Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities

Accepted Manuscript Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities Rui Jia, Bao-Liang Liu, Wen-Rong Feng, Cen Han, Bin Huang, Ji-Lin Lei PII:

S1050-4648(16)30339-4

DOI:

10.1016/j.fsi.2016.05.032

Reference:

YFSIM 3989

To appear in:

Fish and Shellfish Immunology

Received Date: 2 February 2016 Revised Date:

22 May 2016

Accepted Date: 23 May 2016

Please cite this article as: Jia R, Liu B-L, Feng W-R, Han C, Huang B, Lei J-L, Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.05.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities

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Rui Jia a, b , Bao-Liang Liu a, *, Wen-Rong Feng a, Cen Han c, Bin Huang a, Ji-Lin Lei a

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China

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Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science, Qing Dao 266071,

Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China

College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China

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Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, China

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E-mail address: [email protected] (Rui Jia); [email protected] (Bao-Liang Liu)

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Corresponding author. Present address: Yellow Sea Fisheries Research Institute, Chinese

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ACCEPTED MANUSCRIPT Abstract

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Fish skin and its mucus provide the first line of defense against chemical, physical and biological

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stressors, but little is known about the role of skin and its mucus in immune response to crowding

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stress. In the present study, we investigated the stress and immune responses in skin of turbot

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(Scophthalmus maximus) under different stocking densities. Turbot (average weight 185.4 g) were

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reared for 120 days under three densities: low density (LD), medium density (MD), and high

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density (HD). After 120 days, fish were weighed and sampled to obtain blood, mucus and skin

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tissues which were used for analyses of biochemical parameters and genes expression. The results

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showed HD treatment significantly suppressed growth and enhanced plasma cortisol and glucose

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levels (P < 0.05). In mucus, the activities of lysozyme (LZM), alkaline phosphatase (ALP) and

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esterase in HD treatment were lower than LD and MD treatments (P < 0.05) In skin, HD

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treatment resulted in up-regulation in malondialdehyde (MDA) formation and heat shock protein

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70 (HSP 70) mRNA level, and down-regulation in activity of superoxide dismutase (SOD) and the

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transcriptions of glutathione-s-transferase (GST), interleukin-1β (IL-1β), tumor necrosis factor -α

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(TNF-α), insulin-like growth factor-Ι (IGF-Ι) and LZM (P < 0.05). Overall, the data suggested that

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overly high stocking density was a stressor which caused an immunosuppression in skin of turbot.

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Moreover, this information would help to understand the skin immunity and their relation with

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stress and disease in fish.

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Key word: Immune response; Skin; Mucosal immunity; Stocking density; Scophthalmus maximus

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1. Introduction Aquaculture is an important economic activity in many countries, especially in china. Among

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all farmed species, turbot (Scophthalmus maximus) are a high market value fish with an estimated

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production of 59,900 tons in china in 2013 [1]. The species is mainly produced in land-based

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farms including recirculation and flow-through systems [2, 3]. In commercial operation, in order

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to reduce production costs, this species can be adequately cultivated at high stocking density [4, 5].

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Nevertheless, previous investigations have found that overly high stocking density affected the

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fish producing adverse changes in growth and physiological responses [2, 3].

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The stress response is vitally important for all living organisms. In aquaculture activity,

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temporary or chronic stresses are usually inevitable for most fish due to variations of culture

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environment, such as temperature, salinity, stocking density and feed supply [6, 7]. When fish

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confront a stress factor that may threaten its homeostasis, a series of physiological and behavioral

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processes are activated to cope with the adverse situation [8]. However, excessive or prolonged

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activation of stress systems may cause physiological disorder, immunosuppression and growth

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inhibition in farmed fish [9, 10].

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The relationship between stress and immunosuppression has been suggested in fish [11]. Many

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studies have shown that chronic stress resulted in significant immunosuppression, which was

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associated with elevated plasma levels of cortisol [9]. High stocking density has been considered

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as an aquaculture-related chronic stressor invoking primary, secondary and tertiary responses in

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fish [12, 13]. In physiologically, high density not only produces a chronic elevation of plasma

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cortisol, but also increases the formation of reactive oxygen species (ROS) inducing oxidative

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stress [14, 15]. Likewise, high stocking density as a stressor results in alteration of immune-related

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enzyme, protein or gene [7, 16, 17]. Previous report has shown that the levels of immune related

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proteins including lysozyme (LZM), alternative complement pathway (ACP) and peroxidase

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(POD) significantly reduced in plasma of Solea senegalensis subjected to high stocking density

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[18]. Similar results were also reported in Sparus aurata, Oncorhynchus mykiss and Oreochromis

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niloticus reared in high density [12, 19, 20]. The investigations at a molecular level displayed that

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high stocking density affected expressions of genes involved in physiological stress such as

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cytochrome P450 1A (CYP1A), heat shock proteins (HSP 70) and (HSP 90), and innate immune

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system such as interleukin 1β (IL-1β), g-LZM and hepcidin (HAMP) [7, 17, 21, 22].

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ACCEPTED MANUSCRIPT Teleost skin is a multifunctional organ involved in protection, communication, sensory

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perception, locomotion, respiration, ion regulation excretion, and thermal regulation [23-25]. It is

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extremely important as the primary line of defense against a wide variety of chemical, physical

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and biological stressors [26-29]. Disruption of skin barrier homeostasis and dysregulation of skin

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commensals as a result of stress can potentially explain the increased disease susceptibility in

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stressed fish [26, 30]. Unlike terrestrial vertebrates, fish skin secretes mucus through epidermal

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goblet cells, which forms a layer of adherent mucus covering the living epithelial cells [31-33].

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The main components of skin mucus are water and immune substances, such as immunoglobulin

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M (IgM), protease and LZM, which exert inhibitory or lytic activity against different type of

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pathogens [33-35]. To date, several studies have shown that structure and cellular composition of

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fish epidermis can be affected by stressors, such as pathogens, environmental contaminant or

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transportation stress [26, 27, 31]. Furthermore, changes of skin mucus composition and amount

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have also been observed in response to microbial exposure and environmental stress [36, 37].

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Although skin is an important component of the mucosal immune system, there are no

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published reports to investigate the changes of relevant immune parameters in fish under crowding

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conditions. Thus, the aim of the present study is to evaluate the stress and immune responses in

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skin of turbot reared in different densities.

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2. Materials and methods

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2.1. Experimental conditions and animal

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The study was conducted at the farm of Shandong Oriental Ocean Sci-Tech Co., Ltd.

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(Shandong, China) in recirculating aquaculture system (RAS). The RAS unit was consisted of ten

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30-m3 octagonal rearing tanks and a water treatment unit. The water treatment unit contains a filter

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screen, a foam-separation unit, a bio-filter section consisting of 4 separate bio-filters in parallel

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(each 35 m3), a UV sterilizer, and a DO regulating tank. Water (16-18 ℃) supplying the RAS unit

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was pumped from 20 m depth from Laizhou Bay of China, mechanically filtered by two sand

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filters (5 µm filtration) and UV-sterilized before entering the RAS unit.

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Juvenile turbot were obtained from this farm and reared in the RAS for 15 days to acclimatize

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to the experimental environment. After the adaptation period, fish (average weight 185.4 g) were

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randomly distributed into three initial densities: low density (LD) with 1500 fish per tank (9.3

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kg/m2), medium density (MD) with 2200 fish per tank (13.6 kg/m2), and high density (HD) with 4

ACCEPTED MANUSCRIPT 3100 fish per tank (19.1 kg/m2). All treatments were performed in triplicate tanks for a period of

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120 days. No differences in weight and coefficient of variation (CV) for initial weight were found

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among the three densities. During the experimental period, mean temperature was 17 ± 1 °C,

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photoperiod 12 L:12D, salinity 27.3 ± 3 ‰, dissolved oxygen (DO) 8.2 ± 1.1 mg/L and pH 7.6 ±

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0.5. Total ammonium nitrogen (TAN), nitrite and orthophosphates (PO4-P) were measured twice a

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week and never exceeded 0.3 mg/L, 0.25 mg/L and 7.5µM, respectively. The turbot were fed a

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commercial-pellet diet containing 52% crude protein, 12% crude lipids, 16% crude ash, 3% crude

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fiber, 12% water, 5% Ca, 0.5% P, 2.3% lysine, and 3.8% sodium chloride (Ningbo Tech-Bank Co.,

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LTD., Zhejiang, China) two times per day (08:00 and 20:00), and the daily feed rations

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(approximately 1.2% of fish weight/day) were adjusted based on observation of the feeding

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behavior and weight of fish.

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2.2. Sampling

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Fish growth was evaluated biometrically by randomly measuring the individual weight of 15%

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fish population in each tank [38]. The parameters evaluated were as follows: stocking density =

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number of fish × fish weight/ the bottom area of the fish tank; weight gain = (final weight−initial

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weight) × 100/initial weight; stocking density gain = final stocking density– initial stocking

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density.

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After 120 days. Twenty fish were randomly netted from each tank immediately anesthetized in

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0.05% tricaine methane sulfonate (MS-222, Sigma, MO). Mucus collection was performed using

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the methods of Guardiola et al [27] and Jung et al [28] with slight modifications. Briefly, skin

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mucus was collected by gentle scraping the dorsal surface of fish using a soft rubber spatula and

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homogenized with 1 volume of Tris-buffered saline (TBS, 50 mM TriseHCl, 150 mM NaCl, pH

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8.0). The homogenate was centrifuged at 2860 g for 30 min at 4 ℃ and the supernatant was

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lyophilized following overnight freezing at - 80 ℃. Lyophilized skin mucus powder was

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dissolved in Milli-Q water and centrifuged (352 g, 10 min, 4 ℃) to collect supernatant. After

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mucus collection, the skin tissue samples were taken from the dorsal side of turbot as described

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previously [39]. All samples were stored at - 80 ℃ until use.

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2.3. Biochemical parameters assays

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Plasma cortisol was measured using a commercially available ELISA kit (Mlbio, Shanghai,

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China) as a previously described method of Rodríguez et al [40]. Protease activity in mucus was 5

ACCEPTED MANUSCRIPT measured using the azocasein hydrolysis method described by Ross et al[37]. Briefly, skin mucus

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was added in 100 mM ammonium bicarbonate buffer (1:1 v/v) supplemented with 0.7% azocasein

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(Sigma), and incubated for 19 h at 30℃ with shaking. The reaction was stopped by adding

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trichloroacetic acid (4.6% final concentration) and the mixture was centrifuged at 12,000 g for 10

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min. 100 µL of supernatant was transferred to a 96-well plate (Corning Incorporated, USA)

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containing an equal volume of 0.5 M NaOH (Sigma), and the OD read at 450 nm using a plate

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reader (Bio-Rad, USA) with a standard curve using trypsin (Sigma) as reference. Plasma glucose

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and mucus alkaline phosphatase (ALP) were measured in an automatic biochemical analyzer

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Roche CobasC311 (Roche Cobas, Switzerland) using colorimetric test kits purchased from

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Nanjing Jiancheng Biological Engineering Research Institute of China [41]. Activities of

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lysozyme (LZM), esterase and peroxidase (POD) in mucus were determined according to the

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methods described by Dussauze et al [42], Ross et al [37] and Dash et al [43], respectively.

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The activities of superoxide dismutase (SOD) and catalase (CAT) in skin were measured using

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commercially available kits (Jiancheng Institute of Biotechnology, Nanjing, China) and expressed

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as units per milligram protein [44]. The formation of malondialdehyde (MDA) was detected by

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the thiobarbituric acid-reactive substances (TBARS, Sigma) assay [45]. Protein concentration of

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skin homogenate and mucus was determined by the Bradford method, using bovine serum

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albumin as a standard [46]

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2.4. RNA isolation and cDNA synthesis

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The total RNA was isolated from skin tissues using a fast pure RNA kit (Dalian Takara, China)

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according to the manufacturer’s instruction. The amount of RNA was measured using GeneQuant

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1300 (GE Healthcare Biosciences, Piscataway, NJ) and its quality was checked on an agarose gel.

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For each sample, 2 µg of RNA was subjected to cDNA synthesis using PrimeScript RT reagent Kit

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with gDNA Eraser (Takara, Dalian, China) following the manufacturer's protocol. Reverse

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transcription reaction (20 µl) consisted of the following: 2 µg of total RNA, 2 µL of Oligo dT (50

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µM), 4 µL of 5 × M-MLV buffer, 4 µL of dNTP Mixture (10 mM), 0.5 µL of RNase inhibitor (40

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U/µL), 1 µL of Moloney murine leukemia virus reverse transcriptase (200 U/µL), and RNase free

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dH2O up to a final volume of 20 µL. Products (cDNA) were stored at -20℃ for quantitative

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real-time PCR (qRT-PCR).

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2.5. Quantitative real-time PCR protocol

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ACCEPTED MANUSCRIPT The primers used for amplification and gene expression analysis are presented in Table 1. qPCR

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experiments were performed on an ABI PRISM 7500 Detection System (Applied Biosystems,

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USA) by using 96-well plates and SYBR Premix Ex Taq (Dalian Takara, China). For each gene, 4

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µl of cDNA was run in duplicates with the addition of specific primers at 10 µM concentration in

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a final volume of 20 µl. The qRT-PCR cycle was as follows: one cycle at 95 ℃ for 10 s, 40

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cycles at 95 ℃ for 5 s and at 60 ℃ for 34 s. The melting curve was used to ensure that a single

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product was amplified and check for the absence of primer-dimer artifacts. All qRT-PCRs were

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performed at least in triplicate. The results were normalized to β-actin and relative gene

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quantification was performed using the 2-

CT method [47]

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2.6. Statistical analysis

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The statistical analysis was performed using SPSS (version 18.0) software. The results were

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presented as mean ± standard error of the mean (SEM). Data was analyzed for normality

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(Kolmogorov–Smirnov test) and homoscedasticity of variance (Levene’s test) and, when

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necessary, they were log-transformed before being treated statistically. Differences among groups

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were tested by one-way analysis of variance (ANOVA) using density as factor of variance,

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followed by a Tukey test to identify different groups. The differences were considered to be

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significantly different at a level of P < 0.05.

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3. Results

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3.1. Growth performance

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During the course of the experiment, no disease outbreak or other signs of disease were

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observed. Survival was extremely high in all treatments (survival > 96 %) with no significant

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differences between treatments. The established initial stocking densities 9.3 ± 0.11, 13.6 ± 0.21

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and 19.1 ± 0.32 kg/m2 increased up to 26.1 ± 0.16, 38.2 ± 0.18 and 52.3 ± 0.19 kg/m2 at the end of

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experiment, respectively. The final weight and weight gain in the HD group were significantly

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lower than in MD and LD groups (P < 0.05; Table 2).

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3.2. Levels of cortisol and glucose

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Post hoc analysis demonstrated that level of plasma cortisol was markedly higher under the HD

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treatment compared with the LD treatment at day 120 (P < 0.05; Table 3). Similarly, the glucose

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level was apparently higher in HD treatment than LD and MD treatments (P < 0.05; Table 3). 7

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3.3. Enzyme activities in mucus Enzyme activities including protease, LZM, ALP, esterase and POD found in mucus varied

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depending on the stocking density (Fig. 1). At the end of experiment, LZM activity showed

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significantly lower in fish reared in HD group than LD group (P < 0.05). Similarly, the activities

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of ALP and esterase clearly decreased in HD group compared to LD and MD groups (P < 0.05).

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However, the protease and POD activities did not show any differences among the three densities

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during the trial.

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3.4. Oxidative stress parameters in skin

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Compared with MD and LD groups, HD group clearly suppressed SOD activity and improved

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MDA formation in skin (P<0.05; Fig.2A and C). But CAT activity was not affected by stocking

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density.

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3.5. Expressions of stress-related genes in skin

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The expressions of genes involved in the stress responses in the skin of turbot are shown in Fig.

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3. The mRNA level of heat shock protein 70 (HSP 70) showed an obvious increase in fish held at

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HD group compared to fish at MD and LD groups after 120 days (P < 0.05), while the mRNA

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level of glutathione-s-transferase (GST) showed an apparent decrease in HD group (P < 0.05).

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Moreover, metallothionein (MT) mRNA level did not present any differences between different

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treatments.

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3.6. Expressions of immune-related genes in skin

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Skin LZM, interleukin-1β (IL-1β) and insulin-like growth factor-Ι (IGF-Ι) genes expression

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markedly down-regulated in the HD group relative to the LD and MD groups at the end of trial (P

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< 0.05; Fig 4A, B and D). Similarly, the expression of tumor necrosis factor – α (TNF-α) was also

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suppressed by HD treatment compared to LD treatment (P < 0.05; Fig 4C). However, the

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expressions of toll-like receptor 3 (TLR-3) and major histocompatibility complex -Ι (MHC-Ι)

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genes were unchanged by increased stocking density (Fig. 4E, and F).

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4. Discussion

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Stocking density is considered a relevant issue concerning welfare of farmed fish. High

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stocking density as a chronic stressor can negatively affect the growth, welfare, immunologic

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response and behavior [16, 20, 48]. Hence, it is necessary to elucidate the effects of stocking

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density on the immune system and physiology. Up to now, the effects of crowding stress or 8

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stocking density on fish skin have not been extensively studied. To our knowledge, this is the first

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study evaluating the effects of stocking density on stress and immune responses in skin and mucus

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of fish. Generally, reduction of growth and elevation of cortisol and glucose are considered to be

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characteristic indicators of chronic or acute stress, and have been observed in different cultured

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species at high stocking densities [49, 50]. In this work, lower growth (final weight and weight

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gain), and higher cortisol and glucose levels in HD group indicated that turbot were subjected to

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crowding stress when the stocking density reached up to 53.2 kg/m2.

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Fish skin is covered with a layer of mucus which forms an additional barrier against potentially

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harmful substances in water [23]. In the mucus, enzymes, mainly including LZM, POD, ALP,

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esterase and proteases, play important role in the fish immune functions [28]. LZM is a lytic

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protein which causes the lysis of the cell walls of Gram-positive bacteria [51]. Many researches

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have revealed that high stocking density reduced the LZM activity in plasma of fish [12, 18, 19].

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Further studies showed that the mRNA levels of LZM down-regulated in liver and kidney of S.

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senegalensis and O. mykiss farmed under crowding conditions [7, 21, 52]. In line with previous

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studies, the mucus LZM activity clearly decreased in turbot reared in HD group after 120 days.

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Also, in skin the transcription of LZM was markedly down-regulated in HD group. ALP as an

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antibacterial agent is an important enzyme associated with the innate immune system in fish [53].

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Increased activity of ALP was demonstrated in Pentius tetrazona mucus during crowding stress

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[36] and in Scortom barcoo serum during exposure to high density [54]. Conversely, in our study,

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the ALP activity obviously decreased in mucus of turbot reared in high density for 120 days,

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which was consistent with previous report in Salmo salar under high stocking density [55]. Like

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ALP activity, the esterase activity from HD treatment showed a significant decrease at the end of

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trial. Even though the role of esterase in fish mucosal immunity is unclear, it seems that this

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enzyme could act individually or in cooperation with other immune substances to defend against

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pathogens in the mucus [56]. The decreases of these enzymes activities in the mucus might

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suggest an immunodepression due to chronic stress caused by overly high density. The other

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studied enzymes, protease and POD, were also present in skin mucus and related with the defence

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against stress [29]. Their activities are modified by physical or chemical stress suggesting an

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important role in immunity [27, 30]. However, our data showed that both the enzymes activities

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did not change with increasing stocking density. It is reported that high stocking density of fish tended to increase free radical production that

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could damage many biological molecules and antioxidant defense system [14]. SOD and CAT are

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major antioxidants, which are involved in removing the reactive oxide species (ROS) [57].

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Reductions of SOD and CAT are associated with the accumulation of high-living free radical,

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leading to injury of tissue [58]. Sahin et al [59] showed that, when O. mykiss were reared in high

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density, SOD and CAT activities in serum would significantly decrease. In the same way, the

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present study showed that skin SOD activity decreased in HD group. The depression was

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considered to be a response to the continuous stress of stocking density, which might impair the

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proper control of antioxidant defense system [14, 18, 48]. Some studies have showed that stress

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such as high stocking density in fish disturbed the balance between the production of ROS and the

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antioxidant systems, inducing lipid peroxidation [48, 59]. Similarly, our study showed that the

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MDA formation significantly increased in HD group.

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Skin is considered as the largest immunologically active organ, but its molecular mechanism

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remains unclear in fish. The analysis of gene expression patterns would help to understand

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environmental stress responses upon exposure to various stressors at the molecular level, and

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provide a reliable molecular biomarker to estimate the status of the organism [60]. Among several

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molecules involved, HSP 70, GST and MT genes have been used to evaluate the effects of

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stocking density on animal [60-62]. HSP 70 is a most frequently studied heat shock protein in

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response to cellular stress, which assists the folding of nascent polypeptide chains and mediates

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the repair and degradation of altered or denatured proteins [63]. Increased HSP 70 transcription in

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response to stress induced by high density has been documented in fish [60, 64, 65]. The present

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findings confirmed that HD treatment obviously up-regulated HSP 70 mRNA level in skin of

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turbot at the end of trial. The increase in HSP 70 level may be critical for protection from cellular

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damage associated with environmental stressors such as high density [66]. GSTs represent a

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multigene family of primarily soluble enzymes that play a pivotal role not only in detoxification of

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xenobiotics but also in the catalysis of GSH conjugation with nucleophile xenobiotics or

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ROS-impaired nuclear component [67]. Our results displayed that GST gene expression in skin

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was significantly lower in high stocking density. It was possible that the intense oxidative stress

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caused by overly high density induced oxidative damage and suppressed the GST synthesis. MTs

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ACCEPTED MANUSCRIPT are a group of nonenzymatic proteins associated with a variety of functions including metal

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detoxification and antioxidant function [68-70]. Moreover, MT serves as biomarkers of stress and

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is induced by metals and other stress [71, 72]. Gornati et al [22] and Kim et al [60] reported that

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the level of MT mRNA in liver and brain of sea bass and Takifugu obscurus increased after

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exposure to high rearing density. But the results in the present work revealed a lack of induction of

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MT gene expression in skin of turbot reared in different densities. This could mean that the MT

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transcription showed a difference in different species, tissues or environments.

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IL-1β and TNF-α are well-known pro-inflammatory cytokines and mainly secreted by T cells

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and macrophages to stimulate immune response to inflammation in vertebrates [73]. Recent report

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showed that long-term overcrowding stress suppressed the expressions of IL-1β and tumor

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necrosis factor-a (TNF-α) in head kidney of O. mykiss [52]. The reduction of these cytokines

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transcription might be related to elevated cortisol after long-term overcrowding stress [74].

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Similar to previous results, our data exhibited that IL-1β and TNF-α gene expressions reduced in

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skin of turbot rearing in high density for 120 days. Whereas the opposite results were also reported

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in blood of Gadus morhua where overcrowding up-regulated IL-1β gene expression [75].

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The growth hormone/insulin-like growth factor-I (GH/IGF-I) axis regulates many physiological

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functions in fish including growth, osmoregulation, development, and immune response [76, 77].

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IGF-I is a key component of the somatotrophic axis acting as mediator of GH actions to regulate

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somatic growth [78]. Decrease of IGF-1 level may inhibit growth, and even cause physical

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deformity during the early fish body development [79]. In the present work, there was a clear

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down-regulation of IGF-I mRNA in skin of turbot submitted to the highest stocking density. This

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result was in agreement with previous study reported by Salas-Leiton et al [7] where IGF-I mRNA

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levels decreased significantly in liver of S. senegalensis at the highest stocking density. Some

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studies suggested that inhibition IGF-I synthesis was related to high values of cortisol in teleost

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species subjected to different stress sources [79-81].

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Among the examined genes, TLR-3 and MCH-Ι genes also play important role in immune

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response of fish. TLR-3 is one type of innate immunity-related pattern recognition receptor which

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can recognize multiple endogenous and exogenous stress signals [82]. Samanta, Basu [83]

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reported that the activation of TLR-3 signaling induced expression and secretion of cytokines

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TNF-α, IL-1β and IL-8, and played an important role in inducing the anti-viral immunity to the 11

ACCEPTED MANUSCRIPT host. Major histocompatibility complex -Ι (MHC-Ι) encodes highly polymorphic polypeptides,

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which serves the immune system as peptide receptors [84]. Its mRNA level down-regulated in S.

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salar after incubation with PGE2 and LPS [85, 86], and up-regulated in Megalobrama

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amblycephalain, Pseudosciaena crocea and S. salar infected with A. hydrophila [87-89]. Although

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TLR-3 and MHC-Ι have been widely studied in fish, there were no reports on the relationship

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between both genes and stocking density or crowding stress. In the present work, the results

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showed that both genes expressed in the skin of turbot and were no obvious differences among

352

three treatments. It seemed that the two genes were insensitive to crowding stress and were not as

353

potential markers of immune response to chronic stress caused by high stocking density.

354

5 Conclusions

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345

In conclusion, the present study demonstrated, for the first time in fish, the effects of increasing

356

stocking density on stress and immune responses in skin. When the stocking density reached up to

357

52.3 ± 0.34 kg/m2, turbot were subjected to crowding stress accompanied by reduction of growth

358

and elevation of cortisol and glucose. In mucus, overly high density significantly reduced the

359

activities of LZM, ALP and esterase. In skin, the transcription of HSP70 and MDA formation

360

clearly up-regulated, while the activity of SOD and the transcriptions of GST, IL-1β, IGF-Ι,

361

TNF-α and LZM down-regulated in highest stocking density. These results revealed that overly

362

high density was a stressor which caused an immunosuppression in skin of turbot. Moreover, this

363

information will help to understand the skin immunity and their relation with stress and disease in

364

fish.

365

Acknowledgments

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This work was supported by the National Natural Science Foundation of China (No. 31402315

367

and 31240012), the Modern Agriculture Industry System Construction of Special Funds

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(CARS-50-G10), Key R & D program of Jiangsu Province (BE2015328) and the Key Laboratory

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of Mariculture & Stock Enhancement in North ChinaNorth Chinal Science Foundation，P.R.

370

China.

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371 372 373 374 12

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18

ACCEPTED MANUSCRIPT Table 1 Primer utilized for gene expression analysis. Genes

Primer sequence (5’-3’)

Amplicon

Gen Bank

Reference

107

EU686692.1

[90]

139

FJ160587.1

[91]

196

DQ848966.1

[92]

220

EF191027.1

[92]

148

EF406132.1

This study

size (pb) ß-actin

F: TGAACCCCAAAGCCAACAGG

F: TGTACTGTGCGCCTGCCAAGACTA R: TGCTGTGCTGTCCTACGCTCTGT

GST

F: GGGTTCGCATCGCTTTT R: GGCCTGGTCTCGTCTATGTACT

HSP 70

F: CTGTCCCTGGGTATTGAGAC R: GAACACCACGAGGAGCA

MT

F: TGCTCCAAGAGTGGAACCTG R: CGCATGTCTTCCCTTTGCAC

TNF-α

F: GGGCTGGTACAACACCATCTATC

165

FJ654645.1

[93]

AJ295836.2

[91]

191

AJ250732.1

[93]

147

EF032639.1

[93]

100

FJ009111.1

[94]

R: TTCAATTAGTGCCACGACAAAGA F: ACCAGACCTTCAGCATCCAGCGT

81

M AN U

IL-1β

SC

IGF-Ι

RI PT

R: AGAGGCATACAGGGACAGCAC

R: TTCAGTGCCCCATTCCACCTTCCA LZM

F: CTCTCAACGTTCCCACTGGTTCTA R: GGGGTCATGAAGTGTCTGTAGAT

MHC-Ι

F: GGATCCTCTCAAGTCCCAAACTT R:CTCCCAGTACTGTGCATCTGTTG

TLR-3

F: GACGTGCTGATCCTGGTCTTTCTGG

AC C

EP

TE D

R: AGCTCAGGTAGGTCCGCTTGTTCA

19

ACCEPTED MANUSCRIPT Table 1 Primer utilized for gene expression analysis. Genes

Primer sequence (5’-3’)

Amplicon

Gen Bank

Reference

107

EU686692.1

[90]

139

FJ160587.1

[91]

196

DQ848966.1

[92]

220

EF191027.1

[92]

148

EF406132.1

This study

FJ654645.1

[93]

size (pb) ß-actin

F: TGAACCCCAAAGCCAACAGG

F: TGTACTGTGCGCCTGCCAAGACTA R: TGCTGTGCTGTCCTACGCTCTGT

GST

F: GGGTTCGCATCGCTTTT R: GGCCTGGTCTCGTCTATGTACT

HSP 70

F: CTGTCCCTGGGTATTGAGAC R: GAACACCACGAGGAGCA

MT

F: TGCTCCAAGAGTGGAACCTG R: CGCATGTCTTCCCTTTGCAC

TNF-α

F: GGGCTGGTACAACACCATCTATC

165

IL-1β

M AN U

R: TTCAATTAGTGCCACGACAAAGA

SC

IGF-Ι

RI PT

R: AGAGGCATACAGGGACAGCAC

F: ACCAGACCTTCAGCATCCAGCGT

81

AJ295836.2

[91]

191

AJ250732.1

[93]

147

EF032639.1

[93]

100

FJ009111.1

[94]

R: TTCAGTGCCCCATTCCACCTTCCA LZM

F: CTCTCAACGTTCCCACTGGTTCTA R: GGGGTCATGAAGTGTCTGTAGAT

MHC-Ι

F: GGATCCTCTCAAGTCCCAAACTT R:CTCCCAGTACTGTGCATCTGTTG

TLR-3

F: GACGTGCTGATCCTGGTCTTTCTGG

AC C

EP

TE D

R: AGCTCAGGTAGGTCCGCTTGTTCA

ACCEPTED MANUSCRIPT Table 2 Changes of survival, weight and stocking density of turbot for 120 days Parameters

Low density

Medium density

High density

Survival (%)

96.8 ± 6.33

97.7 ± 5.35

98.3 ± 4.87

Initial weight (g)

186.0 ± 0.97

185.2 ± 0.82

185.0 ± 1.57

Final weight (g)

540.4 ± 4.52 a

534.4 ± 2.25

Weight gain (%)

190.5 ± 1.72 a

188.5 ± 1.32 a

Initial stocking density (kg/m2)

9.3 ± 0.11

13.6 ± 0.21

Final stocking density (kg/m2)

26.1 ± 0.16

38.2 ± 0.18

52.3 ± 0.19

Stocking density gain (kg/m2)

16.8 ± 1.54

24.6 ± 2.76

33.2 ± 1.26

b

RI PT

514.2 ± 3.37

177.9 ± 2.45 b 19.1 ± 0.32

M AN U

SC

a

The values are means ± SEM. Different letter denote significant differences between densities (P

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EP

TE D

< 0.05).

ACCEPTED MANUSCRIPT Table 3 Changes of cortisol and glucose of turbot under different stocking density for 120 days Parameters

Low density

Medium density

High density

Cortisol (ng/mL)

1.51 ± 0.05 a

2.12 ± 0.04 ab

2.58 ± 0.05 b

Glucose (mmol/L)

1.28 ± 0.14 a

1.25 ± 0.13 a

1.80 ± 0.03 b

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EP

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M AN U

SC

< 0.05).

RI PT

The values are means ± SEM. Different letter denote significant differences between densities (P

ACCEPTED MANUSCRIPT Figure legends: Fig. 1. Enzyme activities in skin mucus of turbot reared in different densities for 120 days. The values are means ± SEM, n = 20 fish per tank. Different letters denote significant differences between densities (P < 0.05). LD, low density; MD, medium density; HD, high density.

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Fig. 2. Changes of oxidative stress parameters in skin of turbot reared in different densities for 120 days. The values are means ± SEM, n = 20 fish per tank. Different letters denote significant differences between densities (P < 0.05). LD, low density; MD, medium density; HD, high density.

SC

Fig. 3. Expressions of stress-related genes in skin of turbot reared in different densities for 120 days. The values are means ± SEM, n = 20 fish per tank. Different letters denote significant differences between densities (P < 0.05). LD, low density; MD, medium density; HD, high density.

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EP

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M AN U

Fig. 4. Expressions of immune-related genes in skin of turbot reared in different densities for 120 days. The values are means ± SEM, n = 20 fish per tank. Different letters denote significant differences between densities (P < 0.05). LD, low density; MD, medium density; HD, high density.

ACCEPTED MANUSCRIPT Fig.1 12

12

b 8 6 4

b 8 6 4

2

2 0

0 HD

1.4

LD

D

a

a

MD

0.7 0.6

1.0 b 0.8 0.6

0.4 0.3 0.2

M AN U

0.4

0.5

0.2 0.0

HD

SC

1.2

0.1

0

LD

MD

E

HD

12

8 6

LD

4

TE D

POD (U/mgprot)

10

2 0

EP

LD

AC C

Esterase (U/mgprot)

MD

Protease (ng/mgprot)

LD

C

a

a 10

ab

RI PT

10

LZM (ng/mgprot)

B

a

ALP (U/mgprot)

A

MD

HD

MD

HD

ACCEPTED MANUSCRIPT Fig.2 A 120 a a

80

b

60 40 20

14 12 10 8

MD

LD

MD

6 4 2 0

C

12

HD

b

10 8 a 6 4 2 0

MD

AC C

EP

LD

a

TE D

MDA (nmol/mgprot)

HD

SC

20 18 16

LD

M AN U

B CAT (U/mgprot)

0

RI PT

SOD (U/mgprot)

100

HD

ACCEPTED MANUSCRIPT Fig.3

A

3.0 b

2.0 1.5

a

1.0 0.5 0.0

B

1.8

LD

MD

a

a

HD

1.4 1.2

b

1.0 0.8 0.6 0.4 0.2 0.0 LD

C

MD

1.6

1.0 0.8 0.6 0.4 0.2 0.0

HD

TE D

1.2

EP

MT mRNA levels (arbitary units)

1.4

M AN U

GST mRNA levels (arbitary units)

1.6

AC C

LD

MD

RI PT

a

SC

HSP70 mRNA levels (arbitary units)

2.5

HD

ACCEPTED MANUSCRIPT Fig.4

LZM mRNA levels (arbitary units)

1.2

1.2

a

1.0 0.8 b

0.6 0.4 0.2 MD

D

a

b

0.4

1.4 1.2

ab

1.0 0.8

b

0.6 0.4

IGF-1 mRNA levels (arbitary units)

TNF-α mRNA levels (arbitary units)

0.6

LD

1.0 0.8 0.6 0.4 0.2

0.0

a

M AN U

0.2

MD

HD

a

b

0.0

LD

MD

1.6

HD

F

1.4

LD

MD

HD

LD

MD

HD

1.1

MHC-Ι mRNA levels (arbitary units)

1.1

1.2 1.0 0.8 0.6 0.4 0.2 0.0

MD

AC C

EP

LD

TE D

TLR-3 mRNA levels (arbitary units)

0.8

HD

1.4

E

1.0

0.0 LD

1.2

a a

0.2

0.0

C

1.4

RI PT

B

a

SC

1.4

IL-1β mRNA levels (arbitary units)

A

HD

1.0 1.0 0.9 0.9 0.8

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Skin and mucus were key components of the innate immune system against stressor. Overly stocking density inhibits activities of most mucus enzymes. Overly stocking density induces cellular stress response and oxidative damage in skin. Skin immune response gene expressions down-regulate in skin of turbot rearing in highest density.

AC C