Fish & Shellfish Immunology 39 (2014) 326e335
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Full length article
Effects of hot-water extract of banana (Musa acuminata) fruit's peel on the antibacterial activity, and anti-hypothermal stress, immune responses and disease resistance of the giant freshwater prawn, Macrobrachium rosenbegii Wutti Rattanavichai a, Winton Cheng b, * a b
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 22 May 2014 Accepted 26 May 2014 Available online 4 June 2014
The hot-extracts isolated from fruit's peel of banana, Musa acuminata, was evaluated on the antibacterial activity to pathogens from aquatic animals, and immunostimulating potential, disease resistance and anti-hypothermal stress in giant freshwater prawn, Macrobrachium rosenbergii through injection administration. The banana peel extract (BPE) showed good activity against 1 Gram-positive and 3 Gramnegative pathogens, including Lactococcus garvieae, Photobacteria damsella, Vibrio alginolyticus and Vibrio parahemolyticus especially in prawn pathogen of L. garvieae strain, which were carried out by a disk diffusion method. Prawn received BPE via injection administration at 1e6 mg (g prawn)1 significantly increased total haemocyte count (THC), hyaline cell (HC), granular cell (GC), phenoloxidase (PO) activity and phagocytic activity against L. garvieae from 3 to 6 days, and significantly increased clearance efficiency against L. garvieae and a significantly decreased coagulation time of prawn from 1 to 6 days. Prawn injected with BPE at 6.0 mg (g prawn)1 for 6 days showed significantly increased superoxide dismutase (SOD) activity, but significantly decreased respiratory bursts (RBs) of per haemocyte. Survival rates of M. rosenbergii injected with BPE at concentrations of 1, 3 and 6 mg (g prawn)1 were significantly higher than those injected with saline control after challenge with L. garvieae for 4e6 days, and the respective relative survival percentages of prawn were 28.6%, 38.1%, and 47.8%, respectively at 6 days. The sublethal time of prawns that had received saline and BPE at 1, 3 and 6 mg (g prawn)1 for 6 days and then were transferred from 28 C to 14 C were 69.4, 79.8, 83.6, and 90.2 h, respectively. It was concluded that the BPE can be used as the bacteriostat, and immunostimulant and physiological regulator for prawn through injection administration to enhance immunity, physiological responses, and resistance against L. garvieae. © 2014 Published by Elsevier Ltd.
Keywords: Macrobachium rosenbergii Immunostimulant Banana peel extract Musa acuminata Antibacterial activity
1. Introduction The giant freshwater prawn, Macrobrachium rosenbergii, is a commercially important cultured species in Asia. Commercial prawn farming has been severely adversely impacted by epidemics associated with yeasts in the cool season [1] and bacteria in the hot season [2] in Taiwan, which have caused serious economic losses. It is known that the rapid degradation of environments in intensive culture ponds may elevate the increased incidences of diseases that
* Corresponding author. E-mail address: [email protected] (W. Cheng). http://dx.doi.org/10.1016/j.fsi.2014.05.031 1050-4648/© 2014 Published by Elsevier Ltd.
can lead to culture failure. Disease outbreaks result from interactions among the environment, hosts, and pathogens. Therefore, maintaining the health of prawns and enhancing their immunity are primary concerns. The use of antibiotic medications has been successfully applied to mitigate losses from diseases in hatchery- and pond-cultured shrimp. However, the excessive use and misuse of antibiotics have resulted in antibiotic residuals in food and the spread of antibiotic-resistant pathogens in the aquatic environment [3], which threaten the safety and hygiene of food, and control of diseases in humans. Consequently, the use of antibiotics in aquaculture has been banned in many countries. To sustain the development of the shrimp-culture industry, shrimp farmers have
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
begun to examine the potential use of immunostimulants as a more environmentally friendly approach to disease management [4]. The immune stimulatory effects of immunostimulants like glucan, chitosan, and other polysaccharides have been widely studied in fish and crustaceans and were reviewed by Sakai [5] and Ringø et al. [6]. The effect of plant products on innate and adaptive immune response and to prevent and control fish and shellfish diseases was reviewed by Harikrishnan et al. [7]. Dietary administration of polysaccharide gell (from the fruit-rind of Durio zibethinus) significantly increased the immune response and the resistance of Penaeus monodon against Vibrio harveyi and WSSV infection [8]. Administration of Withania somnifera and Eichhornia crassipes extracts through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against Aeromonas hydrophila and Lactococcus garvieae infection, respectively [9,10]. Litopenaeus vannamei that are injected with hot-water extract of Sargassum duplicatum and Gelidium amansii or the shrimp are immersed in hot-water extract have increased immune ability as well as resistance to Vibrio alginolyticus infection [11,12]. Banana is the second largest produced fruit after citrus, contributing about 17% of the world's total fruit production, and is cultured over 130 countries, along the tropics and subtropic of Capricorn [13]. The waste peel of banana or plantains poses the problem of disposal without causing environmental pollution in the countries of the processing industries of banana and plantains [14]. However, Banana peel is a rich source of starch (3%), crude protein (6e9%), crude fat (3.8e11%), total dietary fibre (43.2e49.7%) and vitamins, which could be used as feed for livestock and poultry [15]. Dietary fibre which is the polysaccharides mainly consists of soluble and insoluble fractions such as lignin, pectin, cellulose and hemicellulose are an extremely diverse set of biopolymers in banana peels [16]. Soluble fibres are well known to lower serum cholesterol and to help reduce the risk of colon cancer [17,18]. Additionally, flavonoids, tannins, phlobatannins, alkaloids, glycosides and terpenoids were found to be present in the peels of genus Musa. These phytochemicals have been reported to exert multiple biological and pharmacological effects (antibacterial, antihypertensive, antidiabetic and anti-inflammatory activities) [19]. The presence of these bioactive substances in banana peels therefore suggests that the peels possess valuable medicinal potential and immunostimulant. The immunostimulant can be used by injected, immersed and oral administration. The aim of this study was to examine the immune responses of giant freshwater prawn M. rosenbergii, and its resistance against L. garvieae and the tolerance to hypothermia following injected with hot-water extract of banana, M. acuminata, peel (BPE). The immune parameters of prawns injected with hotwater extract were examined, including the total haemocyte count (THC), different haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity and haemolymph coagulation time, and the phagocytic activity and clearance efficiency of prawn to L. garvieae. In addition, the antibacterial activity was evaluated in BPE against pathogens of abalone and shrimp. 2. Materials and methods 2.1. Preparation of the banana peel extract Banana fruits, M. acuminata, of all green were obtained from the Nepui market, Pingtung, Taiwan. Banana fruits were drench with soda water for 5 min, washed with tap water, rinsed twice with distilled water to remove the contaminant, and then pass normal ripeness until 7 of peel color index (PCI) [20]. At 7th PCI, the flavour
327
and taste of the banana evenly coloured yellow with brown speckles are popular for consumers, so that the peel is obtained easily. The peel was collected and air-dried in an oven at 50 C. The dried peel was ground into a powder with a mortar and pestle. A known weight of powdered samples was extracted in hot water. Briefly, milled peel (100 g) was extracted in hot water (1.0 g in 10 ml distilled water at 90 C). The mixture was mechanically stirred for 5 min. After extraction, the extract was filtered, and the solution was centrifuged at 104 g for 10 min of three times. The supernatant containing the water-soluble extracts was lyophilized using a freeze-dryer (Eyela, FDU-1100, Tokyo, Japan). The extract was composed of crude protein (7.71 ± 0.3%), crude lipids (0.46 ± 0.03%), moisture content (14.45 ± 0.18%), ash (27.21 ± 0.11%) and total carbohydrate 50.17 ± 0.15%, and was stored at 20 C until being used. 2.2. Antibacterial activity of extract from banana peel Four pathogens including Taiwan abalone, Haliotis diversicolor supertexta pathogens: Vibrio parahaemolyticus [21], white shrimp, L. vannamei pathogens: V. alginolyticus [22], tiger shrimp, P. monodon pathogen Photobacteria damsala [23] and giant freshwater prawn, M. rosenbergii pathogen L. garvieae [2] were used in this study (Table 1). The strains of L. garvieae were cultured at 28 ± 0.5 C on tryptic soy agar (TSA, Difco) without supplement of NaCl and the other strains were cultured at 28 ± 0.5 C on TSA medium with 2.0% NaCl. L. garvieae was cultured on tryptic soy agar (TSA, Difco) for 24 h at 28 C before being transferred to 10 ml of tryptic soy broth (TSB, Difco), where it remained for 24 h at 28 C before being centrifuged at 7155 g for 15 min at 4 C. The supernatants were removed, and the bacterial pellets were re-suspended in a saline solution (0.85% NaCl) at 2.0 107 colony-forming units (cfu) ml1 as a stock bacterial suspension for the susceptibility study and at 2.0 108 cfu ml1 for studies of phagocytic activity and clearance efficiency. A disk diffusion method was used to assay the pathogens bactericidal activity of the extract of banana peel. The BPE was dissolved in sterile saline (0.85%) to the concentrations of 100, 50, 25, 12.5, 6.25, 3.13 or 1.56 mg ml1, respectively, before assay. All bacteria were suspended respectively in sterile saline and were diluted to ~107 cfu ml1. The suspension (100 ml) was spread on the medium along the BBL™ blank paper disc (8 mm diameter, Becton, Dickinson and Company, Spark, MD21152, USA) containing 20 ml different concentrations of BPE resulting in 2000, 1000, 500, 250, 125, 62.5 and 31.25 mg disc1. Blank paper disc without, or with saline was served as control. The inoculated plates were incubated at 28 ± 0.5 C for 24 h. Antibacterial activity was evaluated by measuring the distance of inhibition zone of the tested bacteria. Distance of inhibition zone [(Diameter of inhibition zone Disc diameter)/2] was expressed in millimetres. 2.3. Experimental design Prawns, M. rosenbergii were obtained from a commercial farm in Pingtung, Taiwan, shipped to National Pingtung University of Science and Technology (Aquatic Animal Physiology and Immunology Lab), and acclimated at room temperature (28 ± 1 C) and pH 7.1e7.6 in the laboratory for 2 weeks before experimentation. Only prawns in the intermoult stage were used for the study. The moult stage was determined under a stereomicroscope according to retraction of the epithelium within the setal base interface of the antennal scale [24]. During the acclimation period, prawns were fed the commercial prawn diet (Shinta Feed Company, Pingtung, Taiwan) twice daily.
328
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
Table 1 Inhibition zone (cm) of aquatic animal pathogens by hot-water extract of banana peel. Aquatic animal species
Macrobrachium rosenbergii Penaeus monodon Litopenaeus vannamei Haliotis diversicolor supertexta
Pathogens
Disc only
Lactococcous garvieae Photobacteria damsela Vibrio alginolyticus Vibrio parahemolyticus
e e e e
Saline
e e e e
Hot-water extract of banana peel at different concentration (mg disc1) 2000
1000
500
250
125
62.5
31.25
4.57 4.78 4.18 5.82
3.78 4.67 3.74 3.78
2.91 3.61 3.17 2.66
1.87 3.07 1.12 2.01
1.46 1.68 e 1.04
0.89 0.63 e e
0.16 e e e
Inhibition zone were express ¼ (diameter of inhibition zone diameter disc)/2 and disc diameter is 8 mm.
Four studies were conducted. For the susceptibility experiment, test and control groups were comprised of 10 prawns each, and tests were conducted in triplicate. The same experimental design was carried out for the hypothermal (14 C) stress test. To determine THC, DHC, PO activity, RBs, SOD activity, and haemolymph coagulation time, tests and controls were carried out in six replicates. Twelve 180 l glass aquaria containing 100 l of aerated freshwater were used for this study. Each aquarium was used to rear 10 prawns. Groups with four different dosages of BPE (0, 1.0, 3.0, and 6.0 mg (g prawn)1) were established, with each group consisting of three aquaria. Two prawns were randomly sampled from each aquarium after 0, 1, 3, and 6 days of the BPE or saline injection. For studies of phagocytic activity and clearance efficiency, another ten prawns were used in each of the test and control groups. No significant difference in weight was observed among treatments. During the experiments, prawns were fed twice daily with a formulated prawn diet, and the water temperature was maintained at 28 ± 1.0 C and the pH at 7.1e7.6. 2.4. Effect of the BPE on the susceptibility of M. rosenbergii to L. garvieae The PBE was dissolved in sterile saline (0.85% NaCl) to concentrations of 0.5, 1.5 and 3 mg ml1, before injection. M. rosenbergii (15 ± 1.6 g) was injected individually with 0.5, 1.5 and 3 mg ml1 BPE solution (around 80 ml) into the ventral sinus of the cephalothorax to reach doses of 1, 3 and 6 mg (g prawn)1, respectively. Challenge tests were conducted after 6 days with the injection of 10 ml of bacterial suspension (2.0 107) resulting in 2 105 cfu prawn1 into the ventral sinus of the cephalothorax. The prawn that received 80 ml saline, and then received L. garvieae at 2 105 cfu prawn1 served as the challenged controls. The prawn that received saline (80 ml), and then received saline (20 ml), however, served as the unchallenged controls (Table 2). Experimental and control prawns (10 aquarium1) were kept in 180 l glass aquaria containing 100 l of aerated freshwater. Therefore, there were a total of five treatments. Each treatment was conducted with 30 prawns. The experiment lasted 144 h. The relative percentage survival (RPS) of prawn was calculated at the end of the experiment according to Amend [25] using the following formula:
h RPS ¼ 1 ðpercentage mortality in treatmentÞ i ðpercentage mortality in controlÞ1 100:
2.5. Effect of the BPE on the tolerance of M. rosenbergii to hypothermal stress M. rosenbergii (16 ± 1.5 g) that received saline or BPE was the same as those described above. For the hypothermal stress assay, the prawn that received saline and BPE after 6 days, and then were transferred to 14 C from 28 C. The prawn that received saline, and then held at 28 C served as the non-hypothermal stress control. The mortality was counted daily during the experimental period of 96 h. There were five treatments with 30 prawns each. Each treatment was conducted in triplicates, and each replicate had 10 prawns. The sublethal time of prawns was calculated by nonlinear sigmoid regression (time mortality) with the aid of the SPSS statistical software version 10 (SPSS, Inc, Chicago). 2.6. Effects of the BPE on the immune parameters of M. rosenbergii Immune parameters of prawns (35.0 ± 2.1 g) that injected with the BPE at 0, 1.0, 3.0, and 6.0 mg (g prawn)1 were determined at the beginning and after 1, 3, and 6 days of injection. Two prawns from each aquarium were sampled and analysed at each sample time. Six prawns for each treatment and time were used for these studies. In total, 156 prawns were used in this study (78 prawns for examination of THC, DHC, PO activity, RBs, SOD activity, and haemolymph coagulation time, and 78 prawns for phagocytic activity and clearance efficiency). Haemolymph (570 ml) was withdrawn from the ventral sinus of each prawn and divided into three parts. One hundred microliters of haemolymph was placed into 1-ml sterile Eppendorf tubes containing 0.9 ml anticoagulant buffer (0.8 g sodium citrate, 0.34 g EDTA, and 10 ml Tween 80 in 100 ml of distilled water, at pH 7.45 with the osmolality adjusted to 490 mOsm kg1 with NaCl) for the THC, DHC, PO activity, and RB assays. A drop of the anticoagulant-
Table 2 The survival rate and relative percentage survival (RPS) of Macrobrachium rosenbergii challenged with Lactococcus garvieae, when the prawns were injected with different dosage of hot-water extract of banana peel after 6 days. Bacterial dose (cfu prawn1)
Banana peel extract mg (g prawn)1
No. of prawns
Survival (%), time after challenge (h) 24
48
72
96
144
Saline 2 105 2 105 2 105 2 105
Control Saline 1 3 6
30 30 30 30 30
100 76.7 ± 3.3c 83.3 ± 3.3bc 90.0 ± 5.8ab 100.0 ± 0.0a
100 76.7 ± 3.3c 83.3 ± 5.8bc 90.0 ± 3.3ab 100.0 ± 0.0a
100 46.7 ± 3.3c 60.00 ± 5.8b 63.70 ± 3.3b 80.00 ± 0.0a
100 33.3 53.3 56.7 66.7
100 30.0 50.0 56.7 63.3
± ± ± ±
3.3b 8.8a 6.7a 3.3a
Data in the same column with different letters are significantly differ (p < 0.05) among treatments. Values are mean ± S.E. (n ¼ 30 prawns in each). *Values in parentheses are relative percentage survival (RPS).
± ± ± ±
0.0b 5.8a (28.6)* 6.7a (38.1)* 3.3a (47.8)*
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
haemolymph mixture was placed on a haemocytometer to measure the THC and DHC (Leica DMIL, Leica Microsystems, Wetzlar, Germany). The remainder of the haemolymph mixture was used for PO activity and RB assays. Two hundred microliters of haemolymph was added to an Eppendorf tube containing 0.4 ml of anticoagulant buffer for the SOD activity assays. The remainder of the haemolymph (270 ml) was used for the coagulation test. The PO activity of haemocytes was measured spectrophotometrically at 490 nm by recording the formation of dopachrome produced from L-3,4-dihydroxyphenylalanine (L-DOPA, D-9628, Sigma), following a method modified from Mason [26] and pez et al. [27]. The details of measurements were Hern andez-Lo described previously [10]. The optical density (OD) was measured at 490 nm, and the PO activity was expressed as dopachrome formation in 50 ml of haemolymph or per 106 GCs. RBs of haemocytes were quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O 2 ) formation as described previously [28,29]. The OD at 630 nm was measured using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RBs were expressed as NBT reduction in 10 ml of haemolymph or per 107 haemocytes. The haemocytes lysate supernatant (HLS) was prepared as described previously [10]. The SOD activity of HLS was measured by its ability to inhibit superoxide radical-dependent reactions using a Ransod kit (Randox, Crumlin, UK). Details of the measurement were described previously [30]. The OD was measured at 505 nm and 37 C, and the rate of the reaction was estimated from the absorbance readings at 0.5 and 3 min after adding xanthine oxidase. A reference standard for SOD was supplied with the Ransod kit. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units (mg protein)1 [31]. The concentration of protein in the HLS was quantified by the method described by Bradford [32] using a Bio-Rad Protein Assay Kit (no. 500-0006, Bio-Rad Laboratories, Richmond, CA, USA) with bovine serum albumin (BSA) as the standard. Measurement of the haemolymph coagulation time was modified from methods of Tsai et al. [33] and Jussila et al. [34]. Briefly, 270 ml of haemolymph was withdrawn from the ventral sinus, mixed with 30 ml of 20 mM CaCl2 in Eppendorf tubes, and continuously turned upside down in slow motion every 5 s. The motion was repeated until the haemolymph had coagulated, and the time was recorded. 2.7. Phagocytic activity and clearance efficiency of prawns injected with BPE Prawns were injected and sampled as described above. For the phagocytic activity and clearance efficiency tests, 10 ml of a bacterial suspension (2.0 108 cfu ml1) resulting in 2 106 cfu prawn1 was injected into the ventral sinus. After the injection, prawns were held in separate tanks containing 40 l of water at 28 ± 0.5 C for 2 h. Then, 200 ml of haemolymph was collected from the ventral sinus and mixed with 200 ml of a sterile anticoagulant solution. This mixture was then divided into two equal subsamples to respectively evaluate the phagocytic activity and clearance efficiency. Methods for measuring the phagocytic activity and clearance efficiency were described previously [35,36]. Two hundred haemocytes were counted, and the rate of phagocytosis (PR) was calculated as follows:
i h PR ¼ ðphagocytic haemocytesÞðtotal haemocytesÞ1 100:
329
The clearance efficiency, defined as the percentage inhibition (PI) of L. garvieae was calculated as:
i h PI ¼ 100 ðcfu in the test groupÞðcfu in the control groupÞ1 100:
2.8. Statistical analysis A multiple-comparisons (Tukey's) test was conducted to compare significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Before analysis, percentage data were normalized using an arc-sine transformation. Statistically significant differences required that p < 0.05. 3. Results 3.1. Antibacterial activity of BPE The inhibition zone of BPE and saline were presented in Table 1. The BPE showed good activity against all organisms in the test (1 gram-positive and 3 gram-negative pathogen). Of the four strains, L. garvieae strain was more sensitive to BPE, followed by P. damsella and V. parahemolyticus. V. alginolyticus was more resistant to BPE. The lowest dosages of inhibition against L. garvieae, P. damsella, V. parahemolyticus and V. alginolyticus were 31.25, 62.5, 125.0 and 250 mg disc1, respectively. 3.2. Effect of BPE on the resistance of prawn to L. garvieae infection All the unchallenged control prawns that received saline and then injected with saline survived. For the challenge test, survival rates of prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 72 to 144 h. After 144 h of challenge, survival rates of prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 were 20.0%, 26.7.0% and 33.3% higher, respectively, than that of prawns received saline, and the respective relative survival percentages of prawns were 28.6%, 38.1% and 47.8% (Table 2). 3.3. Effect of BPE on the tolerance of hypothermal stress All the non-hypothermal stress control prawns that received saline after 6 days, and then held at 28 C survived. After 96 h of hypothermal (14 C) stress, survival rates of prawns that received BPE at 6.0 mg (g prawn)1 was significantly higher than those of prawns received 1.0 and 3.0 mg (g prawn)1, which were significantly higher than that of prawns received saline control. The sublethal time of prawns that had received saline and BPE at 1, 3 and 6 mg (g prawn)1 for 6 days and then was transferred to 14 C were 69.4, 79.8, 83.6, and 90.2 h, respectively (Table 3). 3.4. Immune parameters of prawns injected with the BPE The THC, HC and GC of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline control from 3 to 6 days. For the prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 after 6 days, the THC, HC, and GC increased by 31.8%, 35.6% and 35.6%; 20.2%, 29.9% and 60.3%; and 57.7%, 66.0% and 67.3%, respectively, compared to prawns that received saline. However, no significant differences were observed in the THC, HC and GCs among prawns that received the BPE at 1.0e6.0 mg (g prawn)1 after 3e6 days (Fig. 1AeC).
330
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
Table 3 The survival rate and sublethal time of Macrobrachium rosenbergii subjected to hypothermal (14 C) stress, when the prawns were injected with different dosage of hot-water extract of banana peel after 6 days. Temperature ( C)
28 14 14 14 14
Banana peel extract mg (g prawn)1
Control Saline 1 3 6
No. of prawns
Survival (%), time after challenge (h)
30 30 30 30 30
24
48
72
100 96.7 ± 3.3a 96.7 ± 3.3a 100 ± 0.0a 100.0 ± 0.0a
100 73.3 ± 3.3b 80.0 ± 5.8ab 83.3 ± 6.7ab 96.7.0 ± 3.3a
100 36.7 53.3 63.3 66.7
96 ± ± ± ±
3.3b 8.8ab 3.3a 6.7a
100 0.0 ± 0.0c (69.4)* 3.3 ± 3.3b (79.8)* 30.0 ± 5.8b (83.6)* 46.7 ± 6.7a (90.18)*
Data in the same column with different letters are significantly differ (p < 0.05) among treatments. Values are mean ± S.E. (n ¼ 6 prawns in each). *Values in parentheses are sublethal time (h).
For the prawns that received BPE at 6.0 mg (g prawn)1 after 1 day, the PO activity was significantly higher than those of prawns received BPE at 1.0 and 3.0 mg (g prawn)1, and saline. PO activity of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were
saline
1
150
3 ab ab
a
a a
6
a
a a a
b 100
A
Banana peel extract (μg (g prawn)-1)
200
THC (x 105 cell -1)
significantly higher than those of prawns received saline from 3 to 6 days. After 6 days of injection, PO activity of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 increased by 69.0%, 72.4% and 72.4%, compared to prawn that received saline. However, no
a
a a
b
b
a
50 0 0
1
3
6
Time elapsed (days) Banana peel extract (μg (g prawn)-1) saline
HC (x 105 cell -1)
120
1
3
100 a
80 60
a
a
a a
a
B
6
a
a
a a a
a
a b
a
b
40 20 0 0
1
3
6
Time elapsed (days)
GC+SGC (x 105 cell -1)
C
Banana peel extract (μg (g prawn)-1)
120
saline
1
3
6 a
90 60
a
a
a a
a
a
a
a a a
b ab
a
b
b 30 0 0
1
3
6
Time elapsed (days) Fig. 1. (A) Total haemocyte count (THC), (B) hyaline cell (HC) and (C) granular cell (granular cell þ seimigranular cell, GC þ SGC) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Each bar represents the mean value from 6 samples with the standard error. Bars with different letters significantly differ (p < 0.05) among treatments.
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
activities of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 had increased by 17.6%, 19.5% and 31.8%, respectively, compared to the saline control (Fig. 5A). Clearance efficiencies that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 1 to 6 days. After 6 days, clearance efficiencies of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 had increased by 153.5%, 152.4% and 161.0%, respectively, as compared to the saline control (Fig. 5B).
significant differences in PO activity were observed among prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 at 3 and 6 days sampling times (Fig. 2A). The same tendency was observed in the PO activity of per GCs (Fig. 2B). RBs of prawn that received BPE at 1.0e6.0 mg (g prawn)1 were significantly higher than that of the saline control at 3 days. However, no significant difference in RBs was observed among four treatments at sampling times of 1 and 6 days (Fig. 3A). RBs per haemocyte of prawns that received BPE at 3.0 and 6.0 mg (g prawn)1 were significantly lower than that of the saline control after 6 days of injection, and had decreased by 16.2%, and 16.1% as compared to prawns that received saline (Fig. 3B). SOD activity of haemocytes of prawns that received BPE at 6.0 mg (g prawn)1 was significantly higher than those of prawns received saline after 6 days. For the prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 after 6 days, the SOD activity increased by 32.0%, 44.0%, and, 62.0%, compared to prawns that received saline control (Fig. 3C). Haemolymph coagulation times of prawns that received BPE at the levels of 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly shorter than those of the saline control for 1e3 days. After 6 days, haemolymph coagulation times of prawns that received BPE at 3.0, and 6.0 mg (g prawn)1 were significantly shorter than those of the saline control, and had significantly decreased by 20.4% and 24.7%, respectively, compared to the saline control prawns (Fig. 4).
4. Discussion Musa is genus from Zingiberales and family Musaceae; it includes bananas and plantains. Effects of the antibacterial activity have been investigated for the extracts of banana peels. Ighodaro [37] demonstrated that the aqueous extract of Musa paradisiaca give an antibacterial effect against Staphylococcus aureus, Escherichia coli and Proteus mirabilis more than antifungal. The aqueous and acetone extracts of three varieties of bananas namely the green banana, M. paradisiaca, yellow banana, Musa Cavendish and red banana, Robusta fruit's peel possess antibacterial and antifungal activities against the skin and gastrointestinal tract diseases causing organisms [38]. Chabuck et al. [19] indicated that the aqueous extract of banana peel (Musa sapientum) exhibit a good antibacterial effect against Gram-positive bacteria of Staphylococcus aureus and Staphylococcus pyogenes, and Gram-negative bacteria of Moraxella catarrhalis, Enterobacter aerogenes and Klebsiella pneumonia. The same antibacterial activity was observed in the hotwater extracts of M. acuminata fruit's peel against the pathogens of aquatic animals, including Gram-positive of L. garvieae and Gram-negative of P. damsella, V. alginolyticus and V. parahemolyticus in the present study.
3.5. Phagocytic activity and clearance efficiency of prawns injected with the BPE
PO activity (O.D. 490 nm)
Phagocytic activities of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 3 to 6 days. After 6 days, phagocytic
A
Banana peel extract (μg (g prawn)-1)
0.8
saline
1
3 a
6
0.6
a
b
b b
a
a a
a a
b
0.4 0.2
331
a
a
a b
a
0 0
1
3
6
PO activity (O.D. 490 nm) per 107 GC+SGC
Time elapsed (days)
B
Banana peel extract (μg (g prawn)-1)
0.6
saline
1
3
6
a 0.4 ab b a a
a
a
b
0.2 a
ab
a
a c
a
b
a
0 0
1
3
6
Time elapsed (days) Fig. 2. (A) Phenoloxidase (PO) activity and (B) PO activity at 105 granular cell (semigranular cell and granular cell, SGC þ GC) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
332
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
RB (O.D. 630 nm)
saline
0.2 0.15
A
Banana peel extract (μg (g prawn)-1)
0.25
1 a a
a a
a a
3
6
a a
a
a
a
b
a
a
a
a
0.1 0.05 0 0
1
3
6
RB (O.D. 630 nm) per 107 THC
Time elapsed (days)
0.2
B
Banana peel extract (μg (g prawn)-1)
0.25
saline a
a
a
a
1
3
6
a a
a
a a
0.15
a a
a
a
ab b
b
0.1 0.05 0 0
1
3
6
Time elapsed (days)
SOD activity (units mg protein-1)
Banana peel extract (μg (g prawn)-1) saline
6
1
3 a
5
C
6 ab
a
4
a
a
a
a
a
ab
a
a
b
3 2 1
a
a a
a
0 0
1
3
6
Time elapsed (days) Fig. 3. (A) Respiratory burst (RB), (B) RB at 107 haemocytes and (C) Superoxide dismutase activity (SOD) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
Several studies examined the use of immunostimulants in aquatic animals to enhance immunity and resistance to pathogens [5,6]. Administration of plant extracts or their products through an oral (diet) or injection route enhanced the innate and adaptive immune response of different freshwater and marine fish and shellfish against bacterial, viral, and parasitic diseases [7]. Injection administration of stevia extracts enhances the innate immune response and resistance of tiger shrimp P. monodon to V. harveyi [39]. Litopenaeus vannamei that injected with the hot-water extracts from Gracilaria tenuistipitata [40], S. duplicatum [11] and G. amansii [12] exhibited enhanced resistance to V. alginolyticus infection. In the present study, injection BPE administration at 1.0e6.0 mg (g prawn)1 for 6 days significantly increased survival rates of the prawn M. rosenbergii against L. garvieae with a dosedependent manner, suggesting that BPE can enhance prawns disease resistance via injection administration. The optimum metabolic temperature range for M. rosenbergii is between 26 and 32 C [41]. Rogers and Fast [42] reported that
prawns were stressed by temperature below 22 C. Yeh et al. (2010) [43] reported that L. vannamei immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited a protective effect against at temperature change as evidenced by the earlier recovery of immune parameters especially in total haemocyte counts. Liu et al. (2010) [44] indicated that M. rosenbergii fed the diets containing Rheum officinale Bail significantly decreased the mortality under high temperature stress, and these were the consequences of the enhanced physiological responses including total haemolymph protein, energy metabolites, etc. The haemolymph protein content is used as an immune parameter indicating re et al. 1997) [45]. whether the prawn is healthy or not (Bache Furthermore, the injection of hot-water extract of G. tenuistipitata was reported to increase the haemocyte number of L. vannamei after 24 h (Yeh and Chen, 2009) [46]. The similar phenomena can be observed in the present study. Prawn injected with BPE significantly increased THC, and the increased survival rate after hypothermal stress was revealed thereafter. Prawns received BPE at
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
333
Banana peel extract (μg (g prawn)-1) saline
Coagulation time (seconds)
350 300
a a
a
1
3
a
250 200
6
a a b bc c
150
a b
b
b
ab
b
b
100 50 0 0
1
3
6
Time elapsed (days) Fig. 4. Coagulation time of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
S3 mg (g prawn)1 for 6 days showed significantly increased survival rate when the prawns transferred to lethally cold water (14 C) after 96 h. These facts suggested that the proliferation of haemocyte and the maturation of haemocyte precursors in haematopoietic tissue may cause earlier recovery of immune responses, and meanwhile, the enhanced haemolymph protein occurred to improve stress responses. Further tests would be conducted on the variation of haemolymph protein levels in M. rosenbergii injected with BPE to clarify the hypothesis. White shrimp L. vannamei that received extracts of G. tenuistipitata [40], S. duplicatum [11] and G. amansii [12] via an injected route showed increased THCs, PO activity, RBs and SOD activity together with increased resistance against L. garvieae challenge. Hyaline cells are young, immature haemocytes of both
large- and small-GC lines that are produced in haematopoietic tissues (HPTs), and can be released into the haemolymph in P. monodon [47]. GCs of the large-GC line mature and accumulate in connective tissues and are easily released into the haemolymph [47]. Litopenaeus vannamei which was fed G. tenuistipitata extractcontaining diets for 14 days showed increased THCs, especially HCs and GCs together with increases in mitotic cells and the mitotic index of HPTs which supported the proliferation of haemocytes [48]. In the present study, THCs, HCs, and GCs significantly increased in prawns injected with BPE at 1.0e3.0 mg (g prawn)1 for 3e6 days. The facts suggest that BPE via injection administration can induce proliferation of haemocytes in HPTs and promote mobilization of mature GCs, which result in an increase in THC of prawns.
Fig. 5. (A) Phagocytotic activity and (B) clearance efficiency of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
334
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
PO is the terminal enzyme of the prophenoloxidase (proPO) system and acts as both a recognition and effector component to promote cell-to-cell communication and subsequently eliminate pathogens [49]. proPO is synthesized and localized in granules, and is released into the plasma by exocytosis which is triggered by the binding of foreign polysaccharides and pattern-recognition proteins (PRPs) [50]. In the present study, the M. rosenbergii that received BPE at 1.0e6.0 mg (g prawn)1 for 3e6 days through injection increased the PO activity and PO activity per GCs. An increase of PO activity for prawns that received BPE may be a consequence of increased GC and its proPO synthesis. Reactive oxygen intermediates (ROIs) are released during RBs of phagocytosis, which represent a defence mechanism against microbial infection [28]. In a normal physiological state, harmful effects of ROIs are effectively neutralized by the antioxidant defence system of organisms, which in general comprises enzymes like SODs, and small antioxidant molecules like ascorbate and sugars. Therefore, the regulation of the ROI system is usually considered a defence mechanism in living systems. In the present study, RBs slightly increased, and SOD activity significantly increased in prawns injected with BPE at 1e6 mg (g prawn)1 for 6 days, indicating that the BPE may promote regulation of the ROI system in prawn. Injection administration BPE at 3e6 mg (g prawn)1 for 6 days significantly decreased O 2 production of single haemocytes, but maintained or slightly increased O 2 production of prawns. The negative regulation per haemocyte maintains homeostasis and prevents disturbance to immunity by restoring immune parameters [48]. However, the unbiased O 2 production of prawns is related with an increase in the THC, and the decreased O 2 production of single haemocytes may be resulting from increased SOD and/or rich antioxidants in BPE. Coagulation of the haemolymph is part of the innate immune response in crustaceans; it prevents leakage of haemolymph and helps protect against dissemination of invaders such as bacteria throughout the body. In shrimp, coagulation is initiated by the release of haemocyte transglutaminases (TGs) which effectively polymerizes the haemolymph clotting protein (CP) to form a stabilized gel [51]. Lightner and Lewis [52] observed a reduction in haemocyte numbers and prolongation of coagulation in Vibrioinfected shrimp. TG-I messenger (m)RNA expression levels and clotting times of haemolymph of the tiger shrimp, P. monodon, fed a diet containing 2.0 g kg1 sodium alginate significantly decreased, but clotting protein and TG-II mRNA expression increased [53]. Chang et al. [10] indicated that M. rosenbergii fed diets containing the extract of water hyacinth (E. crassipes) at 2.0 and 3.0 g kg1 for 12 days significantly increased the TG activity and decreased the clotting time, suggesting acceleration of haemolymph coagulation of prawns is resulting from an increase in TG activities and/or the concentration of clotting proteins. In the present study, prawns injected with BPE at 1.0e6.0 mg (g prawn)1 significantly increased THC and TG activities, and decreased haemolymph clotting times, suggesting that acceleration of haemolymph coagulation of prawns that received BPE is resulting from an increase in TG activities accompanied with THC. However, further research is necessary to determine the TG activity and CP concentration of prawn injected with BPE. Phagocytosis is an important cellular defence mechanism, whereas the clearance efficiency is an important humoral defence mechanism in crustaceans [49]. Clearance from the circulation is induced by humoral factors such as agglutinins, lectins, cytotoxic factors [54], and antimicrobial factors [55], which cause bacterial aggregation in the circulating haemolymph, and enhance the clearance rate [47]. Both phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus increased significantly, and
these levels correlated well with the resistance to V. alginolyticus when the shrimp received hot-water extracts of G. tenuistipitata and S. duplicatum by injection administration [11,40]. In the present study, the phagocytic activity and clearance efficiency of M. rosenbergii to L. garvieae increased, and these correlated well with the resistance against L. garvieae when the prawns were injected with BPE at 1e6 mg (g prawn)1. Banana peel is rich in phytochemical compounds with antioxidant, antibacterial, antihypertensive, antidiabetic and antiinflammatory effects. The lectin from banana peel has been characterized with inhibited HIV replication through interaction with its GP120 coat glycoprotein [56]. Lectins are powerful oral and parenteral immunogens. The primary effects and the potency of lectins as biological signal are the direct result of their specific chemical reactivity with saccharides. As these reactions are predictable, the use of lectins as blockers of pathogens, immune stimulants, hormone modulators and metabolic agents in clinicalmedical applications [57]. Emaga et al. [16] indicated that banana peels are the potential source of dietary fibres and pectins (pectic polysaccharides), and the galacturonic acid and methoxy group contents. Polysaccharide gel from the D. zibethinus is a pectic polysaccharide with inhibited growth of V. harveyi, and increased the immunity and resistance of P. monodon against V. harveyi and WSSV infection through dietary administration [8]. In the present study, the BPE inhibited growth of pathogens from aquatic animals and strengthened anti-hypothermal stress as well as enhanced immune response and resistance of M. rosenbergii to L. garvieae via injection administration, suggesting that BPE is not only an immunostimulant but also physiological regulator and bacteriostat. Further research is needed to evaluate anti-environmental stress ability and immune resistance activity when M. rosenbergii are fed a diet containing BPE. In conclusion, the present study showed that hot-water extract of M. acuminata fruit's peel has a growth-inhibiting effect in vitro against pathogens from aquatic animals. In addition, the giant freshwater prawn M. rosenbergii injected with hot-water extracts of M. acuminata fruit's peel at 1e6 mg (g prawn)1 for 6 days showed increased non-specific immune responses (including reduced clotting times of haemolymph, and increased THC, HC, GC, PO activity, SOD activity, and phagocytic activity and clearance efficiency against L. garvieae) together with increased resistance to L. garvieae infection, and enhanced anti-hypothermal stress ability. Acknowledgement This work was financially supported by a grant (NSC102-2622B-020-004-CC2) from the National Science Council, Taiwan. Special thanks to the budgets support for Wutti Rattanavichai from Rajamangala University of Technology Isan of Thailand to pursue further education in NPUST, Taiwan, ROC. References [1] Hsu JP, Liu CI. Studies on yeast infection in cultured giant freshwater prawn (Macrobrachium rosenbergii). Fish Dis Res 1994;15:55e68. [2] Cheng W, Chen JC. Isolation and characterization of Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan. Dis Aquat Org 1998;34:93e101. [3] Sahul Hameed AS, Rahaman KH, Alagan A, Yoganandhan K. Antibiotic resistance in bacteria isolated from hatchery-reared larvae and post-larvae of Macrobrachium rosenbergii. Aquaculture 2003;217:39e48. [4] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection? Fish Shellfish Immunol 2003;15:71e90. [5] Sakai M. Current research status of fish immunostimulants. Aquaculture 1999;172:63e92. [6] Ringø E, Olsen RE, Vecino JLG, Wadsworth S, Song SK. Use of immunostimulants and nucleotides in aquaculture: a review. J Mar Sci Res Dev 2012;2: 1e22.
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335 [7] Harikrishnan R, Balasundaram C, Heo MS. Impact of plant products on innate and adaptive immune system of cultured finfish and shellfish. Aquaculture 2011;317:1e15. [8] Pholdaeng K, Pongsamart S. Studies on the immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus monodon shrimp against Vibrio harveyi and WSSV. Fish Shellfish Immunol 2010;28: 555e61. [9] Harikrishnan R, Balasundaram C, Jawahar S, Heo MS. Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila. Fish Shellfish Immunol 2012;32:94e100. [10] Chang CC, Tan HC, Cheng W. Effects of dietary administration of water hyacinth (Eichhornia crassipes) extracts on the immune responses and disease resistance of giant freshwater prawn, Macrobrachium rosenbergii. Fish Shellfish Immunol 2013;35:92e100. [11] Yeh ST, Lee CS, Chen JC. Administration of hot-water extract of brown seaweed Sargassum duplicatum via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2006;20:332e45. [12] Fu YW, Hou WY, Yeh ST, Li CH, Chen JC. The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol 2007;22:673e85. [13] FAO (Food and Agriculture Organization, Geneva) 2013: FAOSTAT, http:// faostat.fao.org/site/291/default.aspx. [14] Zhang P, Whistler RL, BeMiller JN, Hamaker BR. Banana starch: production, physicochemical properties, and digestibility e a review. Carbohyd Polym 2005;59:443e58. [15] Mohapatra D, Mishra S, Sutar N. Banana and its by-product ultilisation: an overview. J Sci Ind Res 2010;69:323e9. [16] Emaga TH, Robert C, Ronkart SN, Wathelet B, Paquot M. Dietary fibre components and pectin chemical features of peels during ripening in banana and plantain varieties. Biores Technol 2008;99:4346e54. [17] Burkitt DPJ, Walker ARP, Painter NJ. Dietary fibre and disease. J Am Med Assoc 1974;229:1068e77. [18] Kelsey JL. A review of research on effect of fiber intake on man. Am J Clin Nutr 1978;31:142e59. [19] Chabuck ZAG, Al-Charrakh AH, Hindi NKK, Hindi SKK. Antimicrobial effect of aqueous banana peel extract, Iraq. Res Gate Pharm Sci 2013;1:73e5. [20] Soltani M, Alimardani R, Omid M. Prediction of banana quality during ripening stage using capacitance sensing system. Aust J Crop Sci 2010;4:443e7. [21] Liu PC, Chen YC, Huang CY, Lee KK. Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome. Lett Appl Microbiol 2000;31:433e7. [22] Liu CH, Cheng W, Hsu JP, Chen JC. Vibrio alginolyticus infection in the white shrimp (Litopenaeus vannamei) confirmed by polymerase chain reaction and 16S rDNA sequencing. Dis Aquat Organ 2004;61:169e74. [23] Song YL, Cheng W, Wang CH. Isolation and characterization of Vibrio damsela infectious for cultured shrimp in Taiwan. J Invertebr Pathol 1993;61:24e31. [24] Peebles JB. A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture 1977;12:173e80. [25] Amend DF. Potency testing of fish vaccines. International symposium on fish biologics: serodiagnostics and vaccines. Dev Biol Stand 1981;49:447e54. [26] Mason HS. Structures and functions of the phenolase complex. Nature 1956;177:79e81. ndez-Lo pez J, Gollas-Galv [27] Herna an TS, Vargas-Albores F. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comp Biochem Physiol 1996;113C:61e6. [28] Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Dev Comp Immunol 1993;17:211e9. [29] Song YL, Hsieh YT. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Dev Comp Immunol 1994;18:201e9. [30] Liu KF, Yeh MS, Kou GH, Cheng W, Lo CF. Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages. Dev Comp Immunol 2010;34:935e44. [31] Biagini G, Sala D, Zini I. Diethyldithiocarbamate, a superoxide dismutase inhibitor, counteracts the maturation of ischemic-like lesions caused by endothelin-1 intrastriatal injection. Neurosci Lett 1995;90:212e6. [32] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [33] Tsai IH, Chen YL, Lu PJ. Relationship between haemolymph-coagulation and disease in shrimp: 1. Coagulation rate in vitro and the haemolymph proteins of various shrimp species. COA Fish Ser 1991;29:22e30.
335
[34] Jussila J, McBride S, Jago J, Evans LH. Haemolymph clotting time as an indicator of stress in western rock lobster (Panulirus cygnus George). Aquaculture 2001;199:185e93. [35] Li JT, Lee PP, Chen OC, Cheng W, Kuo CM. Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii. Fish Shellfish Immunol 2005;19: 269e80. [36] Adams A. Response of penaeid shrimps to exposure to Vibrio species. Fish Shellfish Immunol 1991;1:59e70. [37] Ighodaro OM. Evaluation study on Nigerian species of Musa paradisiaca peels: phytochemical screening, proximate analysis, mineral composition and antimicrobial activities. Researcher 2012;4:17e20. [38] Sumathy NH, Sumathy JH. Antibacterial and antifungal activity of Musa fruit peels against skin and gastrointestinal tract diseases. Herb Tech Ind 2011;1: 9e11. ~ o EM, Xi YC, Liao IC. Effects of stevia extract on growth, non-specific [39] Lean immune response and disease resistance of grass prawn, Penaeus monodon (Fabricius), Juveniles. J Fish Soc Taiwan 2007;34:165e75. [40] Hou WY, Chen JC. The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol 2005;19: 127e38. [41] Boyd C, Zimmermann S. Grow-out systems-water quality and soil management. In: New MB, Valenti WC, Tidwell JH, D'Abramo LR, Kutty MN, editors. Freshwater prawns biology and farming. Oxford: Blackwell Science; 2010. pp. 239e55. [42] Rogers GL, Fast AW. Potential benefits of low energy water circulation in Hawaiian prawn ponds. Aqua Eng 1988;7:155e65. [43] Yeh ST, Li CC, Tsui WC, Lin YC, Chen JC. The protective immunity of white shrimp Litopenaeus vannamei that had been immersed in the hot-water extract of Gracilaria tenuistipitata and subjected to combined stresses of Vibrio alginolyticus injection and temperature change. Fish Shellfish Immunol 2010;29:271e8. [44] Liu B, Ge X, He Y, Xie J, Xu P, He Y, et al. Effects of anthraquinones extracted from Rheum officinale Bail on the growth, non-specific immune response of Macrobrachium rosenbergii. Aquaculture 2010;310:13e9. re E, Mialhe E, Rodriguez J. Identification of defence effectors in the [45] Bache haemolymph of Crustaceans with particular reference to the shrimp Penaeus japonicus (Bate): prospects and applications. Fish Shellfish Immunol 1997;5: 597e612. [46] Yeh ST, Chen JC. White shrimp Litopenaeus vannamei that received the hotwater extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection. Fish Shellfish Immunol 2009;5: 724e30. [47] Van de Braak CBT, Botterblom MHA, Liu W, Taverne N, Van de Knaap WPW, Rombout JHWM. The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp (Penaeus monodon). Fish Shellfish Immunol 2002;12:253e72. [48] Sirirustananun N, Chen JC, Lin YC, Yeh ST, Liou CH, Chen LL, et al. Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2011;31:848e55. [49] Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP. Invertebrate immunity: basic concepts and recent advances. Int Rev Cytol 1985;97:183e350. €derha €ll K. Early events in crustacean innate immunity. Fish Shellfish [50] Lee SY, So Immunol 2002;12:421e37. [51] Yeh MS, Kao LR, Huang CJ, Tsai IH. Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus. Biochim Biophys Acta 2006;1764: 1167e78. [52] Lightner DV, Lewis DH. A septicemic bacterial disease syndrome of penaeid shrimp. Mar Fish Rev 1975;17:25e8. [53] Chung MY, Franklin SMT, Lee PP, Cheng W. Shrimp immune responses and disease management by using seaweed extracts: a review. J Int Coop 2011;6: 109e38. [54] Smith VJ, Chisholm JRS. Non-cellular immunity in crustaceans. Fish Shellfish Immunol 1992;2:1e31. [55] Smith VJ. Invertebrate immunology: phylogenetic, ecotoxicological and biomedical implication. Comp Haematol Int 1991;1:60e76. [56] Swanson MD, Winter HG, Goldstein IJ, Markovitz DM. A lectin isolated from banana is a potent inhibitor of HIV replication. J Biol Chem 2010;285: 8646e55. [57] Pusztai A, Bardocz S. Biological effects of plant lectins on the gastrointestinal tract: metabolic consequences and applications. Trends Glycosci Glycotechnol 1996;8:149e65.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Full length article
Effects of hot-water extract of banana (Musa acuminata) fruit's peel on the antibacterial activity, and anti-hypothermal stress, immune responses and disease resistance of the giant freshwater prawn, Macrobrachium rosenbegii Wutti Rattanavichai a, Winton Cheng b, * a b
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 22 May 2014 Accepted 26 May 2014 Available online 4 June 2014
The hot-extracts isolated from fruit's peel of banana, Musa acuminata, was evaluated on the antibacterial activity to pathogens from aquatic animals, and immunostimulating potential, disease resistance and anti-hypothermal stress in giant freshwater prawn, Macrobrachium rosenbergii through injection administration. The banana peel extract (BPE) showed good activity against 1 Gram-positive and 3 Gramnegative pathogens, including Lactococcus garvieae, Photobacteria damsella, Vibrio alginolyticus and Vibrio parahemolyticus especially in prawn pathogen of L. garvieae strain, which were carried out by a disk diffusion method. Prawn received BPE via injection administration at 1e6 mg (g prawn)1 significantly increased total haemocyte count (THC), hyaline cell (HC), granular cell (GC), phenoloxidase (PO) activity and phagocytic activity against L. garvieae from 3 to 6 days, and significantly increased clearance efficiency against L. garvieae and a significantly decreased coagulation time of prawn from 1 to 6 days. Prawn injected with BPE at 6.0 mg (g prawn)1 for 6 days showed significantly increased superoxide dismutase (SOD) activity, but significantly decreased respiratory bursts (RBs) of per haemocyte. Survival rates of M. rosenbergii injected with BPE at concentrations of 1, 3 and 6 mg (g prawn)1 were significantly higher than those injected with saline control after challenge with L. garvieae for 4e6 days, and the respective relative survival percentages of prawn were 28.6%, 38.1%, and 47.8%, respectively at 6 days. The sublethal time of prawns that had received saline and BPE at 1, 3 and 6 mg (g prawn)1 for 6 days and then were transferred from 28 C to 14 C were 69.4, 79.8, 83.6, and 90.2 h, respectively. It was concluded that the BPE can be used as the bacteriostat, and immunostimulant and physiological regulator for prawn through injection administration to enhance immunity, physiological responses, and resistance against L. garvieae. © 2014 Published by Elsevier Ltd.
Keywords: Macrobachium rosenbergii Immunostimulant Banana peel extract Musa acuminata Antibacterial activity
1. Introduction The giant freshwater prawn, Macrobrachium rosenbergii, is a commercially important cultured species in Asia. Commercial prawn farming has been severely adversely impacted by epidemics associated with yeasts in the cool season [1] and bacteria in the hot season [2] in Taiwan, which have caused serious economic losses. It is known that the rapid degradation of environments in intensive culture ponds may elevate the increased incidences of diseases that
* Corresponding author. E-mail address: [email protected] (W. Cheng). http://dx.doi.org/10.1016/j.fsi.2014.05.031 1050-4648/© 2014 Published by Elsevier Ltd.
can lead to culture failure. Disease outbreaks result from interactions among the environment, hosts, and pathogens. Therefore, maintaining the health of prawns and enhancing their immunity are primary concerns. The use of antibiotic medications has been successfully applied to mitigate losses from diseases in hatchery- and pond-cultured shrimp. However, the excessive use and misuse of antibiotics have resulted in antibiotic residuals in food and the spread of antibiotic-resistant pathogens in the aquatic environment [3], which threaten the safety and hygiene of food, and control of diseases in humans. Consequently, the use of antibiotics in aquaculture has been banned in many countries. To sustain the development of the shrimp-culture industry, shrimp farmers have
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
begun to examine the potential use of immunostimulants as a more environmentally friendly approach to disease management [4]. The immune stimulatory effects of immunostimulants like glucan, chitosan, and other polysaccharides have been widely studied in fish and crustaceans and were reviewed by Sakai [5] and Ringø et al. [6]. The effect of plant products on innate and adaptive immune response and to prevent and control fish and shellfish diseases was reviewed by Harikrishnan et al. [7]. Dietary administration of polysaccharide gell (from the fruit-rind of Durio zibethinus) significantly increased the immune response and the resistance of Penaeus monodon against Vibrio harveyi and WSSV infection [8]. Administration of Withania somnifera and Eichhornia crassipes extracts through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against Aeromonas hydrophila and Lactococcus garvieae infection, respectively [9,10]. Litopenaeus vannamei that are injected with hot-water extract of Sargassum duplicatum and Gelidium amansii or the shrimp are immersed in hot-water extract have increased immune ability as well as resistance to Vibrio alginolyticus infection [11,12]. Banana is the second largest produced fruit after citrus, contributing about 17% of the world's total fruit production, and is cultured over 130 countries, along the tropics and subtropic of Capricorn [13]. The waste peel of banana or plantains poses the problem of disposal without causing environmental pollution in the countries of the processing industries of banana and plantains [14]. However, Banana peel is a rich source of starch (3%), crude protein (6e9%), crude fat (3.8e11%), total dietary fibre (43.2e49.7%) and vitamins, which could be used as feed for livestock and poultry [15]. Dietary fibre which is the polysaccharides mainly consists of soluble and insoluble fractions such as lignin, pectin, cellulose and hemicellulose are an extremely diverse set of biopolymers in banana peels [16]. Soluble fibres are well known to lower serum cholesterol and to help reduce the risk of colon cancer [17,18]. Additionally, flavonoids, tannins, phlobatannins, alkaloids, glycosides and terpenoids were found to be present in the peels of genus Musa. These phytochemicals have been reported to exert multiple biological and pharmacological effects (antibacterial, antihypertensive, antidiabetic and anti-inflammatory activities) [19]. The presence of these bioactive substances in banana peels therefore suggests that the peels possess valuable medicinal potential and immunostimulant. The immunostimulant can be used by injected, immersed and oral administration. The aim of this study was to examine the immune responses of giant freshwater prawn M. rosenbergii, and its resistance against L. garvieae and the tolerance to hypothermia following injected with hot-water extract of banana, M. acuminata, peel (BPE). The immune parameters of prawns injected with hotwater extract were examined, including the total haemocyte count (THC), different haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity and haemolymph coagulation time, and the phagocytic activity and clearance efficiency of prawn to L. garvieae. In addition, the antibacterial activity was evaluated in BPE against pathogens of abalone and shrimp. 2. Materials and methods 2.1. Preparation of the banana peel extract Banana fruits, M. acuminata, of all green were obtained from the Nepui market, Pingtung, Taiwan. Banana fruits were drench with soda water for 5 min, washed with tap water, rinsed twice with distilled water to remove the contaminant, and then pass normal ripeness until 7 of peel color index (PCI) [20]. At 7th PCI, the flavour
327
and taste of the banana evenly coloured yellow with brown speckles are popular for consumers, so that the peel is obtained easily. The peel was collected and air-dried in an oven at 50 C. The dried peel was ground into a powder with a mortar and pestle. A known weight of powdered samples was extracted in hot water. Briefly, milled peel (100 g) was extracted in hot water (1.0 g in 10 ml distilled water at 90 C). The mixture was mechanically stirred for 5 min. After extraction, the extract was filtered, and the solution was centrifuged at 104 g for 10 min of three times. The supernatant containing the water-soluble extracts was lyophilized using a freeze-dryer (Eyela, FDU-1100, Tokyo, Japan). The extract was composed of crude protein (7.71 ± 0.3%), crude lipids (0.46 ± 0.03%), moisture content (14.45 ± 0.18%), ash (27.21 ± 0.11%) and total carbohydrate 50.17 ± 0.15%, and was stored at 20 C until being used. 2.2. Antibacterial activity of extract from banana peel Four pathogens including Taiwan abalone, Haliotis diversicolor supertexta pathogens: Vibrio parahaemolyticus [21], white shrimp, L. vannamei pathogens: V. alginolyticus [22], tiger shrimp, P. monodon pathogen Photobacteria damsala [23] and giant freshwater prawn, M. rosenbergii pathogen L. garvieae [2] were used in this study (Table 1). The strains of L. garvieae were cultured at 28 ± 0.5 C on tryptic soy agar (TSA, Difco) without supplement of NaCl and the other strains were cultured at 28 ± 0.5 C on TSA medium with 2.0% NaCl. L. garvieae was cultured on tryptic soy agar (TSA, Difco) for 24 h at 28 C before being transferred to 10 ml of tryptic soy broth (TSB, Difco), where it remained for 24 h at 28 C before being centrifuged at 7155 g for 15 min at 4 C. The supernatants were removed, and the bacterial pellets were re-suspended in a saline solution (0.85% NaCl) at 2.0 107 colony-forming units (cfu) ml1 as a stock bacterial suspension for the susceptibility study and at 2.0 108 cfu ml1 for studies of phagocytic activity and clearance efficiency. A disk diffusion method was used to assay the pathogens bactericidal activity of the extract of banana peel. The BPE was dissolved in sterile saline (0.85%) to the concentrations of 100, 50, 25, 12.5, 6.25, 3.13 or 1.56 mg ml1, respectively, before assay. All bacteria were suspended respectively in sterile saline and were diluted to ~107 cfu ml1. The suspension (100 ml) was spread on the medium along the BBL™ blank paper disc (8 mm diameter, Becton, Dickinson and Company, Spark, MD21152, USA) containing 20 ml different concentrations of BPE resulting in 2000, 1000, 500, 250, 125, 62.5 and 31.25 mg disc1. Blank paper disc without, or with saline was served as control. The inoculated plates were incubated at 28 ± 0.5 C for 24 h. Antibacterial activity was evaluated by measuring the distance of inhibition zone of the tested bacteria. Distance of inhibition zone [(Diameter of inhibition zone Disc diameter)/2] was expressed in millimetres. 2.3. Experimental design Prawns, M. rosenbergii were obtained from a commercial farm in Pingtung, Taiwan, shipped to National Pingtung University of Science and Technology (Aquatic Animal Physiology and Immunology Lab), and acclimated at room temperature (28 ± 1 C) and pH 7.1e7.6 in the laboratory for 2 weeks before experimentation. Only prawns in the intermoult stage were used for the study. The moult stage was determined under a stereomicroscope according to retraction of the epithelium within the setal base interface of the antennal scale [24]. During the acclimation period, prawns were fed the commercial prawn diet (Shinta Feed Company, Pingtung, Taiwan) twice daily.
328
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
Table 1 Inhibition zone (cm) of aquatic animal pathogens by hot-water extract of banana peel. Aquatic animal species
Macrobrachium rosenbergii Penaeus monodon Litopenaeus vannamei Haliotis diversicolor supertexta
Pathogens
Disc only
Lactococcous garvieae Photobacteria damsela Vibrio alginolyticus Vibrio parahemolyticus
e e e e
Saline
e e e e
Hot-water extract of banana peel at different concentration (mg disc1) 2000
1000
500
250
125
62.5
31.25
4.57 4.78 4.18 5.82
3.78 4.67 3.74 3.78
2.91 3.61 3.17 2.66
1.87 3.07 1.12 2.01
1.46 1.68 e 1.04
0.89 0.63 e e
0.16 e e e
Inhibition zone were express ¼ (diameter of inhibition zone diameter disc)/2 and disc diameter is 8 mm.
Four studies were conducted. For the susceptibility experiment, test and control groups were comprised of 10 prawns each, and tests were conducted in triplicate. The same experimental design was carried out for the hypothermal (14 C) stress test. To determine THC, DHC, PO activity, RBs, SOD activity, and haemolymph coagulation time, tests and controls were carried out in six replicates. Twelve 180 l glass aquaria containing 100 l of aerated freshwater were used for this study. Each aquarium was used to rear 10 prawns. Groups with four different dosages of BPE (0, 1.0, 3.0, and 6.0 mg (g prawn)1) were established, with each group consisting of three aquaria. Two prawns were randomly sampled from each aquarium after 0, 1, 3, and 6 days of the BPE or saline injection. For studies of phagocytic activity and clearance efficiency, another ten prawns were used in each of the test and control groups. No significant difference in weight was observed among treatments. During the experiments, prawns were fed twice daily with a formulated prawn diet, and the water temperature was maintained at 28 ± 1.0 C and the pH at 7.1e7.6. 2.4. Effect of the BPE on the susceptibility of M. rosenbergii to L. garvieae The PBE was dissolved in sterile saline (0.85% NaCl) to concentrations of 0.5, 1.5 and 3 mg ml1, before injection. M. rosenbergii (15 ± 1.6 g) was injected individually with 0.5, 1.5 and 3 mg ml1 BPE solution (around 80 ml) into the ventral sinus of the cephalothorax to reach doses of 1, 3 and 6 mg (g prawn)1, respectively. Challenge tests were conducted after 6 days with the injection of 10 ml of bacterial suspension (2.0 107) resulting in 2 105 cfu prawn1 into the ventral sinus of the cephalothorax. The prawn that received 80 ml saline, and then received L. garvieae at 2 105 cfu prawn1 served as the challenged controls. The prawn that received saline (80 ml), and then received saline (20 ml), however, served as the unchallenged controls (Table 2). Experimental and control prawns (10 aquarium1) were kept in 180 l glass aquaria containing 100 l of aerated freshwater. Therefore, there were a total of five treatments. Each treatment was conducted with 30 prawns. The experiment lasted 144 h. The relative percentage survival (RPS) of prawn was calculated at the end of the experiment according to Amend [25] using the following formula:
h RPS ¼ 1 ðpercentage mortality in treatmentÞ i ðpercentage mortality in controlÞ1 100:
2.5. Effect of the BPE on the tolerance of M. rosenbergii to hypothermal stress M. rosenbergii (16 ± 1.5 g) that received saline or BPE was the same as those described above. For the hypothermal stress assay, the prawn that received saline and BPE after 6 days, and then were transferred to 14 C from 28 C. The prawn that received saline, and then held at 28 C served as the non-hypothermal stress control. The mortality was counted daily during the experimental period of 96 h. There were five treatments with 30 prawns each. Each treatment was conducted in triplicates, and each replicate had 10 prawns. The sublethal time of prawns was calculated by nonlinear sigmoid regression (time mortality) with the aid of the SPSS statistical software version 10 (SPSS, Inc, Chicago). 2.6. Effects of the BPE on the immune parameters of M. rosenbergii Immune parameters of prawns (35.0 ± 2.1 g) that injected with the BPE at 0, 1.0, 3.0, and 6.0 mg (g prawn)1 were determined at the beginning and after 1, 3, and 6 days of injection. Two prawns from each aquarium were sampled and analysed at each sample time. Six prawns for each treatment and time were used for these studies. In total, 156 prawns were used in this study (78 prawns for examination of THC, DHC, PO activity, RBs, SOD activity, and haemolymph coagulation time, and 78 prawns for phagocytic activity and clearance efficiency). Haemolymph (570 ml) was withdrawn from the ventral sinus of each prawn and divided into three parts. One hundred microliters of haemolymph was placed into 1-ml sterile Eppendorf tubes containing 0.9 ml anticoagulant buffer (0.8 g sodium citrate, 0.34 g EDTA, and 10 ml Tween 80 in 100 ml of distilled water, at pH 7.45 with the osmolality adjusted to 490 mOsm kg1 with NaCl) for the THC, DHC, PO activity, and RB assays. A drop of the anticoagulant-
Table 2 The survival rate and relative percentage survival (RPS) of Macrobrachium rosenbergii challenged with Lactococcus garvieae, when the prawns were injected with different dosage of hot-water extract of banana peel after 6 days. Bacterial dose (cfu prawn1)
Banana peel extract mg (g prawn)1
No. of prawns
Survival (%), time after challenge (h) 24
48
72
96
144
Saline 2 105 2 105 2 105 2 105
Control Saline 1 3 6
30 30 30 30 30
100 76.7 ± 3.3c 83.3 ± 3.3bc 90.0 ± 5.8ab 100.0 ± 0.0a
100 76.7 ± 3.3c 83.3 ± 5.8bc 90.0 ± 3.3ab 100.0 ± 0.0a
100 46.7 ± 3.3c 60.00 ± 5.8b 63.70 ± 3.3b 80.00 ± 0.0a
100 33.3 53.3 56.7 66.7
100 30.0 50.0 56.7 63.3
± ± ± ±
3.3b 8.8a 6.7a 3.3a
Data in the same column with different letters are significantly differ (p < 0.05) among treatments. Values are mean ± S.E. (n ¼ 30 prawns in each). *Values in parentheses are relative percentage survival (RPS).
± ± ± ±
0.0b 5.8a (28.6)* 6.7a (38.1)* 3.3a (47.8)*
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
haemolymph mixture was placed on a haemocytometer to measure the THC and DHC (Leica DMIL, Leica Microsystems, Wetzlar, Germany). The remainder of the haemolymph mixture was used for PO activity and RB assays. Two hundred microliters of haemolymph was added to an Eppendorf tube containing 0.4 ml of anticoagulant buffer for the SOD activity assays. The remainder of the haemolymph (270 ml) was used for the coagulation test. The PO activity of haemocytes was measured spectrophotometrically at 490 nm by recording the formation of dopachrome produced from L-3,4-dihydroxyphenylalanine (L-DOPA, D-9628, Sigma), following a method modified from Mason [26] and pez et al. [27]. The details of measurements were Hern andez-Lo described previously [10]. The optical density (OD) was measured at 490 nm, and the PO activity was expressed as dopachrome formation in 50 ml of haemolymph or per 106 GCs. RBs of haemocytes were quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O 2 ) formation as described previously [28,29]. The OD at 630 nm was measured using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RBs were expressed as NBT reduction in 10 ml of haemolymph or per 107 haemocytes. The haemocytes lysate supernatant (HLS) was prepared as described previously [10]. The SOD activity of HLS was measured by its ability to inhibit superoxide radical-dependent reactions using a Ransod kit (Randox, Crumlin, UK). Details of the measurement were described previously [30]. The OD was measured at 505 nm and 37 C, and the rate of the reaction was estimated from the absorbance readings at 0.5 and 3 min after adding xanthine oxidase. A reference standard for SOD was supplied with the Ransod kit. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units (mg protein)1 [31]. The concentration of protein in the HLS was quantified by the method described by Bradford [32] using a Bio-Rad Protein Assay Kit (no. 500-0006, Bio-Rad Laboratories, Richmond, CA, USA) with bovine serum albumin (BSA) as the standard. Measurement of the haemolymph coagulation time was modified from methods of Tsai et al. [33] and Jussila et al. [34]. Briefly, 270 ml of haemolymph was withdrawn from the ventral sinus, mixed with 30 ml of 20 mM CaCl2 in Eppendorf tubes, and continuously turned upside down in slow motion every 5 s. The motion was repeated until the haemolymph had coagulated, and the time was recorded. 2.7. Phagocytic activity and clearance efficiency of prawns injected with BPE Prawns were injected and sampled as described above. For the phagocytic activity and clearance efficiency tests, 10 ml of a bacterial suspension (2.0 108 cfu ml1) resulting in 2 106 cfu prawn1 was injected into the ventral sinus. After the injection, prawns were held in separate tanks containing 40 l of water at 28 ± 0.5 C for 2 h. Then, 200 ml of haemolymph was collected from the ventral sinus and mixed with 200 ml of a sterile anticoagulant solution. This mixture was then divided into two equal subsamples to respectively evaluate the phagocytic activity and clearance efficiency. Methods for measuring the phagocytic activity and clearance efficiency were described previously [35,36]. Two hundred haemocytes were counted, and the rate of phagocytosis (PR) was calculated as follows:
i h PR ¼ ðphagocytic haemocytesÞðtotal haemocytesÞ1 100:
329
The clearance efficiency, defined as the percentage inhibition (PI) of L. garvieae was calculated as:
i h PI ¼ 100 ðcfu in the test groupÞðcfu in the control groupÞ1 100:
2.8. Statistical analysis A multiple-comparisons (Tukey's) test was conducted to compare significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Before analysis, percentage data were normalized using an arc-sine transformation. Statistically significant differences required that p < 0.05. 3. Results 3.1. Antibacterial activity of BPE The inhibition zone of BPE and saline were presented in Table 1. The BPE showed good activity against all organisms in the test (1 gram-positive and 3 gram-negative pathogen). Of the four strains, L. garvieae strain was more sensitive to BPE, followed by P. damsella and V. parahemolyticus. V. alginolyticus was more resistant to BPE. The lowest dosages of inhibition against L. garvieae, P. damsella, V. parahemolyticus and V. alginolyticus were 31.25, 62.5, 125.0 and 250 mg disc1, respectively. 3.2. Effect of BPE on the resistance of prawn to L. garvieae infection All the unchallenged control prawns that received saline and then injected with saline survived. For the challenge test, survival rates of prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 72 to 144 h. After 144 h of challenge, survival rates of prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 were 20.0%, 26.7.0% and 33.3% higher, respectively, than that of prawns received saline, and the respective relative survival percentages of prawns were 28.6%, 38.1% and 47.8% (Table 2). 3.3. Effect of BPE on the tolerance of hypothermal stress All the non-hypothermal stress control prawns that received saline after 6 days, and then held at 28 C survived. After 96 h of hypothermal (14 C) stress, survival rates of prawns that received BPE at 6.0 mg (g prawn)1 was significantly higher than those of prawns received 1.0 and 3.0 mg (g prawn)1, which were significantly higher than that of prawns received saline control. The sublethal time of prawns that had received saline and BPE at 1, 3 and 6 mg (g prawn)1 for 6 days and then was transferred to 14 C were 69.4, 79.8, 83.6, and 90.2 h, respectively (Table 3). 3.4. Immune parameters of prawns injected with the BPE The THC, HC and GC of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline control from 3 to 6 days. For the prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 after 6 days, the THC, HC, and GC increased by 31.8%, 35.6% and 35.6%; 20.2%, 29.9% and 60.3%; and 57.7%, 66.0% and 67.3%, respectively, compared to prawns that received saline. However, no significant differences were observed in the THC, HC and GCs among prawns that received the BPE at 1.0e6.0 mg (g prawn)1 after 3e6 days (Fig. 1AeC).
330
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
Table 3 The survival rate and sublethal time of Macrobrachium rosenbergii subjected to hypothermal (14 C) stress, when the prawns were injected with different dosage of hot-water extract of banana peel after 6 days. Temperature ( C)
28 14 14 14 14
Banana peel extract mg (g prawn)1
Control Saline 1 3 6
No. of prawns
Survival (%), time after challenge (h)
30 30 30 30 30
24
48
72
100 96.7 ± 3.3a 96.7 ± 3.3a 100 ± 0.0a 100.0 ± 0.0a
100 73.3 ± 3.3b 80.0 ± 5.8ab 83.3 ± 6.7ab 96.7.0 ± 3.3a
100 36.7 53.3 63.3 66.7
96 ± ± ± ±
3.3b 8.8ab 3.3a 6.7a
100 0.0 ± 0.0c (69.4)* 3.3 ± 3.3b (79.8)* 30.0 ± 5.8b (83.6)* 46.7 ± 6.7a (90.18)*
Data in the same column with different letters are significantly differ (p < 0.05) among treatments. Values are mean ± S.E. (n ¼ 6 prawns in each). *Values in parentheses are sublethal time (h).
For the prawns that received BPE at 6.0 mg (g prawn)1 after 1 day, the PO activity was significantly higher than those of prawns received BPE at 1.0 and 3.0 mg (g prawn)1, and saline. PO activity of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were
saline
1
150
3 ab ab
a
a a
6
a
a a a
b 100
A
Banana peel extract (μg (g prawn)-1)
200
THC (x 105 cell -1)
significantly higher than those of prawns received saline from 3 to 6 days. After 6 days of injection, PO activity of prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 increased by 69.0%, 72.4% and 72.4%, compared to prawn that received saline. However, no
a
a a
b
b
a
50 0 0
1
3
6
Time elapsed (days) Banana peel extract (μg (g prawn)-1) saline
HC (x 105 cell -1)
120
1
3
100 a
80 60
a
a
a a
a
B
6
a
a
a a a
a
a b
a
b
40 20 0 0
1
3
6
Time elapsed (days)
GC+SGC (x 105 cell -1)
C
Banana peel extract (μg (g prawn)-1)
120
saline
1
3
6 a
90 60
a
a
a a
a
a
a
a a a
b ab
a
b
b 30 0 0
1
3
6
Time elapsed (days) Fig. 1. (A) Total haemocyte count (THC), (B) hyaline cell (HC) and (C) granular cell (granular cell þ seimigranular cell, GC þ SGC) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Each bar represents the mean value from 6 samples with the standard error. Bars with different letters significantly differ (p < 0.05) among treatments.
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
activities of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 had increased by 17.6%, 19.5% and 31.8%, respectively, compared to the saline control (Fig. 5A). Clearance efficiencies that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 1 to 6 days. After 6 days, clearance efficiencies of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 had increased by 153.5%, 152.4% and 161.0%, respectively, as compared to the saline control (Fig. 5B).
significant differences in PO activity were observed among prawns that received BPE at 1.0, 3.0 and 6.0 mg (g prawn)1 at 3 and 6 days sampling times (Fig. 2A). The same tendency was observed in the PO activity of per GCs (Fig. 2B). RBs of prawn that received BPE at 1.0e6.0 mg (g prawn)1 were significantly higher than that of the saline control at 3 days. However, no significant difference in RBs was observed among four treatments at sampling times of 1 and 6 days (Fig. 3A). RBs per haemocyte of prawns that received BPE at 3.0 and 6.0 mg (g prawn)1 were significantly lower than that of the saline control after 6 days of injection, and had decreased by 16.2%, and 16.1% as compared to prawns that received saline (Fig. 3B). SOD activity of haemocytes of prawns that received BPE at 6.0 mg (g prawn)1 was significantly higher than those of prawns received saline after 6 days. For the prawns that received BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 after 6 days, the SOD activity increased by 32.0%, 44.0%, and, 62.0%, compared to prawns that received saline control (Fig. 3C). Haemolymph coagulation times of prawns that received BPE at the levels of 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly shorter than those of the saline control for 1e3 days. After 6 days, haemolymph coagulation times of prawns that received BPE at 3.0, and 6.0 mg (g prawn)1 were significantly shorter than those of the saline control, and had significantly decreased by 20.4% and 24.7%, respectively, compared to the saline control prawns (Fig. 4).
4. Discussion Musa is genus from Zingiberales and family Musaceae; it includes bananas and plantains. Effects of the antibacterial activity have been investigated for the extracts of banana peels. Ighodaro [37] demonstrated that the aqueous extract of Musa paradisiaca give an antibacterial effect against Staphylococcus aureus, Escherichia coli and Proteus mirabilis more than antifungal. The aqueous and acetone extracts of three varieties of bananas namely the green banana, M. paradisiaca, yellow banana, Musa Cavendish and red banana, Robusta fruit's peel possess antibacterial and antifungal activities against the skin and gastrointestinal tract diseases causing organisms [38]. Chabuck et al. [19] indicated that the aqueous extract of banana peel (Musa sapientum) exhibit a good antibacterial effect against Gram-positive bacteria of Staphylococcus aureus and Staphylococcus pyogenes, and Gram-negative bacteria of Moraxella catarrhalis, Enterobacter aerogenes and Klebsiella pneumonia. The same antibacterial activity was observed in the hotwater extracts of M. acuminata fruit's peel against the pathogens of aquatic animals, including Gram-positive of L. garvieae and Gram-negative of P. damsella, V. alginolyticus and V. parahemolyticus in the present study.
3.5. Phagocytic activity and clearance efficiency of prawns injected with the BPE
PO activity (O.D. 490 nm)
Phagocytic activities of prawns that received the BPE at 1.0, 3.0, and 6.0 mg (g prawn)1 were significantly higher than those of prawns received saline from 3 to 6 days. After 6 days, phagocytic
A
Banana peel extract (μg (g prawn)-1)
0.8
saline
1
3 a
6
0.6
a
b
b b
a
a a
a a
b
0.4 0.2
331
a
a
a b
a
0 0
1
3
6
PO activity (O.D. 490 nm) per 107 GC+SGC
Time elapsed (days)
B
Banana peel extract (μg (g prawn)-1)
0.6
saline
1
3
6
a 0.4 ab b a a
a
a
b
0.2 a
ab
a
a c
a
b
a
0 0
1
3
6
Time elapsed (days) Fig. 2. (A) Phenoloxidase (PO) activity and (B) PO activity at 105 granular cell (semigranular cell and granular cell, SGC þ GC) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
332
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
RB (O.D. 630 nm)
saline
0.2 0.15
A
Banana peel extract (μg (g prawn)-1)
0.25
1 a a
a a
a a
3
6
a a
a
a
a
b
a
a
a
a
0.1 0.05 0 0
1
3
6
RB (O.D. 630 nm) per 107 THC
Time elapsed (days)
0.2
B
Banana peel extract (μg (g prawn)-1)
0.25
saline a
a
a
a
1
3
6
a a
a
a a
0.15
a a
a
a
ab b
b
0.1 0.05 0 0
1
3
6
Time elapsed (days)
SOD activity (units mg protein-1)
Banana peel extract (μg (g prawn)-1) saline
6
1
3 a
5
C
6 ab
a
4
a
a
a
a
a
ab
a
a
b
3 2 1
a
a a
a
0 0
1
3
6
Time elapsed (days) Fig. 3. (A) Respiratory burst (RB), (B) RB at 107 haemocytes and (C) Superoxide dismutase activity (SOD) of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
Several studies examined the use of immunostimulants in aquatic animals to enhance immunity and resistance to pathogens [5,6]. Administration of plant extracts or their products through an oral (diet) or injection route enhanced the innate and adaptive immune response of different freshwater and marine fish and shellfish against bacterial, viral, and parasitic diseases [7]. Injection administration of stevia extracts enhances the innate immune response and resistance of tiger shrimp P. monodon to V. harveyi [39]. Litopenaeus vannamei that injected with the hot-water extracts from Gracilaria tenuistipitata [40], S. duplicatum [11] and G. amansii [12] exhibited enhanced resistance to V. alginolyticus infection. In the present study, injection BPE administration at 1.0e6.0 mg (g prawn)1 for 6 days significantly increased survival rates of the prawn M. rosenbergii against L. garvieae with a dosedependent manner, suggesting that BPE can enhance prawns disease resistance via injection administration. The optimum metabolic temperature range for M. rosenbergii is between 26 and 32 C [41]. Rogers and Fast [42] reported that
prawns were stressed by temperature below 22 C. Yeh et al. (2010) [43] reported that L. vannamei immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited a protective effect against at temperature change as evidenced by the earlier recovery of immune parameters especially in total haemocyte counts. Liu et al. (2010) [44] indicated that M. rosenbergii fed the diets containing Rheum officinale Bail significantly decreased the mortality under high temperature stress, and these were the consequences of the enhanced physiological responses including total haemolymph protein, energy metabolites, etc. The haemolymph protein content is used as an immune parameter indicating re et al. 1997) [45]. whether the prawn is healthy or not (Bache Furthermore, the injection of hot-water extract of G. tenuistipitata was reported to increase the haemocyte number of L. vannamei after 24 h (Yeh and Chen, 2009) [46]. The similar phenomena can be observed in the present study. Prawn injected with BPE significantly increased THC, and the increased survival rate after hypothermal stress was revealed thereafter. Prawns received BPE at
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
333
Banana peel extract (μg (g prawn)-1) saline
Coagulation time (seconds)
350 300
a a
a
1
3
a
250 200
6
a a b bc c
150
a b
b
b
ab
b
b
100 50 0 0
1
3
6
Time elapsed (days) Fig. 4. Coagulation time of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
S3 mg (g prawn)1 for 6 days showed significantly increased survival rate when the prawns transferred to lethally cold water (14 C) after 96 h. These facts suggested that the proliferation of haemocyte and the maturation of haemocyte precursors in haematopoietic tissue may cause earlier recovery of immune responses, and meanwhile, the enhanced haemolymph protein occurred to improve stress responses. Further tests would be conducted on the variation of haemolymph protein levels in M. rosenbergii injected with BPE to clarify the hypothesis. White shrimp L. vannamei that received extracts of G. tenuistipitata [40], S. duplicatum [11] and G. amansii [12] via an injected route showed increased THCs, PO activity, RBs and SOD activity together with increased resistance against L. garvieae challenge. Hyaline cells are young, immature haemocytes of both
large- and small-GC lines that are produced in haematopoietic tissues (HPTs), and can be released into the haemolymph in P. monodon [47]. GCs of the large-GC line mature and accumulate in connective tissues and are easily released into the haemolymph [47]. Litopenaeus vannamei which was fed G. tenuistipitata extractcontaining diets for 14 days showed increased THCs, especially HCs and GCs together with increases in mitotic cells and the mitotic index of HPTs which supported the proliferation of haemocytes [48]. In the present study, THCs, HCs, and GCs significantly increased in prawns injected with BPE at 1.0e3.0 mg (g prawn)1 for 3e6 days. The facts suggest that BPE via injection administration can induce proliferation of haemocytes in HPTs and promote mobilization of mature GCs, which result in an increase in THC of prawns.
Fig. 5. (A) Phagocytotic activity and (B) clearance efficiency of Macrobrachium rosenbergii that injected with the saline and banana (Musa acuminata) fruit's peel extract at 1, 3 and 6 mg (g prawn)1. Statistical descriptions are the same as those given in the legend to Fig. 1.
334
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335
PO is the terminal enzyme of the prophenoloxidase (proPO) system and acts as both a recognition and effector component to promote cell-to-cell communication and subsequently eliminate pathogens [49]. proPO is synthesized and localized in granules, and is released into the plasma by exocytosis which is triggered by the binding of foreign polysaccharides and pattern-recognition proteins (PRPs) [50]. In the present study, the M. rosenbergii that received BPE at 1.0e6.0 mg (g prawn)1 for 3e6 days through injection increased the PO activity and PO activity per GCs. An increase of PO activity for prawns that received BPE may be a consequence of increased GC and its proPO synthesis. Reactive oxygen intermediates (ROIs) are released during RBs of phagocytosis, which represent a defence mechanism against microbial infection [28]. In a normal physiological state, harmful effects of ROIs are effectively neutralized by the antioxidant defence system of organisms, which in general comprises enzymes like SODs, and small antioxidant molecules like ascorbate and sugars. Therefore, the regulation of the ROI system is usually considered a defence mechanism in living systems. In the present study, RBs slightly increased, and SOD activity significantly increased in prawns injected with BPE at 1e6 mg (g prawn)1 for 6 days, indicating that the BPE may promote regulation of the ROI system in prawn. Injection administration BPE at 3e6 mg (g prawn)1 for 6 days significantly decreased O 2 production of single haemocytes, but maintained or slightly increased O 2 production of prawns. The negative regulation per haemocyte maintains homeostasis and prevents disturbance to immunity by restoring immune parameters [48]. However, the unbiased O 2 production of prawns is related with an increase in the THC, and the decreased O 2 production of single haemocytes may be resulting from increased SOD and/or rich antioxidants in BPE. Coagulation of the haemolymph is part of the innate immune response in crustaceans; it prevents leakage of haemolymph and helps protect against dissemination of invaders such as bacteria throughout the body. In shrimp, coagulation is initiated by the release of haemocyte transglutaminases (TGs) which effectively polymerizes the haemolymph clotting protein (CP) to form a stabilized gel [51]. Lightner and Lewis [52] observed a reduction in haemocyte numbers and prolongation of coagulation in Vibrioinfected shrimp. TG-I messenger (m)RNA expression levels and clotting times of haemolymph of the tiger shrimp, P. monodon, fed a diet containing 2.0 g kg1 sodium alginate significantly decreased, but clotting protein and TG-II mRNA expression increased [53]. Chang et al. [10] indicated that M. rosenbergii fed diets containing the extract of water hyacinth (E. crassipes) at 2.0 and 3.0 g kg1 for 12 days significantly increased the TG activity and decreased the clotting time, suggesting acceleration of haemolymph coagulation of prawns is resulting from an increase in TG activities and/or the concentration of clotting proteins. In the present study, prawns injected with BPE at 1.0e6.0 mg (g prawn)1 significantly increased THC and TG activities, and decreased haemolymph clotting times, suggesting that acceleration of haemolymph coagulation of prawns that received BPE is resulting from an increase in TG activities accompanied with THC. However, further research is necessary to determine the TG activity and CP concentration of prawn injected with BPE. Phagocytosis is an important cellular defence mechanism, whereas the clearance efficiency is an important humoral defence mechanism in crustaceans [49]. Clearance from the circulation is induced by humoral factors such as agglutinins, lectins, cytotoxic factors [54], and antimicrobial factors [55], which cause bacterial aggregation in the circulating haemolymph, and enhance the clearance rate [47]. Both phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus increased significantly, and
these levels correlated well with the resistance to V. alginolyticus when the shrimp received hot-water extracts of G. tenuistipitata and S. duplicatum by injection administration [11,40]. In the present study, the phagocytic activity and clearance efficiency of M. rosenbergii to L. garvieae increased, and these correlated well with the resistance against L. garvieae when the prawns were injected with BPE at 1e6 mg (g prawn)1. Banana peel is rich in phytochemical compounds with antioxidant, antibacterial, antihypertensive, antidiabetic and antiinflammatory effects. The lectin from banana peel has been characterized with inhibited HIV replication through interaction with its GP120 coat glycoprotein [56]. Lectins are powerful oral and parenteral immunogens. The primary effects and the potency of lectins as biological signal are the direct result of their specific chemical reactivity with saccharides. As these reactions are predictable, the use of lectins as blockers of pathogens, immune stimulants, hormone modulators and metabolic agents in clinicalmedical applications [57]. Emaga et al. [16] indicated that banana peels are the potential source of dietary fibres and pectins (pectic polysaccharides), and the galacturonic acid and methoxy group contents. Polysaccharide gel from the D. zibethinus is a pectic polysaccharide with inhibited growth of V. harveyi, and increased the immunity and resistance of P. monodon against V. harveyi and WSSV infection through dietary administration [8]. In the present study, the BPE inhibited growth of pathogens from aquatic animals and strengthened anti-hypothermal stress as well as enhanced immune response and resistance of M. rosenbergii to L. garvieae via injection administration, suggesting that BPE is not only an immunostimulant but also physiological regulator and bacteriostat. Further research is needed to evaluate anti-environmental stress ability and immune resistance activity when M. rosenbergii are fed a diet containing BPE. In conclusion, the present study showed that hot-water extract of M. acuminata fruit's peel has a growth-inhibiting effect in vitro against pathogens from aquatic animals. In addition, the giant freshwater prawn M. rosenbergii injected with hot-water extracts of M. acuminata fruit's peel at 1e6 mg (g prawn)1 for 6 days showed increased non-specific immune responses (including reduced clotting times of haemolymph, and increased THC, HC, GC, PO activity, SOD activity, and phagocytic activity and clearance efficiency against L. garvieae) together with increased resistance to L. garvieae infection, and enhanced anti-hypothermal stress ability. Acknowledgement This work was financially supported by a grant (NSC102-2622B-020-004-CC2) from the National Science Council, Taiwan. Special thanks to the budgets support for Wutti Rattanavichai from Rajamangala University of Technology Isan of Thailand to pursue further education in NPUST, Taiwan, ROC. References [1] Hsu JP, Liu CI. Studies on yeast infection in cultured giant freshwater prawn (Macrobrachium rosenbergii). Fish Dis Res 1994;15:55e68. [2] Cheng W, Chen JC. Isolation and characterization of Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan. Dis Aquat Org 1998;34:93e101. [3] Sahul Hameed AS, Rahaman KH, Alagan A, Yoganandhan K. Antibiotic resistance in bacteria isolated from hatchery-reared larvae and post-larvae of Macrobrachium rosenbergii. Aquaculture 2003;217:39e48. [4] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection? Fish Shellfish Immunol 2003;15:71e90. [5] Sakai M. Current research status of fish immunostimulants. Aquaculture 1999;172:63e92. [6] Ringø E, Olsen RE, Vecino JLG, Wadsworth S, Song SK. Use of immunostimulants and nucleotides in aquaculture: a review. J Mar Sci Res Dev 2012;2: 1e22.
W. Rattanavichai, W. Cheng / Fish & Shellfish Immunology 39 (2014) 326e335 [7] Harikrishnan R, Balasundaram C, Heo MS. Impact of plant products on innate and adaptive immune system of cultured finfish and shellfish. Aquaculture 2011;317:1e15. [8] Pholdaeng K, Pongsamart S. Studies on the immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus monodon shrimp against Vibrio harveyi and WSSV. Fish Shellfish Immunol 2010;28: 555e61. [9] Harikrishnan R, Balasundaram C, Jawahar S, Heo MS. Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila. Fish Shellfish Immunol 2012;32:94e100. [10] Chang CC, Tan HC, Cheng W. Effects of dietary administration of water hyacinth (Eichhornia crassipes) extracts on the immune responses and disease resistance of giant freshwater prawn, Macrobrachium rosenbergii. Fish Shellfish Immunol 2013;35:92e100. [11] Yeh ST, Lee CS, Chen JC. Administration of hot-water extract of brown seaweed Sargassum duplicatum via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2006;20:332e45. [12] Fu YW, Hou WY, Yeh ST, Li CH, Chen JC. The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol 2007;22:673e85. [13] FAO (Food and Agriculture Organization, Geneva) 2013: FAOSTAT, http:// faostat.fao.org/site/291/default.aspx. [14] Zhang P, Whistler RL, BeMiller JN, Hamaker BR. Banana starch: production, physicochemical properties, and digestibility e a review. Carbohyd Polym 2005;59:443e58. [15] Mohapatra D, Mishra S, Sutar N. Banana and its by-product ultilisation: an overview. J Sci Ind Res 2010;69:323e9. [16] Emaga TH, Robert C, Ronkart SN, Wathelet B, Paquot M. Dietary fibre components and pectin chemical features of peels during ripening in banana and plantain varieties. Biores Technol 2008;99:4346e54. [17] Burkitt DPJ, Walker ARP, Painter NJ. Dietary fibre and disease. J Am Med Assoc 1974;229:1068e77. [18] Kelsey JL. A review of research on effect of fiber intake on man. Am J Clin Nutr 1978;31:142e59. [19] Chabuck ZAG, Al-Charrakh AH, Hindi NKK, Hindi SKK. Antimicrobial effect of aqueous banana peel extract, Iraq. Res Gate Pharm Sci 2013;1:73e5. [20] Soltani M, Alimardani R, Omid M. Prediction of banana quality during ripening stage using capacitance sensing system. Aust J Crop Sci 2010;4:443e7. [21] Liu PC, Chen YC, Huang CY, Lee KK. Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome. Lett Appl Microbiol 2000;31:433e7. [22] Liu CH, Cheng W, Hsu JP, Chen JC. Vibrio alginolyticus infection in the white shrimp (Litopenaeus vannamei) confirmed by polymerase chain reaction and 16S rDNA sequencing. Dis Aquat Organ 2004;61:169e74. [23] Song YL, Cheng W, Wang CH. Isolation and characterization of Vibrio damsela infectious for cultured shrimp in Taiwan. J Invertebr Pathol 1993;61:24e31. [24] Peebles JB. A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture 1977;12:173e80. [25] Amend DF. Potency testing of fish vaccines. International symposium on fish biologics: serodiagnostics and vaccines. Dev Biol Stand 1981;49:447e54. [26] Mason HS. Structures and functions of the phenolase complex. Nature 1956;177:79e81. ndez-Lo pez J, Gollas-Galv [27] Herna an TS, Vargas-Albores F. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comp Biochem Physiol 1996;113C:61e6. [28] Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Dev Comp Immunol 1993;17:211e9. [29] Song YL, Hsieh YT. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Dev Comp Immunol 1994;18:201e9. [30] Liu KF, Yeh MS, Kou GH, Cheng W, Lo CF. Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages. Dev Comp Immunol 2010;34:935e44. [31] Biagini G, Sala D, Zini I. Diethyldithiocarbamate, a superoxide dismutase inhibitor, counteracts the maturation of ischemic-like lesions caused by endothelin-1 intrastriatal injection. Neurosci Lett 1995;90:212e6. [32] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [33] Tsai IH, Chen YL, Lu PJ. Relationship between haemolymph-coagulation and disease in shrimp: 1. Coagulation rate in vitro and the haemolymph proteins of various shrimp species. COA Fish Ser 1991;29:22e30.
335
[34] Jussila J, McBride S, Jago J, Evans LH. Haemolymph clotting time as an indicator of stress in western rock lobster (Panulirus cygnus George). Aquaculture 2001;199:185e93. [35] Li JT, Lee PP, Chen OC, Cheng W, Kuo CM. Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii. Fish Shellfish Immunol 2005;19: 269e80. [36] Adams A. Response of penaeid shrimps to exposure to Vibrio species. Fish Shellfish Immunol 1991;1:59e70. [37] Ighodaro OM. Evaluation study on Nigerian species of Musa paradisiaca peels: phytochemical screening, proximate analysis, mineral composition and antimicrobial activities. Researcher 2012;4:17e20. [38] Sumathy NH, Sumathy JH. Antibacterial and antifungal activity of Musa fruit peels against skin and gastrointestinal tract diseases. Herb Tech Ind 2011;1: 9e11. ~ o EM, Xi YC, Liao IC. Effects of stevia extract on growth, non-specific [39] Lean immune response and disease resistance of grass prawn, Penaeus monodon (Fabricius), Juveniles. J Fish Soc Taiwan 2007;34:165e75. [40] Hou WY, Chen JC. The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol 2005;19: 127e38. [41] Boyd C, Zimmermann S. Grow-out systems-water quality and soil management. In: New MB, Valenti WC, Tidwell JH, D'Abramo LR, Kutty MN, editors. Freshwater prawns biology and farming. Oxford: Blackwell Science; 2010. pp. 239e55. [42] Rogers GL, Fast AW. Potential benefits of low energy water circulation in Hawaiian prawn ponds. Aqua Eng 1988;7:155e65. [43] Yeh ST, Li CC, Tsui WC, Lin YC, Chen JC. The protective immunity of white shrimp Litopenaeus vannamei that had been immersed in the hot-water extract of Gracilaria tenuistipitata and subjected to combined stresses of Vibrio alginolyticus injection and temperature change. Fish Shellfish Immunol 2010;29:271e8. [44] Liu B, Ge X, He Y, Xie J, Xu P, He Y, et al. Effects of anthraquinones extracted from Rheum officinale Bail on the growth, non-specific immune response of Macrobrachium rosenbergii. Aquaculture 2010;310:13e9. re E, Mialhe E, Rodriguez J. Identification of defence effectors in the [45] Bache haemolymph of Crustaceans with particular reference to the shrimp Penaeus japonicus (Bate): prospects and applications. Fish Shellfish Immunol 1997;5: 597e612. [46] Yeh ST, Chen JC. White shrimp Litopenaeus vannamei that received the hotwater extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection. Fish Shellfish Immunol 2009;5: 724e30. [47] Van de Braak CBT, Botterblom MHA, Liu W, Taverne N, Van de Knaap WPW, Rombout JHWM. The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp (Penaeus monodon). Fish Shellfish Immunol 2002;12:253e72. [48] Sirirustananun N, Chen JC, Lin YC, Yeh ST, Liou CH, Chen LL, et al. Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2011;31:848e55. [49] Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP. Invertebrate immunity: basic concepts and recent advances. Int Rev Cytol 1985;97:183e350. €derha €ll K. Early events in crustacean innate immunity. Fish Shellfish [50] Lee SY, So Immunol 2002;12:421e37. [51] Yeh MS, Kao LR, Huang CJ, Tsai IH. Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus. Biochim Biophys Acta 2006;1764: 1167e78. [52] Lightner DV, Lewis DH. A septicemic bacterial disease syndrome of penaeid shrimp. Mar Fish Rev 1975;17:25e8. [53] Chung MY, Franklin SMT, Lee PP, Cheng W. Shrimp immune responses and disease management by using seaweed extracts: a review. J Int Coop 2011;6: 109e38. [54] Smith VJ, Chisholm JRS. Non-cellular immunity in crustaceans. Fish Shellfish Immunol 1992;2:1e31. [55] Smith VJ. Invertebrate immunology: phylogenetic, ecotoxicological and biomedical implication. Comp Haematol Int 1991;1:60e76. [56] Swanson MD, Winter HG, Goldstein IJ, Markovitz DM. A lectin isolated from banana is a potent inhibitor of HIV replication. J Biol Chem 2010;285: 8646e55. [57] Pusztai A, Bardocz S. Biological effects of plant lectins on the gastrointestinal tract: metabolic consequences and applications. Trends Glycosci Glycotechnol 1996;8:149e65.
