Accepted Manuscript The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: activity and gene transcription Wutti Rattanavichai, Ying-Nan Chen, Chin-Chyuan Chang, Associate Professor, Winton Cheng, PhD, Professor PII:

S1050-4648(15)30059-0

DOI:

10.1016/j.fsi.2015.06.031

Reference:

YFSIM 3522

To appear in:

Fish and Shellfish Immunology

Received Date: 29 April 2015 Revised Date:

17 June 2015

Accepted Date: 23 June 2015

Please cite this article as: Rattanavichai W, Chen Y-N, Chang C-C, Cheng W, The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: activity and gene transcription, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.06.031. 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.

ACCEPTED MANUSCRIPT The effect of banana (Musa acuminata) peels hot-water extract on the immunity

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and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary

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administration for a long term: activity and gene transcription Wutti Rattanavichaia, Ying-Nan Chenb, Chin-Chyuan Chang b*, Winton Chengb*

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Department of Tropical Agriculture and International Cooperation, and bDepartment of

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Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201,

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Taiwan, ROC

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Abstract

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The non-specific immune parameters, disease resistance and immune genes expressions

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in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets

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containing the extracts of banana, Musa acuminate, fruit’s peel (banana peels extract, BPE) at

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0, 1.0, 3.0 and 6.0 g kg-1. Results showed that prawns fed with a diet containing BPE at the

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level of 1.0, 3.0 and 6.0 g kg-1 for 120 days had a significantly higher survival rate (30.0%,

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40.0% and 56.7%, respectively) than those fed with the control diet after challenge with

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Lactococcus garvieae for 144 hours, and the respective relative survival percentages were

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22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg-1

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for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC),

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superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG)

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activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and

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meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts

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(RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of

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prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP),

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peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We

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therefore recommend that BPE can be used as an immunomodulator for prawns through

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dietary administration at 6.0 g kg-1 for a long term (over 120 days) to modify immune

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responses and genes expression following the enhanced resistance against pathogens.

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Keywords: Macrobrachium rosenbergii; dietary administration; banana peels extract; immunity; disease resistance

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1. Introduction

The giant freshwater prawn, Macrobrachium rosenbergii, is commercially important in

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Taiwan as well as the world as a primary freshwater cultured prawn species. The intensive

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culture of which is strikingly developed in Taiwan, which accelerate the degradation of ponds

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environment lead to increased incidences of diseases caused serious economic losses [1,2].

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Disease outbreaks are result from the interactions of the three facts of poor environment,

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offensive pathogens and weakened hosts. However, it is difficult to control the stress of

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highly polluted pond environments and constantly existent pathogens in the intensive

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cultured ponds. Therefore, enhance the health of hosts as a prophylactic measure is of

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primary concern.

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The application of antibiotics and chemical disinfectants is a traditional measure to

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control and prevent diseases in aquaculture, but they are no longer recommended due to the

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potential for enhanced microbial resistance, and the accumulation of residues in the

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environment and in non-target organisms. The application of immunostimulants has been

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developed in aquaculture as a more environmentally friendly approach to disease

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management. These compounds are derived from bacteria, fungi, animal and plant extracts,

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nutritional factors, and synthetic drugs that stimulate the immune system resulting in an

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immune response and increase resistance disease of aquatic animals were reviewed by Sakai

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[3] and Ringø et al. [4]. Vibrio harveyi and white spot syndrome virus (WSSV) infection may

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be prevented by the enhancement of immune responses thought the dietary application of

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polysaccharide gel derived from the fruit-rind of Durio zibethinus [5]. Moreover,

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administration of Withania somnifera and Eichhornia crassipes extracts through

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supplementation diet positively enhances the immunity and increase survival rate in M.

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rosenbergii against Aeromonas hydrophila and Lactococcus garvieae infection, respectively

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[6,7].

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Plant extracts have been demonstrated as anti-stress, growth promotion, appetite

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stimulation, tonic and immunostimulation, and to have aphrodisiac and antimicrobial

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properties in fish and shrimp culture due to the active principles such as polysaccharides,

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alkaloids, flavonoids, pigments, phenolics, terpenoids, steroids, and essential oils and were

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reviewed by Harikrishnan et al. [8]. Banana peel could be potentially administrated in diets

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for livestock and poultry because of its rich source of starch (3%), crude protein (6~9%),

ACCEPTED MANUSCRIPT crude fat (3.8~11%), total dietary fiber (43.2~49.7%) and vitamins [9]. Dietary fiber which is

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the polysaccharides mainly consists of soluble and insoluble fractions such as lignin, pectin,

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cellulose and hemicellulose are an extremely diverse set of biopolymers in banana peels [10].

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Moreover, soluble fibers are well known to lower serum cholesterol and helps to reduce the

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risk of colon cancer [11,12]. Additionally, the phytochemicals including flavonoids, tannins,

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phlobatannins, alkaloids, glycosides and terpenoids were found to be present in the peels of

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genus Musa, and had been reported to exert multiple biological and pharmacological effects

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(antibacterial, antihypertensive, antidiabetic and anti-inflammatory activities) [13]. Therefore,

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we consider that the peels possess valuable medicinal potential and can be applied as an

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immunostimulant.

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The prophylactic measures for control disease are the major concern in aquaculture

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nowadays, and most of them are immunostimulants including natural products and probiotic.

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In previously study, we demonstrated that the banana (Musa acuminate) peels extract (BPE)

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not only had an antimicrobial effect, but also can enhance immune responses and disease

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resistance of prawns via both injection for 6 days and dietary administrations for 32 days

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[14,15]. Furthermore, the enhanced growth performance was found in prawns fed with BPE-

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containing diet especially at 6.0 g kg−1 from 30 to 120 days [15]. It, therefore, was speculated

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that the enhanced growth performance might result from the continuously up-regulated

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immunogenes expressions, and immune responses without immunostimulatory fatigue

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occurred in M. rosenbergii fed with BPE-containing diet for 120 days of feeding trial.

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Against this background, the objectives of the present study were aimed to evaluate the

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dietary administration of banana peels extract from M. acuminate in M. rosenbergii upon

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long term feeding trial (120 days) on: (1) the immune parameters including the total

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haemocyte count (THC), different haemocyte count (DHC), phenoloxidase (PO) activity,

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respiratory bursts (RBs), superoxide dismutase (SOD) activity, transglutaminase activity,

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haemolymph clotting times, (2) the immune genes expressions including proPO,

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lipopolysaccharide

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transglutaminase (TG), and crustin, (3) the phagocytic activity and clearance efficiency to L.

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garvieae and, (4) the resistance against L. garvieae infection following dietary administration

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of banana peels extract from M. acuminate.

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2. Materials and methods

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and

β-1,3-glucan

binding

protein

(LGBP),

peroxinectin

(PE),

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2.1. Preparation of the banana peels extract and diets Banana fruits, M. acuminate, of all green were obtained from the Neipu market,

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Pingtung, Taiwan. Subsequently, banana fruits were drenched with soda water for 5 min,

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washed with tap water, and later rinsed twice with distilled water to remove the contaminants.

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The banana peel was collected, and the banana peels extract (BPE) was manufactured as

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described previously [15]. The proximate analysis of BPE and the basal diet were conducted

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according to the AOAC method [16]. The BPE was composed of crude protein (7.71 ±

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0.30%), crude lipids (0.46 ± 0.03%), moisture content (14.45 ± 0.18%), ash (27.21 ± 0.11%) and total carbohydrate 50.17 ± 0.15 %, and was stored at -20 °C until being further used.

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Four diets containing different concentrations of BPE were prepared as described in

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Table 1. The basal diet contained 40.5% crude protein, 5.1% crude lipids, 18.3% ash, and

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5.0% moisture. For the experiment diet with banana peels extract, the BPE was added to the

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test diets at levels of 0, 1.0, 3.0, and 6.0 g kg-1 with a corresponding decrease in the amount

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of cellulose. Ingredients were made to ground using Hammer mill until they passed through

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an 80-mesh screen. Experimental diets were prepared by mixing the dry ingredients with fish

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oil and later water was added until it becomes stiff dough. Each diet was then passed through

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a mincer with a die, and the resulting spaghetti-like strings were dried in a drying cabinet

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using an air blower at 50°C until moisture levels were lower than 10%. After drying, the

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finished pellets were stored in plastic bins at 4 °C until being further used.

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2.2. Lactococcus garvieae

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A known pathogenic strain, L. garvieae, isolated from diseased M. rosenbergii, which

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displayed clinical signs of opaque and whitish musculature, was used for the study [17]. The

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pathogen was cultured and collected according to the method of previous description [15].

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The bacterial pellets were re-suspended in a saline solution (0.85% NaCl) at 2.0 × 107

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colony-forming units (cfu) ml-1 as a stock bacterial suspension for the susceptibility study and

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at 1.0 × 108 cfu ml-1 for studies on phagocytic activity and clearance efficiency. The

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concentration of bacterial suspension was measured by its absorbance at 601 nm optical

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density using a spectrophotometer (Hitachi U-2001), and was calculated as a standard curve

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based on a series of different concentrations of bacterial suspension. The diluted bacterial

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suspension was plated on tryptic soy agar plates to determine viable cell counts.

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2.3. Experimental design Prawns (3.6 ± 0.4 g), M. rosenbergii, were obtained from a commercial farm in

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Pingtung, Taiwan, and were shipped to National Pingtung University of Science and

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Technology (Aquatic Animal Physiology and Immunology Laboratory), and acclimatized at

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room temperature (27 ± 1°C) and pH 7.0~7.5 in the laboratory for 2 weeks before being used

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for experimentation. Only prawns in the intermoult stage were used in this study. The moult

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stage was determined under a stereomicroscope according to retraction of the epithelium

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within the setal base interface of the antennal scale [18]. During the acclimatization period,

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prawns were fed with the control diet twice daily.

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For the banana peels extract supplementations in the diet, four studies were conducted.

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Twelve 9-ton fiber reinforced plastic (FRP) tank containing 5 ton of aerated freshwater were

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used in this study. Each tank was used to rear 300 prawns (3.6 ± 0.4 g). Groups with four

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different dosages of BPE (0, 1.0, 3.0, and 6.0 g kg-1) were established, with each group

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consisting of three tanks. Ten, two, two, two and two prawns were randomly sampled from

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each tank after 120 days of feeding trial for the susceptibility, immune parameters,

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haemolymph clotting times, immune genes expression, and phagocytic activity and clearance

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efficiency tests, respectively. For the susceptibility experiment, test and control groups were

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comprised of 10 prawns each, and tests were conducted in triplicate. To determine THC,

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DHC, PO activity, RBs, SOD activity, TG activity, haemolymph clotting times, and genes

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expressions, tests and controls were carried out on six replicates. For studies on phagocytic

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activity and clearance efficiency, six prawns were used in the test and control groups. No

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significant difference in weight was observed among different treatments. During the

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experiments, prawns were continuously fed with their respective diets, and the water

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temperature was maintained at 27 ± 1°C and the pH at 6.9~7.5.

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2.4. Effect of oral BPE on the susceptibility of M. rosenbergii to L. garvieae

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Prawns were fed with the BPE-containing diets at 0, 1.0, 3.0, and 6.0 g kg-1 for 120 days

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before the challenge tests were conducted. Ten microlitres of bacterial suspension (2.0 × 107

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cfu ml-1) resulting in 2 × 105 cfu per prawn was injected slowly into the ventral sinus located

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between the ventral nerve cord/cephalothoracic ganglia associated with 4th and 5th pereiopod

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and striated muscle of the cephalothorax from the first segment of abdomen with a sterile

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syringe. Prawns fed with the control diet and then received 10 µl saline were served as the

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unchallenged control (Table 2). There were five treatments with 30 prawns each. After the

ACCEPTED MANUSCRIPT injection, each group of ten prawns was kept in 60-l glass aquaria (10 prawns each)

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containing 40 L of freshwater and 27 ± 1.0°C. During the experiment, prawns were

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continually fed with their respective diets, and 30% of the water was exchanged daily. The

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mortality was counted on daily basis until the total experimental period of 144 h of post

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challenge. The relative percent survival (RPS) of prawn was calculated at the end of the

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experiment according to Amend [19] using the following formula: (12.5 ± 1.3 g)

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RPS= [1 – [(percentage mortality in treatment) (percentage mortality in control)-1]] ×

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100.

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2.5. Effects of oral BPE on the immune parameters of M. rosenbergii

Immune parameters of prawns that were fed with the BPE-containing diets at 0, 1.0, 3.0,

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and 6.0 g kg-1 were determined after 120 days of feeding. Six prawns (20.0 ± 1.0 g) for each

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treatment were used for examination of THC, DHC, PO activity, RBs, SOD activity, TG

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activity, and another six prawns were used for determination of haemolymph clotting times.

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After 120 days of feeding, haemolymph (300 µl) was withdrawn from the ventral sinus

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of each prawn and were divided into two parts. One hundred microliters of haemolymph was

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placed into 1-ml sterile eppendorf tubes containing 0.9 ml anticoagulant buffer (0.8 g sodium

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citrate, 0.34 g EDTA, and 10 µl Tween 80 in 100 ml of distilled water, at pH 7.45 with the

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osmolality adjusted to 490 mOsm kg-1 with NaCl) for the THC, DHC, PO activity and RB

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assays. A drop of the anticoagulant-haemolymph mixture was placed on a haemocytometer in

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order to measure the THC and DHC (Leica DMIL, Leica Microsystems, Wetzlar, Germany).

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The remainder of the haemolymph mixture was used for PO activity and RB assays. Two

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hundred microliters of haemolymph was added to an eppendorf tube containing 0.4 ml of

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anticoagulant buffer for the SOD, and TG activity assays.

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The PO activity of haemocytes was measured spectrophotometrically at 490 nm by

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recording the formation of dopachrome produced from L-3,4-dihydroxyphenylalanine (L-

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DOPA, D-9628, Sigma), following a method modified from Mason [20] and Hernández-

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López et al. [21]. The details of measurements were described previously [6]. The optical

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density (OD) was measured at 490 nm, and the PO activity was expressed as dopachrome

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formation in 50 µl of haemolymph or per 107 the sum of granular cells (GCs) and semi-GCs

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(SGCs).

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RBs of haemocytes were quantified using the reduction of nitroblue tetrazolium (NBT)

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to formazan as a measure of superoxide anion (O2−) formation as described previously

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[22,23]. The OD at 630 nm was measured using a microplate reader (Model VERSAmax,

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Molecular Devices, Sunnyvale, CA, USA). RBs were expressed as NBT reduction in 10 µl of

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haemolymph or per 107 haemocytes. The haemocytes lysate supernatant (HLS) was prepared as described previously [7]. The

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SOD activity of HLS was measured by its ability to inhibit superoxide radical-dependent

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reactions using a Ransod kit (Randox, Crumlin, UK). Details of the measurement were

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described previously [24]. The OD was measured at 505 nm and 37°C, and the rate of the

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reaction was estimated from the absorbance readings at 0.5 and 3 min after adding xanthine

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oxidase. A reference standard for SOD was supplied with the Ransod kit. One unit of SOD

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was defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific

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activity was expressed as SOD units (mg protein)-1 [25].

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TG activity was measured by the colorimetric hydroxamate procedure according to Folk

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and Cole [26] with modifications. Details of the measurement were described previously [27].

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The OD of hydroxamate was measured at 525 nm, and one unit of TG activity was defined as

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the amount of enzyme needed to produce 1 mmole of hydroxamate per minute. The specific

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activity was expressed as TG activity units (mg protein)-1.

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The concentration of protein in the HLS was quantified by the method described by

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Bradford [28] using a Bio-Rad Protein Assay Kit (no. 500-0006, Bio-Rad Laboratories,

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Richmond, CA, USA) with bovine serum albumin (BSA) as the standard.

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Measurement of the haemolymph clotting times was modified from the methods of

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Tsai et al. [29] and Jussila et al. [30]. Briefly, 270 µl of haemolymph was withdrawn from the

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ventral sinus, mixed with 30 µl of 20 mM CaCl2 in eppendorf tubes, and continuously turned

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upside down in slow motion every 5 s. The motion was repeated until the haemolymph had

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coagulated, and the time was recorded.

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2.6. Effects of oral BPE on the phagocytic activity and clearance efficiency of M.

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For the phagocytic activity and clearance efficiency tests, 20 µl of a bacterial

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suspension (1.0 × 108 cfu ml-1) resulting in 2 × 106 cfu prawn-1 was injected into the ventral

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sinus. After the injection, prawns were held in separate tanks containing 40 L of water at 27 ±

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1.0°C for 2 h. Then, 200 µl of haemolymph was collected from the ventral sinus and mixed

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with 200 µl of a sterile anticoagulant solution. This mixture was then divided into two equal

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subsamples to respectively evaluate the phagocytic activity and clearance efficiency.

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Methods for measuring the phagocytic activity and clearance efficiency were described previously [31,32]. Two hundred haemocytes were counted, and the rate of phagocytosis (PR)

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was calculated as follows:

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PR = [(phagocytic haemocytes) (total haemocytes)-1] × 100.

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The clearance efficiency, defined as the percentage inhibition (PI) of L. garvieae was

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calculated as:

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PI = 100 – [(cfu in the test group) (cfu in the control group)-1] × 100. 2.7. Effects of oral BPE on the immune genes of M. rosenbergii

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The haemocytes was collected as described by Hsu et al. [33] and total RNA was

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extracted and further purified using the ULTRASPEC™ RNA, Total RNA Isolation Reagent

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(Biotecx, Houston, TX, USA) following the manufacturer's instructions. First-strand

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complementary (c)DNA synthesis in reverse transcription (RT) was accomplished as

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described previously [34]. Messenger (m)RNA expressions of immune genes including

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proPO, LGBP, PE, TG, and CT of haemocytes of M. rosenbergii were measured using an

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SYBR green I real-time RT-polymerase chain reaction (PCR) assay in an ABI PRISM 7900

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Sequence Detection System (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA), and

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the details of the measurement were previously described [35]. Specific primers of immune

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genes and the β-actin primer were used for the quantitative RT-PCR (Table 3). After

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amplification, data acquisition and analysis were performed using Sequence Detection

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Software (SDS vers. 2.1, Applied Biosystems). The 2-∆∆CT method was chosen as the

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calculation method [36]. The difference in the cycle threshold (CT) value of the individual

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immune gene and its housekeeping gene (β-actin), called ∆CT, was calculated. ∆∆CT = (∆CT

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of prawn fed diet containing BPE at 1, 3, and 6 g kg-1 for immune genes) - (∆CT of the

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control).

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2.8. Statistical analysis

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A multiple-comparisons (Tukey’s) test was conducted to compare significant differences

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among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Before

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analysis, percentage data were normalized using an arc-sine transformation. Statistically

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significant differences were calculated when p < 0.05.

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3. Results

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3.1. Effect of oral BPE for 120 days on the resistance of prawn to L. garvieae infection No mortality was recorded in the unchallenged control prawns. Survival rates of prawns

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that fed with the BPE-containing at 1.0, 3.0 and 6.0 g kg-1 were significantly higher than

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those of prawns fed with the control diet from 72 to 144 h. After 144 h of challenge, survival

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rates of prawns that fed with the BPE-containing diet at 1.0, 3.0 and 6.0 g kg-1 were 30.0%,

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40.0% and 56.7%, respectively and the respective relative survival percentages of prawns

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were 22.2%, 33.3% and 51.9% (Table 2).

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3.2. Effect of oral BPE for 120 days on the immune parameters of prawns

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The THC, HC, and GC of prawns fed with the BPE-containing diets at 6.0 g kg-1 were

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significantly higher than those of prawns fed with the control diet for 120 days, and had

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increased by 152.6%, 235.7%, and 125.9%, respectively, compared to prawns fed with the

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control diet. However, no significant differences were observed in SGC among prawns that

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fed with the BPE at 0.0-6.0 g kg-1, and in THC, HC and GC among prawns fed with the BPE

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at 0.0~3.0 g kg-1 after 120 days (Fig. 1A-D).

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The PO activity of prawns fed with the diets containing BPE at 3.0 and 6.0 g kg-1 was

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significantly higher than those of prawns fed with the control diet at 120 days of post feeding,

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and had significantly increased by 63.2%, and 89.5%, compared to prawn fed with the control

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diet (Fig. 2A). The similar phenomenon revealed in PO activity per GCs (GC + SGC)

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among prawns that fed with control diet and BPE-containing diets. The PO activity per GCs

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of prawns that fed with BPE-containing diets at 3.0 and 6.0 g kg-1 increased by 91.9%, and

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64.9%, compared to prawn fed with the control diet (Fig. 2B).

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No significant differences in RBs were observed among prawns fed with BPE-

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containing diets and the control diet (Fig. 3A). The significantly decreased RBs per

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haemocyte was observed in prawn fed with BPE-containing diets at 6.0 g kg-1 for 120 days,

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and had decreased by 32.4%, compared to prawn fed with the control diet (Fig. 3B). SOD

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activity in haemocytes of prawns fed with the BPE-containing diets at 6.0 g kg-1 for 120 days

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was higher than those of prawns fed with the diet at 0-3.0 g kg-1, and had increased by

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735.7%, compared to prawns that fed with the control diet (Fig. 3C). TGs activities in haemocytes of prawns fed with the BPE-containing diets for 120

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days were significantly higher than those of prawns fed the control diet. The increase in TGs

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activities of prawns fed with the BPE-containing diets appeared to have been dose dependent.

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TGs activities of prawns fed with the BPE-containing diets at 1.0, 3.0 and 6.0 g kg-1 had

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significantly increased by 352.4%, 480.6% and 770.0% compared to prawns fed with the

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control diet (Fig. 4A).

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Haemolymph clotting times of prawns directly decreased with fed dosage of BPE in

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the range of 0.0-6.0 g kg-1 after 120 h. For the prawns fed with the BPE-containing diets at

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6.0 g kg-1, haemolymph clotting times had significantly decreased by 43.7%, compared to the

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prawns fed with the control diet (Fig. 4B).

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3.3. Effect of oral BPE on phagocytic activity and clearance efficiency of prawns

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Phagocytic activities and clearance efficiencies of prawns fed with the BPE-containing

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diets separately at 3.0 and 6.0 g kg-1, and at 1.0, 3.0 and 6.0 g kg-1 were significantly higher

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than those of prawns fed with the control diet for 120 days. Phagocytic activities and

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clearance efficiencies of prawns fed with the BPE-containing diets at 1.0, 3.0 and 6.0 g kg-1

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had increased by 10.9%, 41.3% and 45.4%, and 42.9%, 44.2% and 66.0%, respectively,

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compared to prawns fed with the control diet (Fig. 5A,B).

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3.4. Effect of oral BPE on the immune genes expressions of prawns

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A real-time RT-PCR was used to determine the immune genes expression. The proPO,

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LGBP, TG and crustin mRNA expression levels in haemocytes of prawn fed with the BPE-

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containing diets at 6.0 g kg-1 at 120 days of post feeding were significantly higher than those

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of prawns fed with the control diet, and had increased by 141.7%, 167.3%, 56.5% and

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202.4%, respectively, compared to the prawns fed with the control diet. The increase in

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proPO, PE and TG mRNA expression levels of prawns following administrative dosage of

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BPE appeared to have been dose dependent. However, no significant difference in PE mRNA

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expression were observed among the prawns fed with the BPE-containing diets at 0, 1.0, 3.0

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and 6.0 g kg-1 after 120 days of feeding (Fig. 6A,B,C,D,E).

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4. Discussion

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ACCEPTED MANUSCRIPT Immunostimulants can be applied via injection, bathing or oral administration to

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enhance the immunity and disease resistance in aquatic animals, and its effects depend on

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time, dosage and method of administration, and the physiological condition of the animals.

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Matsuo and Miyazono [37] indicated that dietary peptidoglycan administration at 0.2 and 2.0

308

mg kg-1 for 28 days enhanced resistance of rainbow trout against Vibrio anguillarum, but not

309

protective effect for 56 days of feeding. Tiger shrimp P. monodon fed diet containing 1.0, 2.0

310

and 3.0% polysaccharide gel (PG) extracted from D. zibethinus for 12 weeks significantly

311

enhanced the immune responses and resistance against WSSV and V. harveyi infection, and

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has the highest effect at 2.0% of supplementation [5]. Giant freshwater prawn fed diet

313

containing hot-water extracts from water hyacinth E. crassipes at 2 and 3 g kg-1 for 12 days

314

exhibited increased resistance against L. garvieae infection [7]. Survival rates of M.

315

rosenbergii injected with BPE at concentrations of 1.0, 3.0 and 6.0 µg (g prawn)-1 were

316

significantly higher than those injected with saline control after challenge with L. garvieae

317

for 4~6 days [14], and in oral route, the significantly increased survival rates of the M.

318

rosenbergii against L. garvieae revealed when they were fed with diets supplemented at

319

1.0~6.0 g kg-1 BPE for 32 days of feeding [15]. The similar phenomena were observed when

320

BPE were administrated in diets at 1.0~6.0 g kg-1 to fed M. rosenbergii for 120 days in the

321

present study. These suggested that prawns disease resistance can be improved via BPE

322

dietary administrations at 6.0 g kg-1 especially lasting for a long term feeding.

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Polysaccharide biological response modifiers are usually strong mitogens, which

324

stimulate proliferation of immune cells [38]. Sirirustananun et al. [39] reported that the

325

evidence of haemocytes proliferation in L. vannamei fed with G. tenuistipitata extract-

326

containing diets were the increases of HCs, GCs and THCs, together with increases in mitotic

327

cells and the mitotic index of haematopoietic tissues (HPTs). Rattanavichaia and Cheng [15]

328

reported that M. rosenbergii fed with BPE containing diets for 32 days of feeding showed

329

that the THC, DHC, PO activity, RBs, and SOD activity significantly increased, and

330

meanwhile, no significant differences in PO activity per granulocyte and RBs per haemocyte

331

were observed. M. rosenbergii that received BPE via an injected route also showed increased

332

THCs, GCs, PO activity, PO activity per granulocyte, and SOD activity and decreased RBs

333

per haemocyte [14]. In the present study, prawn fed with the BPE containing diets at 3 or 6 g

334

kg-1 after 120 days of feeding trial revealed the increases of THC, HC, GC, PO activity, PO

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activity per granulocyte (SGC+GC) and SOD activity, and the decrease of RBs per

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haemocyte. The facts suggest that BPE might also be capable to increase THC of prawns with

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ACCEPTED MANUSCRIPT 337

induced proliferation of haemocytes in HPTs via both oral and injection routes. Further

338

research would be conducted to clarify the statements. The respiratory burst (RB) activity is an indicator of the status of phagocytes activation.

340

During phagocytosis, ROIs derived from the processes of RBs are generated by plasma

341

membrane NADPH oxidase. The ROIs are effectively neutralised by the antioxidant defence

342

system of organisms to maintain homoeostasis, which included enzymes like SOD, catalase

343

and various peroxidase, and small antioxidant molecules like ascorbate, sugars and

344

polyunsaturated fatty acids. Chang et al. [7] indicated that RBs, and SOD and phagocytic

345

activity significantly increased, but O2- production per haemocyte significantly decreased in

346

prawns fed with E. crassipes leaves extracts-containing diets at 2-3 g kg-1 for 12 days. The

347

similar results were also observed in prawns that received BPE by injection at 3-6 µg (g

348

prawn)-1 for 6 days [14] and by dietary administration at 6 g kg-1 for 120 days in this study.

349

The facts suggest that BPE via dietary administration for a long term feeding trial may

350

promote both phagocytic activity of phagocytes and regulation of ROI system in prawn, and

351

the decreased O2- production of single haemocyte may be resulting from an increase of SOD

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activity to maintain homoeostasis in prawn.

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Several pattern recognition proteins (PRPs), such as lipopolysaccharide- and β-glucan-

354

binding protein (LGBP), play an important role in crustaceans-pathogen interaction. They

355

recognise and respond to microbial intruders, and are involved in activation of the proPO

356

system, the coagulation cascade, and expression for antibacterial effector proteins [40], and

357

act as an opsonin to increase the rate of phagocytosis [41]. Peroxinectin (PE), an associated

358

protein of the proPO system, has multiple functions of degranulation [42], encapsulation

359

enhancement [43], opsonification [44], and peroxidation [45], which is essential in crustacean

360

cellular defence reaction. These biological activities of PE are generated concomitant with

361

activation of the proPO system [46]. In the present study, PO activity, PO activity per GCs,

362

and proPO, LGBP and PE genes expression of prawns fed with the BPE at 6.0 g kg-1

363

increased after 120 days of feeding trial. The increase PO activity is considered to be

364

associated with up-expression of proPO, LGBP and PE genes to increase resistance against

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pathogen.

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In crustaceans, haemolymph coagulation is triggered by the release of haemocyte TGs to

367

polymerise the clotting proteins (CPs), which is involved in the innate immune responses due

368

to it prevents leakage of haemolymph and helps protect against dissemination of invaders

ACCEPTED MANUSCRIPT [47]. M. rosenbergii fed with water hyacinth, E. crassipes, extract containing diets at 2.0-3.0

370

g kg-1 for 12 days and fed with BPE-containing diets at 3.0-6.0 g kg-1 for 32 days

371

significantly increased THC and TG activity, and decreased the clotting time [7,15]. The

372

similar results is observed in prawns injected with BPE at 1.0-6.0 µg (g prawn)-1 [14]. In the

373

present study, the TG activity and gene expression directly increased, and haemolymph

374

clotting times directly decreased of prawns fed with the BPE in the range of 1.0-6.0 g kg-1 for

375

120 days. The facts suggest that acceleration of haemolymph coagulation of prawns received

376

BPE via injection and oral routes are relate to increase of TG gene expression accompany TG

377

activity.

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Clearance of invading pathogens in circulating haemolymph is associated with humoral

379

factors such as agglutinins, lectins, cytotoxic factors, antimicrobial factors, and clotting

380

factors. Antimicrobial peptides (AMPs) are small cationic molecules that can play an

381

important role in the innate immune defence against bacterial and fungal pathogens. The

382

crustin peptide is a cysteine-rich antimicrobial peptide in the blood circulation system of

383

crustacean [48]. In kuruma shrimp, TG silencing causes significant down-regulation of the

384

expressions of crustin and lysozyme, but does not affect the expression of proPO [49]. L.

385

vannamei that received LvTGII dsRNA showed significant decrease in clearance efficiency,

386

but did not affect the PO activity in our previous study [50]. In the present study, the

387

increased clearance efficiency of M. rosenbergii to L. garvieae was correlated well with the

388

increased resistance against L. garvieae and TG activity, and decreased clotting times of

389

haemolymph, when the prawns received BPE via dietary administration after 120 days. The

390

facts suggest that the elevation of clearance efficiency is related to the increases of TG

391

activity, TG and crustin genes expression, and the increased crustin expression was resulting

392

from the increased TG gene expression accompany TG activity.

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In aquaculture the plant extracts and products are known to possess anti-stress

394

characteristics, promote growth, stimulate appetite, tonic and immunostimulation, and

395

aphrodisiac and anti-microbial properties in fish and shrimp culture [8]. The phytochemical

396

compounds rich in banana peel possess multiple biological and pharmacological effects.

397

Additionally, the various parts of banana plant performed the inhibitory effect against food

398

borne pathogens to be considered to be a potential natural source of antimicrobial and

399

antioxidant agents [51]. In our previous study, the BPE inhibited growth of aquatic animals’

400

pathogens, and strengthened anti-hypothermal stress as well as enhanced immune responses

ACCEPTED MANUSCRIPT and resistance of M. rosenbergii to L. garvieae via injection and oral administration for 32

402

days of feeding trial [14,15]. In the present study, the similar outcomes were recorded

403

through enhanced immunological responses and resistance to pathogen infection as well as

404

increase in immune genes expressions when M. rosenbergii fed with the diets containing BPE

405

for 120 days of feeding trial, and meantime, the growth performance was improved [15]. The

406

results suggest that, BPE as an immunostimulant not only to improve the status of

407

immunological responses and physiological regulation via injection or diet administration,

408

but also to provide a better diet administration for a long term feeding trial which is an

409

important issue in the present aquaculture industry.

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In conclusion, the results demonstrated that M. rosenbergii fed with the diets containing

411

6.0 g kg-1 BPE for 120 days showed an increase in the immune responses by increasing its

412

haemocytes count of circulation, SOD activity, PO activity and proPO system-related genes

413

expression, TG activity and gene expression, and crustin mRNA expression as well as

414

phagocytic activity and clearance efficiency against L. garvieae, which bring an increased

415

resistance to L. garvieae infection.

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Acknowledgement

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This work was financially supported by a grant (NSC102-2622-B-020 -004 -CC2) from

418

the National Science Council, Taiwan. Special thanks to the budgets support for Wutti

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Rattanavichai from Rajamangala University of Technology Isan of Thailand to pursue further

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education in NPUST, Taiwan , ROC.

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ACCEPTED MANUSCRIPT

Table 1. Composition of the basal diet (g kg-1) for Macrobrachium rosenbergii. Banana Musa acuminate hot-water extract in diet (g kg-1) Ingredients Control 1.0 3.0 6.0 470

470

470

470

α-starch

130

130

130

130

Squid cream

50

50

50

50

Shrimp meal

50

50

50

50

Gluten

30

30

Fermented soybean meal

140

140

Wheat flour

100

100

Cellulose

6

5

M. acuminata extract

0

1

Pre-mix

30

30

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Fish meal

30

140

140

100

100

3

0

3

6

30

30

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30

100±0.0a

83.3±3.33a

2×10 5

3

30

100±0.0a

93.3±3.33a

2×10 5

6

30

100±0.0a

100.0±0.0a

70.0±0.0b

60.0±3.3b

57.0±3.3b

30.0±0.0b(22.2)p

90.0±0.0a

80.0±5.8a

70.0±0.0a

40.0±10.0ab(33.3)p

90.0±3.3a

83.3±3.3a

70.0±0.0a

56.7±3.3a(51.9) p

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Table 2. The survival rate and relative percentage survival (RPS) of Macrobrachium rosenbegii fed with the diets containing hot-water extract of banana peel for 120 days and then challenged with Lactococcus garvieae. Survival ratio and RPS(%) time after challenge (hrs) Challenge dose Banana peel extract No. of (cfu prawn-1) (g kg-1) Prawn 12 24 48 72 96 144 saline 0 (control) 30 100 100 100 100 100 100 5 a a b c c 2×10 0 (control) 30 100±0.0 80.0±0.00 70.0±0.0 40.0±0.0 10.0±0.0 10.0±0.0c

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Data in the same column with different letters are significantly differ (p Long term feeding with the diet containing banana peels extract enhanced resistance of prawns. > The immunological responses up-regulated post feeding the diet containing banana peels extract. > Diet supplemented with banana peels extract increased the immune genes expressions.

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> Dietary banana peels extract administration was applicable for long term feeding trial.

The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: Activity and gene transcription.

The non-specific immune parameters, disease resistance and immune genes expressions in Macrobrachium rosenbergii were evaluated at 120 days of post fe...
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