Elmada, Effects of dietary fucoidan on the blood constituents,
836
anti-oxidation and innate immunity of juvenile yellow catfish (Pelteobagrus fulvidraco), Fish Shellfish Immunol. 41
837
(2014) 264-270.
839 840 841 842 843 844 845
[52] H. Ji, L. Jie, P. Liu, Regulation of growth performance and lipid metabolism by dietary n-3 highly unsaturated fatty acids in juvenile grass carp, Ctenopharyngodon idellus, Comp. Biochem. Physiol. 159 (2011) 49–56.
AC C
838
EP
835
[53] X. Lu, C. Wang, B. Liu, The role of Cu/Zn-SOD and Mn-SOD in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix, Fish Shellfish Immunol. 42 (2015) 58-65. [54] X.D. Zhang, Y.F. Zhu, L.S. Cai, T.X. Wu, Effects of fasting on the meat quality and antioxidant defenses of market-size farmed large yellow croaker (Pseudosciaena crocea), Aquaculture 280 (2008) 136-139. [55] Z. Zhang, R.P. Wilson, A modified enzymatic assay for quantifying choline in fish tissue and common feed ingredients, J. Agr. Food Chem. 46 (1998) 3673-3676.
846
[56] J.B. Luo, L. Feng, W.D. Jiang, Y. Liu, P. Wu, J. Jiang, et al., The impaired intestinal mucosal immune system by valine
847
deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating
848
tight junction proteins transcript abundance in the intestine, Fish Shellfish Immunol. 40 (2014) 197-207.
849 850
[57] K.J. Livak, T.D. Schmittgen, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 −∆∆ C T Method, Methods 25 (2001) 402-408.
851
[58] NRC. Nutrient requirements of fish and shrimp: National academies press; 2011.
852
[59] B.C. Peterson, E. Peatman, D.D. Ourth, G.C. Waldbieser, Effects of a phytogenic feed additive on growth performance,
853
susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin, Fish Shellfish Immunol.
854
44 (2015) 21-25.
855
[60] B. Magnadóttir, Innate immunity of fish (overview), Fish Shellfish Immunol. 20 (2006) 137-151.
856
[61] Y. Liu, R.C.-M. Ho, A. Mak, Interleukin (IL)-6, tumour necrosis factor alpha (TNF-α) and soluble interleukin-2 ACCEPTED MANUSCRIPT
857
receptors (sIL-2R) are elevated in patients with major depressive disorder: a meta-analysis and meta-regression, J. Affect.
858
Disorders 139 (2012) 230-239.
859
[62] R. Wei, G.M. Jonakait, Neurotrophins and the anti-inflammatory agents interleukin-4 (IL-4), IL-10, IL-11 and
860
transforming growth factor-β1 (TGF-β1) down-regulate T cell costimulatory molecules B7 and CD40 on cultured rat
861
microglia, J. Neuroimmunol. 95 (1999) 8-18.
863
[63] B.M.L.V.V. Kemenade, E.H. Stolte, J.R. Metz, M. Chadzinska, Chapter 7 Neuroendocrine–Immune Interactions in Teleost Fish, Fish Physiol. 28 (2009) 313-364.
RI PT
862
864
[64] X. Huang, Y. Huang, Z. Ouyang, J. Cai, Y. Yan, Q. Qin, Roles of stress-activated protein kinases in the replication of
865
Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells, J. Gen. Virol. 92 (2011)
866
1292-1301.
869 870 871 872 873 874
23 p19 and p40 in lesional skin of patients with psoriasis vulgaris, J. Exp. Med. 199 (2004) 125-130.
SC
868
[65] E. Lee, W.L. Trepicchio, J.L. Oestreicher, D. Pittman, F. Wang, F. Chamian, et al., Increased expression of interleukin
[66] M. Ohtani, N. Hayashi, K. Hashimoto, T. Nakanishi, J.M. Dijkstra, Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish, Immunogenetics 60 (2008) 383-397.
[67] O. Jaillon, J.M. Aury, J.L. Petit, N. Stangethomann, E. Mauceli, L. Bouneau, et al., Genome duplication in the teleost
M AN U
867
fish : Tetraodon nigroviridis: reveals the early vertebrate proto-karyotype : Article : Nature, Nat. (2004). [68] J.H. Li, J.Z. Shao, L.X. Xiang, Y. Wen, Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: The first evidence of Th2-type cytokine in fish, Mol. Cell. Biol. 44 (2007) 2078-2086. [69] Y.D. Min, C.H. Choi, H. Bark, H.Y. Son, H.H. Park, S. Lee, et al., Quercetin inhibits expression of inflammatory
876
cytokines through attenuation of NF-κB and p38 MAPK in HMC-1 human mast cell line, Inflamm. Res. 56 (2007)
877
210-215.
878 879 880 881
TE D
875
[70] P. Viatour, M.P. Merville, V. Bours, A. Chariot, Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation, Trends Biochem. Sci. 30 (2005) 43-52.
[71] S. Tegethoff, J. Behlke, C. Scheidereit, Tetrameric oligomerization of IκB kinase γ (IKKγ) is obligatory for IKK complex activity and NF-κB activation, Mol. Cell. Biol. 23 (2003) 2029-2041. [72] Y.-P. Chen, W.-D. Jiang, Y. Liu, J. Jiang, P. Wu, J. Zhao, et al., Exogenous phospholipids supplementation improves
883
growth and modulates immune response and physical barrier referring to NF-κB, TOR, MLCK and Nrf2 signaling factors
884
in the intestine of juvenile grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 47 (2015) 46-62.
886
AC C
885
EP
882
[73] T. Weichhart, G. Costantino, M. Poglitsch, M. Rosner, M. Zeyda, K.M. Stuhlmeier, et al., The TSC-mTOR signaling pathway regulates the innate inflammatory response, Immunity 29 (2008) 565-577.
887
[74] N. Hay, N. Sonenberg, Upstream and downstream of mTOR, Genes. Dev. 18 (2004) 1926-1945.
888
[75] J. Deng, B. Kang, L. Tao, H. Rong, X. Zhang, Effects of dietary cholesterol on antioxidant capacity, non-specific
889
immune response, and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean
890
meal-based diets, Fish Shellfish Immunol. 34 (2013) 324-331.
891
[76] S.C. Lu, Glutathione synthesis, Biochim. Biophys. Acta-Gen. Subjects 1830 (2013) 3143-3153.
892
[77] M. Li, W. Wu, P. Zhou, F. Xie, Q. Zhou, K. Mai, et al., Comparison effect of dietary astaxanthin and Haematococcus
893
pluvialis on growth performance, antioxidant status and immune response of large yellow croaker Pseudosciaena crocea,
894
Aquaculture 434 (2014) 227–232.
895
[78] H. Elabd, H.P. Wang, A. Shaheen, H. Yao, A. Abbass, Feeding Glycyrrhiza glabra (liquorice) and Astragalus
896
membranaceus (AM) alters innate immune and physiological responses in yellow perch (Perca flavescens), Fish Shellfish
897
Immunol. 54 (2016) 374-384.
898
[79] K. Mukaigasa, L.T. Nguyen, L. Li, H. Nakajima, M. Yamamoto, M. Kobayashi, Genetic evidence of an evolutionarily
899
conserved role for Nrf2 in the protection against oxidative stress, Mol. Cell. Biol. 32 (2012) 4455-4461.
900
[80] H.F. Zhao, W.D. Jiang, Y. Liu, J. Jiang, P. Wu, S.Y. Kuang, et al., Dietary choline regulates antibacterial activity,
901
inflammatory response and barrier function in the gills of MANUSCRIPT grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. ACCEPTED
902
52 (2016) 139–150.
903
[81] M.G. Grütter, Caspases: key players in programmed cell death, Curr. Opin. Struct. Biol. 10 (2000) 649-655.
904
[82] Y. Xiao-Ming, Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and
905
mitochondria apoptosis pathways, Cell Res. 10 (2000) 161-167. [83] Q.L. Deveraux, J.C. Reed, IAP family proteins—suppressors of apoptosis, Genes. Dev. 13 (1999) 239-252.
907
[84] B.J. Kim, S.W. Ryu, B.J. Song, JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and
908
mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells, J. Biol. Chem. 281 (2006) 21256-21265.
909
[85] Z.Q. Yuan, R.I. Feldman, M. Sun, N.E. Olashaw, D. Coppola, G.E. Sussman, et al., Inhibition of JNK by cellular stress-
910
and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells,
911
J. Biol. Chem. 277 (2002) 29973-29982.
RI PT
906
[86] K. Shin, V.C. Fogg, B. Margolis, Tight junctions and cell polarity, Annu. Rev. Cell Dev. Bi. 22 (2006) 207-235.
913
[87] Y. Kiuchi-Saishin, S. Gotoh, M. Furuse, A. Takasuga, Y. Tano, S. Tsukita, Differential expression patterns of claudins,
914
SC
912
tight junction membrane proteins, in mouse nephron segments, J. Am. Soc. Nephrol. 13 (2002) 875-886. [88] C.M. Van Itallie, J.M. Anderson, Claudins and epithelial paracellular transport, Annu. Rev. Physiol. 68 (2006) 403-429.
916
[89] T.Y. Ma, D. Tran, N. Hoa, D. Nguyen, M. Merryfield, A. Tarnawski, Mechanism of extracellular calcium regulation of
917
intestinal epithelial tight junction permeability: Role of cytoskeletal involvement, Microsc. Res. Techniq. 51 (2000)
918
156-168.
920 921 922 923 924
[90] A. Steensberg, C.P. Fischer, C. Keller, K. Møller, B.K. Pedersen, IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans, Am. J. Physiol-Endoc 285 (2003) E433-437.
[91] H. Chasiotis, S.P. Kelly, Effect of cortisol on permeability and tight junction protein transcript abundance in primary cultured gill epithelia from stenohaline goldfish and euryhaline trout, Gen. Comp. Endocr. 172 (2011) 494-504.
TE D
919
M AN U
915
[92] P. Bui, B.L.M. Sp, Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia, Mol. Cell. Endocrino. 317 (2010) 120-126.
[93] J. Lake, C. Gravel, G.K.D. Koko, C. Robert, G.W. Vandenberg, Combining suppressive subtractive hybridization and
926
cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney,
927
Comp. Biochem. Physiol. D: Genom. Proteom. 5 (2010) 24-35.
EP
925
[94] M.C. Bossus, S.S. Madsen, C.K. Tipsmark, Functional dynamics of claudin expression in Japanese medaka (Oryzias
929
latipes): Response to environmental salinity, Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol. 187 (2015) 74-85.
930
[95] G. Liu, Y. Zhang, A.M. Bode, W.Y. Ma, Z. Dong, Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in
931 932 933
AC C
928
response to UVB irradiation, J. Biol. Chem. 277 (2002) 8810-8816. [96] F.J. Paula, A.E. Plens, M.C. Foss, Effects of hypophosphatemia on glucose tolerance and insulin secretion, Horm. Metab. Res. 30 (1998) 281-284.
934
[97] B.X. Peng, P.Z. Xu, M.L. Chen, A. Hahnwindgassen, J. Skeen, Dwarfism, impaired skin development, skeletal muscle
935
atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev 17, (2010).
936
[98] E. Takeda, Y. Taketani, K, M. Nomoto, E. Shuto, N. Sawada, H. Yamamoto, et al., A novel function of
937 938 939
phosphate-mediated intracellular signal transduction pathways, Adv. Enzyme. Regul. 46 (2006) 154-161. [99] E. Shuto, Y. Taketani, R. Tanaka, N. Harada, M. Isshiki, M. Sato, et al., Dietary phosphorus acutely impairs endothelial function, J. Am. Soc. Nephrol. 20 (2009) 1504-1512.
940
[100] A. Liu, J.L. Arbiser, A. Holmgren, G. Klein, E. Klein, PSK and Trx80 inhibit B-cell growth in EBV-infected cord
941
blood mononuclear cells through T cells activated by the monocyte products IL-15 and IL-12, Blood 105 (2005)
942
1606-1613.
943
[101] I. Perez-Cruz, J.M. Cárcamo, D.W. Golde, Caspase-8 dependent TRAIL-induced apoptosis in cancer cell lines is
944
inhibited by vitamin C and catalase, Apoptosis 12 (2007) 225-234.
945
[102] M. Benahmed, B. Meresse, B. Arnulf, U. Barbe, J.J. Mention, V. Verkarre, et al., Inhibition of TGF-β signaling by
946
IL-15: a new role for IL-15 in the loss ofACCEPTED immune homeostasis in celiac disease, Gastroenterology 132 (2007) 994-1008. MANUSCRIPT
947
[103] M.F. Holick, Holick MF.Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and
948
cardiovascular disease. Am J Clin Nutr 80:1678S-1688SS.
949
[104] Y.Y. Zeng, W.D. Jiang, Y. Liu, P. Wu, J. Zhao, J. Jiang, et al., Optimal dietary alpha-linolenic acid/linoleic acid ratio
950
improved digestive and absorptive capacities and target of rapamycin gene expression of juvenile grass carp
951
(Ctenopharyngodon idellus), Aquac. Nutr. (2015). [105] L. Wang, N. Shang, H. Feng, Q. Guo, H. Dai, Molecular cloning of grass carp (Ctenopharyngodon idellus) T-bet and
953
GATA-3, and their expression profiles with IFN-γ in response to grass carp reovirus (GCRV) infection, Fish Physiol.
954
Biochem. 39 (2013) 793-805.
RI PT
952
955
AC C
EP
TE D
M AN U
SC
956
Table 1 Composition and nutrients content of basal diet.
958
ACCEPTED MANUSCRIPT
Ingredients
g/kg
Nutrients content
40.00
Fish meal
Crude protein
Casein
146.00
Crude lipid
Gelatin
66.00
n-3 5
g/kg
4
307.10
4
55.10 10.40
5
153.80
n-6
α-starch
240.00
Total phosphorus 6
2.30
Corn starch
162.20
Available phosphorus 6
0.95
Fish oil
29.00
Soybean oil
9.30
Cellulose
50.00
Vitamin premix 1
10.00
Mineral premix 2
20.00
Monosodium phosphate mixture 3
60.00
Choline chloride (50%)
10.00
DL-Met (99%)
2.60
L-Trp (99%)
0.60
Ethoxyquin (30%)
0.50
9.60
RI PT
Rice gluten meal
SC
957
959 960
1
961
L-α-tocopherol acetate (50%), 12.58; menadione (22.9%), 0.83; cyanocobalamin (1%), 0.94; D-biotin (2%), 0.75; folic acid
962
(95%), 0.42; thiamine nitrate (98%), 0.09; ascorhyl acetate (95%), 4.31; niacin (99%), 4.04; meso-inositol (98%), 19.39;
963
calcium-D-pantothenate (98%), 3.85; riboflavin (80%), 0.73; pyridoxine hydrochloride (98%), 0.62. All ingredients were
964
diluted with corn starch to 1 kg.
965
2
M AN U
Per kilogram of vitamin premix (g/kg): retinyl acetate (500,000 IU/g), 2.10; cholecalciferol (500,000 IU/g), 0.40; D,
Per kilogram of mineral premix (g/kg): MnSO4⋅H2O (31.8% Mn), 2.6590; MgSO4⋅H2O (15.0% Mg), 200.0000;
FeSO4⋅H2O (30.0% Fe), 12.2500; ZnSO4⋅H2O (34.5% Zn), 8.2460; CuSO4⋅5H2O (25.0% Cu), 0.9560; KI (76.9% I), 0.0650;
967
Na2SeO3 (44.7% Se), 0.0168. All ingredients were diluted with corn starch to 1 kg.
968
3
Monosodium phosphate mixture: premix was added to obtain graded level of phosphorus.
969
4
Crude protein and crude lipid contents were measured value.
970
5
n-3 and n-6 contents were calculated according to Zeng et al. [104].
971
6
Total phosphorus content were determined according to the method of the AOAC (2000) [47], the value of available
972
phosphorus was calculated based on the digestibility of basal diet as determined in the digestibility trial according to NRC
973
(2011) [58].
975
EP
AC C
974
TE D
966
Table 2 Real-time PCR primer sequences 1.
977
ACCEPTED MANUSCRIPT Target gene
Primer sequence Forward (5’→3’)
hepcidin
AGCAGGAGCAGGATGAGC
GCCAGGGGATTTGTTTGT
59.3
Accession number JQ246442.1
LEAP-2A
TGCCTACTGCCAGAACCA
AATCGGTTGGCTGTAGGA
59.3
FJ390414
LEAP-2B
TGTGCCATTAGCGACTTCTGAG
ATGATTCGCCACAAAGGGG
59.3
KT625603
β-defensin-1
TTGCTTGTCCTTGCCGTCT
AATCCTTTGCCACAGCCTAA
58.4
KT445868
IFN-γ2
TGTTTGATGACTTTGGGATG
TCAGGACCCGCAGGAAGAC
60.4
JX657682
TNF-α
CGCTGCTGTCTGCTTCAC
CCTGGTCCTGGTTCACTC
58.4
HQ696609
IL-1β
AGAGTTTGGTGAAGAAGAGG
TTATTGTGGTTACGCTGGA
57.1
JQ692172 KC535507.1
Primer sequence Reverse (5’→3’)
Temperature(°C)
CAGCAGAATGGGGGAGTTATC
CTCGCAGAGTCTTGACATCCTT
62.3
ATGAGTCTTAGAGGTCTGGGT
ACAGTGAGGGCTAGGAGGG
60.3
JN663841
IL-10
AATCCCTTTGATTTTGCC
GTGCCTTATCCTACAGTATGTG
61.4
HQ388294
IL-11
GGTTCAAGTCTCTTCCAGCGAT
TGCGTGTTATTTTGTTCAGCCA
57.0
KT445870
IL-12p35
TGGAAAAGGAGGGGAAGATG
AGACGGACGCTGTGTGAGTGTA
55.4
KF944667.1
IL-12p40
ACAAAGATGAAAAACTGGAGGC
GTGTGTGGTTTAGGTAGGAGCC
59.0
KF944668.1
IL-15
CCTTCCAACAATCTCGCTTC
AACACATCTTCCAGTTCTCCTT
61.4
KT445872
IL-17D
GTGTCCAGGAGAGCACCAAG
GCGAGAGGCTGAGGAAGTTT
62.3
KF245426.1
IL-4/13A
CTACTGCTCGCTTTCGCTGT
CCCAGTTTTCAGTTCTCTCAGG
55.9
KT445871
IL-4/13B
TGTGAACCAGACCCTACATAACC
TTCAGGACCTTTGCTGCTTG
55.9
KT625600
TGF-β1
TTGGGACTTGTGCTCTAT
AGTTCTGCTGGGATGTTT
55.9
EU099588
TGF-β2
TACATTGACAGCAAGGTGGTG
TCTTGTTGGGGATGATGTAGTT
55.9
KM279716
GATA-3
AGCCCACACCTCTTCACCTT
CACTCTTTGTCCTCCTGCCG
58.5
JX021295, [105]
NF-κB p52
TCAGTGTAACGACAACGGGAT
ATACTTCAGCCACACCTCTCTTAG
58.4
KM279720
NF-κB p65
GAAGAAGGATGTGGGAGATG
TGTTGTCGTAGATGGGCTGAG
62.3
KJ526214
c-Rel
GCGTCTATGCTTCCAGATTTACC
ACTGCCACTGTTCTTGTTCACC
59.3
KT445865
IκBα
TCTTGCCATTATTCACGAGG
TGTTACCACAGTCATCCACCA
62.3
KJ125069
IKKα
GGCTACGCCAAAGACCTG
CGGACCTCGCCATTCATA
60.3
KM279718
SC
RI PT
IL-6 IL-8
M AN U
976
GTGGCGGTGGATTATTGG
GCACGGGTTGCCAGTTTG
60.3
KP125491
AGAGGCTCGTCATAGTGG
CTGTGATTGGCTTGCTTT
58.4
KM079079
TOR
TCCCACTTTCCACCAACT
ACACCTCCACCTTCTCCA
61.4
JX854449
S6K1
TGGAGGAGGTAATGGACG
ACATAAAGCAGCCTGACG
54.0
EF373673
4E-BP1
GCTGGCTGAGTTTGTGGTTG
CGAGTCGTGCTAAAAAGGGTC
60.3
KT757305
4E-BP2
CACTTTATTCTCCACCACCCC
TTCATTGAGGATGTTCTTGCC
60.3
KT757306
occludin
TATCTGTATCACTACTGCGTCG
CATTCACCCAATCCTCCA
59.4
KF193855
ZO-1
CGGTGTCTTCGTAGTCGG
CAGTTGGTTTGGGTTTCAG
59.4
KJ000055
ZO-2
TACAGCGGGACTCTAAAATGG
TCACACGGTCGTTCTCAAAG
60.3
KM112095
claudin-b
GAGGGAATCTGGATGAGC
ATGGCAATGATGGTGAGA
57.0
KF193860
claudin-c
GAGGGAATCTGGATGAGC
claudin-f
GCTGGAGTTGCCTGTCTTATTC
claudin-3c claudin-7a
EP
TE D
IKKβ IKKγ
59.4
KF193859
57.1
KM112097
ATCACTCGGGACTTCTA
CAGCAAACCCAATGTAG
57.0
KF193858
ACTTACCAGGGACTGTGGATGT
CACTATCATCAAAGCACGGGT
59.3
KT625604
claudin-7b
CTAACTGTGGTGGTGATGAC
AACAATGCTACAAAGGGCTG
59.3
KT445866
claudin-11
TCTCAACTGCTCTGTATCACTGC
TTTCTGGTTCACTTCCGAGG
62.3
KT445867
claudin-12
CCCTGAAGTGCCCACAA
GCGTATGTCACGGGAGAA
55.4
KF998571
claudin-15a
TGCTTTATTTCTTGGCTTTC
CTCGTACAGGGTTGAGGTG
59.0
KF193857
claudin-15b
AGTGTTCTAAGATAGGAGGGGAG
AGCCCTTCTCCGATTTCAT
62.3
KT757304
NaPi-IIb
CTGGACTCAAGAGAACCAAACG
GCAATGACAGAGCCAACAAAAT
62.7
KX823957
MLCK
GAAGGTCAGGGCATCTCA
GGGTCGGGCTTATCTACT
53.0
KM279719
FasL
AGGAAATGCCCGCACAAATG
AACCGCTTTCATTGACCTGGAG
61.4
KT445873
p38 MAPK
TGGGAGCAGACCTCAACAAT
TACCATCGGGTGGCAACATA
60.4
KM112098
JNK
ACAGCGTAGATGTGGGTGATT
GCTCAAGGTTGTGGTCATACG
62.3
KT757312
Bcl-2
AGGAAAATGGAGGTTGGGAT
CTGAGCAAAAAAGGCGATG
60.3
JQ713862.1
Mcl-1
TGGAAAGTCTCGTGGTAAAGCA
ATCGCTGAAGATTTCTGTTGCC
58.4
KT757307
Bax
CATCTATGAGCGGGTTCGTC
TTTATGGCTGGGGTCACACA
60.3
JQ793788.1
Apaf-1
AAGTTCTGGAGCCTGGACAC
AACTCAAGACCCCACAGCAC
61.4
KM279717
IAP
CACAATCCTGGTATGCGTCG
GGGTAATGCCTCTGGTGCTC
58.4
FJ593503.1
caspase-2
CGCTGTTGTGTGTTTACTGTCTCA
ACGCCATTATCCATCTCCTCTC
60.3
KT757313
AC C
CTGTTATGAAAGCGGCAC ACCAATCTCCCTCTTTTGTGTC
caspase-3
GCTGTGCTTCATTTGTTTG
TCTGAGATGTTATGGCTGTC
55.9
JQ793789
caspase-7
GCCATTACAGGATTGTTTCACC
CCTTATCTGTGCCATTGCGT
57.1
KT625601
caspase-8
ATCTGGTTGAAATCCGTGAA
TCCATCTGATGCCCATACAC
59.0
KM016991
caspase-9
CTGTGGCGGAGGTGAGAA
59.0
JQ793787
CuZnSOD
CGCACTTCAACCCTTACA
ACTTTCCTCATTGCCTCC
61.5
GU901214
MnSOD
ACGACCCAAGTCTCCCTA
ACCCTGTGGTTCTCCTCC
60.4
GU218534
CAT
GAAGTTCTACACCGATGAGG
CCAGAAATCCCAAACCAT
58.7
FJ560431
GPx1a
GGGCTGGTTATTCTGGGC
AGGCGATGTCATTCCTGTTC
61.5
EU828796
GPx1b
TTTTGTCCTTGAAGTATGTCCGTC
GGGTCGTTCATAAAGGGCATT
60.3
KT757315
GPx4a
TACGCTGAGAGAGGTTTACACAT
CTTTTCCATTGGGTTGTTCC
60.4
KU255598
GPx4b
CTGGAGAAATACAGGGGTTACG
CTCCTGCTTTCCGAACTGGT
60.3
KU255599
ACCEPTED MANUSCRIPT GTGCTGGAGGACATGGGAAT
TCTCAAGGAACCCGTCTG
CCAAGTATCCGTCCCACA
58.4
EU107283
GSTP1
ACAGTTGCCCAAGTTCCAG
CCTCACAGTCGTTTTTTCCA
59.3
KM112099
GSTO1
GGTGCTCAATGCCAAGGGAA
CTCAAACGGGTCGGATGGAA
GR
GTGTCCAACTTCTCCTGTG
ACTCTGGGGTCCAAAACG
Nrf2
CTGGACGAGGAGACTGGA
ATCTGTGGTAGGTGGAAC
Keap1a
TTCCACGCCCTCCTCAA
TGTACCCTCCCGCTATG
Keap1b
TCTGCTGTATGCGGTGGGC
CTCCTCCATTCATCTTTCTCG
β-actin
GGCTGTGCTGTCCCTGTA
GGGCATAACCCTCGTAGAT
58.4
KT757314
59.4
JX854448
62.5
KF733814
63.0
KF811013
57.9
KJ729125
61.4
M25013
SC
978
RI PT
GSTR
979
1
980
TGF-β, transforming growth factor β; NF-κB, nuclear factor kappa B; IκBα, inhibitor of κBα; IKK, IκB kinase; TOR, target
981
of rapamycin; S6K1, ribosomal protein S6 kinases 1; 4E-BP, eIF4E-binding proteins; ZO, zonula occludens; NaPi-IIb,
982
type-IIb sodium-phosphate cotransporter; MLCK, myosin light chain kinase; FasL, fatty acid synthetase ligand; p38 MAPK,
983
p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal protein kinase; Bcl-2, B-cell lymphoma protein-2; Mcl-1,
984
myeloid cell leukemia-1; Bax, Bcl-2 associated X protein; Apaf-1, apoptotic protease activating factor-1; IAP, inhibitor of
985
apoptosis proteins; caspase, cysteinyl aspartic acid-protease; CuZnSOD, copper, zinc superoxide dismutase; MnSOD,
986
manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR,
987
glutathione reductase; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like-ECH-associated protein 1.
M AN U
TE D
EP
989
AC C
988
LEAP-2, liver expressed antimicrobial peptide 2; IFN-γ2, interferon γ2; TNF-α, tumor necrosis factor α; IL, interleukin;
990
Table 3 Growth performance, head kidney and spleen weight (g), index (%) and serum phosphorus
991
concentration (mmol/L) and serum alkaline phosphatase activity (AKP, King’s unit/100mL) of young grass
992
carp (Ctenopharyngodon idellus) fed diets containing graded levels of available phosphorus for 60 days.
ACCEPTED MANUSCRIPT
993 Available P in the diet（ （g/kg diet） ） 2.46
3.96
5.68
7.10
8.75
IBW1
256.44±0.38a
256.00±0.67a
256.67±0.67a
256.22±0.38a
255.78±1.02a
254.56±0.51a
FBW1
610.87±12.09a
734.33±21.83b
880.63±16.57c
884.63±15.50c
898.23±18.11c
889.10±9.30c
PWG1
138.20±4.39a
186.86±9.04b
243.11±6.87c
245.26±6.39c
251.20±8.44c
247.00±4.09c
SGR1
1.45±0.03a
1.76±0.05b
2.05±0.03c
2.07±0.03c
2.09±0.04c
2.07±0.02c
FI1
688.48±0.99a
780.57±0.26b
851.03±0.69c
850.72±0.63c
850.52±0.81c
851.02±0.38c
FE1
51.48±1.67a
61.28±2.87b
73.32±1.92c
73.87±1.86c
75.51±2.31c
74.37±1.15c
Weight2
0.688±0.067a
1.087±0.092b
1.542±0.138c
1.578±0.086c
1.583±0.141c
1.559±0.159c
Index2
0.140±0.008a
0.161±0.012b
0.178±0.012c
0.174±0.013c
0.177±0.010c
0.176±0.015c
Weight2
0.842±0.067a
1.117±0.103c
1.417±0.103e
1.358±0.124de
1.292±0.138d
0.936±0.103b
Index2
0.173±0.018c
0.165±0.016c
1.49±0.13a
1.79±0.09b
13.20±0.90a
15.46±1.10b
Serum phosphorus3 Serum AKP
3
Regression YFBW = 89.6147x + 521.7909
0.150±0.015b
0.145±0.014b
0.105±0.008a
2.19±0.14c
2.48±0.14d
2.42±0.19d
2.51±0.14d
16.79±1.15b
20.14±1.51c
19.55±1.74c
19.60±1.43c
R2 = 0.9974
P < 0.05
Yhead kidney weight = 0.2838x + 0.4082 Yhead kidney index = 0.0125x + 0.1288 Yspleen weight = -0.0342x2 + 0.3477x + 0.5227
EP
Y Serum phosphorus = 0.2127x + 1.2948
TE D
Ymax = 2.07
YFE = 7.2550x + 44.2025
994
0.164±0.013c
Ymax = 888.15
YSGR = 0.2021x + 1.2559
Y Serum AKP = 1.4189x + 11.7693
M AN U
Spleen
SC
Head kidney
RI PT
0.95
2
P < 0.05
2
R = 0.9999
Ymax = 74.27
R = 0.9963
P < 0.05
Ymax = 1.565
R2 = 0.9984
P < 0.05
Ymax = 0.176
Ymax = 2.47 Ymax = 19.76
2
R = 0.9968
P < 0.05
R2 = 0.9704
P < 0.01
R2 = 0.9923
P < 0.01
2
R = 0.9802
P = 0.01
995
1
996
rate (%/day); FI: feed intake (g/fish); FE: feed efficiency (%).
997
Values are means ± SD for three replicate groups, with 30 fish in each group, and different superscripts in the same row are
998
significantly different (P < 0.05).
999
2
Values are means ± SD (n = 12), and different superscripts in the same row are significantly different (P < 0.05).
1000
3
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1001
1002
AC C
IBW: Initial body weight (g/fish); FBW: final body weight (g/fish); PWG: percent weight gain (%); SGR: specific growth
1003
Table 4 Immune parameters in the head kidney, spleen and skin of young grass carp (Ctenopharyngodon
1004
idella) fed diets containing graded levels of available phosphorus for 60 days, and then challenge with
1005
Aeromonas hydrophila for 14 days1
ACCEPTED MANUSCRIPT
1006 Available P in the diet（ （g/kg diet） ） 0.95
2.46
3.96
5.68
7.10
8.75
Head kidney 192.88±11.50a
213.24±20.70a
242.65±13.78b
266.16±23.39c
265.25±17.12c
271.09±19.62c
ACP
432.01±41.72a
552.73±52.40b
611.05±31.51c
656.39±30.62c
663.46±65.80c
664.11±44.34c
C3
14.96±1.44a
17.77±1.64b
20.06±1.09c
23.32±2.10d
26.25±1.73e
26.68±2.03e
C4
9.50±0.81a
10.31±0.45a
11.84±1.11b
13.50±1.13c
15.73±1.49d
15.82±1.45d
IgM
70.06±6.54a
79.88±7.10b
82.23±4.34b
82.28±7.02b
80.89±4.39b
79.74±7.70b
Spleen LZ
143.81±12.11a
165.89±11.45b
a
b
443.83±43.04
168.35±5.92b 532.75±51.50
177.59±11.89b c
548.63±38.33
c
RI PT
LZ
179.28±14.23b
173.35±11.75b
c
556.85±17.38c
553.14±53.41
380.01±29.75
C3
28.85±2.42a
32.19±2.85a
38.43±3.38b
40.77±2.11bc
42.43±3.96c
43.34±3.31c
C4
4.77±0.47a
5.95±0.38b
7.64±0.72c
8.51±0.63d
8.73±0.71d
8.58±0.66d
IgM
31.79±2.83a
44.80±3.89b
51.98±4.74c
52.67±4.78cd
57.76±5.61d
55.90±4.31cd
LZ
53.61±2.00a
103.36±7.33b
170.37±17.45c
159.47±15.52c
174.36±17.52c
162.33±15.80c
ACP
182.00±14.68a
251.22±22.56b
273.36±21.94b
316.40±13.24c
307.62±20.18c
313.29±26.68c
C3
25.46±2.16a
30.23±2.98b
35.96±2.75c
42.24±3.02d
43.34±3.94d
41.52±3.40d
C4
5.75±0.54a
8.43±0.83b
11.94±1.13c
12.59±1.26c
14.09±1.06d
15.24±1.08d
IgM
49.87±4.96a
90.36±5.94b
119.19±9.55c
117.89±9.75c
124.01±11.39c
125.78±12.31c
Ymax = 648.75
R2 = 0.9618
P = 0.125
YACP in head kidney = 59.5039x + 385.7503 YC3 in head kidney = 1.8124x + 13.1685 YC4 in head kidney = 1.0073x + 8.1159 YACP in spleen = 50.7319x + 327.5648 YC3 in spleen = 2.6641x + 26.3691 2
TE D
Regression
M AN U
Skin
SC
ACP
Ymax = 26.46 Ymax = 15.77 Ymax = 547.84 Ymax = 42.88
YLZ in skin = 38.7853x + 13.8321 YACP in skin = 27.0312x + 167.5545
AC C
YC3 in skin = 3.5755x + 21.8066 YC4 in skin = 1.3383x + 5.1679
YIgM in skin = 23.0345x + 29.8872
1007
EP
YIgM in spleen = -0.6184x + 8.8951x + 25.0505
2
P < 0.01
2
P < 0.01
2
P = 0.061
2
P < 0.05
2
P < 0.01
2
P = 0.055
2
P < 0.05
2
P < 0.01
2
P < 0.01
2
P = 0.060
R = 0.9978 R = 0.9740 R = 0.9907 R = 0.9632 R = 0.9668
Ymax = 166.63 Ymax = 313.43 Ymax = 42.36 Ymax = 14.67 Ymax = 121.72
R = 0.9924 R = 0.9529 R = 0.9986 R = 0.9347 R = 0.9910
1008
1
1009
Lysozyme (U/mg protein); ACP: acid phosphatase (U/mg protein); C3: complement component 3 (mg/g protein); C4:
1010
complement component 4 (mg/g protein); IgM: immunoglobulin M (mg/g protein).
1011
1012
Values are means ± SD (n = 6), and superscripted different letters in the same row are significantly different (P < 0.05). LZ:
1013
Table 5 Antioxidant related parameters in the head kidney, spleen and skin of young grass carp
1014
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus for 60 days, and then
1015
challenge with Aeromonas hydrophila for 14 days1
ACCEPTED MANUSCRIPT
1016 Available P in the diet（ （g/kg diet） ） 0.95
2.46
3.96
5.68
7.10
8.75
Head kidney 100.00±6.13d d
69.84±2.94c
60.52±1.68b
43.09±3.70a
65.79±2.56c
65.95±2.37c
c
b
a
a
34.35±1.64a
MDA
53.12±3.29
PC
6.87±0.57d
5.47±0.32c
4.43±0.42b
3.50±0.26a
AHR
60.91±3.76a
63.30±2.10a
68.03±1.90b
73.33±3.34c
ASA
40.71±3.61a
54.98±5.39b
62.56±5.34c
72.78±5.09d
a
46.45±4.19
d
3.98±0.35
MnSOD
3.80±0.36a
4.36±0.29a
5.47±0.51b
5.68±0.48b
CAT
0.75±0.06a
0.92±0.09bc
1.08±0.08d
1.11±0.09d
GPx
172.60±16.35a
186.34±14.03ab
193.41±13.57bc
210.04±18.98c
GST
82.94±7.61a
101.60±10.49b
129.87±9.30c
155.11±11.37d
74.98±6.08c
72.00±4.72d
74.33±4.86d
d
7.48±0.46d
5.72±0.51b
5.64±0.50b
0.98±0.09c
0.85±0.04b
207.80±13.43c
208.57±14.78c
157.08±13.62d
157.04±9.90d 22.77±1.56a
GSH
1.37±0.12a
1.76±0.14b
2.57±0.23c
2.51±0.19c
2.58±0.25c
2.62±0.20c
Choline
88.40±8.34a
111.24±8.17b
124.51±11.28b
128.77±10.18b
114.49±8.06b
119.45±7.43b
ROS
100.00±7.91e
70.00±4.61c
52.03±2.73b
39.92±2.68a
68.10±4.10c
76.86±5.08d
MDA
87.55±7.97d
79.99±3.90c
70.93±5.27b
63.63±5.66a
64.18±5.46ab
65.81±3.47ab
PC
5.44±0.50c
3.80±0.33b
2.46±0.20a
2.51±0.25a
2.72±0.14a
2.66±0.19a
AHR
39.74±2.50a
42.90±3.33ab
45.24±2.48bc
46.97±1.92c
49.07±2.97c
49.05±4.48c
ASA
47.85±4.18a
60.17±5.48b
73.64±5.35c
83.28±7.11d
95.53±6.48e
94.72±5.99e
CuZnSOD
4.87±0.45a
5.87±0.29b
5.54±0.32b
5.51±0.40b
5.49±0.50b
5.70±0.46b
MnSOD
4.78±0.41a
6.02±0.17b
7.48±0.56c
7.43±0.38c
7.49±0.72c
7.31±0.69c
CAT
0.50±0.04a
0.51±0.04a
0.52±0.05a
0.52±0.04a
0.53±0.04a
0.53±0.05a
GPx
213.54±19.80a
244.72±15.39b
284.86±19.13c
288.08±19.03c
282.21±24.96c
289.47±25.70c
GST
106.96±7.57a
116.92±5.75a
128.67±5.81b
150.84±8.86d
140.47±9.60c
129.81±12.08b
GR
24.96±1.93c
22.53±1.44b
20.35±1.75a
19.62±1.17a
19.53±1.74a
19.83±1.18a
GSH
1.02±0.08a
1.83±0.13b
3.05±0.27c
3.04±0.18c
3.12±0.21c
3.16±0.24c
Choline
93.60±4.99a
96.12±4.05a
102.29±7.51ab
112.07±7.24b
106.49±9.31ab
105.21±5.91ab
TE D
EP
AC C
23.25±0.93
a
29.20±1.66
Skin
21.57±2.15
a
GR
Spleen
22.56±1.15
a
3.67±0.27a
74.25±3.48c
7.60±0.30
M AN U
23.19±1.47
a
7.51±0.24
35.11±1.67 3.40±0.32a
CuZnSOD
b
6.21±0.41
c
34.47±0.75
SC
5.05±0.39
b
40.48±2.00
RI PT
ROS
ROS
100.00±3.27d
82.79±4.57c
78.40±7.35bc
66.30±6.52a
62.67±3.59a
74.82±7.32b
MDA
23.53±1.85c
20.17±1.96b
15.24±1.42a
16.22±0.93a
18.66±1.83b
19.90±1.97b
PC
6.15±0.50d
5.50±0.39c
4.43±0.35b
3.88±0.38a
3.71±0.37a
3.74±0.31a
AHR
27.80±1.80a
30.72±1.52a
47.09±1.41b
48.46±3.93b
45.71±3.63b
47.22±2.46b
ASA
36.36±3.52a
58.09±2.53b
97.29±9.17c
99.19±8.64c
96.62±6.00c
97.59±9.03c
CuZnSOD
4.89±0.48a
5.97±0.50b
7.39±0.37c
8.15±0.40d
9.62±0.35e
9.57±0.16e
MnSOD
4.61±0.44a
5.86±0.52b
6.38±0.59b
7.59±0.61c
7.72±0.71c
7.70±0.75c
CAT
0.72±0.07a
0.82±0.08b
0.92±0.09c
0.92±0.07c
0.84±0.07bc
0.77±0.07ab
GPx
99.69±6.82a
126.22±7.21b
148.66±13.75c
141.26±10.14c
143.52±10.83c
144.84±8.88c
GST
71.65±1.87a
82.60±5.31b
95.82±5.99c
118.42±11.14d
116.41±9.81d
116.70±6.39d
GR
18.63±1.94a
29.49±2.18b
42.17±2.28c
41.26±3.59c
39.68±2.05c
39.41±1.58c
a
GSH
4.85±0.47
Choline
54.42±4.70a
6.30±0.50
b
59.96±4.03ab
9.67±0.55
c
64.49±4.52bc
9.31±0.68
c
71.78±6.07c
9.44±0.68
c
9.41±0.60c
65.81±5.54bc
64.74±3.35bc
R2 = 0.8716
P < 0.05
Regression YROS in head kidney = 2.0516x2 - 23.5161x + 118.6128
YPC in head kidney = -0.7069x + 7.3761
R2 = 0.9845
Ymin = 3.53
YAHR in head kidney = 2.6843x + 57.6346
Ymax = 74.19
YCuZnSOD in head kidney = 0.7478x + 3.2482
Ymax = 5.63
P < 0.01
2
P < 0.01
2
P < 0.01
2
P = 0.120
2
P < 0.05
2
P < 0.05
2
P < 0.05
2
P < 0.01
2
P < 0.05
2
P < 0.05
2
P = 0.053
2
P < 0.05
R = 0.9642
YGpx in head kidney = 7.6283x + 165.7090
Ymax = 208.80
YGST in head kidney = 15.6009x + 66.4829
R = 0.9850
Ymax = 156.41
YGSH in head kidney = 0.3986x + 0.9198
R = 0.9947
Ymax = 2.57
R = 0.9624
2
YROS in spleen = 2.7772x - 29.3119x + 124.8207
R = 0.9016
YPC in spleen = -0.9880x + 6.3272
Ymax = 48.36
YASA in spleen = 7.6202x + 41.3830
Ymax = 95.13
YMnSOD in spleen = 0.8946x + 3.8958
Ymax = 7.43
YGpx in spleen = 23.6900x + 189.5043
Ymax = 286.16
2
YGST in spleen = -1.4327x + 17.6660x + 87.2845
R = 0.9966
RI PT
Ymin = 2.59
YAHR in spleen = 1.5229x + 38.7456
R = 0.9737
R = 0.9951 R = 0.9977
R = 0.9945
R = 0.8582
Ymin = 19.84
R = 0.9991
SC
YGR in spleen = -1.5319x + 26.3782 YGSH in spleen = 0.6718x + 0.3157
Ymax = 3.09
2
YROS in skin = 1.1271x - 14.5238x + 113.2893
2
P = 0.076
2
P < 0.05
2
P < 0.05
2
P = 0.010
2
P < 0.05
2
P < 0.01
2
P = 0.010
2
R = 0.9470
P < 0.05
R2 = 0.9979
P < 0.05
R = 0.9860 R = 0.9414
2
M AN U
YMDA in skin = 0.3858x - 4.1251x + 27.1300 YPC in skin = -0.4997x + 6.6210
Ymin = 3.78
YASA in skin = 20.2353x + 14.2006
Ymax = 97.67
YCuZnSOD in skin = 0.7465x + 4.1952
Ymax = 9.59
YMnSOD in skin = 0.6022x + 4.1449
Ymax = 7.67
2
YCAT in skin = -0.0117x + 0.1188x + 0.6135 YGpx in skin = 16.2702x + 84.8885
Ymax = 144.57
YGST in skin = 9.8306x + 60.0501
Ymax = 117.18
TE D
1017
P = 0.120
2
R = 0.9653
YCAT in head kidney = -0.0196x + 0.2025x + 0.5670
YGSH in skin = 1.6017x + 3.0042
P < 0.01
2
R = 0.9996
2
YGR in skin = 7.8204x + 10.8856
P = 0.010
2
R = 0.9809
Ymax = 7.53
YMnSOD in head kidney = 0.5563x + 3.1772
P < 0.01
2
R = 0.9817
Ymax = 73.04 ACCEPTED MANUSCRIPT
YASA in head kidney = 6.5996x + 36.2278
Ymax = 40.63 Ymax = 9.46
P < 0.01
2
R = 0.8740
R = 0.9794 R = 0.9727 R = 0.9884 R = 0.9801
2
P = 0.010
2
P < 0.05
2
P < 0.05
R = 0.9801 R = 0.9978 R = 0.9485
1018
1
1019
ROS, reactive oxygen species (% DCF florescence); MDA, malondialdehyde (nmol/g tissue); PC, protein carbonyl
1020
(nmol/mg protein); ASA, anti-superoxide anion (U/g protein); AHR, anti-hydroxyl radical (U/mg protein); CuZnSOD,
1021
copper/zinc superoxide dismutase (U/mg protein); MnSOD, manganese superoxide dismutase (U/mg protein); CAT,
1022
catalase (U/mg protein); GPx, glutathione peroxidase (U/mg protein); GST, glutathione-S-transferase (U/mg protein); GR,
1023
glutathione reductase (U/g protein); GSH, glutathione (mg/g protein); Choline (ug/g tissue).
1025
EP
AC C
1024
Values are means ± SD (n = 6), and superscripted different letters in the same row are significantly different (P < 0.05).
1027
1028
Table 6 Correlation coefficient of parameters in the head kidney and spleen.
c-Rel
IL-1β IκBα
TOR
Nrf2
FasL caspase-9
caspase-3 caspase-7 p38 MAPK
MLCK
< 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 = 0.072 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.05
RI PT
NF-κB p52
+0.991 +0.990 +0.951 +0.844 +0.934 +0.936 +0.944 +0.953 +0.948 +0.973 +0.928 +0.974 +0.961 +0.959 +0.962 +0.979 +0.942 +0.906 +0.944 +0.984 +0.972 +0.944 -0.978 -0.999 -0.989 -0.954 -0.994 +0.998 +0.986 +0.995 +0.959 +0.993 +0.993 +0.969 +0.987 +0.992 +0.957 +0.991 +0.926 +0.965 +0.964 +0.866 -0.983 -0.993 +0.772 +0.958 +0.919 -0.978 -0.917 -0.941 +0.972 +0.947 +0.988 +0.945 +0.982 -0.961 -0.979 -0.949 -0.986 -0.918 -0.993 -0.964 -0.948 -0.829 -0.984 +0.982 -0.933
Correlation coefficients +0.922 +0.966 +0.986 +0.919 +0.989 +0.999 +0.992 +0.935 +0.961 +0.961 +0.891 +0.988 +0.985 +0.999 +0.928 +0.968 +0.979 +0.820 +0.981 +0.974 +0.962 +0.907 -0.991 -0.974 -0.975 -0.976 -0.919 +0.979 +0.957 +0.953 +0.919 +0.956 +0.933 +0.996 +0.996 +0.966 +0.914 +0.990 +0.942 +0.951 +0.987 +0.997 -0.998 -0.948 +0.879 +0.946 +0.955 -0.962 -0.942 -0.772 +0.972 +0.993 +0.925 +0.885 +0.983 -0.895 -0.879 -0.905 -0.881 -0.879 -0.888 -0.976 -0.910 -0.802 -0.982 +0.971 -0.851
SC
IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-1β IL-6 IL-12p35 IL-15 IL-17D TNF-α IFN-γ2 IL-12p35 NF-κB p65 NF-κB p52 c-Rel IKKβ IKKγ IL-4/13A IL-10 IL-11 TGF-β1 TGF-β2 CuZnSOD MnSOD CAT GPx1a GPx1b GPx4a GPx4b GSTR GSTO1 GSTP1 GR Keap1a caspase-8 Apaf-1 Bax Bcl-2 Mcl-1 IAP caspase-9 caspase-9 FasL Apaf-1 Bax Bcl-2 Mcl-1 occludin ZO-1 ZO-2 claudin-c claudin-f claudin-7a claudin-7b claudin-11 claudin-12 claudin-15a
Spleen
M AN U
NF-κB p65
Head kidney
Correlation MANUSCRIPT ACCEPTED P coefficients
TE D
Dependent parameters
EP
Independent parameters
AC C
1026
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.01 < 0.05 = 0.055 < 0.01 < 0.01 < 0.05
1029
Table 7 The available phosphorus (AP) requirements based on PWG, FI, skin hemorrhage and lesions
1030
morbidity, head kidney LZ activity and MDA content, spleen C4 and MDA contents of young grass carp
1031
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus.
ACCEPTED MANUSCRIPT
1032 Indices
Regressive equation
R2
P
AP requirement
Y = 34.8502x + 103.7754
Ymax = 246.65
0.9805
< 0.05
4.10 g/kg
FI
Y = 54.0100x + 640.6770
Ymax = 850.82
0.9943
< 0.05
3.89 g/kg
Y = -5.3136x + 27.2760
Ymin = 5.33
0.8498
< 0.01
4.13 g/kg
Y = 15.8684x + 176.9600
Ymax = 267.50
0.7385
< 0.01
5.71 g/kg
Y = -3.9404x + 56.4860
Ymin = 34.64
0.8722
Y = 0.8200x + 4.0421
Ymax = 8.61
0.8704
Y = -5.1413x + 92.2952
Ymin = 64.54
0.7358
Morbidity LZ in head kidney MDA in head kidney C4 in spleen MDA in spleen
< 0.01
5.54 g/kg
= 0.01
5.57 g/kg
< 0.01
5.40 g/kg
SC
1033
RI PT
PWG
AC C
EP
TE D
M AN U
1034
Fig. 1.
ACCEPTED MANUSCRIPT
RI PT
1035
1036
Fig. 1. Effects of available phosphorus levels on skin hemorrhage and lesions morbidity in young grass
1038
carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila. Values having different
1039
letters are significantly different (P < 0.05).
SC
1037
1040
1042
EP
TE D
M AN U
Fig. 2.
AC C
1041
1043
Fig. 2. Phosphorus deficiency led to obviously skin hemorrhage and lesions, compared to available
1044
phosphorus 3.96 g/kg diet, in young grass carp (Ctenopharyngodon idella) after infection with
1045
Aeromonas hydrophila.
1046
Fig. 3.
ACCEPTED MANUSCRIPT
RI PT
1047
M AN U
SC
1048
AC C
EP
TE D
1049
1050
1051
Fig. 3. Relative expression of LEAP-2A, LEAP-2B, hepcidin and β-defensin-1 in the head kidney (A),
1052
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1053
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1054
Values having different letters are significantly different (P < 0.05). LEAP-2, liver-expressed antimicrobial
1055
peptide 2.
1056
Fig. 4.
ACCEPTED MANUSCRIPT
RI PT
1057
M AN U
SC
1058
AC C
EP
TE D
1059
1060
1061
Fig. 4. Relative expression of pro-inflammatory cytokines in the head kidney (A), spleen (B) and skin
1062
(C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of available
1063
phosphorus. Data represent means of six fish in each group, error bars indicate S.D. Values having different
1064
letters are significantly different (P < 0.05). IL, interleukin; IFN-γ2, interferon γ2 and TNF-α, tumor necrosis
1065
factor α.
1066
Fig. 5.
ACCEPTED MANUSCRIPT
RI PT
1067
M AN U
SC
1068
EP AC C
1070
TE D
1069
1071
Fig. 5. Relative expression of anti-inflammatory cytokines and GATA-3 in the head kidney (A), spleen
1072
(B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1073
available phosphorus. Data represent means of six fish in each group, error bars indicate S.D. Values
1074
having different letters are significantly different (P < 0.05). TGF-β, transforming growth factor β.
1075
1076
Fig. 6.
ACCEPTED MANUSCRIPT
RI PT
1077
M AN U TE D EP AC C
1079
SC
1078
1080
1081
Fig. 6. Relative expression of NF-κB p52, NF-κB p65, c-Rel, IκBα, IKKα, IKKβ, IKKγ, TOR, S6K1,
1082
4E-BP1 and 4E-BP2 in the head kidney (A), spleen (B) and skin (C) of young grass carp
1083
(Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus. Data represent
1084
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1085
different (P < 0.05). NF-κB, nuclear factor kappa B; IκBα, inhibitor of κBα; IKK, IκB kinase; TOR, target of
1086
rapamycin; S6K1, ribosomal protein S6 kinases 1; 4E-BP, eIF4E-binding proteins.
1087
1088
Fig. 7.
ACCEPTED MANUSCRIPT
RI PT
1089
M AN U
SC
1090
EP AC C
1092
TE D
1091
1093
Fig. 7. Relative expression of antioxidant enzymes, Nrf2, Keap1a and Keap1b in the head kidney (A),
1094
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1095
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1096
Values having different letters are significantly different (P < 0.05). CuZnSOD, copper, zinc superoxide
1097
dismutase; MnSOD, manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST,
1098
glutathione-S-transferase;
1099
Kelch-like-ECH-associated protein 1.
1100
1101
GR,
glutathione
reductase;
Nrf2,
NF-E2-related
factor
2;
Keap1,
Fig. 8.
ACCEPTED MANUSCRIPT
RI PT
1102
M AN U TE D EP AC C
1104
SC
1103
1105
1106
Fig. 8. Relative expression of apoptotic parameters in the head kidney (A), spleen (B) and skin (C) of
1107
young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of available phosphorus.
1108
Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1109
significantly different (P < 0.05). caspase, cysteinyl aspartic acid-protease; Apaf-1, apoptotic protease
1110
activating factor-1; Bax, Bcl-2 associated X protein; FasL, Fas ligand; Bcl-2, B-cell lymphoma protein-2;
1111
IAP, inhibitor of apoptosis proteins; Mcl-1, myeloid cell leukemia-1; p38 MAPK, p38 mitogen-activated
1112
protein kinase; JNK, c-Jun N-terminal protein kinase.
1113
Fig. 9.
ACCEPTED MANUSCRIPT
RI PT
1114
M AN U
SC
1115
EP AC C
1117
TE D
1116
1118
Fig. 9. Relative expression of tight junction complexes, transporter and MLCK in the head kidney (A),
1119
spleen (B) and skin (C) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1120
levels of available phosphorus. Data represent means of six fish in each group, error bars indicate S.D.
1121
Values having different letters are significantly different (P < 0.05). ZO, zonula occludens; NaPi-IIb:
1122
type-IIb sodium-phosphate cotransporter; MLCK, myosin light chain kinase.
1123
Fig. 10.
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
1124
1125
Fig. 10. The potential action pathways of phosphorus in the head kidney and spleen immune function
1127
and structural integrity of fish.
EP AC C
1128
TE D
1126
ACCEPTED MANUSCRIPT Highlights 1、 Phosphorus deficiency decreased immune function of fish immune organs. 2、 Phosphorus deficiency induced oxidative damage and apoptosis, and thus impaired cellular integrity of fish immune organs.
RI PT
3、 Phosphorus deficiency damaged tight junction, and thus weakened intercellular integrity of fish immune organs.
4、 Dietary phosphorus regulated NF-κB, TOR, Nrf2, p38 MAPK and MLCK signaling of fish
AC C
EP
TE D
M AN U
SC
immune oregans.