Accepted Manuscript The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: activity and gene transcription Wutti Rattanavichai, Ying-Nan Chen, Chin-Chyuan Chang, Associate Professor, Winton Cheng, PhD, Professor PII:
To appear in:
Fish and Shellfish Immunology
Received Date: 29 April 2015 Revised Date:
17 June 2015
Accepted Date: 23 June 2015
Please cite this article as: Rattanavichai W, Chen Y-N, Chang C-C, Cheng W, The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: activity and gene transcription, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.06.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The effect of banana (Musa acuminata) peels hot-water extract on the immunity
and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary
administration for a long term: activity and gene transcription Wutti Rattanavichaia, Ying-Nan Chenb, Chin-Chyuan Chang b*, Winton Chengb*
Department of Tropical Agriculture and International Cooperation, and bDepartment of
Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201,
The non-specific immune parameters, disease resistance and immune genes expressions
in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets
containing the extracts of banana, Musa acuminate, fruit’s peel (banana peels extract, BPE) at
0, 1.0, 3.0 and 6.0 g kg-1. Results showed that prawns fed with a diet containing BPE at the
level of 1.0, 3.0 and 6.0 g kg-1 for 120 days had a significantly higher survival rate (30.0%,
40.0% and 56.7%, respectively) than those fed with the control diet after challenge with
Lactococcus garvieae for 144 hours, and the respective relative survival percentages were
22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg-1
for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC),
superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG)
activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and
meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts
(RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of
prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP),
peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We
therefore recommend that BPE can be used as an immunomodulator for prawns through
dietary administration at 6.0 g kg-1 for a long term (over 120 days) to modify immune
responses and genes expression following the enhanced resistance against pathogens.
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Keywords: Macrobrachium rosenbergii; dietary administration; banana peels extract; immunity; disease resistance
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The giant freshwater prawn, Macrobrachium rosenbergii, is commercially important in
Taiwan as well as the world as a primary freshwater cultured prawn species. The intensive
culture of which is strikingly developed in Taiwan, which accelerate the degradation of ponds
environment lead to increased incidences of diseases caused serious economic losses [1,2].
Disease outbreaks are result from the interactions of the three facts of poor environment,
offensive pathogens and weakened hosts. However, it is difficult to control the stress of
highly polluted pond environments and constantly existent pathogens in the intensive
cultured ponds. Therefore, enhance the health of hosts as a prophylactic measure is of
The application of antibiotics and chemical disinfectants is a traditional measure to
control and prevent diseases in aquaculture, but they are no longer recommended due to the
potential for enhanced microbial resistance, and the accumulation of residues in the
environment and in non-target organisms. The application of immunostimulants has been
developed in aquaculture as a more environmentally friendly approach to disease
management. These compounds are derived from bacteria, fungi, animal and plant extracts,
nutritional factors, and synthetic drugs that stimulate the immune system resulting in an
immune response and increase resistance disease of aquatic animals were reviewed by Sakai
 and Ringø et al. . Vibrio harveyi and white spot syndrome virus (WSSV) infection may
be prevented by the enhancement of immune responses thought the dietary application of
polysaccharide gel derived from the fruit-rind of Durio zibethinus . Moreover,
administration of Withania somnifera and Eichhornia crassipes extracts through
supplementation diet positively enhances the immunity and increase survival rate in M.
rosenbergii against Aeromonas hydrophila and Lactococcus garvieae infection, respectively
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Plant extracts have been demonstrated as anti-stress, growth promotion, appetite
stimulation, tonic and immunostimulation, and to have aphrodisiac and antimicrobial
properties in fish and shrimp culture due to the active principles such as polysaccharides,
alkaloids, flavonoids, pigments, phenolics, terpenoids, steroids, and essential oils and were
reviewed by Harikrishnan et al. . Banana peel could be potentially administrated in diets
for livestock and poultry because of its rich source of starch (3%), crude protein (6~9%),
ACCEPTED MANUSCRIPT crude fat (3.8~11%), total dietary fiber (43.2~49.7%) and vitamins . Dietary fiber 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 .
Moreover, soluble fibers are well known to lower serum cholesterol and helps to reduce the
risk of colon cancer [11,12]. Additionally, the phytochemicals including flavonoids, tannins,
phlobatannins, alkaloids, glycosides and terpenoids were found to be present in the peels of
genus Musa, and had been reported to exert multiple biological and pharmacological effects
(antibacterial, antihypertensive, antidiabetic and anti-inflammatory activities) . Therefore,
we consider that the peels possess valuable medicinal potential and can be applied as an
The prophylactic measures for control disease are the major concern in aquaculture
nowadays, and most of them are immunostimulants including natural products and probiotic.
In previously study, we demonstrated that the banana (Musa acuminate) peels extract (BPE)
not only had an antimicrobial effect, but also can enhance immune responses and disease
resistance of prawns via both injection for 6 days and dietary administrations for 32 days
[14,15]. Furthermore, the enhanced growth performance was found in prawns fed with BPE-
containing diet especially at 6.0 g kg−1 from 30 to 120 days . It, therefore, was speculated
that the enhanced growth performance might result from the continuously up-regulated
immunogenes expressions, and immune responses without immunostimulatory fatigue
occurred in M. rosenbergii fed with BPE-containing diet for 120 days of feeding trial.
Against this background, the objectives of the present study were aimed to evaluate the
dietary administration of banana peels extract from M. acuminate in M. rosenbergii upon
long term feeding trial (120 days) on: (1) the immune parameters including the total
haemocyte count (THC), different haemocyte count (DHC), phenoloxidase (PO) activity,
respiratory bursts (RBs), superoxide dismutase (SOD) activity, transglutaminase activity,
haemolymph clotting times, (2) the immune genes expressions including proPO,
transglutaminase (TG), and crustin, (3) the phagocytic activity and clearance efficiency to L.
garvieae and, (4) the resistance against L. garvieae infection following dietary administration
of banana peels extract from M. acuminate.
2. Materials and methods
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2.1. Preparation of the banana peels extract and diets Banana fruits, M. acuminate, of all green were obtained from the Neipu market,
Pingtung, Taiwan. Subsequently, banana fruits were drenched with soda water for 5 min,
washed with tap water, and later rinsed twice with distilled water to remove the contaminants.
The banana peel was collected, and the banana peels extract (BPE) was manufactured as
described previously . The proximate analysis of BPE and the basal diet were conducted
according to the AOAC method . The BPE was composed of crude protein (7.71 ±
0.30%), crude lipids (0.46 ± 0.03%), moisture content (14.45 ± 0.18%), ash (27.21 ± 0.11%) and total carbohydrate 50.17 ± 0.15 %, and was stored at -20 °C until being further used.
Four diets containing different concentrations of BPE were prepared as described in
Table 1. The basal diet contained 40.5% crude protein, 5.1% crude lipids, 18.3% ash, and
5.0% moisture. For the experiment diet with banana peels extract, the BPE was added to the
test diets at levels of 0, 1.0, 3.0, and 6.0 g kg-1 with a corresponding decrease in the amount
of cellulose. Ingredients were made to ground using Hammer mill until they passed through
an 80-mesh screen. Experimental diets were prepared by mixing the dry ingredients with fish
oil and later water was added until it becomes stiff dough. Each diet was then passed through
a mincer with a die, and the resulting spaghetti-like strings were dried in a drying cabinet
using an air blower at 50°C until moisture levels were lower than 10%. After drying, the
finished pellets were stored in plastic bins at 4 °C until being further used.
2.2. Lactococcus garvieae
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A known pathogenic strain, L. garvieae, isolated from diseased M. rosenbergii, which
displayed clinical signs of opaque and whitish musculature, was used for the study . The
pathogen was cultured and collected according to the method of previous description .
The bacterial pellets were re-suspended in a saline solution (0.85% NaCl) at 2.0 × 107
colony-forming units (cfu) ml-1 as a stock bacterial suspension for the susceptibility study and
at 1.0 × 108 cfu ml-1 for studies on phagocytic activity and clearance efficiency. The
concentration of bacterial suspension was measured by its absorbance at 601 nm optical
density using a spectrophotometer (Hitachi U-2001), and was calculated as a standard curve
based on a series of different concentrations of bacterial suspension. The diluted bacterial
suspension was plated on tryptic soy agar plates to determine viable cell counts.
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2.3. Experimental design Prawns (3.6 ± 0.4 g), M. rosenbergii, were obtained from a commercial farm in
Pingtung, Taiwan, and were shipped to National Pingtung University of Science and
Technology (Aquatic Animal Physiology and Immunology Laboratory), and acclimatized at
room temperature (27 ± 1°C) and pH 7.0~7.5 in the laboratory for 2 weeks before being used
for experimentation. Only prawns in the intermoult stage were used in this study. The moult
stage was determined under a stereomicroscope according to retraction of the epithelium
within the setal base interface of the antennal scale . During the acclimatization period,
prawns were fed with the control diet twice daily.
For the banana peels extract supplementations in the diet, four studies were conducted.
Twelve 9-ton fiber reinforced plastic (FRP) tank containing 5 ton of aerated freshwater were
used in this study. Each tank was used to rear 300 prawns (3.6 ± 0.4 g). Groups with four
different dosages of BPE (0, 1.0, 3.0, and 6.0 g kg-1) were established, with each group
consisting of three tanks. Ten, two, two, two and two prawns were randomly sampled from
each tank after 120 days of feeding trial for the susceptibility, immune parameters,
haemolymph clotting times, immune genes expression, and phagocytic activity and clearance
efficiency tests, respectively. For the susceptibility experiment, test and control groups were
comprised of 10 prawns each, and tests were conducted in triplicate. To determine THC,
DHC, PO activity, RBs, SOD activity, TG activity, haemolymph clotting times, and genes
expressions, tests and controls were carried out on six replicates. For studies on phagocytic
activity and clearance efficiency, six prawns were used in the test and control groups. No
significant difference in weight was observed among different treatments. During the
experiments, prawns were continuously fed with their respective diets, and the water
temperature was maintained at 27 ± 1°C and the pH at 6.9~7.5.
2.4. Effect of oral BPE on the susceptibility of M. rosenbergii to L. garvieae
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Prawns were fed with the BPE-containing diets at 0, 1.0, 3.0, and 6.0 g kg-1 for 120 days
before the challenge tests were conducted. Ten microlitres of bacterial suspension (2.0 × 107
cfu ml-1) resulting in 2 × 105 cfu per prawn was injected slowly into the ventral sinus located
between the ventral nerve cord/cephalothoracic ganglia associated with 4th and 5th pereiopod
and striated muscle of the cephalothorax from the first segment of abdomen with a sterile
syringe. Prawns fed with the control diet and then received 10 µl saline were served as the
unchallenged control (Table 2). There were five treatments with 30 prawns each. After the
ACCEPTED MANUSCRIPT injection, each group of ten prawns was kept in 60-l glass aquaria (10 prawns each)
containing 40 L of freshwater and 27 ± 1.0°C. During the experiment, prawns were
continually fed with their respective diets, and 30% of the water was exchanged daily. The
mortality was counted on daily basis until the total experimental period of 144 h of post
challenge. The relative percent survival (RPS) of prawn was calculated at the end of the
experiment according to Amend  using the following formula: (12.5 ± 1.3 g)
RPS= [1 – [(percentage mortality in treatment) (percentage mortality in control)-1]] ×
2.5. Effects of oral BPE on the immune parameters of M. rosenbergii
Immune parameters of prawns that were fed with the BPE-containing diets at 0, 1.0, 3.0,
and 6.0 g kg-1 were determined after 120 days of feeding. Six prawns (20.0 ± 1.0 g) for each
treatment were used for examination of THC, DHC, PO activity, RBs, SOD activity, TG
activity, and another six prawns were used for determination of haemolymph clotting times.
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After 120 days of feeding, haemolymph (300 µl) was withdrawn from the ventral sinus
of each prawn and were divided into two 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 µl Tween 80 in 100 ml of distilled water, at pH 7.45 with the
osmolality adjusted to 490 mOsm kg-1 with NaCl) for the THC, DHC, PO activity and RB
assays. A drop of the anticoagulant-haemolymph mixture was placed on a haemocytometer in
order 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, and TG activity assays.
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  and Hernández-
López et al. . The details of measurements were described previously . The optical
density (OD) was measured at 490 nm, and the PO activity was expressed as dopachrome
formation in 50 µl of haemolymph or per 107 the sum of granular cells (GCs) and semi-GCs
RBs of haemocytes were quantified using the reduction of nitroblue tetrazolium (NBT)
to formazan as a measure of superoxide anion (O2−) formation as described previously
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[22,23]. The OD at 630 nm was measured using a microplate reader (Model VERSAmax,
Molecular Devices, Sunnyvale, CA, USA). RBs were expressed as NBT reduction in 10 µl of
haemolymph or per 107 haemocytes. The haemocytes lysate supernatant (HLS) was prepared as described previously . 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 . 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 .
TG activity was measured by the colorimetric hydroxamate procedure according to Folk
and Cole  with modifications. Details of the measurement were described previously .
The OD of hydroxamate was measured at 525 nm, and one unit of TG activity was defined as
the amount of enzyme needed to produce 1 mmole of hydroxamate per minute. The specific
activity was expressed as TG activity units (mg protein)-1.
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The concentration of protein in the HLS was quantified by the method described by
Bradford  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 clotting times was modified from the methods of
Tsai et al.  and Jussila et al. . Briefly, 270 µl of haemolymph was withdrawn from the
ventral sinus, mixed with 30 µl 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.
2.6. Effects of oral BPE on the phagocytic activity and clearance efficiency of M.
For the phagocytic activity and clearance efficiency tests, 20 µl of a bacterial
suspension (1.0 × 108 cfu ml-1) resulting in 2 × 106 cfu prawn-1 was injected into the ventral
sinus. After the injection, prawns were held in separate tanks containing 40 L of water at 27 ±
1.0°C for 2 h. Then, 200 µl of haemolymph was collected from the ventral sinus and mixed
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with 200 µl of a sterile anticoagulant solution. This mixture was then divided into two equal
subsamples to respectively evaluate the phagocytic activity and clearance efficiency.
Methods for measuring the phagocytic activity and clearance efficiency were described previously [31,32]. Two hundred haemocytes were counted, and the rate of phagocytosis (PR)
was calculated as follows:
PR = [(phagocytic haemocytes) (total haemocytes)-1] × 100.
The clearance efficiency, defined as the percentage inhibition (PI) of L. garvieae was
PI = 100 – [(cfu in the test group) (cfu in the control group)-1] × 100. 2.7. Effects of oral BPE on the immune genes of M. rosenbergii
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The haemocytes was collected as described by Hsu et al.  and total RNA was
extracted and further purified using the ULTRASPEC™ RNA, Total RNA Isolation Reagent
(Biotecx, Houston, TX, USA) following the manufacturer's instructions. First-strand
complementary (c)DNA synthesis in reverse transcription (RT) was accomplished as
described previously . Messenger (m)RNA expressions of immune genes including
proPO, LGBP, PE, TG, and CT of haemocytes of M. rosenbergii were measured using an
SYBR green I real-time RT-polymerase chain reaction (PCR) assay in an ABI PRISM 7900
Sequence Detection System (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA), and
the details of the measurement were previously described . Specific primers of immune
genes and the β-actin primer were used for the quantitative RT-PCR (Table 3). After
amplification, data acquisition and analysis were performed using Sequence Detection
Software (SDS vers. 2.1, Applied Biosystems). The 2-∆∆CT method was chosen as the
calculation method . The difference in the cycle threshold (CT) value of the individual
immune gene and its housekeeping gene (β-actin), called ∆CT, was calculated. ∆∆CT = (∆CT
of prawn fed diet containing BPE at 1, 3, and 6 g kg-1 for immune genes) - (∆CT of the
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
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analysis, percentage data were normalized using an arc-sine transformation. Statistically
significant differences were calculated when p < 0.05.
3.1. Effect of oral BPE for 120 days on the resistance of prawn to L. garvieae infection No mortality was recorded in the unchallenged control prawns. Survival rates of prawns
that fed with the BPE-containing at 1.0, 3.0 and 6.0 g kg-1 were significantly higher than
those of prawns fed with the control diet from 72 to 144 h. After 144 h of challenge, survival
rates of prawns that fed with the BPE-containing diet at 1.0, 3.0 and 6.0 g kg-1 were 30.0%,
40.0% and 56.7%, respectively and the respective relative survival percentages of prawns
were 22.2%, 33.3% and 51.9% (Table 2).
3.2. Effect of oral BPE for 120 days on the immune parameters of prawns
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The THC, HC, and GC of prawns fed with the BPE-containing diets at 6.0 g kg-1 were
significantly higher than those of prawns fed with the control diet for 120 days, and had
increased by 152.6%, 235.7%, and 125.9%, respectively, compared to prawns fed with the
control diet. However, no significant differences were observed in SGC among prawns that
fed with the BPE at 0.0-6.0 g kg-1, and in THC, HC and GC among prawns fed with the BPE
at 0.0~3.0 g kg-1 after 120 days (Fig. 1A-D).
The PO activity of prawns fed with the diets containing BPE at 3.0 and 6.0 g kg-1 was
significantly higher than those of prawns fed with the control diet at 120 days of post feeding,
and had significantly increased by 63.2%, and 89.5%, compared to prawn fed with the control
diet (Fig. 2A). The similar phenomenon revealed in PO activity per GCs (GC ＋ SGC)
among prawns that fed with control diet and BPE-containing diets. The PO activity per GCs
of prawns that fed with BPE-containing diets at 3.0 and 6.0 g kg-1 increased by 91.9%, and
64.9%, compared to prawn fed with the control diet (Fig. 2B).
No significant differences in RBs were observed among prawns fed with BPE-
containing diets and the control diet (Fig. 3A). The significantly decreased RBs per
haemocyte was observed in prawn fed with BPE-containing diets at 6.0 g kg-1 for 120 days,
and had decreased by 32.4%, compared to prawn fed with the control diet (Fig. 3B). SOD
activity in haemocytes of prawns fed with the BPE-containing diets at 6.0 g kg-1 for 120 days
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was higher than those of prawns fed with the diet at 0-3.0 g kg-1, and had increased by
735.7%, compared to prawns that fed with the control diet (Fig. 3C). TGs activities in haemocytes of prawns fed with the BPE-containing diets for 120
days were significantly higher than those of prawns fed the control diet. The increase in TGs
activities of prawns fed with the BPE-containing diets appeared to have been dose dependent.
TGs activities of prawns fed with the BPE-containing diets at 1.0, 3.0 and 6.0 g kg-1 had
significantly increased by 352.4%, 480.6% and 770.0% compared to prawns fed with the
control diet (Fig. 4A).
Haemolymph clotting times of prawns directly decreased with fed dosage of BPE in
the range of 0.0-6.0 g kg-1 after 120 h. For the prawns fed with the BPE-containing diets at
6.0 g kg-1, haemolymph clotting times had significantly decreased by 43.7%, compared to the
prawns fed with the control diet (Fig. 4B).
3.3. Effect of oral BPE on phagocytic activity and clearance efficiency of prawns
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Phagocytic activities and clearance efficiencies of prawns fed with the BPE-containing
diets separately at 3.0 and 6.0 g kg-1, and at 1.0, 3.0 and 6.0 g kg-1 were significantly higher
than those of prawns fed with the control diet for 120 days. Phagocytic activities and
clearance efficiencies of prawns fed with the BPE-containing diets at 1.0, 3.0 and 6.0 g kg-1
had increased by 10.9%, 41.3% and 45.4%, and 42.9%, 44.2% and 66.0%, respectively,
compared to prawns fed with the control diet (Fig. 5A,B).
3.4. Effect of oral BPE on the immune genes expressions of prawns
A real-time RT-PCR was used to determine the immune genes expression. The proPO,
LGBP, TG and crustin mRNA expression levels in haemocytes of prawn fed with the BPE-
containing diets at 6.0 g kg-1 at 120 days of post feeding were significantly higher than those
of prawns fed with the control diet, and had increased by 141.7%, 167.3%, 56.5% and
202.4%, respectively, compared to the prawns fed with the control diet. The increase in
proPO, PE and TG mRNA expression levels of prawns following administrative dosage of
BPE appeared to have been dose dependent. However, no significant difference in PE mRNA
expression were observed among the prawns fed with the BPE-containing diets at 0, 1.0, 3.0
and 6.0 g kg-1 after 120 days of feeding (Fig. 6A,B,C,D,E).
ACCEPTED MANUSCRIPT Immunostimulants can be applied via injection, bathing or oral administration to
enhance the immunity and disease resistance in aquatic animals, and its effects depend on
time, dosage and method of administration, and the physiological condition of the animals.
Matsuo and Miyazono  indicated that dietary peptidoglycan administration at 0.2 and 2.0
mg kg-1 for 28 days enhanced resistance of rainbow trout against Vibrio anguillarum, but not
protective effect for 56 days of feeding. Tiger shrimp P. monodon fed diet containing 1.0, 2.0
and 3.0% polysaccharide gel (PG) extracted from D. zibethinus for 12 weeks significantly
enhanced the immune responses and resistance against WSSV and V. harveyi infection, and
has the highest effect at 2.0% of supplementation . Giant freshwater prawn fed diet
containing hot-water extracts from water hyacinth E. crassipes at 2 and 3 g kg-1 for 12 days
exhibited increased resistance against L. garvieae infection . Survival rates of M.
rosenbergii injected with BPE at concentrations of 1.0, 3.0 and 6.0 µg (g prawn)-1 were
significantly higher than those injected with saline control after challenge with L. garvieae
for 4~6 days , and in oral route, the significantly increased survival rates of the M.
rosenbergii against L. garvieae revealed when they were fed with diets supplemented at
1.0~6.0 g kg-1 BPE for 32 days of feeding . The similar phenomena were observed when
BPE were administrated in diets at 1.0~6.0 g kg-1 to fed M. rosenbergii for 120 days in the
present study. These suggested that prawns disease resistance can be improved via BPE
dietary administrations at 6.0 g kg-1 especially lasting for a long term feeding.
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Polysaccharide biological response modifiers are usually strong mitogens, which
stimulate proliferation of immune cells . Sirirustananun et al.  reported that the
evidence of haemocytes proliferation in L. vannamei fed with G. tenuistipitata extract-
containing diets were the increases of HCs, GCs and THCs, together with increases in mitotic
cells and the mitotic index of haematopoietic tissues (HPTs). Rattanavichaia and Cheng 
reported that M. rosenbergii fed with BPE containing diets for 32 days of feeding showed
that the THC, DHC, PO activity, RBs, and SOD activity significantly increased, and
meanwhile, no significant differences in PO activity per granulocyte and RBs per haemocyte
were observed. M. rosenbergii that received BPE via an injected route also showed increased
THCs, GCs, PO activity, PO activity per granulocyte, and SOD activity and decreased RBs
per haemocyte . In the present study, prawn fed with the BPE containing diets at 3 or 6 g
kg-1 after 120 days of feeding trial revealed the increases of THC, HC, GC, PO activity, PO
activity per granulocyte (SGC+GC) and SOD activity, and the decrease of RBs per
haemocyte. The facts suggest that BPE might also be capable to increase THC of prawns with
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induced proliferation of haemocytes in HPTs via both oral and injection routes. Further
research would be conducted to clarify the statements. The respiratory burst (RB) activity is an indicator of the status of phagocytes activation.
During phagocytosis, ROIs derived from the processes of RBs are generated by plasma
membrane NADPH oxidase. The ROIs are effectively neutralised by the antioxidant defence
system of organisms to maintain homoeostasis, which included enzymes like SOD, catalase
and various peroxidase, and small antioxidant molecules like ascorbate, sugars and
polyunsaturated fatty acids. Chang et al.  indicated that RBs, and SOD and phagocytic
activity significantly increased, but O2- production per haemocyte significantly decreased in
prawns fed with E. crassipes leaves extracts-containing diets at 2-3 g kg-1 for 12 days. The
similar results were also observed in prawns that received BPE by injection at 3-6 µg (g
prawn)-1 for 6 days  and by dietary administration at 6 g kg-1 for 120 days in this study.
The facts suggest that BPE via dietary administration for a long term feeding trial may
promote both phagocytic activity of phagocytes and regulation of ROI system in prawn, and
the decreased O2- production of single haemocyte may be resulting from an increase of SOD
activity to maintain homoeostasis in prawn.
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Several pattern recognition proteins (PRPs), such as lipopolysaccharide- and β-glucan-
binding protein (LGBP), play an important role in crustaceans-pathogen interaction. They
recognise and respond to microbial intruders, and are involved in activation of the proPO
system, the coagulation cascade, and expression for antibacterial effector proteins , and
act as an opsonin to increase the rate of phagocytosis . Peroxinectin (PE), an associated
protein of the proPO system, has multiple functions of degranulation , encapsulation
enhancement , opsonification , and peroxidation , which is essential in crustacean
cellular defence reaction. These biological activities of PE are generated concomitant with
activation of the proPO system . In the present study, PO activity, PO activity per GCs,
and proPO, LGBP and PE genes expression of prawns fed with the BPE at 6.0 g kg-1
increased after 120 days of feeding trial. The increase PO activity is considered to be
associated with up-expression of proPO, LGBP and PE genes to increase resistance against
In crustaceans, haemolymph coagulation is triggered by the release of haemocyte TGs to
polymerise the clotting proteins (CPs), which is involved in the innate immune responses due
to it prevents leakage of haemolymph and helps protect against dissemination of invaders
ACCEPTED MANUSCRIPT . M. rosenbergii fed with water hyacinth, E. crassipes, extract containing diets at 2.0-3.0
g kg-1 for 12 days and fed with BPE-containing diets at 3.0-6.0 g kg-1 for 32 days
significantly increased THC and TG activity, and decreased the clotting time [7,15]. The
similar results is observed in prawns injected with BPE at 1.0-6.0 µg (g prawn)-1 . In the
present study, the TG activity and gene expression directly increased, and haemolymph
clotting times directly decreased of prawns fed with the BPE in the range of 1.0-6.0 g kg-1 for
120 days. The facts suggest that acceleration of haemolymph coagulation of prawns received
BPE via injection and oral routes are relate to increase of TG gene expression accompany TG
Clearance of invading pathogens in circulating haemolymph is associated with humoral
factors such as agglutinins, lectins, cytotoxic factors, antimicrobial factors, and clotting
factors. Antimicrobial peptides (AMPs) are small cationic molecules that can play an
important role in the innate immune defence against bacterial and fungal pathogens. The
crustin peptide is a cysteine-rich antimicrobial peptide in the blood circulation system of
crustacean . In kuruma shrimp, TG silencing causes significant down-regulation of the
expressions of crustin and lysozyme, but does not affect the expression of proPO . L.
vannamei that received LvTGII dsRNA showed significant decrease in clearance efficiency,
but did not affect the PO activity in our previous study . In the present study, the
increased clearance efficiency of M. rosenbergii to L. garvieae was correlated well with the
increased resistance against L. garvieae and TG activity, and decreased clotting times of
haemolymph, when the prawns received BPE via dietary administration after 120 days. The
facts suggest that the elevation of clearance efficiency is related to the increases of TG
activity, TG and crustin genes expression, and the increased crustin expression was resulting
from the increased TG gene expression accompany TG activity.
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In aquaculture the plant extracts and products are known to possess anti-stress
characteristics, promote growth, stimulate appetite, tonic and immunostimulation, and
aphrodisiac and anti-microbial properties in fish and shrimp culture . The phytochemical
compounds rich in banana peel possess multiple biological and pharmacological effects.
Additionally, the various parts of banana plant performed the inhibitory effect against food
borne pathogens to be considered to be a potential natural source of antimicrobial and
antioxidant agents . In our previous study, the BPE inhibited growth of aquatic animals’
pathogens, and strengthened anti-hypothermal stress as well as enhanced immune responses
ACCEPTED MANUSCRIPT and resistance of M. rosenbergii to L. garvieae via injection and oral administration for 32
days of feeding trial [14,15]. In the present study, the similar outcomes were recorded
through enhanced immunological responses and resistance to pathogen infection as well as
increase in immune genes expressions when M. rosenbergii fed with the diets containing BPE
for 120 days of feeding trial, and meantime, the growth performance was improved . The
results suggest that, BPE as an immunostimulant not only to improve the status of
immunological responses and physiological regulation via injection or diet administration,
but also to provide a better diet administration for a long term feeding trial which is an
important issue in the present aquaculture industry.
In conclusion, the results demonstrated that M. rosenbergii fed with the diets containing
6.0 g kg-1 BPE for 120 days showed an increase in the immune responses by increasing its
haemocytes count of circulation, SOD activity, PO activity and proPO system-related genes
expression, TG activity and gene expression, and crustin mRNA expression as well as
phagocytic activity and clearance efficiency against L. garvieae, which bring an increased
resistance to L. garvieae infection.
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This work was financially supported by a grant (NSC102-2622-B-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
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Table 1. Composition of the basal diet (g kg-1) for Macrobrachium rosenbergii. Banana Musa acuminate hot-water extract in diet (g kg-1) Ingredients Control 1.0 3.0 6.0 470
Fermented soybean meal
M. acuminata extract
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Table 2. The survival rate and relative percentage survival (RPS) of Macrobrachium rosenbegii fed with the diets containing hot-water extract of banana peel for 120 days and then challenged with Lactococcus garvieae. Survival ratio and RPS(%) time after challenge (hrs) Challenge dose Banana peel extract No. of (cfu prawn-1) (g kg-1) Prawn 12 24 48 72 96 144 saline 0 (control) 30 100 100 100 100 100 100 5 a a b c c 2×10 0 (control) 30 100±0.0 80.0±0.00 70.0±0.0 40.0±0.0 10.0±0.0 10.0±0.0c
Data in the same column with different letters are significantly differ (p Long term feeding with the diet containing banana peels extract enhanced resistance of prawns. > The immunological responses up-regulated post feeding the diet containing banana peels extract. > Diet supplemented with banana peels extract increased the immune genes expressions.
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> Dietary banana peels extract administration was applicable for long term feeding trial.