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Effects of traditional medical herbs “minor bupleurum decoction” on the non-specific immune responses of white shrimp (Litopenaeus vannamei)
Accepted Manuscript Effects of traditional medical herbs “minor bupleurum decoction” on the non-specific immune responses of white shrimp (Litopenaeus vannamei) Yu-Sheng Wu, Meng-Chou Lee, Cheng-Ting Huang, Tzu-Chi Kung, Chi-Yang Huang, Fan-Hua Nan PII:
S1050-4648(17)30137-7
DOI:
10.1016/j.fsi.2017.03.018
Reference:
YFSIM 4487
To appear in:
Fish and Shellfish Immunology
Received Date: 6 February 2017 Revised Date:
6 March 2017
Accepted Date: 7 March 2017
Please cite this article as: Wu Y-S, Lee M-C, Huang C-T, Kung T-C, Huang C-Y, Nan F-H, Effects of traditional medical herbs “minor bupleurum decoction” on the non-specific immune responses of white shrimp (Litopenaeus vannamei), Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of Traditional Medical Herbs “Minor bupleurum decoction “on the
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Non-specific Immune Responses of White Shrimp (Litopenaeus vannamei)
3 Yu-Sheng Wu1, Meng-Chou, Lee1, Cheng-Ting Huang1, Tzu-Chi Kung1, Chi-Yang
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Huang1, Fan-Hua Nan1,*
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1. Department of Aquaculture, National Taiwan Ocean University, Keelung 20248, Taiwan
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*Correspondence Author: Ph.D. Fan-Hua Nan (Department of Aquaculture, National
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Taiwan Ocean University, No.2, Beining Rd., Jhong-jheng District, Keelung City 202)
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Taiwan (R.O.C);
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Phone: +886-2-2462-2192#5231
E-mail address: [email protected]
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Conflict of interest: None of the authors has a financial relationship with a
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commercial entity that has an interest in the subject of this manuscript.
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Abstract
2 This study is investigating the effect of minor bupleurum decoction (Xiao-Chai-Hu
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decoction) on the non-specific immune response of white shrimp (Litopenaeus
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vannamei). To determine prophenoloxidase activity (proPO), reactive oxygen species
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production (ROS), superoxide anion production (O2-), nitric oxide production (NO),
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phagocytic rate (PR), phagocytic index (PI), superoxide dismutase activity (SOD), total
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haemocyte count (THC) and differential haemocyte count (DHC). In this experiment,
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treating with different dosages (0, 0.25, 0.5 and, 1 %) of minor bupleurum decoction
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to detect immune parameters on day 0, 1, 2, 4, 7, 14, 21 and 28.
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Result is shown that 0.25 % treatment significantly enhanced the superoxide
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dismutase (SOD) activity and, 0.25 and 1 % treatment significantly increased the ROS
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production, nitric oxide (NO) production and phagocytic rate (PR) moreover, 0.5 and
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1 % treatment induced the proPO activity and superoxide anion (O2-) production.
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the non-specific immunity responses of white shrimp via in vivo examination.
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Evidence exactly indicated that minor bupleurum decoction is able to enhance
Keyword: Litopenaeus vannamei, Minor Bupleurum Decoction, Non-specific immune response, Flow cytometry.
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Introduction Medical herbs have been used as an alternative therapy in humans for
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thousands of years in the East and West. Previous studies have reported the effects
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of herbs on animals, including the recent use of immune-stimulant herbs in
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aquaculture. In common carp (Cyprinus carpio) and large yellow croaker
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(Pseudosciena crocea), the respiratory burst activity of phagocytic cells and plasma
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lysozyme activity significantly increased after the administration of medical herbs [1].
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Direkbusarakom et al. used guava and balsam pear extracts to inhibit Vibrio
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parahaemolyticus and Vibrio harveyi [2], and Sivaram et al. reported that Ocimum
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sanctum, Withania somnifera, and Myristica fragrans suppress V. harveyi [3]. In grass
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shrimp, the ethanol extract of the Thai traditional medical herb Clinacanthus nutans
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is able to inhibit yellow-head virus (YHV) [4]. These findings reported the using of
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medical herb is able to enhance the immune response in various other fish species
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[5-7].
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Minor bupleurum decoction is from Treatise on Febrile and Miscellaneous
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Diseases of Zhongjin [8], which is the famous representative prescription of
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traditional medical herbs and composed of bupleurum, scutellaria baicalensis,
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liquorice, rhizoma pinellinae praeparata, ginseng and ginger. Studies carried out the
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evidence of the Bupleurum plant have the function in the organism physiological
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response by the ingredients of triterpenoid, complex polysaccharides, and lignan[9].
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In the clinical therapy, it is used to treat the disease including of cholecystitis,
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pancreatitis, nephritis, acute tonsillitis, parotitis and stomatitis[10].
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Crustaceans defence the pathogen infection by the nonspecific immune system.
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In the crustacean nonspecific immune, the haemocyte is the main cell which involved
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into the immune response including of the phagocytosis activity, PO system
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activation, etc. According to their morphology and functions, shrimp haemocytes are 3
ACCEPTED MANUSCRIPT classified into hyaline, semi-granular, and granular cells [11]. Hyaline cell is the
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smallest shrimp haemocytes which has a high nuclear–cytoplasmic ratio and major
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role in phagocytosis. Hyaline cell is recognizing of infective pathogen then to induce
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the reactive oxygen reaction [12, 13]. The nuclear mass ratio of semi-granular cells
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ranges between those of granulocytes and hyaline cells. Semi-granular cells contain a
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small number of particles, which are mainly participating in cytotoxicity,
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encapsulation, and the prophenoloxidase (proPO) system [14-16]. Granulocytes
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contain numerous particles and proPO and are activated by lipopolysaccharide- and
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glucan-binding proteins [17, 18].
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In this investigation, using of the traditional medical herbs “minor bupleurum
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decoction” on the effect of the white shrimp non-specific immune response. The
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examined parameters are including of the prophenoloxidase activity (proPO),
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reactive oxygen species production (ROS), superoxide anion production (O2-), nitric
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oxide production (NO), phagocytic rate (PR), phagocytic index (PI), superoxide
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dismutase activity (SOD), total haemocyte count (THC) and differential haemocyte
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count (DHC).
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Material and methods
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Experimental Animals For this experiment, 160 white shrimps were obtained from Aquatic Animal
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Center, National Taiwan Ocean University, Taiwan. After being transferred to the
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laboratory, the shrimp were cultured until their body mass was 12.36±2.67 g. They
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were subsequently divided into nine groups of 20 individuals and stocked in 10 L
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glass tank at 25 ± 1°C. The groups were treated with the examined feed including of
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different concentrations of control (0%), 0.25%, 0.5% and, 1.0% minor bupleurum
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decoction (Sun Ten Pharmaceutical Co., Ltd) to observe the nonspecific immune
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response on the 0, 1, 2, 4, 7, 14, 21 and 28 days.
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Experimental parameters
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Haemocytes was withdrawn from each shrimp with a 1 ml sterile syringe and
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mixture with 200 μL anticoagulant (30 mM trisodium citrate, 0.34 M sodium chloride,
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10 mM EDTA, 0.12 M glucose, pH 7.4) following to centrifuge with 800 x g for 10 min
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at 4 °C to remove the supernatant and the cell pellet was collected to dissolved and
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quantitative as 1 × 106 cells/MCHBSS-ml.
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Phenoloxidase activity
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Phenoloxidase activity was measured spectrophotometrically by recording the
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formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) [19]. 3
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× 106 cells/PBS-ml was deposited in into the 1.5 ml tube and the collected cell pellet
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dissolved in iced Tris-HCl buffer (250 mM Tris, pH 6.5) was ultrasonicated and the cell
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homogenate was then ultracentrifuged at 48000 x g for 30 min at 4°C and the clear
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supernatant, representing haemocyte lysate supernatant (HLS), was used 5
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immediately in all assays. 50 μl of haemocyte lysate supernatant was reacted with 50
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μl of 3 mg/ml Trypsin and 50 μl of 3 mg/ml L-DOPA (L-3,2-dihydroxyphenylalanine)
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and was measured by microplate reader at O.D 492 nm.
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Phagocytic activity was measured following the method described by [20].
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Briefly, 200μl of 3 × 106 cells/ MCHBSS -ml was deposited in into the 96 microplate
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for 1 hour and 200 μl of 0.8 μm latex beads (1.5 × 107 latex beads/ml, LB8 SIGMA)
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was added to examine the phagocytic activity under microscope observation.
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Phagocytic ratio (PR) = No. of phagocytic cells with engulfed bacteria / No. of
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phagocytes
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Phagocytic index (PI) = No. of engulfed bacteria / Phagocytic cell
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ROS production
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ROS production was detected by DCFH-DA (2,7-dichlorofluorescin diacetate,
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SIGMA) probe. 300 μl of the examined cell was incubated with 300 μl of 100 mM
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DCFH-DA without light in the culture tube for 30 min and the fluorescence of the
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cells from each tube was measured by the flow cytometry, BD FACS Diva software v
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6.1.2.
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Superoxide anion (O2-) production
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The examination protocol was modified by the previous study[21]. 100 μl of 3 ×
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106 cells/PBS-ml diluted haemocytes solution was deposited in microplates and were
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centrifuged with 800 x g for 20 min at 4 °C. After incubation, 100 μl of 1 mg/ml
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zymosan was added to affect haemocyte for 30 min at 26~27 °C. The treatment was
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discarded and the haemocytes were reaction with 100 μl NBT solution (0.3%) for 30
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min at room temperature. The NBT solution was removed and the haemocytes were 6
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fixed with 100% methanol, and washed three times with 100 μl 70% methanol and
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air-dried. The formazan was dissolved by the addition of 120 μl 2 M KOH and 140 μl
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dimethyl sulphoxide (DMSO) and measured by microplate reader at O.D 630 nm.
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Superoxide Dismutase (SOD) Activity The activity of SOD was measured by the RANSOD kit (Randox) and the formula
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was presented as:
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(A2-A1) / 3 = △A / min of standard or sample
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100-(△Astd/min × 100 / △Ast/min or sample min) × 100 % = % inhibition
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Nitric oxide (NO) production NO
production
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detected
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DAF-FM
(4-amino-5-methylamino-2’,7’-difluorescein, SIGMA) probe. 300 μl of the examined
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cell was incubated with 300 μl of 10 μM DAF-FM without light in the culture tube for
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30 min and the fluorescence of the cells from each tube was measured by the flow
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cytometry, BD FACS Diva software v 6.1.2.
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Total haemocyte count (THC) and classification The 485 μL of prepared haemocytes were incubated with the 15 μl AccuCount
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Fluorescent Particles (Spherotech, SPHEROTM) and analyzed the flow cytometry, BD
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FACS Diva software v 6.1.2.
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Statistical Analysis
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Tukey’s Honestly Significant Difference Test（Tukey’s HSD） and one-way ANOVA
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were used to analyze the statistical significance between treatment and control
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groups.
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P value less than 0.05 considered to be statistically significant. Results were
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presented as means ± SD. 7
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proPO Detection
Continuous treated white shrimp with different concentrations of minor
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bupleurum decoction for 28 days, the impact of its proPO activity was as shown in
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Table 1. The experimental result was shown that the 0.5 and 1% treatment group,
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the activity of proPO was significantly higher than the control group on day 1 to 14 (p
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< 0.05). 0.25% treatment was presented with significantly increased on day 4 to 14 (p
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< 0.05), however there was no significant difference between each group after the
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21st day observation.
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ROS production
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Treated with different concentrations of minor bupleurum decoction for 28 days,
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the ROS production rate was shown in Table 2. Experimental results show that the
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1% treatment group, the ROS production rate was significantly higher than control
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group on the 2nd day (p < 0.05) moreover, significantly increased than the control and
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0.25% treatment group on the 4th day. The 0.5% treatment group was significantly
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higher than the control group and 0.25% treatment group on the 4th day (p < 0.05),
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however there was no significant difference between each group after the 28th day
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observation.
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O2- production
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Treated with different concentrations of minor bupleurum decoction for 28 days,
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the O2- production rate was shown in Table 3. Experimental results show that the 0.5
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and 1% treatment group, the ROS production rate was significantly higher than 8
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control group on the 2nd to 28th day (p < 0.05). The 0.25% treatment group was
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significantly higher than the control group on the 4th to 28th day (p < 0.05).
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NO production
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The NO production was significantly higher in the 0.5 and 1% treatment groups
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than in the control and 0.25% treatment groups on the 4th day (p < 0.05). The 0.25%
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treatment group was significantly higher than the control group on the 7th day (p <
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0.05). There were significant differences between the control group and the
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treatment groups on the 21st day. On the 28th day, the 0.5% was presented with
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significantly higher than the control (p < 0.05) as presented as Table 4.
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SOD observation
Treatment of different concentration minor bupleurum decoction for 28 days
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and, the SOD activity was as shown in Table 8. Experimental results show that
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treatment of 0.25% minor bupleurum decoction, the SOD activity was significantly (p
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< 0.05) in the 1st day compared to the control and the other treatment groups and,
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0.5% and 1% treatment groups were significantly higher than the control (p < 0.05).
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0.25% treatment was significantly increased from 7th to the 14th days (p < 0.05) than
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the control group but, not significantly different compared to the other treatment
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groups (p > 0.05) as presented as Table 5.
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Total Haemocyte Count (THC)
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The results showed that the THC of 0.5% treatment group was significantly
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higher than the control and 1% treatment group in the 2nd day (p < 0.05) but not
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significant different compared to the 0.25% treatment (p > 0.05). On the 14th day,
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0.25% treatment was significantly different compared to the control (p < 0.05) and 9
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to the control and the other treatment group from the 4th to 14th days (p < 0.05) as
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presented as Table 6.
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Differential Haemocyte Count (DHC)
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On the granular cell (GC) observation, the 1% treatment was presented with a
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significant difference compared to the control and the other treatment groups from
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the 14th to the 28th days (p < 0.05) as presented as Table 7. On the hyaline cell (HC)
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observation, 0.5% treatment was with a significant difference to the control and the
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other treatment groups from the 7th and 28th day (p < 0.05) as presented as Table 8.
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On the semi-granular cells (SGC) observation, 0.5% treatment was significantly
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different to the control on the 1st and 7th day (p < 0.05) moreover, 0.5% and 1% were
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significantly different to the control on the 28th day (p < 0.05) as presented as Table
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Phagocytic rate (PR) and Phagocytic index (PI)
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Treatment of different concentration minor bupleurum decoction for 28 days and,
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the PR and PI activity was as shown in Table 10 and 11. Experimental results show
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that treatment of 0.25% minor bupleurum decoction, the PR activity was significantly
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(p < 0.05) higher compared to the control and the other treatment groups from the
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2nd to the 21st day however there was no significant difference in the 28th day. 0.5%
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and 1% treatment groups were significantly higher than the control (p < 0.05) but not
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in the 14th and 21st days. In the PI observation, 0.5% treatment was significantly
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increased in the 2nd day (p < 0.05) than the control group.
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5 6 7 8 Discussion
Rodriguez and Le Moullac pointed out that PO activity can be used as indicators
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of crustacean immunity ability [22]. Pro-phenoloxidase is known as the initial enzyme
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in the phenoloxidase (PO) producing system and the end of production is the PO
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enzyme in the most of the invertebrate animals [23, 24]. Soderhall et al. illustrated
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that in the key of the defense mechanism in crustaceans, it is using of releasing the
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bactericidal substance which is including of the melanin and the intermediates after
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activating the proPO system [25]. In addition, the proPO system is activated to
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release the peroxinectin, which stimulates and contacts a variety of haemocytes
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involved in the cellular immune response [26]. The proPO system is activated by the
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lipopolysaccharide (LPS) of gram-negative bacteria [25], the composition of the
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fungal or bacterial cell wall [27]. In vitro treating prawn haemocytes with Cyanodon
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dactylon is able to induce the proPO activity [28]. This result shown that treating
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white shrimps with minor bupleurum decoction exactly induce the proPO activity.
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Invertebrate facing to the foreign pathogens is also releasing the oxygen
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substances to defend the infection. Synthesis of the bactericidal ROS as the free
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radicals, including superoxide anion (O2-), hydrogen peroxide, ect., is function to
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oxidize the pathogens. Based on this mechanism, ROS generation is role as the
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indicator of the crustacean cellular defense mechanism [29, 30]. In this investigation,
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treating of the minor bupleurum decoction is exactly able to induce the ROS
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synthesis in the short-term observation however, it is not presented with a significant 11
ACCEPTED MANUSCRIPT increase while a long-term treating. In the previous study, Bricknell and Dalmo (2005)
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pointed out that the long-term continuous use of immune activator to the fish, the
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immune system may gradually lower the sensitivity to the immune activator to make
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the immune ability with a decline phenomenon after the peak [31]. NO production
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was significantly higher in the treatment of the 0.5 and 1% groups than the control
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and other groups from the 4th day. On the 28th day observation, all of the treatment
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was significantly increased in the NO production than the control group.
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Balasubramanian et al. investigated that treating the Penaeus monodon with the
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extract of Cyanodon dactylon to observe the NO production. The phenomenon of the
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NO production is increased while treatment of the Cyanodon dactylon [28].
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Compared to our findings, the NO production is exactly activated after the minor
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bupleurum decoction treat. Research indicated that injection of the sodium alginate
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to the common carp is inducing the phagocytosis cell counts however it is declined
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with the elapsed time [32].
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Oncorhynchus mykiss with Zingiber officinale is increasing the activity of the
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phagocytosis with the ROS production [33]. Prevention of the excessive ROS
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production damage the host, the SOD was active and to transfer the harmful O2- into
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the hydrogen peroxide etc. [34]. Therefore, SOD is role as an important antioxidant
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enzyme, which can be used as an indicator of antioxidant capacity indicator [35]. This
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evidence showed that SOD activity in the low-concentration treatment at the 1st, 7th
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and 14th days were significantly different to the control group, and O2- generated at
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the 1,7,14 days with a high enhance. According to the SOD and O2- generation, it is
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illustrating that the SOD activation is based to the O2- production and using of the
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minor bupleurum decoction is exactly able to enhance the crustacean SOD synthesis
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to prevent the high concentration of ROS. In this experiment, the treatment groups
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of phagocytosis rate were higher than the control group from the 2nd to the 14th day
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Dügenci et al. also pointed out that treating
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than the control group. It is indicating that phagocytosis rate may be increased with
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the counts of hyaline cell. The proPO activity of each treatment group was higher
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than the control group from the 4th to the 14th day however, the counts of granular
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cell (GC) and semi-granular cell (SGC) were not significantly increased which
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indicated that using of minor bupleurum decoction could promote the proPO activity
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of shrimp.
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Hyaline cells are typically phagocytic cells [36] and major function with releasing
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the transglutaminase [23]. In the previous research, hyaline cell is lacking of proPO
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activity. A series of reactive oxygen species (ROS) are produced while the hyaline cell
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is recognizing and phagocytosing the foreign pathogens. The produced superoxide
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anion (O2-) substances are function of bactericidal activity [37]. In this research, the
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phagocytic rate was with an increase trend in the treating groups. Furthermore,
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contradistinguishing to the quantity of the hyaline cell, it is exact to realize the effect
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of minor bupleurum decoction to increase the white shrimp hyaline cell quantity
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moreover observing the enhanced phagocytic ability. Previous study indicated that
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injection with the latex beads and, the SGC mounted the strongest phagocytic
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response [38]. In this research, treating of 1% minor bupleurum decoction is inducing
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the SGC percentage in the haemocyte component. Treating of the minor bupleurum
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decoction altering the haemocyte component is with the time. Treating of the minor
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bupleurum decoction from 1st to 7th days, the hyaline cell is exactly increased.
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Furthermore, semi-granular cell and granular cell is exactly increased from 14th to
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28th days. As our findings, we proposed that treating of minor bupleurum decoction,
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the hyaline cell is the early activation then to induce the semi-granular and granular
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cells.
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More, we summarize the recent research which using of the traditional medical 13
ACCEPTED MANUSCRIPT herbs in the aquatic animal to improve the non-specific immune response including
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of the non-specific immune response as phagocytic activity as presented in Table 12.
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By these evidence, medical herbs are in the regulation of human beings for a long
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term and has been evidenced with improving the host to induce the non-specific
5
immune response as this finding.
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Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity. Developmental & Comparative Immunology. 2014 44:116-23. 16. Wu Y-S, Chang C-H, Nan F-H. Steroid hormone “cortisone” and “20-hydroxyecdysone” involved in the non-specific immune responses of white
20 21 22 23
shrimp (Litopenaeus vannamei). Fish & shellfish immunology. 2016 56:272-7 17. Gallo C, Schiavon F, Ballarin L. Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda). Fish & shellfish immunology. 2011 31:423-31.
24 25
18. Song Y-L, Yu C-I, Lien T-W, Huang C-C, Lin M-N. Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus.
26 27
Fish & shellfish immunology. 2003 14:317-31. 19. Asokan R, Arumugam M, Mullainadhan P. Activation of prophenoloxidase in the
28 29 30 31 32
plasma and haemocytes of the marine mussel Perna viridis Linnaeus. Developmental & Comparative Immunology. 1997 21:1-12. 20. Chen H, Mai K, Zhang W, Liufu Z, Xu W, Tan B. Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai Ino. Fish & shellfish immunology. 2005 19:241-52.
33 34 35
21. Wu Y-S, Tseng T-Y, Nan F-H. Beta-1, 3-1, 6-glucan modulate the non-specific immune response to enhance the survival in the Vibrio alginolyticus infection of Taiwan abalone (Haliotis diversicolor supertexta). Fish & shellfish immunology. 2016
36 37 38
54:556-63. 22. Rodrıguez J, Le Moullac G. State of the art of immunological tools and health control of penaeid shrimp. Aquaculture. 2000 191:109-19.
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1
15
ACCEPTED MANUSCRIPT 23. Söderhäll K, Cerenius L. Crustacean immunity. Annual Review of Fish Diseases.
2 3 4
1992 2:3-23. 24. Jackson AD, Smith VJ, Peddie CM. In vitro phenoloxidase activity in the blood of Ciona intestinalis and other ascidians. Developmental & Comparative Immunology.
5 6 7 8
1993 17:97-108. 25. Söderhäll K, Aspan A, Duvic B. The pro-PO-system and associated proteins; role in cellular communication in arthropods. Research in immunology. 1990 141:896-907.
9 10 11 12
26. Cerenius L, Jiravanichpaisal P, Liu H-p, Soderhall I. Crustacean immunity. Invertebrate Immunity: Springer; 2010, p. 239-59. 27. Ashida M, Ishizaki Y, Iwahana H. Activation of pro-phenoloxidase by bacterial cell walls or β-1, 3-glucans in plasma of the silkworm, Bombyx mori. Biochemical and
13 14 15
biophysical research communications. 1983 113:562-8. 28. Balasubramanian G, Sarathi M, Venkatesan C, Thomas J, Hameed AS. Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus
16 17 18 19
monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV). Fish & shellfish immunology. 2008 25:820-8. 29. Xian J-A, Zhang X-X, Guo H, Wang D-M, Wang A-L. Cellular responses of the tiger shrimp Penaeus monodon haemocytes after lipopolysaccharide injection. Fish &
20 21 22 23
shellfish immunology. 2016 54:385-90. 30. Wu Y-S, Liau S-Y, Huang C-T, Nan F-H. Beta 1, 3/1, 6-glucan and vitamin C immunostimulate the non-specific immune response of white shrimp (Litopenaeus vannamei). Fish & shellfish immunology. 2016 57:269-77.
24 25
31. Bricknell I, Dalmo RA. The use of immunostimulants in fish larval aquaculture. Fish & shellfish immunology. 2005 19:457-72.
26 27
32. Fujiki K, Yano T. Effects of sodium alginate on the non-specific defence system of the common carp (Cyprinus carpioL.). Fish & shellfish immunology. 1997 7:417-27.
28 29 30 31 32
33. Dügenci SK, Arda N, Candan A. Some medicinal plants as immunostimulant for fish. Journal of Ethnopharmacology. 2003 88:99-106. 34. Holmblad T, Söderhäll K. Cell adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture. 1999 172:111-23. 35. Harris ED. Copper as a Cofactor and Regulator of Copper, Zinc Superoxide
33 34 35
Dismutase1, 2. The Journal of nutrition. 1992 122:636. 36. Johansson MW, Keyser P, Sritunyalucksana K, Söderhäll K. Crustacean haemocytes and haematopoiesis. Aquaculture. 2000 191:45-52.
36 37 38
37. Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Developmental & Comparative Immunology. 1993 17:211-9.
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1
16
ACCEPTED MANUSCRIPT 38. Giulianini PG, Bierti M, Lorenzon S, Battistella S, Ferrero EA. Ultrastructural and
2 3 4
functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge. Micron. 2007 38:49-57.
5 6 7 8
39. Jian J, Wu Z. Influences of traditional Chinese medicine on non-specific immunity of Jian carp (Cyprinus carpio var. Jian). Fish & shellfish immunology. 2004 16:185-91. 40. Rao YV, Das B, Jyotyrmayee P, Chakrabarti R. Effect of Achyranthes aspera on the
9 10 11 12
immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish & shellfish immunology. 2006 20:263-73. 41. Watanuki H, Ota K, Tassakka ACMA, Kato T, Sakai M. Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio. Aquaculture. 2006 258:157-63.
13 14 15
42. Yin G, Jeney G, Racz T, Xu P, Jun X, Jeney Z. Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus. Aquaculture. 2006 253:39-47.
16 17 18 19
43. Sahu S, Das B, Mishra B, Pradhan J, Sarangi N. Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Journal of Applied Ichthyology. 2007 23:80-6. 44. Yin G, Ardó L, Thompson K, Adams A, Jeney Z, Jeney G. Chinese herbs
20 21 22 23
(Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila. Fish & shellfish immunology. 2009 26:140-5. 45. Harikrishnan R, Balasundaram C, Heo M-S. Herbal supplementation diets on
24 25
hematology and innate immunity in goldfish against Aeromonas hydrophila. Fish & shellfish immunology. 2010 28:354-61.
26 27
46. Wu Y-S, Chen Y-Y, Ueng P-S, Nan F-H. Effects of medicinal herbs “Plantago asiatica”,“Houttuynia cordata” and “Mentha haplocalyx” on non-specific immune
28 29 30 31 32
responses of cobia (Rachycentron canadum). Fish & shellfish immunology. 2016 58:406-14. 47. Samad APA, Santoso U, Lee M-C, Nan F-H. Effects of dietary katuk (Sauropus androgynus L. Merr.) on growth, non-specific immune and diseases resistance against Vibrio alginolyticus infection in grouper Epinephelus coioides. Fish & shellfish
33 34 35
immunology. 2014 36:582-9.
AC C
EP
TE D
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1
36 37 38 17
ACCEPTED MANUSCRIPT 1 2 3 4
RI PT
5 6 7 8
SC
9 10 11 12
16 17 18 19
M AN U
13 14 15 Table Legends
Table 1. The prophenoloxidase (proPO) activity (in O.D. 492 nm) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented by mean±S.D.
24 25
Table 2. The reactive oxygen species (ROS) production ratio (in DIFC-DA intensity) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different
26 27
doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different
28 29 30 31 32 33 34
(p<0.05). All values are examined by flow cytometry and represented by mean±S.D. Table 3. The superoxide anion (O2-) production ratio (in O.D. 620 nm) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
35 36 37
Table 4. The nitric oxide (NO) production ratio (in DAF-FM intensity) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of
38
minor bupleurum decoction.
AC C
EP
TE D
20 21 22 23
18
ACCEPTED MANUSCRIPT 1
a~c: mean in the same column with the different latter are significantly different
2 3 4
(p<0.05). All values are examined by flow cytometry and represented mean±S.D.
5 6 7 8
hepaptopancreas of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
9 10 11 12
Table 6. The total haemocyte count (THC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction.
13 14 15
a~c: mean in the same column with the different latter are significantly different (p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
16 17 18 19
Table 7. The granular cell count (GC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction.
M AN U
SC
RI PT
Table 5. The superoxide dismutase (SOD) activity (U/mg protein) in the
a~b: mean in the same column with the different latter are significantly different (p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
24 25
Table 8. The hyaline cell count (HC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of
26 27
minor bupleurum decoction. a~b: mean in the same column with the different latter are significantly different
28 29
(p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
30 31 32 33 34
Table 9. The semi-granular cell count (SGC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~b: mean in the same column with the different latter are significantly different
AC C
EP
TE D
20 21 22 23
35 36 37
(p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
38
Table 10. The phagocytic rate (PR: phagocytic cells/total cells) in the haemocytes of 19
ACCEPTED MANUSCRIPT white shrimp (Litopenaeus vannamei), treating with different doses of minor
2 3 4
bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
5 6 7 8
Table 11. The phagocytic index (PI: 0.8 μm latex beads/phagocytic cells) in the haemocytes of white shrimp (Litopenaeus vannamei), treatng with different doses of minor bupleurum decoction.
RI PT
1
a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
13 14 15
Table 12. Effect of traditional medical herbs on the fish immune response.
Table 12 Treatment
Species
M AN U
16 17
SC
9 10 11 12
Time
Parameters
Astragalus radix Angelicae sinensis
Cyprinus carpio 30 (Common Carp) days
EP
28
ROIs ↑
(Labeo)
days
Lysozyme activity ↑
Spirulina platensis
Cyprinus carpio 5 (Common Carp) days
Astragalus radix
Oreochromis niloticus (Tilapia)
Allium sativum
Labeo rohita (Labeo)
Phagocytic activity ↑ ROIs ↑
Lysozyme activity ↑ 4 Phagocytosis ↑ weeks
60 days
Dügenci et al (2003)[33]
ROIs ↑ Complement activity ↑ Jian and Wu (2004)[39] Lysozyme activity ↑
Labeo rohita
AC C
Achyranthes aspera
TE D
Zingiber officinale
Oncorhynchus ROIs ↑ 3 mykiss Phagocytosis ↑ weeks (Rainbow trout)
References
Rao et al (2006)[40] Watanuki et al (2006)[41] Yin et al (2006)[42]
Phagocytic activity ↑ ROIs ↑ Sahu et al (2006)[43] Complement activity ↑ Lysozyme activity ↑
20
ACCEPTED MANUSCRIPT
(1:1:1) Plantago asiatica, Houttuynia cordata, Mentha haplocalyx Sauropus androgynus L. Merr
Carassius auratus (goldfish) Rachycentron canadum (Cobia) Epinephelus coioides (grouper)
Harikrishnan (2010)[45]
Lysozyme activity ↑ 32 days 30 days
O2- ↑ Lysozyme activity↑ Phagocytosis↑ phagocytosis↑ ROIs↑
AC C
EP
TE D
M AN U
1
WBC↑ 4 RB ↑ weeks Phagocytic activity ↑
Yin et al (2009)[44]
RI PT
Azadirachta indica Ocimum sanctum Curcuma longa
RB ↑ Cyprinus carpio 5 Phagocytosis ↑ (Common Carp) weeks Lysozyme activity ↑
Wu et al (2016)[46]
Samad APA et al (2014)[47]
SC
Astragalus radix Ganoderma lucidum
21
ACCEPTED MANUSCRIPT
RI PT
ble 1 Time elapsed (days)
D. 492 nm 0
1
0.042±0.009
0.026±0.001c
2
4
7
14
21
28
M AN U
SC
se (%)
0.016±0.004b 0.070±0.017b 0.022±0.002a
0.016±0.
0.25
0.397±0.075bc 0.092±0.018b 0.081±0.024a
0.204±0.009a 0.210±0.011a 0.049±0.021a
0.025±0.
0.5
0.848±0.200a
0.245±0.088a 0.228±0.043a 0.062±0.008a
0.027±0.
0.276±0.010a 0.288±0.027a 0.101±0.041a
0.027±0.
EP
0.880±0.091a 0.118±0.004a
0.659±0.047ab 0.835±0.040a 0.093±0.004a
AC C
1
TE D
0.025±0.008b 0.015±0.006b
0
ACCEPTED MANUSCRIPT
Time elapsed (days)
Ratio 1
2
4
7
14
21
28
17.66±3.84
22.80±10.47a
17.55±4.17b
17.05±0.07c
13.35±0.35a
25.25±5.44a
26.35±1.06a
20.10±0.42b
0.25
25.55±7.99a
18.30±3.54b
18.40±0.57b
10.40±0.14a
32.25±1.91a
21.85±0.35a
27.95±1.63a
0.5
33.65±5.59a
21.55±1.48b
12.60±1.27a
23.5±3.96a
28.00±8.77a
19.50±0.57b
1
41.65±9.97a
41.80±4.38a
14.50±1.56a
33.25±3.61a
23.75±0.64a
20.5±0.14b
TE D
M AN U
se (%)
SC
0
28.15±0.07a
EP
AC C
0
RI PT
ble 2
27.10±0.14a
ACCEPTED MANUSCRIPT
Ratio
Time elapsed (days) 1
2
4
7
14
21
0.028±0.006
0.060±0.026a
0.059±0.006b
0.022±0.006c
0.024±0.013c
0.039±0.006b
0.031±0.001b
0.030±0.0
25
0.072±0.049a
0.091±0.018b
0.081±0.011b
0.184±0.016b
0.259±0.013b
0.179±0.027a
0.171±0.0
.5
0.427±0.199a
0.221±0.012a
0.419±0.007a
0.670±0.092a 0.118±0.025ab
0.181±0.0
1
0.429±0.081a
0.298±0.042a
0.388±0.039a
0.667±0.073a 0.096±0.023ab
0.159±0.0
TE D
M AN U
e (%)
0.082±0.001b
EP
AC C
SC
0
0
RI PT
ble 3
0.241±0.013a
28
ACCEPTED MANUSCRIPT
RI PT
ble 4
Time elapsed (days) 1
2
4
6.50±2.68
6.18±3.15a
12.78±4.60a
.25
7.53±4.14a
17.84±4.19a
0.5
6.77±2.24a
25.66±12.50a
1
6.78±3.85a
22.90±14.60a
14
21
9.92±2.06b
15.74±10.15b
7.54±2.78a
4.62±0.90b
7.28±2
4.51±1.41b
39.66±9.68a
22.24±3.75a
49.35±4.27a
37.40±
23.91±5.73a
14.11±1.04b
21.45±11.61a
31.34±14.46a
27.83±
21.62±6.21a
20.87±3.34ab
18.07±0.21a
45.40±2.76a
39.92±
TE D
EP
AC C
0
7
M AN U
0
se (%)
SC
Ratio
28
ACCEPTED MANUSCRIPT
U/mg
RI PT
ble 5 Time elapsed (days) 1
2
4
1.474±0.233
1.805±0.183c
1.821±0.507a
3.163±0.724a
0.25
4.546±0.289a
2.474±0.555a
3.716±0.553a
0.5
3.063±0.409b
1.736±0.313a
1
14
21
2.302±0.743b
2.309±0.151b
2.005±0.274a
2.035±0.
3.838±0.649a
3.144±0.286a
2.571±0.395a
2.283±0.
2.862±0.463a 2.885±0.140ab 2.685±0.323ab 3.046±0.886a
3.432±1.
3.168±0.374a 2.655±0.044ab 2.502±0.359ab 2.330±0.485a
2.340±0.
M AN U
TE D
EP
AC C
0
7
28
SC
0
se (%)
2.456±0.171bc 1.966±0.198a
ACCEPTED MANUSCRIPT
cell/ml
Time elapsed (days) 0
1
2
4
7
14
21
28
SC
6
RI PT
ble 6
M AN U
se(%) 16.08±1.82b
19.60±1.56ab
24.52±3.50b
22.01±1.10c
12.37±2.33ab
19.78±1.14
0.25
17.66±6.07a
18.74±2.29ab
14.96±1.88b
31.85±1.97a
21.42±1.93c
11.26±0.59b
20.43±1.74
0.5
9.33±1.63b
21.25±1.26a
17.90±3.98b
24.14±4.02b
28.27±1.96b
13.35±0.45ab
21.69±3.43
1
9.80±0.53b
16.84±1.53b
33.88±2.41a
37.27±2.71a
15.23±2.05a
21.38±0.12
EP
AC C
10.17±1.73
TE D
13.46±4.25ab
0
23.28±2.17a
ACCEPTED MANUSCRIPT
cell/ml
Time elapsed (days) 1
2
4
2.53±0.26
3.20±1.44a
3.90±0.83a
3.30±1.50a
25
2.57±0.97a
2.57±1.22a
2.39±0.59a
.5
2.36±1.08a
3.17±0.65a
1
2.12±0.23a
2.64±1.38a
14
21
4.68±2.33a
3.52±1.75b
2.08±1.79a
1.93±0.
4.97±0.99a
3.25±1.58b
1.52±0.71a
0.88±0.
2.04±0.71a
2.52±0.18a
3.32±0.81b
1.92±0.70a
2.14±0.
2.02±0.09a
3.75±0.27a
7.36±2.82a
3.26±0.72a
3.15±0.
TE D
EP
AC C
0
7
M AN U
0
e (%)
SC
6
RI PT
ble 7
28
ACCEPTED MANUSCRIPT
RI PT
ble 8
06 cell/ml 1
2
4
1.02±0.17
2.31±1.07ab
2.13±1.05a
0.25
3.74±2.37a
1.29±0.59a
0.5
0.91±0.37b
2.37±0.45a
1
0.92±0.18b
2.27±0.63a
14
21
2.13±0.27a
2.91±0.42b
5.74±1.80bc
3.06±1.35ab
4.62±2.9
3.00±1.56a
3.60±0.77b
1.63±0.25c
1.09±0.53b
3.78±1.9
1.65±0.75a
11.85±2.08a
10.93±4.19a
4.60±1.65a
12.90±3.5
3.50±0.80a
5.12±1.61b
8.26±2.37ab
5.59±1.84a
8.64±5.40
TE D
EP
AC C
0
7
M AN U
0
ose(%)
SC
Time elapsed (days)
28
ACCEPTED MANUSCRIPT
06 cell/ml
Time elapsed (days) 1
2
4
14
21
7.64±1.30
13.97±4.99a
11.43±1.44ab
14.17±2.58ab
20.22±6.58a
12.74±1.75a
6.48±2.92a
20.37±
0.25
11.32±6.15ab
8.17±2.05b
11.76±2.55b
27.29±6.90a
20.96±9.30a
7.25±1.99a
15.47±0
0.5
4.32±2.01b
13.58±3.78a
9.23±4.28b
6.99±2.65b
12.93±7.20a
7.41±3.50a
6.65±2
1
6.76±0.28ab
9.70±1.35ab
17.76±1.36a
25.01±0.99a
23.90±3.02a
6.39±2.67a
9.59±4
AC C
EP
TE D
M AN U
ose(%)
7
SC
0
0
RI PT
ble 9
28
ACCEPTED MANUSCRIPT
phagocytic rate
RI PT
ble 10 Time elapsed (days) 1
2
4
7
14
21
15.05±3.87
21.71±7.39b
22.09±3.39c
0.25
25.99±4.14b
0.5 1
28
20.75±3.54b
19.26±4.12b
22.53±6.20b
23.63±3.90c
23.91±
41.55±9.86b
42.11±3.33a
39.53±3.06a
40.33±2.69a
46.86±0.89a
31.35±4
47.15±5.26a
63.63±1.42a
39.64±4.34a
33.61±1.76a
29.59±5.72ab
39.49±5.41ab
37.46±
43.74±0.26a
51.60±1.41ab
37.29±2.82a
33.21±2.78a
32.31±8.02ab
33.74±4.54bc
38.87±
EP
AC C
0
TE D
ose(%)
M AN U
SC
0
ACCEPTED MANUSCRIPT
phagocytic
Time elapsed (days) 1
2
4
1.25±0.43
1.47±0.50
1.15±0.13b
1.23±0.09a
0.25
1.44±0.16a
1.64±0.18ab
1.21±0.30a
0.5
1.95±0.15a
1.87±0.29a
1
1.71±0.01a
1.57±0.20ab
14
21
1.18±0.17a
1.14±0.25a
1.00±0.00a
1.16±0
1.40±0.23a
1.21±0.13a
1.19±0.17a
1.29±0
1.30±0.09a
1.21±0.04a
1.55±0.41a
1.31±0.02a
1.21±0
1.42±0.13a
1.22±0.18a
1.21±0.11a
1.33±0.23a
1.30±0
AC C
EP
TE D
se (%) 0
7
28
SC
0
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index
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ACCEPTED MANUSCRIPT Highlights 1. Minor bupleurum decoction exactly induce the white shrimp non-specific immune response.
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2. Minor bupleurum decoction exactly alter the hyaline cell function and ratio. 3. Minor bupleurum decoction is able to enhance the ROS and NO production.
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4. Minor bupleurum decoction increase the phagocytic rate
S1050-4648(17)30137-7
DOI:
10.1016/j.fsi.2017.03.018
Reference:
YFSIM 4487
To appear in:
Fish and Shellfish Immunology
Received Date: 6 February 2017 Revised Date:
6 March 2017
Accepted Date: 7 March 2017
Please cite this article as: Wu Y-S, Lee M-C, Huang C-T, Kung T-C, Huang C-Y, Nan F-H, Effects of traditional medical herbs “minor bupleurum decoction” on the non-specific immune responses of white shrimp (Litopenaeus vannamei), Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.03.018. 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 1
Effects of Traditional Medical Herbs “Minor bupleurum decoction “on the
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Non-specific Immune Responses of White Shrimp (Litopenaeus vannamei)
3 Yu-Sheng Wu1, Meng-Chou, Lee1, Cheng-Ting Huang1, Tzu-Chi Kung1, Chi-Yang
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Huang1, Fan-Hua Nan1,*
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1. Department of Aquaculture, National Taiwan Ocean University, Keelung 20248, Taiwan
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*Correspondence Author: Ph.D. Fan-Hua Nan (Department of Aquaculture, National
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Taiwan Ocean University, No.2, Beining Rd., Jhong-jheng District, Keelung City 202)
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Taiwan (R.O.C);
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Phone: +886-2-2462-2192#5231
E-mail address: [email protected]
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Conflict of interest: None of the authors has a financial relationship with a
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commercial entity that has an interest in the subject of this manuscript.
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Abstract
2 This study is investigating the effect of minor bupleurum decoction (Xiao-Chai-Hu
4
decoction) on the non-specific immune response of white shrimp (Litopenaeus
5
vannamei). To determine prophenoloxidase activity (proPO), reactive oxygen species
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production (ROS), superoxide anion production (O2-), nitric oxide production (NO),
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phagocytic rate (PR), phagocytic index (PI), superoxide dismutase activity (SOD), total
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haemocyte count (THC) and differential haemocyte count (DHC). In this experiment,
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treating with different dosages (0, 0.25, 0.5 and, 1 %) of minor bupleurum decoction
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to detect immune parameters on day 0, 1, 2, 4, 7, 14, 21 and 28.
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Result is shown that 0.25 % treatment significantly enhanced the superoxide
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dismutase (SOD) activity and, 0.25 and 1 % treatment significantly increased the ROS
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production, nitric oxide (NO) production and phagocytic rate (PR) moreover, 0.5 and
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1 % treatment induced the proPO activity and superoxide anion (O2-) production.
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the non-specific immunity responses of white shrimp via in vivo examination.
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Evidence exactly indicated that minor bupleurum decoction is able to enhance
Keyword: Litopenaeus vannamei, Minor Bupleurum Decoction, Non-specific immune response, Flow cytometry.
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Introduction Medical herbs have been used as an alternative therapy in humans for
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thousands of years in the East and West. Previous studies have reported the effects
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of herbs on animals, including the recent use of immune-stimulant herbs in
5
aquaculture. In common carp (Cyprinus carpio) and large yellow croaker
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(Pseudosciena crocea), the respiratory burst activity of phagocytic cells and plasma
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lysozyme activity significantly increased after the administration of medical herbs [1].
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Direkbusarakom et al. used guava and balsam pear extracts to inhibit Vibrio
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parahaemolyticus and Vibrio harveyi [2], and Sivaram et al. reported that Ocimum
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sanctum, Withania somnifera, and Myristica fragrans suppress V. harveyi [3]. In grass
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shrimp, the ethanol extract of the Thai traditional medical herb Clinacanthus nutans
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is able to inhibit yellow-head virus (YHV) [4]. These findings reported the using of
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medical herb is able to enhance the immune response in various other fish species
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[5-7].
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Minor bupleurum decoction is from Treatise on Febrile and Miscellaneous
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Diseases of Zhongjin [8], which is the famous representative prescription of
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traditional medical herbs and composed of bupleurum, scutellaria baicalensis,
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liquorice, rhizoma pinellinae praeparata, ginseng and ginger. Studies carried out the
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evidence of the Bupleurum plant have the function in the organism physiological
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response by the ingredients of triterpenoid, complex polysaccharides, and lignan[9].
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In the clinical therapy, it is used to treat the disease including of cholecystitis,
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pancreatitis, nephritis, acute tonsillitis, parotitis and stomatitis[10].
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Crustaceans defence the pathogen infection by the nonspecific immune system.
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In the crustacean nonspecific immune, the haemocyte is the main cell which involved
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into the immune response including of the phagocytosis activity, PO system
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activation, etc. According to their morphology and functions, shrimp haemocytes are 3
ACCEPTED MANUSCRIPT classified into hyaline, semi-granular, and granular cells [11]. Hyaline cell is the
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smallest shrimp haemocytes which has a high nuclear–cytoplasmic ratio and major
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role in phagocytosis. Hyaline cell is recognizing of infective pathogen then to induce
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the reactive oxygen reaction [12, 13]. The nuclear mass ratio of semi-granular cells
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ranges between those of granulocytes and hyaline cells. Semi-granular cells contain a
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small number of particles, which are mainly participating in cytotoxicity,
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encapsulation, and the prophenoloxidase (proPO) system [14-16]. Granulocytes
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contain numerous particles and proPO and are activated by lipopolysaccharide- and
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glucan-binding proteins [17, 18].
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In this investigation, using of the traditional medical herbs “minor bupleurum
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decoction” on the effect of the white shrimp non-specific immune response. The
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examined parameters are including of the prophenoloxidase activity (proPO),
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reactive oxygen species production (ROS), superoxide anion production (O2-), nitric
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oxide production (NO), phagocytic rate (PR), phagocytic index (PI), superoxide
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dismutase activity (SOD), total haemocyte count (THC) and differential haemocyte
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count (DHC).
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Material and methods
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Experimental Animals For this experiment, 160 white shrimps were obtained from Aquatic Animal
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Center, National Taiwan Ocean University, Taiwan. After being transferred to the
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laboratory, the shrimp were cultured until their body mass was 12.36±2.67 g. They
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were subsequently divided into nine groups of 20 individuals and stocked in 10 L
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glass tank at 25 ± 1°C. The groups were treated with the examined feed including of
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different concentrations of control (0%), 0.25%, 0.5% and, 1.0% minor bupleurum
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decoction (Sun Ten Pharmaceutical Co., Ltd) to observe the nonspecific immune
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response on the 0, 1, 2, 4, 7, 14, 21 and 28 days.
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Experimental parameters
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Haemocytes was withdrawn from each shrimp with a 1 ml sterile syringe and
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mixture with 200 μL anticoagulant (30 mM trisodium citrate, 0.34 M sodium chloride,
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10 mM EDTA, 0.12 M glucose, pH 7.4) following to centrifuge with 800 x g for 10 min
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at 4 °C to remove the supernatant and the cell pellet was collected to dissolved and
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quantitative as 1 × 106 cells/MCHBSS-ml.
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Phenoloxidase activity
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Phenoloxidase activity was measured spectrophotometrically by recording the
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formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) [19]. 3
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× 106 cells/PBS-ml was deposited in into the 1.5 ml tube and the collected cell pellet
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dissolved in iced Tris-HCl buffer (250 mM Tris, pH 6.5) was ultrasonicated and the cell
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homogenate was then ultracentrifuged at 48000 x g for 30 min at 4°C and the clear
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supernatant, representing haemocyte lysate supernatant (HLS), was used 5
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immediately in all assays. 50 μl of haemocyte lysate supernatant was reacted with 50
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μl of 3 mg/ml Trypsin and 50 μl of 3 mg/ml L-DOPA (L-3,2-dihydroxyphenylalanine)
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and was measured by microplate reader at O.D 492 nm.
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Phagocytic activity was measured following the method described by [20].
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Briefly, 200μl of 3 × 106 cells/ MCHBSS -ml was deposited in into the 96 microplate
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for 1 hour and 200 μl of 0.8 μm latex beads (1.5 × 107 latex beads/ml, LB8 SIGMA)
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was added to examine the phagocytic activity under microscope observation.
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Phagocytic ratio (PR) = No. of phagocytic cells with engulfed bacteria / No. of
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phagocytes
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Phagocytic index (PI) = No. of engulfed bacteria / Phagocytic cell
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ROS production
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ROS production was detected by DCFH-DA (2,7-dichlorofluorescin diacetate,
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SIGMA) probe. 300 μl of the examined cell was incubated with 300 μl of 100 mM
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DCFH-DA without light in the culture tube for 30 min and the fluorescence of the
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cells from each tube was measured by the flow cytometry, BD FACS Diva software v
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6.1.2.
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Superoxide anion (O2-) production
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The examination protocol was modified by the previous study[21]. 100 μl of 3 ×
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106 cells/PBS-ml diluted haemocytes solution was deposited in microplates and were
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centrifuged with 800 x g for 20 min at 4 °C. After incubation, 100 μl of 1 mg/ml
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zymosan was added to affect haemocyte for 30 min at 26~27 °C. The treatment was
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discarded and the haemocytes were reaction with 100 μl NBT solution (0.3%) for 30
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min at room temperature. The NBT solution was removed and the haemocytes were 6
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fixed with 100% methanol, and washed three times with 100 μl 70% methanol and
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air-dried. The formazan was dissolved by the addition of 120 μl 2 M KOH and 140 μl
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dimethyl sulphoxide (DMSO) and measured by microplate reader at O.D 630 nm.
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Superoxide Dismutase (SOD) Activity The activity of SOD was measured by the RANSOD kit (Randox) and the formula
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was presented as:
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(A2-A1) / 3 = △A / min of standard or sample
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100-(△Astd/min × 100 / △Ast/min or sample min) × 100 % = % inhibition
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Nitric oxide (NO) production NO
production
was
detected
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DAF-FM
(4-amino-5-methylamino-2’,7’-difluorescein, SIGMA) probe. 300 μl of the examined
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cell was incubated with 300 μl of 10 μM DAF-FM without light in the culture tube for
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30 min and the fluorescence of the cells from each tube was measured by the flow
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cytometry, BD FACS Diva software v 6.1.2.
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Total haemocyte count (THC) and classification The 485 μL of prepared haemocytes were incubated with the 15 μl AccuCount
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Fluorescent Particles (Spherotech, SPHEROTM) and analyzed the flow cytometry, BD
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FACS Diva software v 6.1.2.
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Statistical Analysis
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Tukey’s Honestly Significant Difference Test（Tukey’s HSD） and one-way ANOVA
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were used to analyze the statistical significance between treatment and control
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groups.
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P value less than 0.05 considered to be statistically significant. Results were
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presented as means ± SD. 7
ACCEPTED MANUSCRIPT 1 2 3 4 Results
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proPO Detection
Continuous treated white shrimp with different concentrations of minor
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bupleurum decoction for 28 days, the impact of its proPO activity was as shown in
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Table 1. The experimental result was shown that the 0.5 and 1% treatment group,
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the activity of proPO was significantly higher than the control group on day 1 to 14 (p
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< 0.05). 0.25% treatment was presented with significantly increased on day 4 to 14 (p
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< 0.05), however there was no significant difference between each group after the
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21st day observation.
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ROS production
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Treated with different concentrations of minor bupleurum decoction for 28 days,
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the ROS production rate was shown in Table 2. Experimental results show that the
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1% treatment group, the ROS production rate was significantly higher than control
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group on the 2nd day (p < 0.05) moreover, significantly increased than the control and
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0.25% treatment group on the 4th day. The 0.5% treatment group was significantly
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higher than the control group and 0.25% treatment group on the 4th day (p < 0.05),
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however there was no significant difference between each group after the 28th day
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observation.
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O2- production
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Treated with different concentrations of minor bupleurum decoction for 28 days,
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the O2- production rate was shown in Table 3. Experimental results show that the 0.5
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and 1% treatment group, the ROS production rate was significantly higher than 8
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control group on the 2nd to 28th day (p < 0.05). The 0.25% treatment group was
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significantly higher than the control group on the 4th to 28th day (p < 0.05).
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NO production
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The NO production was significantly higher in the 0.5 and 1% treatment groups
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than in the control and 0.25% treatment groups on the 4th day (p < 0.05). The 0.25%
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treatment group was significantly higher than the control group on the 7th day (p <
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0.05). There were significant differences between the control group and the
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treatment groups on the 21st day. On the 28th day, the 0.5% was presented with
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significantly higher than the control (p < 0.05) as presented as Table 4.
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SOD observation
Treatment of different concentration minor bupleurum decoction for 28 days
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and, the SOD activity was as shown in Table 8. Experimental results show that
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treatment of 0.25% minor bupleurum decoction, the SOD activity was significantly (p
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< 0.05) in the 1st day compared to the control and the other treatment groups and,
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0.5% and 1% treatment groups were significantly higher than the control (p < 0.05).
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0.25% treatment was significantly increased from 7th to the 14th days (p < 0.05) than
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the control group but, not significantly different compared to the other treatment
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groups (p > 0.05) as presented as Table 5.
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Total Haemocyte Count (THC)
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The results showed that the THC of 0.5% treatment group was significantly
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higher than the control and 1% treatment group in the 2nd day (p < 0.05) but not
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significant different compared to the 0.25% treatment (p > 0.05). On the 14th day,
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0.25% treatment was significantly different compared to the control (p < 0.05) and 9
ACCEPTED MANUSCRIPT also lower than the 1% treatment (p < 0.05). 1% treatment was significantly different
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to the control and the other treatment group from the 4th to 14th days (p < 0.05) as
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presented as Table 6.
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Differential Haemocyte Count (DHC)
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On the granular cell (GC) observation, the 1% treatment was presented with a
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significant difference compared to the control and the other treatment groups from
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the 14th to the 28th days (p < 0.05) as presented as Table 7. On the hyaline cell (HC)
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observation, 0.5% treatment was with a significant difference to the control and the
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other treatment groups from the 7th and 28th day (p < 0.05) as presented as Table 8.
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On the semi-granular cells (SGC) observation, 0.5% treatment was significantly
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different to the control on the 1st and 7th day (p < 0.05) moreover, 0.5% and 1% were
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significantly different to the control on the 28th day (p < 0.05) as presented as Table
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9.
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Phagocytic rate (PR) and Phagocytic index (PI)
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Treatment of different concentration minor bupleurum decoction for 28 days and,
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the PR and PI activity was as shown in Table 10 and 11. Experimental results show
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that treatment of 0.25% minor bupleurum decoction, the PR activity was significantly
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(p < 0.05) higher compared to the control and the other treatment groups from the
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2nd to the 21st day however there was no significant difference in the 28th day. 0.5%
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and 1% treatment groups were significantly higher than the control (p < 0.05) but not
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in the 14th and 21st days. In the PI observation, 0.5% treatment was significantly
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increased in the 2nd day (p < 0.05) than the control group.
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5 6 7 8 Discussion
Rodriguez and Le Moullac pointed out that PO activity can be used as indicators
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of crustacean immunity ability [22]. Pro-phenoloxidase is known as the initial enzyme
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in the phenoloxidase (PO) producing system and the end of production is the PO
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enzyme in the most of the invertebrate animals [23, 24]. Soderhall et al. illustrated
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that in the key of the defense mechanism in crustaceans, it is using of releasing the
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bactericidal substance which is including of the melanin and the intermediates after
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activating the proPO system [25]. In addition, the proPO system is activated to
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release the peroxinectin, which stimulates and contacts a variety of haemocytes
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involved in the cellular immune response [26]. The proPO system is activated by the
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lipopolysaccharide (LPS) of gram-negative bacteria [25], the composition of the
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fungal or bacterial cell wall [27]. In vitro treating prawn haemocytes with Cyanodon
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dactylon is able to induce the proPO activity [28]. This result shown that treating
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white shrimps with minor bupleurum decoction exactly induce the proPO activity.
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Invertebrate facing to the foreign pathogens is also releasing the oxygen
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substances to defend the infection. Synthesis of the bactericidal ROS as the free
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radicals, including superoxide anion (O2-), hydrogen peroxide, ect., is function to
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oxidize the pathogens. Based on this mechanism, ROS generation is role as the
27
indicator of the crustacean cellular defense mechanism [29, 30]. In this investigation,
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treating of the minor bupleurum decoction is exactly able to induce the ROS
29
synthesis in the short-term observation however, it is not presented with a significant 11
ACCEPTED MANUSCRIPT increase while a long-term treating. In the previous study, Bricknell and Dalmo (2005)
2
pointed out that the long-term continuous use of immune activator to the fish, the
3
immune system may gradually lower the sensitivity to the immune activator to make
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the immune ability with a decline phenomenon after the peak [31]. NO production
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was significantly higher in the treatment of the 0.5 and 1% groups than the control
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and other groups from the 4th day. On the 28th day observation, all of the treatment
7
was significantly increased in the NO production than the control group.
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Balasubramanian et al. investigated that treating the Penaeus monodon with the
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extract of Cyanodon dactylon to observe the NO production. The phenomenon of the
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NO production is increased while treatment of the Cyanodon dactylon [28].
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Compared to our findings, the NO production is exactly activated after the minor
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bupleurum decoction treat. Research indicated that injection of the sodium alginate
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to the common carp is inducing the phagocytosis cell counts however it is declined
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with the elapsed time [32].
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Oncorhynchus mykiss with Zingiber officinale is increasing the activity of the
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phagocytosis with the ROS production [33]. Prevention of the excessive ROS
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production damage the host, the SOD was active and to transfer the harmful O2- into
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the hydrogen peroxide etc. [34]. Therefore, SOD is role as an important antioxidant
19
enzyme, which can be used as an indicator of antioxidant capacity indicator [35]. This
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evidence showed that SOD activity in the low-concentration treatment at the 1st, 7th
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and 14th days were significantly different to the control group, and O2- generated at
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the 1,7,14 days with a high enhance. According to the SOD and O2- generation, it is
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illustrating that the SOD activation is based to the O2- production and using of the
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minor bupleurum decoction is exactly able to enhance the crustacean SOD synthesis
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to prevent the high concentration of ROS. In this experiment, the treatment groups
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of phagocytosis rate were higher than the control group from the 2nd to the 14th day
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than the control group. It is indicating that phagocytosis rate may be increased with
3
the counts of hyaline cell. The proPO activity of each treatment group was higher
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than the control group from the 4th to the 14th day however, the counts of granular
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cell (GC) and semi-granular cell (SGC) were not significantly increased which
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indicated that using of minor bupleurum decoction could promote the proPO activity
7
of shrimp.
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Hyaline cells are typically phagocytic cells [36] and major function with releasing
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the transglutaminase [23]. In the previous research, hyaline cell is lacking of proPO
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activity. A series of reactive oxygen species (ROS) are produced while the hyaline cell
11
is recognizing and phagocytosing the foreign pathogens. The produced superoxide
12
anion (O2-) substances are function of bactericidal activity [37]. In this research, the
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phagocytic rate was with an increase trend in the treating groups. Furthermore,
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contradistinguishing to the quantity of the hyaline cell, it is exact to realize the effect
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of minor bupleurum decoction to increase the white shrimp hyaline cell quantity
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moreover observing the enhanced phagocytic ability. Previous study indicated that
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injection with the latex beads and, the SGC mounted the strongest phagocytic
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response [38]. In this research, treating of 1% minor bupleurum decoction is inducing
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the SGC percentage in the haemocyte component. Treating of the minor bupleurum
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decoction altering the haemocyte component is with the time. Treating of the minor
21
bupleurum decoction from 1st to 7th days, the hyaline cell is exactly increased.
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Furthermore, semi-granular cell and granular cell is exactly increased from 14th to
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28th days. As our findings, we proposed that treating of minor bupleurum decoction,
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the hyaline cell is the early activation then to induce the semi-granular and granular
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cells.
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More, we summarize the recent research which using of the traditional medical 13
ACCEPTED MANUSCRIPT herbs in the aquatic animal to improve the non-specific immune response including
2
of the non-specific immune response as phagocytic activity as presented in Table 12.
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By these evidence, medical herbs are in the regulation of human beings for a long
4
term and has been evidenced with improving the host to induce the non-specific
5
immune response as this finding.
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Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity. Developmental & Comparative Immunology. 2014 44:116-23. 16. Wu Y-S, Chang C-H, Nan F-H. Steroid hormone “cortisone” and “20-hydroxyecdysone” involved in the non-specific immune responses of white
20 21 22 23
shrimp (Litopenaeus vannamei). Fish & shellfish immunology. 2016 56:272-7 17. Gallo C, Schiavon F, Ballarin L. Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda). Fish & shellfish immunology. 2011 31:423-31.
24 25
18. Song Y-L, Yu C-I, Lien T-W, Huang C-C, Lin M-N. Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus.
26 27
Fish & shellfish immunology. 2003 14:317-31. 19. Asokan R, Arumugam M, Mullainadhan P. Activation of prophenoloxidase in the
28 29 30 31 32
plasma and haemocytes of the marine mussel Perna viridis Linnaeus. Developmental & Comparative Immunology. 1997 21:1-12. 20. Chen H, Mai K, Zhang W, Liufu Z, Xu W, Tan B. Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai Ino. Fish & shellfish immunology. 2005 19:241-52.
33 34 35
21. Wu Y-S, Tseng T-Y, Nan F-H. Beta-1, 3-1, 6-glucan modulate the non-specific immune response to enhance the survival in the Vibrio alginolyticus infection of Taiwan abalone (Haliotis diversicolor supertexta). Fish & shellfish immunology. 2016
36 37 38
54:556-63. 22. Rodrıguez J, Le Moullac G. State of the art of immunological tools and health control of penaeid shrimp. Aquaculture. 2000 191:109-19.
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1
15
ACCEPTED MANUSCRIPT 23. Söderhäll K, Cerenius L. Crustacean immunity. Annual Review of Fish Diseases.
2 3 4
1992 2:3-23. 24. Jackson AD, Smith VJ, Peddie CM. In vitro phenoloxidase activity in the blood of Ciona intestinalis and other ascidians. Developmental & Comparative Immunology.
5 6 7 8
1993 17:97-108. 25. Söderhäll K, Aspan A, Duvic B. The pro-PO-system and associated proteins; role in cellular communication in arthropods. Research in immunology. 1990 141:896-907.
9 10 11 12
26. Cerenius L, Jiravanichpaisal P, Liu H-p, Soderhall I. Crustacean immunity. Invertebrate Immunity: Springer; 2010, p. 239-59. 27. Ashida M, Ishizaki Y, Iwahana H. Activation of pro-phenoloxidase by bacterial cell walls or β-1, 3-glucans in plasma of the silkworm, Bombyx mori. Biochemical and
13 14 15
biophysical research communications. 1983 113:562-8. 28. Balasubramanian G, Sarathi M, Venkatesan C, Thomas J, Hameed AS. Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus
16 17 18 19
monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV). Fish & shellfish immunology. 2008 25:820-8. 29. Xian J-A, Zhang X-X, Guo H, Wang D-M, Wang A-L. Cellular responses of the tiger shrimp Penaeus monodon haemocytes after lipopolysaccharide injection. Fish &
20 21 22 23
shellfish immunology. 2016 54:385-90. 30. Wu Y-S, Liau S-Y, Huang C-T, Nan F-H. Beta 1, 3/1, 6-glucan and vitamin C immunostimulate the non-specific immune response of white shrimp (Litopenaeus vannamei). Fish & shellfish immunology. 2016 57:269-77.
24 25
31. Bricknell I, Dalmo RA. The use of immunostimulants in fish larval aquaculture. Fish & shellfish immunology. 2005 19:457-72.
26 27
32. Fujiki K, Yano T. Effects of sodium alginate on the non-specific defence system of the common carp (Cyprinus carpioL.). Fish & shellfish immunology. 1997 7:417-27.
28 29 30 31 32
33. Dügenci SK, Arda N, Candan A. Some medicinal plants as immunostimulant for fish. Journal of Ethnopharmacology. 2003 88:99-106. 34. Holmblad T, Söderhäll K. Cell adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture. 1999 172:111-23. 35. Harris ED. Copper as a Cofactor and Regulator of Copper, Zinc Superoxide
33 34 35
Dismutase1, 2. The Journal of nutrition. 1992 122:636. 36. Johansson MW, Keyser P, Sritunyalucksana K, Söderhäll K. Crustacean haemocytes and haematopoiesis. Aquaculture. 2000 191:45-52.
36 37 38
37. Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Developmental & Comparative Immunology. 1993 17:211-9.
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1
16
ACCEPTED MANUSCRIPT 38. Giulianini PG, Bierti M, Lorenzon S, Battistella S, Ferrero EA. Ultrastructural and
2 3 4
functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge. Micron. 2007 38:49-57.
5 6 7 8
39. Jian J, Wu Z. Influences of traditional Chinese medicine on non-specific immunity of Jian carp (Cyprinus carpio var. Jian). Fish & shellfish immunology. 2004 16:185-91. 40. Rao YV, Das B, Jyotyrmayee P, Chakrabarti R. Effect of Achyranthes aspera on the
9 10 11 12
immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish & shellfish immunology. 2006 20:263-73. 41. Watanuki H, Ota K, Tassakka ACMA, Kato T, Sakai M. Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio. Aquaculture. 2006 258:157-63.
13 14 15
42. Yin G, Jeney G, Racz T, Xu P, Jun X, Jeney Z. Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus. Aquaculture. 2006 253:39-47.
16 17 18 19
43. Sahu S, Das B, Mishra B, Pradhan J, Sarangi N. Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Journal of Applied Ichthyology. 2007 23:80-6. 44. Yin G, Ardó L, Thompson K, Adams A, Jeney Z, Jeney G. Chinese herbs
20 21 22 23
(Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila. Fish & shellfish immunology. 2009 26:140-5. 45. Harikrishnan R, Balasundaram C, Heo M-S. Herbal supplementation diets on
24 25
hematology and innate immunity in goldfish against Aeromonas hydrophila. Fish & shellfish immunology. 2010 28:354-61.
26 27
46. Wu Y-S, Chen Y-Y, Ueng P-S, Nan F-H. Effects of medicinal herbs “Plantago asiatica”,“Houttuynia cordata” and “Mentha haplocalyx” on non-specific immune
28 29 30 31 32
responses of cobia (Rachycentron canadum). Fish & shellfish immunology. 2016 58:406-14. 47. Samad APA, Santoso U, Lee M-C, Nan F-H. Effects of dietary katuk (Sauropus androgynus L. Merr.) on growth, non-specific immune and diseases resistance against Vibrio alginolyticus infection in grouper Epinephelus coioides. Fish & shellfish
33 34 35
immunology. 2014 36:582-9.
AC C
EP
TE D
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1
36 37 38 17
ACCEPTED MANUSCRIPT 1 2 3 4
RI PT
5 6 7 8
SC
9 10 11 12
16 17 18 19
M AN U
13 14 15 Table Legends
Table 1. The prophenoloxidase (proPO) activity (in O.D. 492 nm) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented by mean±S.D.
24 25
Table 2. The reactive oxygen species (ROS) production ratio (in DIFC-DA intensity) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different
26 27
doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different
28 29 30 31 32 33 34
(p<0.05). All values are examined by flow cytometry and represented by mean±S.D. Table 3. The superoxide anion (O2-) production ratio (in O.D. 620 nm) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
35 36 37
Table 4. The nitric oxide (NO) production ratio (in DAF-FM intensity) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of
38
minor bupleurum decoction.
AC C
EP
TE D
20 21 22 23
18
ACCEPTED MANUSCRIPT 1
a~c: mean in the same column with the different latter are significantly different
2 3 4
(p<0.05). All values are examined by flow cytometry and represented mean±S.D.
5 6 7 8
hepaptopancreas of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
9 10 11 12
Table 6. The total haemocyte count (THC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction.
13 14 15
a~c: mean in the same column with the different latter are significantly different (p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
16 17 18 19
Table 7. The granular cell count (GC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction.
M AN U
SC
RI PT
Table 5. The superoxide dismutase (SOD) activity (U/mg protein) in the
a~b: mean in the same column with the different latter are significantly different (p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
24 25
Table 8. The hyaline cell count (HC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of
26 27
minor bupleurum decoction. a~b: mean in the same column with the different latter are significantly different
28 29
(p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
30 31 32 33 34
Table 9. The semi-granular cell count (SGC) of differential haemocyte count (DHC) in the haemocytes of white shrimp (Litopenaeus vannamei), treating with different doses of minor bupleurum decoction. a~b: mean in the same column with the different latter are significantly different
AC C
EP
TE D
20 21 22 23
35 36 37
(p<0.05). All values are examined by flow cytometry and represented mean±S.D. in 106 cell/ml.
38
Table 10. The phagocytic rate (PR: phagocytic cells/total cells) in the haemocytes of 19
ACCEPTED MANUSCRIPT white shrimp (Litopenaeus vannamei), treating with different doses of minor
2 3 4
bupleurum decoction. a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
5 6 7 8
Table 11. The phagocytic index (PI: 0.8 μm latex beads/phagocytic cells) in the haemocytes of white shrimp (Litopenaeus vannamei), treatng with different doses of minor bupleurum decoction.
RI PT
1
a~c: mean in the same column with the different latter are significantly different (p<0.05). Value represented mean±S.D.
13 14 15
Table 12. Effect of traditional medical herbs on the fish immune response.
Table 12 Treatment
Species
M AN U
16 17
SC
9 10 11 12
Time
Parameters
Astragalus radix Angelicae sinensis
Cyprinus carpio 30 (Common Carp) days
EP
28
ROIs ↑
(Labeo)
days
Lysozyme activity ↑
Spirulina platensis
Cyprinus carpio 5 (Common Carp) days
Astragalus radix
Oreochromis niloticus (Tilapia)
Allium sativum
Labeo rohita (Labeo)
Phagocytic activity ↑ ROIs ↑
Lysozyme activity ↑ 4 Phagocytosis ↑ weeks
60 days
Dügenci et al (2003)[33]
ROIs ↑ Complement activity ↑ Jian and Wu (2004)[39] Lysozyme activity ↑
Labeo rohita
AC C
Achyranthes aspera
TE D
Zingiber officinale
Oncorhynchus ROIs ↑ 3 mykiss Phagocytosis ↑ weeks (Rainbow trout)
References
Rao et al (2006)[40] Watanuki et al (2006)[41] Yin et al (2006)[42]
Phagocytic activity ↑ ROIs ↑ Sahu et al (2006)[43] Complement activity ↑ Lysozyme activity ↑
20
ACCEPTED MANUSCRIPT
(1:1:1) Plantago asiatica, Houttuynia cordata, Mentha haplocalyx Sauropus androgynus L. Merr
Carassius auratus (goldfish) Rachycentron canadum (Cobia) Epinephelus coioides (grouper)
Harikrishnan (2010)[45]
Lysozyme activity ↑ 32 days 30 days
O2- ↑ Lysozyme activity↑ Phagocytosis↑ phagocytosis↑ ROIs↑
AC C
EP
TE D
M AN U
1
WBC↑ 4 RB ↑ weeks Phagocytic activity ↑
Yin et al (2009)[44]
RI PT
Azadirachta indica Ocimum sanctum Curcuma longa
RB ↑ Cyprinus carpio 5 Phagocytosis ↑ (Common Carp) weeks Lysozyme activity ↑
Wu et al (2016)[46]
Samad APA et al (2014)[47]
SC
Astragalus radix Ganoderma lucidum
21
ACCEPTED MANUSCRIPT
RI PT
ble 1 Time elapsed (days)
D. 492 nm 0
1
0.042±0.009
0.026±0.001c
2
4
7
14
21
28
M AN U
SC
se (%)
0.016±0.004b 0.070±0.017b 0.022±0.002a
0.016±0.
0.25
0.397±0.075bc 0.092±0.018b 0.081±0.024a
0.204±0.009a 0.210±0.011a 0.049±0.021a
0.025±0.
0.5
0.848±0.200a
0.245±0.088a 0.228±0.043a 0.062±0.008a
0.027±0.
0.276±0.010a 0.288±0.027a 0.101±0.041a
0.027±0.
EP
0.880±0.091a 0.118±0.004a
0.659±0.047ab 0.835±0.040a 0.093±0.004a
AC C
1
TE D
0.025±0.008b 0.015±0.006b
0
ACCEPTED MANUSCRIPT
Time elapsed (days)
Ratio 1
2
4
7
14
21
28
17.66±3.84
22.80±10.47a
17.55±4.17b
17.05±0.07c
13.35±0.35a
25.25±5.44a
26.35±1.06a
20.10±0.42b
0.25
25.55±7.99a
18.30±3.54b
18.40±0.57b
10.40±0.14a
32.25±1.91a
21.85±0.35a
27.95±1.63a
0.5
33.65±5.59a
21.55±1.48b
12.60±1.27a
23.5±3.96a
28.00±8.77a
19.50±0.57b
1
41.65±9.97a
41.80±4.38a
14.50±1.56a
33.25±3.61a
23.75±0.64a
20.5±0.14b
TE D
M AN U
se (%)
SC
0
28.15±0.07a
EP
AC C
0
RI PT
ble 2
27.10±0.14a
ACCEPTED MANUSCRIPT
Ratio
Time elapsed (days) 1
2
4
7
14
21
0.028±0.006
0.060±0.026a
0.059±0.006b
0.022±0.006c
0.024±0.013c
0.039±0.006b
0.031±0.001b
0.030±0.0
25
0.072±0.049a
0.091±0.018b
0.081±0.011b
0.184±0.016b
0.259±0.013b
0.179±0.027a
0.171±0.0
.5
0.427±0.199a
0.221±0.012a
0.419±0.007a
0.670±0.092a 0.118±0.025ab
0.181±0.0
1
0.429±0.081a
0.298±0.042a
0.388±0.039a
0.667±0.073a 0.096±0.023ab
0.159±0.0
TE D
M AN U
e (%)
0.082±0.001b
EP
AC C
SC
0
0
RI PT
ble 3
0.241±0.013a
28
ACCEPTED MANUSCRIPT
RI PT
ble 4
Time elapsed (days) 1
2
4
6.50±2.68
6.18±3.15a
12.78±4.60a
.25
7.53±4.14a
17.84±4.19a
0.5
6.77±2.24a
25.66±12.50a
1
6.78±3.85a
22.90±14.60a
14
21
9.92±2.06b
15.74±10.15b
7.54±2.78a
4.62±0.90b
7.28±2
4.51±1.41b
39.66±9.68a
22.24±3.75a
49.35±4.27a
37.40±
23.91±5.73a
14.11±1.04b
21.45±11.61a
31.34±14.46a
27.83±
21.62±6.21a
20.87±3.34ab
18.07±0.21a
45.40±2.76a
39.92±
TE D
EP
AC C
0
7
M AN U
0
se (%)
SC
Ratio
28
ACCEPTED MANUSCRIPT
U/mg
RI PT
ble 5 Time elapsed (days) 1
2
4
1.474±0.233
1.805±0.183c
1.821±0.507a
3.163±0.724a
0.25
4.546±0.289a
2.474±0.555a
3.716±0.553a
0.5
3.063±0.409b
1.736±0.313a
1
14
21
2.302±0.743b
2.309±0.151b
2.005±0.274a
2.035±0.
3.838±0.649a
3.144±0.286a
2.571±0.395a
2.283±0.
2.862±0.463a 2.885±0.140ab 2.685±0.323ab 3.046±0.886a
3.432±1.
3.168±0.374a 2.655±0.044ab 2.502±0.359ab 2.330±0.485a
2.340±0.
M AN U
TE D
EP
AC C
0
7
28
SC
0
se (%)
2.456±0.171bc 1.966±0.198a
ACCEPTED MANUSCRIPT
cell/ml
Time elapsed (days) 0
1
2
4
7
14
21
28
SC
6
RI PT
ble 6
M AN U
se(%) 16.08±1.82b
19.60±1.56ab
24.52±3.50b
22.01±1.10c
12.37±2.33ab
19.78±1.14
0.25
17.66±6.07a
18.74±2.29ab
14.96±1.88b
31.85±1.97a
21.42±1.93c
11.26±0.59b
20.43±1.74
0.5
9.33±1.63b
21.25±1.26a
17.90±3.98b
24.14±4.02b
28.27±1.96b
13.35±0.45ab
21.69±3.43
1
9.80±0.53b
16.84±1.53b
33.88±2.41a
37.27±2.71a
15.23±2.05a
21.38±0.12
EP
AC C
10.17±1.73
TE D
13.46±4.25ab
0
23.28±2.17a
ACCEPTED MANUSCRIPT
cell/ml
Time elapsed (days) 1
2
4
2.53±0.26
3.20±1.44a
3.90±0.83a
3.30±1.50a
25
2.57±0.97a
2.57±1.22a
2.39±0.59a
.5
2.36±1.08a
3.17±0.65a
1
2.12±0.23a
2.64±1.38a
14
21
4.68±2.33a
3.52±1.75b
2.08±1.79a
1.93±0.
4.97±0.99a
3.25±1.58b
1.52±0.71a
0.88±0.
2.04±0.71a
2.52±0.18a
3.32±0.81b
1.92±0.70a
2.14±0.
2.02±0.09a
3.75±0.27a
7.36±2.82a
3.26±0.72a
3.15±0.
TE D
EP
AC C
0
7
M AN U
0
e (%)
SC
6
RI PT
ble 7
28
ACCEPTED MANUSCRIPT
RI PT
ble 8
06 cell/ml 1
2
4
1.02±0.17
2.31±1.07ab
2.13±1.05a
0.25
3.74±2.37a
1.29±0.59a
0.5
0.91±0.37b
2.37±0.45a
1
0.92±0.18b
2.27±0.63a
14
21
2.13±0.27a
2.91±0.42b
5.74±1.80bc
3.06±1.35ab
4.62±2.9
3.00±1.56a
3.60±0.77b
1.63±0.25c
1.09±0.53b
3.78±1.9
1.65±0.75a
11.85±2.08a
10.93±4.19a
4.60±1.65a
12.90±3.5
3.50±0.80a
5.12±1.61b
8.26±2.37ab
5.59±1.84a
8.64±5.40
TE D
EP
AC C
0
7
M AN U
0
ose(%)
SC
Time elapsed (days)
28
ACCEPTED MANUSCRIPT
06 cell/ml
Time elapsed (days) 1
2
4
14
21
7.64±1.30
13.97±4.99a
11.43±1.44ab
14.17±2.58ab
20.22±6.58a
12.74±1.75a
6.48±2.92a
20.37±
0.25
11.32±6.15ab
8.17±2.05b
11.76±2.55b
27.29±6.90a
20.96±9.30a
7.25±1.99a
15.47±0
0.5
4.32±2.01b
13.58±3.78a
9.23±4.28b
6.99±2.65b
12.93±7.20a
7.41±3.50a
6.65±2
1
6.76±0.28ab
9.70±1.35ab
17.76±1.36a
25.01±0.99a
23.90±3.02a
6.39±2.67a
9.59±4
AC C
EP
TE D
M AN U
ose(%)
7
SC
0
0
RI PT
ble 9
28
ACCEPTED MANUSCRIPT
phagocytic rate
RI PT
ble 10 Time elapsed (days) 1
2
4
7
14
21
15.05±3.87
21.71±7.39b
22.09±3.39c
0.25
25.99±4.14b
0.5 1
28
20.75±3.54b
19.26±4.12b
22.53±6.20b
23.63±3.90c
23.91±
41.55±9.86b
42.11±3.33a
39.53±3.06a
40.33±2.69a
46.86±0.89a
31.35±4
47.15±5.26a
63.63±1.42a
39.64±4.34a
33.61±1.76a
29.59±5.72ab
39.49±5.41ab
37.46±
43.74±0.26a
51.60±1.41ab
37.29±2.82a
33.21±2.78a
32.31±8.02ab
33.74±4.54bc
38.87±
EP
AC C
0
TE D
ose(%)
M AN U
SC
0
ACCEPTED MANUSCRIPT
phagocytic
Time elapsed (days) 1
2
4
1.25±0.43
1.47±0.50
1.15±0.13b
1.23±0.09a
0.25
1.44±0.16a
1.64±0.18ab
1.21±0.30a
0.5
1.95±0.15a
1.87±0.29a
1
1.71±0.01a
1.57±0.20ab
14
21
1.18±0.17a
1.14±0.25a
1.00±0.00a
1.16±0
1.40±0.23a
1.21±0.13a
1.19±0.17a
1.29±0
1.30±0.09a
1.21±0.04a
1.55±0.41a
1.31±0.02a
1.21±0
1.42±0.13a
1.22±0.18a
1.21±0.11a
1.33±0.23a
1.30±0
AC C
EP
TE D
se (%) 0
7
28
SC
0
M AN U
index
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ble 11
ACCEPTED MANUSCRIPT Highlights 1. Minor bupleurum decoction exactly induce the white shrimp non-specific immune response.
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2. Minor bupleurum decoction exactly alter the hyaline cell function and ratio. 3. Minor bupleurum decoction is able to enhance the ROS and NO production.
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4. Minor bupleurum decoction increase the phagocytic rate