Interactive effects of dietary cholesterol and phospholipids on the growth performance, expression of immune-related genes and resistance against Vibrio alginolyticus in white shrimp (Litopenaeus vannamei)

Journal Pre-proof Interactive effects of dietary cholesterol and phospholipids on the growth performance, expression of immune-related genes and resistance against Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) Minglei Yan, Weilong Wang, Xuxiong Huang, Xinlei Wang, Yi Wang PII:

S1050-4648(19)31091-5

DOI:

https://doi.org/10.1016/j.fsi.2019.11.048

Reference:

YFSIM 6617

To appear in:

Fish and Shellfish Immunology

Received Date: 12 June 2019 Revised Date:

15 November 2019

Accepted Date: 18 November 2019

Please cite this article as: Yan M, Wang W, Huang X, Wang X, Wang Y, Interactive effects of dietary cholesterol and phospholipids on the growth performance, expression of immune-related genes and resistance against Vibrio alginolyticus in white shrimp (Litopenaeus vannamei), Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.11.048. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Interactive effects of dietary cholesterol and phospholipids on

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the growth performance, expression of immune-related genes

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and resistance against Vibrio alginolyticus in white shrimp

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(Litopenaeus vannamei)

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Minglei Yan1, Weilong Wang1,3, Xuxiong Huang1,2,3*, Xinlei Wang1, Yi Wang1

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Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of the

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Ministry of Agriculture, Shanghai Ocean University, Shanghai, China.

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Shanghai Engineering Research Center of Aquaculture, Shanghai, China.

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Demonstration Center for Experimental Fisheries Science Education (Shanghai

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Ocean University), Lingang New City, Shanghai, China.

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Minglei Yan and Weilong Wang these authors contributed equally to this work.

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*Corresponding author: Xuxiong Huang, Key Laboratory of Freshwater Aquatic

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Genetic Resources, Ministry of Agriculture, Lingang New City, Shanghai, China.

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Email address: [email protected]

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Abstract:

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A 56-day feeding trial was done to investigate the interactive effects of cholesterol

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(CHO) and phospholipids (PL) on the growth performance, immune response,

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expression of immune-related genes, and resistance against Vibrio alginolyticus of

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freshwater cultured white shrimp (Litopenaeus vannamei). A 3 × 3 experimental

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design was conducted with nine experimental diets containing three levels of CHO (0,

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0.2%, and 0.4%) and three levels of PL (0, 2%, and 4%). The results indicated that the

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growth performance significantly (P < 0.05) increased with the increase in dietary

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CHO levels. Interactive effects between dietary CHO and PL on the growth

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parameters were not observed. Superoxide dismutase (SOD) and lysozyme activities

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were also significantly affected by dietary CHO levels. Furthermore, the interaction

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between these two additives was only detected in SOD activity. Shrimp fed

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experimental diet with CHO and PL supplementation showed better tolerance against

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Vibrio alginolyticus compared to the control, interactive effects (P < 0.05) were also

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detected on these two factors. The expression of immune deficiency (IMD) and

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lysozyme mRNA was up-regulated in shrimp fed diets with CHO and PL. The

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expression level of Toll-like receptor mRNA directly reflected the dietary CHO levels,

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which was not affected by dietary PL. The interaction between dietary CHO and PL

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was shown as the significant factor (P < 0.05) both in the expression of IMD and

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lysozyme mRNA, which indicated that different dietary levels of CHO and PL could

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strongly affect expression levels of some immune-relevant genes of the juvenile

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freshwater cultured L. vannamei. 2

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Keywords: interaction, cholesterol, phospholipids, freshwater, Litopenaeus vannamei.

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1.

Introduction

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Litopenaeus vannamei is one of the three largest farmed species of shrimp in the

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world (Morris et al., 2011). Given the deterioration of the aquatic aquaculture

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environment, the degradation of germplasm resources, the frequent occurrence of

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disease, and the abuse of aquatic drugs (Zhu et al., 2018), the L. vannamei aquaculture

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industry is facing severe challenges. In particular, primary and secondary bacterial

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diseases have caused large-scale mortalities of farmed L. vannamei (Wang and Chen,

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2005). Therefore, the immunity ability of L. vannamei is of primary concern when

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they are subjected to environmental stressors (Li and Chen, 2008).

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Invertebrates, such as crustaceans, do not possess an adaptive immune response

60

based on immunoglobulin (Underwood et al., 2013). The defense mechanisms of

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crustaceans depend entirely on the innate immune system, which is activated once the

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pathogen-associated molecular patterns (PAMPs) are recognized by host pattern

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recognition receptors (Vazquez et al., 2009). The innate immune system then relies on

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cellular or humoral immune mechanisms to destroy the invasive pathogen (Vazquez et

65

al., 2009). The immune response to foreign pathogens is triggered by the immune

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recognition of foreign substances, leading to the transduction of recognized signals,

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and the activation of a series of cascade reactions (Li and Chen, 2008). Finally, the

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innate immune system generates and releases immune effect factors (such as

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lysozymes or antimicrobial peptides), targeting and eliminating the invasive substance

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(Wang et al., 2008). Signaling pathways (e.g., the Toll and IMD pathways), which 3

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regulate intracellular and extracellular signal transduction, play an important role in

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the transduction of immune signals (Shrestha and Kim, 2010). The Toll receptor is a

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transmembrane protein, and the immune deficiency (IMD) protein is the first agent to

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receive the glucan recognition protein signal. Both the Toll receptor and the IMD

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protein transmit signals about specific types of foreign invaders from the exterior to

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the interior of the cell, activating a series of cascade reactions to produce immune

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effect factors (Li and Xiang, 2013). Superoxide dismutase (SOD) is an antioxidant

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enzyme that forms the first line of defense against free radicals in organisms (Li et al.,

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2018). Lysozyme acts as part of the nonspecific immune system by hydrolyzing the

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β-1, 4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine

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(Jeune et al., 2010). Therefore, signal pathways-related gene expression, SOD, and

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lysozyme activity are commonly used as an indicator for evaluating a host’s immunity

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and health status (Yang et al., 2017; Li et al., 2018).

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Adjustment of nutrients in the feed may not only improve the growth performance

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but also the resistance to disease of shrimp (Zhang et al., 2013; Guo et al., 2016; Lee

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et al., 2017). Cholesterol (CHO) is an important sterol which serves as a precursor for

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many physiologically active compounds such as sex and molting hormones, adrenal

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corticoids, bile acids, and vitamin D (Sheen, 2000). Phospholipids (PL) are known to

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comprise a group of polar lipids as a source of energy added to the diet of crustaceans

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and are believed to play a vital role to facilitate the storage of lipids in the

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hepatopancreas (Gong et al., 2001; Wu et al., 2007). Both CHO and PL are

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bio-membrane components that associated with maintaining cellular structure and

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function (Gonzalez-Felix et al., 2002; Zhu et al., 2018). However, crustaceans cannot

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synthesize CHO de novo or synthesize PL sufficiently (Teshima et al., 1997; Wu et al.,

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2007). In addition, limited investigations have examined cholesterol-related and 4

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phospholipid-related antioxidant and immune functions in fish and crustaceans (Gu et

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al., 2017; Jafari et al.,2018)

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Previous studies have shown the effects of dietary CHO and PL (such as soybean

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lecithin) on growth performance (Smith et al., 2015; Sánchez et al., 2014), lipid

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content and fractions in shrimp tissues (Thongrod et al., 1998; Gong et al., 2000a).

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However, information about the influence of these two dietary components on

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immune response particularly for freshwater cultured L. vannamei is still lacking.

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Therefore, an 8-week experiment was conducted to determine the (1) effects of

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dietary CHO and PL on survival, growth, expression of immune-related genes, and

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resistance against Vibrio alginolyticus of juvenile L. vannamei in the freshwater; (2)

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possible interactions between dietary CHO and PL on these parameters.

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2.

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2.1. Diet preparation

Materials and methods

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Nine isonitrogenous experimental diets were formulated with three levels of CHO

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(0, 0.2%, and 0.4%) and three levels of PL (0, 2%, and 4%) in a 3 × 3 experimental

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design as shown in Table 1. Different PL levels in diets were achieved by adding

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soybean lecithin in different levels at the expense of soybean oil. Dry ingredients of

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each diet were ground, screened through an 80-mesh sieve, and mixed thoroughly.

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The lipid ingredients were added drop by drop to the dry mixture while blending.

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After that, the mixture was formed into a dough by adding water. Then the dough was

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extruded through 1.5 mm orifice die and dried at 90°C for 20 min. The dried pellets

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were stored at -20°C in a refrigerator until use.

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2.2. Test shrimp and feeding protocol 5

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Juvenile L. vannamei were obtained from the local hatchery (Shanghai, China),

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where the salinity was already decreased from 20‰ to 2‰ during the post-larval

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stage.

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Juveniles were maintained in an indoor cement pool fed with a commercial diet for 14

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days. After acclimatizing to laboratory conditions (1‰), 1800 healthy shrimp with

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similar size (0.23 ± 0.01 g initial average wet weight) were selected and randomly

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allocated to 36 PVC net cages (1 m × 1 m × 1.2 m) located in a cement pool (10 m ×

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20 m × 1.5 m) corresponding to quadruplicate cages of the nine dietary treatments.

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Juveniles were fed four times a day (5:30, 10:30, 16:30, and 22:00), at a daily ration

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amount of 5-8% of body weight. Each morning, the uneaten feed was collected, while

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fecal matters were siphoned from the bottom. All uneaten feed was freeze-dried to

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calculate feed intake and the feed efficiency ratio. During the feeding trial, the

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experimental water was changed 50% every three days from the impounding reservoir

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nearby. The water quality parameters were tested everyday including temperature, pH,

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dissolved oxygen, and ammonia nitrogen were maintained at 30-32°C, 8.0-8.5, > 5

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mg L-1, and <0.05 mg L-1, respectively.

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2.3. Sample collection and biochemical analysis

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At the end of the 8-week feeding trial, all treatments were fasted for 24 h prior to

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final sampling. The survivors and individual body weight from each cage were

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measured using the following formulae:

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Survival (%) = (final number of shrimp/initial number of shrimp) × 100

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Specific growth rate (SGR, % day-1) = [(Ln final weight-Ln initial weight)/duration] ×

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100

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Feed conversion ratio (FCR) = dry weight of feed consumed (g)/live weight gain (g) 6

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Proximate compositions of experimental diets and shrimp body samples were

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measured per the standard methods of Association of Official Analytical Chemists

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(AOAC, 1995). Moisture was determined by oven-drying at 105℃ to a constant

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weight. Ash was determined by incinerating in a muffle furnace at 550℃ for 6 h.

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Crude protein contents of diets and bodies were determined by the Kjeldahl method

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(2300-Autoanalyzer, Foss Tecator, Sweden). Total lipids contents of them were

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individually extracted using chloroform-methanol (2:1, v/v) containing 0.01%

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butylated hydroxytoluene (BHT) as an antioxidant per Cejas et al. (2004). Total lipid

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was separated to Neural lipid and Polar lipid fractions by Sep-pak silica cartridge

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(Waters Corporation, Milford, Massachusettes, USA) as described by Juaneda and

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Rocquelin (1985). Both fractions were analyzed to quantify the content of CHO and

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PL from lipid class per the method of Thongrod et al. (1998).

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Five juveniles from each tank were obtained for immunological analysis.

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Hemolymph was collected from the ventral sinus of shrimp using 1 mL syringes, and

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then centrifugated at 2500 rpm for 20 min at 4°C to obtain serum. The serum was

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used to measured lysozyme and superoxide dismutase (SOD) activities. SOD was

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measured using diagnostic reagent kits (Nanjing Jiancheng Bioengineering Institute,

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China). And lysozyme activity in the serum was determined per turbidimetric assay

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(Lygren et al, 1999).

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2.4. Susceptibility of shrimp to Vibrio alginolyticus

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The V. alginolyticus strains were obtained from the National Pathogen Collection

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Center for Aquatic Animals (Shanghai Ocean University, Shanghai, China) and

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activated by infecting the L. vannamei. Hemolymph samples were inoculated from the

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diseased shrimp on thiosulfate citrate bile salts sucrose (TCBS) agar culture medium 7

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plates to obtain virulent clones for the formal infection test.

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At the end of the eight-week feeding trial, thirty juveniles in similar size were

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selected from each treatment and used for susceptibility assay to V. alginolyticus in

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triplicates. The challenge test was carried out by injecting 25 µL activated V.

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alginolyticus (6.25×107 CFU mL-1). Control shrimp were injected with 25 µL of

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physiological shrimp saline. All 30 cages (50 cm × 30 cm × 80 cm) were suspended in

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a cement pool with continuous aeration, a water temperature of 29-30°C, a salinity of

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0 ppt, and dissolved oxygen > 6 mg L-1. The water was renewed daily, and the

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experiment lasted for four days.

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2.5. Total RNA extraction, reverse transcription and quantitative real-time PCR

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Seventy-five juveniles in similar sizes from each treatment were selected and

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injected 25 µL inactivated V. alginolyticus (1.25×106 CFU mL-1) of each shrimp. The

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control group was injected with physiological shrimp saline. All cages were

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suspended in a cement pool as described earlier for activated V. alginolyticus

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challenge. For 48 h after injection, the gill tissues of three shrimp were sampled from

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each cage every 6 h. The samples were quickly placed into 1.5 mL centrifuge tubes

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containing precooled RNA preservation solution (Tiangen Biochemical Technology

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Co., Ltd., China).

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Total RNA was extracted from the gill tissues with Total RNA Extraction Kits

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(Tiangen, China), according to the manufacturer's instructions. Isolated RNA quantity

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and quality were determined via ultra-micro spectrophotometer and on a 1% agarose

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gel electrophoresis, respectively. cDNA for qRT-PCR was synthesized via reverse

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transcription using PrimeScriptTM RT Reagent Kits (Takara, Japan). Reaction

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conditions were those recommended by the manufacturer. Gene-specific primers were 8

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designed using Primer Premier 5.0 software based on the cDNA sequences in

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GenBank [lysozyme (GenBank: AY170126.2); Toll-like receptor (GenBank:

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DQ923424.1); IMD (GenBank: FJ592176.1)] (Table 2). qRT-PCR was performed

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using the SYBR@Premix ExTaq™ Kit (Takara, Japan) in the CFX96™ real-time

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system (Bio-Rad, Hercules, CA). Amplification was performed in a total volume of

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20 µL, containing 10 µL SYBR Green Premix ExTaq, 0.8 µL of each primer, 1.6 µL

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cDNA, and 6.8 µL DEPC-H2O. The cycling condition was as: 95°C for 30s, followed

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by 39 cycles of 95°C for 10s, 60°C for 30s, and 72°C for 20s. The housekeeping gene

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β-actin was amplified as an internal reference. The experiment was repeated three

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times. The relative mRNA expression of target genes was calculated using the 2-∆∆Ct

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method (Livak and Schmittgen, 2001).

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2.6. Statistical analysis

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All data were presented as mean values ± standard error of the mean (S.E.M., n=3).

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The statistical analysis was performed by using an analysis of variance (SPSS 20.0)

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after exploring the normality and homogeneity of data. Two-way ANOVA was

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employed to test the effects of dietary CHO and PL levels as well as their interactions.

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Once the interaction between these two factors is found, all the six groups were tested

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by one-way ANOVA to test all levels of the two factors together. Moreover, data from

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each treatment were compared to the control using Duncan's new multiple range test

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Differences between treatments were considered significant when P < 0.05.

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2.7. Ethical statement

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The present experimental procedures were carried out in strict accordance with the

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recommendations in the ethical guidelines of EU Directive 2010/63/EU for animal 9

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experiments.

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3.

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3.1. Growth performances and survival

Results

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Growth performance, survival, and feed conversion ratio of the test shrimp fed

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with experimental diets for 56 days are provided in Table 3. In two-way ANOVA, no

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interactive effect was found on all parameters. Survival was not significantly affected

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by either dietary CHO or PL. Meanwhile, a similar trend could be also found on the

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feed conversion ratio (FCR). Dietary CHO was the only factor that significantly (P <

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0.05) affected final body weight (FBW) and specific growth rate (SGR). The FBW

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and SGR showed an increasing trend with increased dietary CHO.

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3.2. Shrimp muscle proximate analysis

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The muscle proximate analysis of L. vannamei fed with experimental diets for 56

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days is shown in Table 4. Dietary CHO and PL supplementations and the interaction

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between these two additives did not significantly (P > 0.05) affect moisture. Although

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no interactive effect was found, both CHO and PL levels significantly (P < 0.05)

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affect ash. Dietary CHO and the interaction between dietary CHO and PL were the

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only significant factor in total lipid and crude protein, respectively.

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3.3. Immune parameters

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The superoxide dismutase (SOD) and lysozyme activities in serum of L. vannamei

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fed with experimental diets for 56 days are shown in Table 5. Dietary CHO and the

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interaction between dietary CHO and PL significantly affected SOD activity. Dietary

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CHO was the only significant factor in lysozyme activity. 10

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The low dietary CHO three groups (CHO0-PL0, CHO0-PL2, and CHO0-PL4)

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groups showed significantly lower (P < 0.05) SOD activity than intermediate dietary

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CHO two groups (CHO0.2-PL0 and CHO0.2-PL2) and high dietary CHO two groups

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(CHO0.4-PL2 and CHO0.4-PL4). Furthermore, the CHO0.4-PL4 showed the highest SOD

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activity among all the groups.

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3.4. Immune related gene expression

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The expressions of genes related to Toll-like receptor, immune deficiency (IMD),

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and lysozyme from gill tissues of L. vannamei are shown in Table 6, 7, and 8,

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respectively.

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For the expression level of the Toll-like receptor gene, the interaction between

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dietary CHO and PL was observed at 12h, 24h, and 36h. Dietary CHO significantly

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(P < 0.05) affected the mRNA expression at 24h and 36h. For expression level of

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IMD gene, dietary CHO and PL significantly affected the mRNA expression at 24h.

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Meanwhile, the interaction between dietary these two additives was not detected

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during the challenge period. For the lysozyme gene, the interaction between dietary

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CHO and PL was observed during the whole challenge period. Furthermore, dietary

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CHO and PL were the significant factors at 24h, 36h, and 48h.

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The peaks of relative mRNA expression of the Toll-like receptor and lysozyme

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genes were all detected at 24h and 36h, respectively. CHO0.2-PL2 and CHO0.4-PL2

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showed significantly higher expression levels of the Toll-like receptor gene when

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compared to the control group. And CHO0.2-PL4 and CHO0.4-PL4 showed significantly

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higher expression levels of the lysozyme gene when compared to the control. The

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peaks of relative mRNA expression of the IMD gene were all detected at 24h except

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the control group. 11

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3.5. Cumulative mortality of L. vannamei upon challenge with V. alginolyticus

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Monitoring the mortality of shrimp post pathogen challenge (Fig. 1) revealed

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that

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the interaction between dietary CHO and PL was observed at 24h, 48h, 72h, and 96h.

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Furthermore, CHO0.4-PL0 and CHO0.4-PL4 showed significantly better performance

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when compared to the control (CHO0-PL0) at 48h, 72h, and 96h.

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4.

Discussion

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CHO is a major dietary sterol and nutritionally superior to other sterols for shrimp

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(Teshima, 1997). The requirement of dietary CHO has been generally established

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through observation of reduced growth response in crustaceans (NRC, 2011).

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Previous studies reported CHO requirements for shrimp commonly range from 0.2-1%

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(Kanazawa et al., 1971; Kean et al., 1985; Gong et al., 2000a). On the other hand,

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excess cholesterol might cause some adverse effects (Thongrod et al., 1998). Gong et

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al. (2004) suggested that incorporating PL and CHO could improve osmoregulatory

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capacity in L. vannamei, thus leading to better survival and growth under low salinity

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conditions (Paibulkichakul et al., 1998; Coutteau et al., 2000). Therefore, dietary

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supplementation of CHO and PL is crucial to meet metabolic requirements during the

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juvenile and younger stages, especially for inland culture of shrimp (Kanazawa, 1985;

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Gong et al., 2000b; Roy et al., 2006).

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These were consistent with the results in the current study which showed that

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dietary CHO is a significant factor in growth performances. Although dietary PL has 12

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been found to have a beneficial effect on the growth and survival of marine shrimp in

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several studies (Kanazawa et al., 1985; Gonzalez et al., 2002; Roy et al., 2006),

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dietary PL was not detected as the significant factor on either survival or growth

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performances in current study. This indicated that the non-PL supplemented diet

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which already contained 1% PL, might have satisfied the requirement of L. vannamei

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in freshwater. Furthermore, the efficacy of PL on growth and survival may vary with

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the type and source of PL used (Han et al., 2016). The major PL in animals is

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phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI),

304

and phosphatidylserine (PS) (Gong et al., 2001). In most investigations, the source of

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PL is provided by soybean lecithin (Chen and Jenn, 1991; Thongrod et al., 1998).

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Dietary PL such as soybean lecithin and PC have been demonstrated to be required

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for growth and survival for juveniles and larval forms of shrimp (Jafari et al., 2018).

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Since soybean lecithin is available in different purity and composition of PL, the

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required quantitative and qualitative amount of dietary soybean lecithin is difficult to

310

state. For PC requirement, a relatively conservative estimate based upon previous

311

results is between 0.5-1.5% (NRC, 2011). The interaction between PL and CHO has

312

been of great interest in crustacean nutrition (Roy et al., 2006). PL was well known to

313

facilitate the transport of CHO, which implied an interaction between them.

314

Furthermore, Gong et al. (2000a) and Holme et al. (2007) had reported that there was

315

a synergistic effect between dietary PL and CHO in marine L. vannamei and Scylla

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serrata. However, no interactions were detected in the present study. Similar results

317

were observed in Penaeus monodon (Chen, 1993), Macrobrachium rosenbergii

318

(Briggs et al., 1988) and Portunus trituberculatus (Han et al., 2016). Hence, the

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interaction between dietary CHO and PL in the diet of crustaceans may not be

320

universal for improving growth performance. 13

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Organisms produce superoxide anions (O2-) to kill or eliminate harmful

322

substances in response to bacterial invasions or stress (Gu et al., 2017). However,

323

excessive O2- accumulation can also pose a threat to the organism itself. By catalyzing

324

superoxide anions via disproportionation reactions, superoxide dismutase (SOD) can

325

reduce O2- accumulation (Maher and Schubert, 2000). Therefore, SOD levels are not

326

only as an indicator of the antioxidant but also help to characterize immune status. In

327

the current study, dietary supplementation with CHO had significant effects on serum

328

SOD activity in L. vannamei (P < 0.05), as did the interaction between dietary CHO

329

and PL. In the high dietary CHO (0.4%) supplementation three groups, the SOD

330

activity increased with the increase in dietary PL levels. Furthermore, the highest

331

level of SOD activity was observed in CHO0.4-PL4 group. Han et al. (2015) elucidated

332

that swimming crabs (Portunus trituberculatus) fed 1.4% CHO had significantly

333

higher SOD activity levels than those fed low levels of CHO (0.2% and 0.6%). Thus,

334

it might be concluded that there was a synergistic effect between dietary PL and

335

high-level CHO on the antioxidant activity of freshwater cultured L. vannamei in the

336

current study. However, the detailed mechanisms underlying the increase in SOD

337

activity in response to increased dietary CHO levels in shrimp remain unclear.

338

Lysozyme is one of the most important non-specific immune factors in organisms

339

(Liu et al., 2019). Lysozyme activity has been used frequently as an important

340

indicator of non-specific immunity for crustaceans. In the current study, lysozyme

341

activity in serum was enhanced with the increase in dietary CHO levels. Moreover,

342

the lysozyme transcript levels in gills were significantly affected not only by dietary

343

CHO, but also by dietary PL and their interaction. Burge et al. (2007) have found that

344

the lysozyme gene was expressed in all tissues of shrimp with no tissue specificity.

345

Thus, it can be hypothesized that dietary CHO would alter organisms’ susceptibility 14

346

to pathogeny both in the transcription level and protein level.

347

Defense mechanisms in crustacean depend entirely on the innate immune system

348

to destroy invasive pathogens (Vazquez et al., 2009). The pathogen invasion signal is

349

transmitted from the exterior to the interior of the cell via various signaling pathways

350

on the cell membrane (Li et al., 2018). The Toll, IMD, and JAK/STAT signaling

351

pathways regulate the nonspecific immunity of shrimp (Li and Xiang, 2013). An

352

increase in the transcription of immune effectors associated with invasion-signaling

353

pathways directly indicates a sufficient and effective immune response to pathogen

354

invasion (Liu et al., 2016). This present study showed that administration of dietary

355

CHO significantly affected the expression of Toll-like receptor and IMD mRNA in

356

gills of experimental shrimp 24-hour post V. alginolyticus infection. Furthermore, the

357

maximum relative mRNA expression levels of IMD increased with the increasing of

358

dietary PL levels. These results implied that the dietary PL and CHO had different

359

effects on Toll-like receptor gene expression in shrimp, but the effects of these

360

supplements on IMD and lysozyme gene expression were both positive and

361

dose-related. Nonetheless, the current study proved that dietary CHO, PL, and their

362

interaction contributed to increasing the immune response of shrimp which in turn

363

could result in better protection against pathogen infection.

364

It is well known that shrimp living in pond water has always been affected by

365

infectious diseases, mainly of bacterial and viral etiology (Flegel, 2012). V.

366

alginolyticus is a conditional pathogen of shrimp (Huang et al., 2015): the

367

pathogenicity of this bacterium is associated with the presence of various virulence

368

factors and the synergistic regulation of the expression of these factors by

369

environmental stimuli (Cai et al., 2018). The present study showed that shrimp fed

370

with CHO and PL supplemented diets were less susceptible to V. alginolyticus 15

371

infection. The higher pathogen resistance of shrimp is related to its immune status

372

(Lin et al., 2013). Moreover, the cumulative mortality results were consistent with the

373

changes in lysozyme gene expression and lysozyme activity, supporting previous

374

reports that lysozyme is an important index of bacterial resistance in shrimp.

375

In conclusion, dietary CHO and PL functioned interactively on muscle crude

376

protein content, SOD, Toll-like receptor and lysozyme mRNA expression, and

377

resistance against Vibrio alginolyticus injection in freshwater cultured L. vannamei.

378

However, no interactive effect was shown in the other parameters. Moreover,

379

improved dietary PL levels did not result in less expenditure of CHO in the current

380

study.

381

Acknowledgments

382 383 384

This study was financially supported by a Shanghai Science and Technology Committee project (No. 14320502000).

385 386

Conflict of interest

387 388

The authors have no conflict of interest to declare.

389 390

Data availability statement

391 392

The authors declare that the data supporting the findings of this study are available

393

within the article.

394 395

Authors’ contributions 16

396 397

Minglei Yan conducted the feeding experiment and determined the body composition,

398

immune response, and stress resistance test. Weilong Wang drafted the manuscript,

399

Xinlei Wang and Yi Wang helped to do analysis and interpretation of data, Xuxiong

400

Huang designed the research. All authors read and gave final approval of the

401

manuscript.

402 403 404 405 406

References

407

Association of Official Analytical Chemists (AOAC), Official Methods of Analysis of

408

Official Analytical Chemists International, 16th ed. Association of Official

409

Analytical Chemists, Arlington, VA, 1995.

410

M. R. P. Briggs, K. Jauncey, J. H. Brown, The cholesterol and lecithin requirements of

411

juvenils

412

Aquaculture 70(1988) 121-129.

prawn

(Macrobrachium

rosenbergii)

fed

semi-purified

diets,

413

E. J. Burge, D. J. Madigan, L. E. Burnett, K. G. Burnett, Lysozyme gene expression

414

by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection

415

with Vibrio, Fish Shellfish Immunol. 22(2007) 327-339.

416

S. H. Cai, H. Y. Cheng, H. Y. Pang, J. C. Jian, Z. H. Wu, AcfA is an essential regulator

417

for pathogenesis of fish pathogen Vibrio alginolyticus, Vet. Microbiol. 213(2018)

418

35-41. 17

419

J. R. Cejas, E. Almansa, S. Jerez, A. Bolanos, B. Felipe, A. Lorenzo, Changes in lipid

420

class and fatty acid composition during development in white seabream

421

(diplodus sargus) eggs and larvae, Comp. Biochem. Physiol. B Biochem. Mol.

422

Biol. 139(2004) 209-216.

423

H. Y. Chen, J. S. Jenn, Combined effects of dietary phosphatidylcholine and

424

cholesterol on the growth, survival, and body composition of marine shrimp,

425

Penaeus penicillatus, Aquaculture 96(1991) 167-178.

426 427

H. Y. Chen, Requirements of marine shrimp, Penaeus monodon, juveniles for phosphatidylcholine and cholesterol, Aquaculture 109(1993) 165-176.

428

P. Coutteau, E. K. M. Kontara, P. Sorgeloos, Comparison of phosphatidylcholine

429

purified from soybean and marine fish roe in the diet of postlarval Penaeus

430

vannamei Boone, Aquaculture 181(2000) 331-345.

431 432

D. W. Flegel, Historic emergence, impact and current status of shrimp pathogens in Asia, J. Invertebr. Pathol. 110(2012) 166-173.

433

H. Gong, A. L. Lawrence, D. H. Jiang, F. L. Castille, D. M. Gatlin III, Lipid nutrition

434

of juvenile Litopenaeus vannamei: I. Dietary cholesterol and de-oiled soy

435

lecithin requirements and their interaction, Aquaculture 190(2000a) 305-324.

436

H. Gong, A. L. Lawrence, D. H. Jiang, F. L. Castille, D. M. Gatlin III, Lipid nutrition

437

of juvenile Litopenaeus vannamei: II. Active components of soybean lecithin,

438

Aquaculture 190(2000b) 325-342.

439

H. Gong, A. L. Lawrence, D. M. Gatlin III, D. H. Jiang, F. Zhang, Comparison of

440

different types and levels of commercial soybean lecithin supplemented in

441

semipurified diets for juvenile Litopenaeus vannamei Boone, Aquaculture Nutr.

442

7(2001) 11-17.

443

H. Gong, D. H. Jiang, D. V. Lightner, C. Collins, D. Brock, A dietary modification 18

444

approach to improve the osmoregulatory capacity of Litopenaeus vannamei

445

cultured in the Arizona Desert, Aquaculture Nutr. 10(2004) 227-236.

446

M. L. Gonzalez-Felix, D. M. Gatlin III, A. L. Lawrence, M. Perez-Velazquez, Effect

447

of dietary phospholipids on essential fatty acid requirements and tissue lipid

448

composition of Litopenaeus vannamei juveniles, Aquaculture 207(2002)

449

151-167.

450

J. P. Guo, B. Y. Guo, H. L. Zhang, W. Xu, W. B. Zhang, K. S. Mai, Effects of

451

nucleotides on growth performance, immune response, disease resistance and

452

intestinal morphology in shrimp Litopenaeus vannamei fed with a low fish meal

453

diet, Aquaculture Int. 24(2016) 1007-1023.

454

X. Z. Gu, H. T. Fu, S. M. Sun, H. Qiao, W. Y. Zhang, S. F. Jiang, Y. W. Xiong, S. B.

455

Jin, Y. S. Gong, Y. Wu, Effects of cholesterol on growth, feed utilization, body

456

composition and immune parameters in juvenile oriental river prawn,

457

Macrobrachium nipponense (De Haan), Aquaculture Res. 48(2017) 4262-4271.

458

T. Han, J. T. Wang, X. Y. Li, Y. X. Yang, J. X. Wang, S. X. Hu, Y. D. Jiang, C. K. Mu,

459

C. L. Wang, Effects of dietary cholesterol levels on the growth, molt

460

performance, and immunity of juvenile swimming crab, Portunus trituberculatus,

461

Isr. J. Aquacult-Bamid. 67(2015) 1065-1069.

462

T. Han, J. T. Wang, X. Y. Li, Y. X. Yang, M. Yang, H. L. Tian, P. Q. Zheng, C. L.

463

Wang, Effects of dietary phospholipid and cholesterol levels on growth and fatty

464

acid composition of juvenile swimming crab, Portunus trituberculatus,

465

Aquaculture Nutr. 24(2016) 164-172.

466

M. H. Holme, P. C. Southgate, C. Zeng, Assessment of dietary lecithin and cholesterol

467

requirements of mud crab, Scylla serrata, megalopa using semi-purified

468

microbound diets, Aquaculture Nutr. 13(2007) 413-423. 19

469

M. Z. Huang, Y. Liu, C. Y. Xie, W. N. Wang, LvDJ-1 plays an important role in

470

resistance against Vibrio alginolyticus in Litopenaeus vannamei, Fish Shellfish

471

Immunol. 44(2015) 180-186.

472

F. Jafari, N. Agh, F. Noori, A. Tokmachi, E. Gisbert, Effects of dietary soybean

473

lecithin on growth performance, blood chemistry and immunity in juvenile

474

stellate sturgeon (Acipenser stellatus), Fish Shellfish Immunol. 80(2018)

475

487-496.

476

A. L. Jeune, R. Torelli, M. Sanguinetti, J. C. Giard, A. Hartke, Y. Auffray, A.

477

Benachour, The extracytoplasmic function sigma factor sigV plays a key role in

478

the original model of lysozyme resistance and virulence of Enterococcus faecalis,

479

Plos One 5(2010) e9658.

480

P. Juaneda, G. Rocquelin, Rapid and convenient separation of phospholipids and

481

non-phosphorus lipids from rat heart using silica cartridges, Lipids 20(1985)

482

40-41.

483 484

A. Kanazawa, N. Tanaka, S. Teshima, Nutritional requirements of prawn-II. Requirement for sterols, B. Jpn. Soc. Sci. Fish. 37(1971) 211-215.

485

A. Kanazawa, S. Teshima, M. Sakamoto, Effects of dietary lipids, fatty acids and

486

phospholipids on growth and survival of the prawn (Penaeus japonicus) larvae,

487

Aquaculture 50(1985) 39-49.

488

J. C. Kean, J. D. Castell, A. G. Boghen, L. R. D’Abramo, D. E. Conklin, A

489

re-evaluation of the lecithin and cholesterol requirements of juvenile lobster

490

(Homarus americanus) using crab protein-based diets, Aquaculture 47(1985)

491

143-149.

492

C. Lee, S. Kim, S. J. Lim, K. J. Lee, Supplemental effects of biofloc powder on

493

growth performance, innate immunity, and disease resistance of Pacific white 20

494

shrimp Litopenaeus vannamei, Can J Fish Aquat Sci. 20(2017) 15.

495

C. C. Li, J. C. Chen, The immune response of white shrimp Litopenaeus vannamei

496

and its susceptibility to Vibrio alginolyticus under low and high pH stress, Fish

497

Shellfish Immunol. 25(2008) 701-709.

498

C. Li, Y. Zhao, T. T. Liu, J. Huang, Q. Zhang, B. K. Liu, S. Xiao, H. Wang, B. N. Liu,

499

J. H. Wang, L. N. Cong, The distribution and function characterization of the i

500

type lysozyme from Apostichopus japonicas, Fish Shellfish Immunol. 74(2018)

501

419-425.

502

Y. Li, H. Liu, X. L. Dai, J. J. Li, D. J. Ding, Effects of dietary inulin and mannan

503

oligosaccharide on immune related genes expression and disease resistance of

504

Pacific white shrimp, Litopenaeus vannamei, Fish Shellfish Immunol. 76(2018)

505

78-92.

506 507

F. H. Li, J. H. Xiang, Recent advances in researches on the innate immunity of shrimp in China, Dev. Comp. Immunol. 39(2013) 11-26.

508

S. Lin, X. Lin, Y. Yang, F. Li, L. Luo, Comparison of chelated zinc and zinc sulfate as

509

zinc sources for growth and immune response of shrimp (Litopenaeus vannamei),

510

Aquaculture 406-407(2013) 79-84.

511

T. Liu, G. G. Zhang, Y. Feng, C. Kong, C. L. Ayisi, X. X. Huang, X. M. Hua, Dietary

512

soybean antigen impairs growth and health through stress-induced non-specific

513

immune responses in Pacific white shrimp, Litopenaeus vannamei, Fish Shellfish

514

Immunol. 84(2019) 124-129.

515

Y. Liu, L. Song, Y. Sun, T. Liu, F. Hou, X. L. Liu, Comparison of immune response in

516

Pacific white shrimp, Litopenaeus vannamei, after knock down of Toll and IMD

517

gene in vivo, Dev. Comp. Immunol. 60(2016) 41-52.

518

K. J. Livak, T. D. Schmittgen, Analysis of relative gene expression data using 21

519

real-time quantitative PCR and the 2-∆∆Ct method, Method 25(2001) 402-408.

520

B. Lygren, H. Sveier, B. Hjeltnes, R. Waagbo, Examination of the immunomodulatory

521

properties and the effect on disease resistance of dietary bovine lactoferrin and

522

vitamin C fed to Atlantic salmon (Salmo salar) for a short-term period. Fish

523

Shellfish Immunol. 9(1999) 95-107.

524 525

P. Maher, D. Schubert, Signaling by reactive oxygen species in the nervous system, Cell. Mol. Life. Sci. 57(2000) 1287-1305.

526

T. Morris, S. Tzachim, D. Dallen, F. Joem, Cholesterol supplements for Litopenaeus

527

vannamei reared on plant based diets in the presence of natural productivity,

528

Aquaculture 314(2011) 140-144.

529 530

National Research Council (NRC), Nutrient Requirements of Fish and Shrimp. DC. National Academies Press, Washington, 2011, pp. 112-121.

531

C. Paibulkichakul, S. Piyatiratitivorakul, P. Kittakoop, V. Viyakarn, A. W. Fast, P.

532

Menasveta, Optimal dietary level of lecithin and cholesterol for black tiger

533

prawn Penaeus monodon larvae and postlarvae, Aquaculture 167(1998) 273-281.

534

L. A. Roy, D. A. Davis, I. P. Saoud, Effects of lecithin and cholesterol

535

supplementation to practical diets for Litopenaeus vannamei reared in low

536

salinity waters, Aquaculture 257(2006) 446-452.

537

D. R. Sánchez, J. M. Fox, D. Gatlin, A. L. Lawrence, Dietary effect of fish oil and

538

soybean lecithin on growth and survival of juvenile Litopenaeus vannamei in the

539

presence or absence of phytoplankton in an indoor system, Aquaculture Res.

540

45(2014) 1367–1379.

541 542 543

S. Sheen, Dietary cholesterol requirements of juvenile mud crab Scylla serrate, Aquaculture 189(2000) 277-285. S. Shrestha, Y. Kim, Activation of immune-associated phospholipase A2 is 22

544

functionally linked to Toll/IMD signal pathways in the red flour beetle,

545

Tribolium castaneum, Dev. Comp. Immunol. 34(2010) 530-537.

546 547

D. M. Smith, S. J. Tabrett, M. C. Barclay, Cholesterol requirement of subadult black tiger shrimp Penaeus monodon (Fabricius), Aquaculture Res. 32(2015) 399-405.

548

S. Teshima, M. Ishikawa, S. Koshio, A. Kanazawa, Assessment of cholesterol

549

requirements in the prawn, Penaeus japonicas, Aquaculture Nutr. 3(1997)

550

247-253.

551

S. Thongrod, M. Boonyaratpalin, R. P. Wilson, Cholesterol and lecithin requirement

552

of juvenile banana shrimp, Penaeus merguiensis, Aquaculture 161(1998)

553

315-321.

554 555

D. J. Underwood, J. A. Cowley, K. N. Johnson, Antiviral immunity and protection in penaeid shrimp, Invert. Immu. 1(2013) 2-14.

556

L. Vazquez, J. Alpuche, G. Maldonado, C. Agundis, A. Pereyramorales, E. Zenteno,

557

Review: Immunity mechanisms in crustaceans, Innate Immun. 15(2009)

558

179-188.

559

L. U. Wang, J. C. Chen, The immune response of white shrimp Litopenaeus vannamei

560

and its susceptibility to Vibrio alginolyticus at different salinity levels, Fish

561

Shellfish Immunol. 18(2005) 269-278.

562

Y. C. Wang, P. S. Chang, H. Y. Chen, Differential time-series expression of

563

immune-related genes of Pacific white shrimp Litopenaeus vannamei in response

564

to dietary inclusion of β-1, 3-glucan, Fish Shellfish Immunol. 24(2008) 113-121.

565

W. L. Wang, M. Ishikawa, S. Koshio, S. Yokoyama, M. A. O. Dawood, Y. K. Zhang,

566

Effects of dietary astaxanthin supplementation on survival, growth and stress

567

resistance in larval and post-larval kuruma shrimp, Marsupenaeus japonicas,

568

Aquaculture Res. 49(2018a) 2225-2232. 23

569

X. G. Wu, Y. X. Cheng, L. Y. Sui, C. S. Zeng, P. C. Southgate, X. Z. Yang, Effect of

570

dietary supplementation of phospholipids and highly unsaturated fatty acids on

571

reproductive performance and offspring quality of Chinese mitten crab,

572

Eriocheir sinensis (H. Milne-Edwards), female broodstock, Aquaculture

573

273(2007) 602-613.

574

D. L. Yang, Q. Wang, L. Z. Chen, Y. L. Liu, R. W. Cao, H. F. Wu, F. Li, C. L. Ji, M.

575

Cong, J. M. Zhao, Molecular characterization and antibacterial activity of a

576

phage-type lysozyme from the Manila clam, Ruditapes philippinarum, Fish

577

Shellfish Immunol. 65(2017) 17-24.

578

S. P. Zhang, J. F. Li, X. C. Wu, W. J. Zhong, J. A. Xian, S. A. Liao, S. A. Miao, A. L.

579

Wang, Effects of different dietary lipid level on the growth, survival and

580

immune-relating genes expression in Pacific white shrimp, Litopenaeus

581

vannamei, Fish Shellfish Immunol. 34(2013) 1131-1138.

582

M. Zhu, X. H. Long, S. J. Wu, Effects of dietary trehalose on the growth performance

583

and nonspecific immunity of white shrimps (Litopenaeus vannamei), Fish

584

Shellfish Immunol. 78(2018) 127-130.

585

24

1

Table 1. Composition of the experimental diets.

Ingredients (%)

CHO0-P CHO0-P CHO0-P CHO0.2- CHO0.2- CHO0.2- CHO0.4- CHO0.4- CHO0.4L0 L2 L4 PL0 PL2 PL4 PL0 PL2 PL4

Brown fishmeala 10.00 Meat meala 4.00 a Soybean meal 34.00 Peanut meala 8.00 a Wheat flour 23.60 a Blood powder 3.00 a Ca(H2PO4)2 2.00 b Cholesterol 0.00 Soybean lecithina 0.00 Fish oila 2.00 a Beer yeast cell 3.00 Soybean oila 4.40 Mineral premixc 2.50 d 0.50 Vitamin premix e Yeast extract 3.00 Total 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.00 2.00 2.00 3.00 2.40 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.00 4.00 2.00 3.00 0.40 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.20 0.00 2.00 3.00 4.20 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.20 2.00 2.00 3.00 2.20 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.20 4.00 2.00 3.00 0.20 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.40 0.00 2.00 3.00 4.00 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.40 2.00 2.00 3.00 2.00 2.50 0.50 3.00 100.00

10.00 4.00 34.00 8.00 23.60 3.00 2.00 0.40 4.00 2.00 3.00 0.00 2.50 0.50 3.00 100.00

Crude protein 39.14 39.04 39.40 Crude lipid 6.52 6.43 6.46 Ash 9.02 9.03 9.29 -1 Energy (kJ g ) 17.15 17.12 17.10 Phospholipids 1.25 1.74 2.24 Cholesterol 0.06 0.05 0.06 a 2 Yuehai Feed Group, Zhejiang, China.

39.04 6.60 9.02 17.16 1.12 0.22

39.09 6.66 9.10 17.16 1.56 0.19

39.22 6.70 9.37 17.13 2.06 0.18

39.28 7.36 9.01 17.34 1.34 0.33

39.01 7.51 9.15 17.33 1.55 0.35

39.11 7.43 9.24 17.31 2.08 0.39

Proximate compositions (%)

3

b

Shanghai Gaoxin chemical glass, ≥ 95%.

4

c

Mineral premix: Co 100 mg Kg-1; Cu 1400 mg Kg-1; Zn 900 mg Kg-1; Mn 450 mg Kg-1.

5

d

Vitamin premix: Vitamin A 1500000 IU Kg-1; Vitamin B1 5000 mg Kg-1; Vitamin B2 2500 mg

6

Kg-1; Vitamin D3 190000 IU Kg-1; Vitamin E 180000000 IU Kg-1; Vitamin B6 8000 mg Kg-1;

7

Vitamin K3 2000 mg Kg-1; Nicotinic acid 2600 mg Kg-1; Pantothenic acid 2000 mg Kg-1; Folic

8

acid 250 mg Kg-1.

9

e

Angel Yeast co., LTD, China.

10 11 -1-

12 13 14

Table 2. Real-time quantitative PCR primers for immune related genes and β-actin of white

15

shrimp (Litopenaeus vannamei).

Primer Lysozyme

Nucleotide sequence (5′-3′)

GenBank reference

F: CGACCTCGATCAGTACATGG AY170126 R: GTAACCCTGGTGACAAGCCT

Toll-like receptor

F: TGGTGCTTTCGTCAAACTTC DQ923424 R: AACCTGGCCATACACAATGA

IMD

F: ATCGAGGAACGAGACAAGGT FJ592176 R: CGTACACTCGGTCGACATTC

β-actin

F: CGCGACCTCACAGACTACCT AF300705 R: CTCGTAGGACTTCTCCAGCG

16

-2-

17 18

Table 3. Growth parameters in Litopenaeus vannamei fed with experimental diets for 56 daysa. CHO (%)

PL (%)

Survival (%)

FBW (g)b

FCR

SGR (% day-1)

0 0

0 2

86.00±4.55 93.50±0.96

10.33±0.46 10.32±0.79

1.40±0.07 1.35±0.07

6.58±0.02 6.66±0.13

0

4

94.00±2.45

10.61±1.02

1.40±0.07

6.71±0.12

0.2 0.2 0.2 0.4

0 2 4 0

93.50±2.50 92.50±3.50 96.50±0.96 96.00±1.83

10.59±0.61 10.52±0.44 10.78±0.26 11.49±0.70

1.35±0.09 1.35±0.07 1.28±0.05 1.19±0.06

6.71±0.08 6.68±0.08 6.80±0.05 6.90±0.09

0.4 0.4

2 4

98.00±0.82 89.00±3.11

11.50±0.65 12.04±0.06

1.30±0.07 1.39±0.07

6.95±0.11 6.86±0.06

Probability (P value)c

19

NS * NS CHO PL NS NS NS NS NS NS CHO × PL a Data were expressed as mean ± S.E.M. from triplicate groups.

* NS NS

20

b

FBW, final body weight; FCR, feed conversion ratio; SGR, specific growth rate.

21

c

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

22

0.05). NS: not significant, CHO × PL: interaction.

23

.

-3-

24 25 26

Table 4. Muscle proximate analysis (% dry matter basis, except moisture) of Litopenaeus

27

vannamei fed with experimental diets for 56 daysa. CHO (%) 0 0 0 0.2 0.2 0.2 0.4 0.4 0.4

PL (%) 0 2 4 0 2 4 0 2 4

Probability (P value)b CHO PL

Moisture (%) 76.12±0.12 75.91±0.17 75.76±0.33 75.56±0.21 76.28±0.28 76.39±0.36 75.57±0.40 75.45±0.19 75.59±0.31

Ash 5.90±0.05 6.16±0.05 6.21±0.03 6.03±0.21 5.99±0.12 6.01±0.06 6.25±0.27 6.96±0.09 6.69±0.06

Total lipid 4.48±0.07 4.62±0.10 4.48±0.08 4.48±0.07 4.69±0.08 4.57±0.10 5.08±0.06 4.95±0.11 4.71±0.08

Crude protein 88.54±0.21c 87.33±0.36ab 88.03±0.30bc 87.28±0.27ab 87.03±0.13a 88.51±0.26c 87.86±0.17bc 87.93±0.31bc 86.83±0.14a

NS NS

* *

* NS

NS NS

28

CHO × PL NS NS NS * a Data were expressed as mean ± S.E.M. from triplicate groups. Data with different superscript

29

letters in one column represent significant difference from control group (P < 0.05).

30

b

31

0.05). NS: not significant, CHO × PL: interaction.

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

32 33

-4-

34 35

Table 5. Superoxide dismutase (SOD) and lysozyme activities in serum of Litopenaeus vannamei

36

fed with experimental diets for 56 daysa. CHO（%） 0 0 0 0.2 0.2 0.2 0.4 0.4 0.4

PL（%） 0 2 4 0 2 4 0 2 4

SOD（unit/ml） 317.81±12.04a 323.27±23.35a 325.61±9.08a 369.11±13.33b 381.35±9.69b 356.22±8.68ab 345.06±9.95ab 371.28±8.61b 418.96±8.66c

Lysozyme（unit/ml） 111.71±11.45 125.07±5.17 130.29±8.43 139.54±8.29 144.72±4.71 146.75±9.37 152.06±3.71 140.56±11.29 149.82±9.92

* NS *

* NS NS

37

Probability (P value)b CHO PL CHO × PL a Data were expressed as mean ± S.E.M.

38

letters in one column represent significant difference from control group (P < 0.05).

39

b

40

0.05). NS: not significant, CHO × PL: interaction.

from triplicate groups. Data with different superscript

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

41 42

-5-

43 44

Table 6. Effect of dietary cholesterol and soybean lecithin supplementation on relative expression

45

of Toll-like receptor mRNA in Litopenaeus vannamei challenged to Vibrio alginolyticusa. CHO (%) 0 0 0 0.2 0.2 0.2 0.4 0.4 0.4

PL (%) 0 2 4 0 2 4 0 2 4

0h 1.07±0.27A 0.70±0.11A 1.02±0.12A 1.10±0.33A 1.20±0.49A 1.02±0.13A 1.01±0.25A 1.33±0.68AB 1.23±0.58A

12h 0.98±0.82aA 0.96±0.36aAB 1.40±0.79abA 1.69±0.30abAB 3.66±0.71cC 1.87±0.23abB 2.46±0.19bB 1.82±0.24abB 1.42±0.29abAB

24h 1.90±0.54abcB 1.02±0.22aB 2.93±0.40bcdB 2.22±0.52abcB 3.85±0.70dC 2.81±0.46bcdC 3.40±0.82cdC 3.94±0.08dC 1.72±0.31abC

36h 1.89±0.52bcB 0.78±0.06aA 1.90±0.13bcAB 2.02±0.12cB 2.12±0.69cB 1.55±0.24abB 1.82±0.22bcAB 1.50±0.24abAB 1.41±0.64abAB

48h 0.81±0.26A 0.92±0.01AB 1.01±0.22A 1.23±0.26A 1.38±0.12A 0.96±0.41A 0.75±0.05A 1.18±0.47A 1.16±0.16A

46

Probability (P value)b NS NS * CHO NS NS NS PL NS * * CHO × PL a Data were expressed as mean ± S.E.M. from triplicate groups. Data

47

lowercase letters (a, b, c, and d) in one column represent significant difference from control group

48

(P < 0.05). Values in the same line with different superscript capital letters (A, B, C, and D) are

49

significantly different (P < 0.05).

50

b

51

0.05). NS: not significant, CHO × PL: interaction.

* NS NS NS * NS with different superscript

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

52 53

-6-

54 55

Table 7. Effect of dietary cholesterol and soybean lecithin supplementation on relative expression

56

of immune deficiency (IMD) mRNA in Litopenaeus vannamei challenged to Vibrio alginolyticusa. CHO (%) 0 0 0 0.2 0.2 0.2 0.4 0.4 0.4

PL (%) 0 2 4 0 2 4 0 2 4

0h 1.01±0.05AB 0.69±0.01A 0.85±0.07A 0.72±0.07A 0.86±0.10A 1.08±0.25A 0.75±0.32A 1.11±0.36A 0.67±0.01A

12h 1.01±0.04AB 1.11±0.06AB 1.40±0.30AB 1.08±0.17AB 1.56±0.14AB 1.11±0.18A 1.21±0.03AB 1.27±0.22AB 1.25±0.19AB

24h 1.08±0.15AB 1.42±0.16B 2.19±0.52C 2.27±0.27C 1.96±0.77B 2.93±0.20C 2.06±0.02C 2.26±0.37C 3.63±0.18C

36h 1.71±0.38B 1.04±0.37AB 1.16±0.39AB 0.80±0.16A 1.23±0.20AB 1.50±0.14B 1.55±0.22BC 1.41±0.30AB 1.46±0.22B

57

Probability (P value)b CHO PL CHO × PL a Data were expressed

58

different superscript capital letters (A, B, C, and D) are significantly different (P < 0.05).

59

b

60

0.05). NS: not significant, CHO × PL: interaction.

NS NS * NS NS NS * NS NS NS NS NS as mean ± S.E.M. from triplicate groups. Values in the same

48h 0.70±0.06A 1.09±0.16AB 0.94±0.16A 1.62±0.33B 0.87±0.31A 1.05±0.49A 1.08±0.07AB 1.34±0.29AB 1.12±0.17AB

NS NS NS line with

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

-7-

61 62

Table 8. Effect of dietary cholesterol and soybean lecithin supplementation on relative expression

63

of lysozyme mRNA in Litopenaeus vannamei challenged to Vibrio alginolyticusa. CHO (%) 0 0 0 0.2 0.2 0.2 0.4 0.4 0.4

PL (%) 0 2 4 0 2 4 0 2 4

0h 1.03±0.23AB 0.83±0.18A 0.62±0.12A 1.08±0.26A 0.78±0.04A 0.84±0.07A 1.01±0.11A 1.00±0.03A 1.00±0.01A

NS CHO NS PL NS CHO × PL a 64 Data were expressed as mean ±

12h 0.72±0.33aA 1.72±0.14bAB 1.01±0.23abAB 1.50±0.30abAB 1.53±0.22abB 1.48±0.23abAB 1.85±0.32bAB 0.98±0.22abA 1.75±0.39bB

24h 1.21±0.68aAB 0.98±0.22aA 1.24±0.29aAB 1.37±0.22aAB 1.59±0.15abB 1.77±0.40abABC 2.06±0.17bBC 1.25±0.29aAB 2.51±0.11cC

36h 1.83±0.65aB 2.48±0.27abB 1.73±0.42aB 2.52±0.33abB 2.42±0.49abC 3.17±0.09bcD 2.46±0.37abC 1.91±0.31aB 3.99±0.34cD

48h 0.42±0.01aA 1.44±0.17bAB 0.80±0.08abA 1.50±0.25bAB 0.69±0.02abA 2.56±0.01cCD 1.04±0.31abA 1.20±0.52abAB 1.32±0.43bAB

NS * * * NS * * * * * * * S.E.M. from triplicate groups. Data with different superscript

65

lowercase letters (a, b, c, and d) in one column represent significant difference from control group

66

(P < 0.05). Values in the same line with different superscript capital letters (A, B, C, and D) are

67

significantly different (P < 0.05).

68

b

69

0.05). NS: not significant, CHO × PL: interaction.

Significant effects determined by two-way ANOVA. Asterisks indicate the significance (P <

70

-8-

1

Figure legends

2 3

Fig. 1. Interactive effects of dietary cholesterol and phospholipids on the resistance

4

against Vibrio alginolyticus injection in freshwater cultured white shrimp

5

(Litopenaeus vannamei).

1

6 7

Fig. 1. Interactive effects of dietary cholesterol and phospholipids on the resistance

8

against Vibrio alginolyticus injection in freshwater cultured white shrimp

9

(Litopenaeus vannamei). Data with different superscript lowercase letters (a, b, and c)

10

in one column represent significant difference from control group (P < 0.05).

2

Highlight 1. The currrent study was conducted to provide the information about the influcence of dietary cholesterol and phospholipids on immune response, expression of immune-related genes, and resistance against Vibrio alginolyticus particularly for freshwater cultured Litopenaeus vannamei. 2. The current study indicated that different dietary levels of cholesterol and phospholipids could strongly affect expression levels of some immune-relevant genes of the juvenile freshwater cultured Litopenaeus vannamei. 3. Dietary cholesterol and phospholipids functioned interactively on muscle crude protein content, immune response, and immune related gene expression in L. vannamei.