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Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors
Fish & Shellfish Immunology 27 (2009) 341–348
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Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors Huynh Minh Sang*, Le Trung Ky, Ravi Fotedar Aquatic Sciences, Agriculture and Environment, Curtin University of Technology, 1 Turner Avenue, Technology Park, Bentley, Western Australia 6102, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2009 Received in revised form 1 June 2009 Accepted 4 June 2009 Available online 16 June 2009
Three trials were conducted to determine the effects of mannan oligosaccharide (Bio-MosÒ) on the immune responses of marron. In the first trial marron were challenged with Vibrio mimicus infection, in the second with NH3 exposure and in the third, the marron were exposed to air during a simulated live transportation trial. For V. mimicus infection and live transportation trials, marron (10.44 0.20 g and 4.44 0.20 g initial weights, respectively) were fed three different diets containing 0% (control diet), 0.2% and 0.4% Bio-MosÒ for 30 days and 112 days respectively before challenge, whereas for the NH3 exposure trial, marron (94 2.17 g initial weight) were reared with the control diet and 0.4% Bio-MosÒ diet for 42 days before exposure to NH3. Marron were examined for survival and total haemocyte count (THC), differential haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability as indicators of immune responses during the course of the challenge. Survival of marron infected with bacteria and exposed to NH3, were significantly improved when fed Bio-MosÒ. THCs were significantly reduced in marron fed the control diet when they were infected with bacteria and subjected to live transportation while it remained unchanged in the marron fed the Bio-MosÒ supplemented diets. THCs of marron fed any of the diets were reduced when they were exposed to NH3 but the THCs were higher (P < 0.05) in marron fed Bio-MosÒ diets. Vibrio spp. in haemolymph of marron fed the control diet significantly increased when they were infected with V. mimicus and challenged with NH3 but it remained unchanged in the marron fed the Bio-MosÒ diets. Haemolymph clotting time was higher in marron fed the control diets when subjected to live transportation and 3 days of exposure to NH3. After 96 h infection marron fed the Bio-MosÒ diets had longer NRR time than those fed the control diet. All the findings demonstrated the ability of Bio-MosÒ to improve the survival, health status and immunity of marron under the bacterial infection and stress conditions caused by air and NH3 exposures. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: Bio-MosÒ Immune response Mannan oligosaccharide Marron Prebiotic
1. Introduction Bacterial and viral diseases are known to be the major constraints in the aquaculture industry in recent years [1] and are impeding the development and sustainability of the industry throughout the world [2]. Thus, the need to control the loss due to disease outbreaks in aquaculture is becoming increasingly important. In most cases, water is disinfected before use and antibiotics are used to protect cultured species from microbial infections [3]. Such practices have led to promote the spread of antibiotic-resistant pathogens in both cultured species and in the environment * Corresponding author. Aquatic Science Research Unit, Curtin University of Technology, Brodie Hall Building, 1 Turner Avenue, Technology Park, Bentley, Western Australia 6102, Australia. Tel.: þ61 92664508; fax: þ61 92664422. E-mail address: [email protected] (H.M. Sang).
[4,5]. In addition, the application of such practices is expensive and undesirable due to having harmful effects to the environment and human health [6]. In addition, the global demand for safe food has prompted the search for natural, alternative growth promoters to be used in aquatic animal feeds. Recently, immunostimulants such as probiotics and prebiotics have shown promise as preventive and environmentallyfriendly alternatives to antibiotics in aquaculture, especially for fishes and crustacea [7–9]. However, the application of prebiotics, classified as non-digestible food ingredients that beneficially affect the host by stimulating growth and/or activity of a limited number of bacteria in the intestine, is in its infancy with crustacea, compared with the progress that has been made in the development of prebiotics for poultry [10]. Thus, there is an increased attention on the use of prebiotics as a key strategy to maintain and expand the aquaculture industries in recent years [11].
1050-4648/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.06.003
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H.M. Sang et al. / Fish & Shellfish Immunology 27 (2009) 341–348
As a common prebiotic, Mannan oligosaccharide (MOS) derived from the cell wall of Saccharomyces cerevisiae, has recently been used in poultry husbandry and in aquaculture as a diet supplement [12]. This compound has been shown to affect gut health by improved adsorption and immune modulation in the target species. In finfish aquaculture, incorporation of MOS in the diet has promoted resistance to bacterial infection and stressors of common carp (Cyprinus carpio) [13], rainbow trout (Salmo gairdneri irideus G.) [14], channel catfish (Ictalurus punctatus) [15], rainbow trout (Oncorhynchus mykiss) [16] and sea bass (Dicentrarchus labrax) [12]. In crustacean aquaculture the use of MOS has been studied by Genc et al. [11] on Penaeus semisulcatus. Marron (Cherax tenuimanus Smith) is one of the world’s largest freshwater crayfish found in Australia and capable of reaching 2 kg in weight [17]. Native to Western Australia, it has been recognised as a potential aquaculture species due to its attractive attributes [18]. Recently, marron was introduced into South Africa, Zimbabwe, Japan, USA, China and the Caribbean as a commercial aquaculture species [19]. As a result of the national and international interest in marron farming, its distribution has been widely extended [20] and research is in progress related to its aquaculture potential. The area of nutrient manipulations in the formulated diets in order to improve marron productivity has attracted many researchers [21–27]. Despite the available information on the macronutrient requirements of marron, the use of supplemented elements in the diet to improve the health status and immunity of marron is still unknown and there is no record on the use of MOS in the diet of marron. In addition, air exposure has been used as a common stressor to evaluate the health of crab (Cancer pagurus) [28], lobster (Panulirus cygnus) [29], western king prawn (Penaeus latisulcatus) and brown tiger shrimp (Penaeus esculentus) [30]. NH3 at toxic level has been proved to alter the physiological and immunological status of different crustacea such as mud crabs (Scylla serrata) [31], blue swimmer crabs (Portunus pelagicus) [32], etc. However, the effects of this stressor on marron have not been investigated. The present paper aims to evaluate the effectiveness of MOS in enhancing the survival and immunity of marron when exposed to bacteria, NH3 and air. Total haemocyte count (THC), different haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability of haemocyte were used as tools to evaluate the immune responses of marron.
Table 1 Ingredients of the test diets. Ingredients
Test diets Control (%)
0.2% Bio-MosÒ (%)
0.4% Bio-MosÒ (%)
Fish oil Soybean meal Fishmeal Wheat flour Ascorbic acid Betaine Calcium ascobate Premix Cholesterol Wheat starch Bio-MosÒ Total
3.20 10.15 33.78 49.35 0.05 1.20 0.02 0.15 0.25 1.85 0.00 100
3.23 10.14 33.83 49.08 0.05 1.20 0.02 0.15 0.25 1.85 0.20 100
3.23 10.14 33.86 48.85 0.05 1.20 0.02 0.15 0.25 1.85 0.40 100
2.2. Experimental animals Marron were supplied by Aquatic Resource Management Pty Ltd, Western Australia and shipped to the Curtin Aquatic Research Laboratory (CARL). The marron were stocked in tanks provided with aerated, recirculating filtered freshwater and were then acclimated to the culture conditions for 2 weeks. During the acclimation period, the marron were fed with a commercial diet supplied by Enviroplus, Australia (26% protein, 47–50% carbohydrate, 9% fats and 8.9% ash) at a rate of 3% total biomass per two days. 2.3. Culture system Plastic cylindrical tanks (800 mm diameter, 500 mm high, and 250 L capacity) were used for the trials. Sufficient PVC pipes and oyster net of appropriate sizes were placed in each tank to provide shelters for the marron. The tank was supplied with aerated recirculating freshwater at a rate of approximately 3 L min1. The water in each tank was filtered through both mechanical and biological filters. 2.4. Feeding and challenge of marron with various stressors A total of 3 independent trials were conducted one after the other. Each trial involved the exposure of the selected stressor to marron under laboratory conditions.
2. Materials and methods 2.1. Test diets Mannan oligosaccharide (Bio-MosÒ, Alltech Inc, USA) which is derived from the cell wall of S. cerevisiae, containing minimum of 30% protein, 1.4% crude fat and maximum of 13% crude fiber was supplemented at two levels of 0.2% and 0.4% to a basal control diet (0% Bio-MosÒ). The concentrations of Bio-MosÒ at 0.2% and 0.4% are the most effective on performance of marron (unpublished data). All test diets contained approximately 34% crude protein, 8% crude lipid, 6% ash, which is considered nutritionally adequate for the growth of juvenile marron [23]. The proximate compositions of the ingredients (supplied by Specialty Feeds Pty Ltd, Western Australia) and Bio-MosÒ (supplied by Alltech Inc, USA) were used as a basis to form the required diets using the software FeedLIVE version 1.52 (Table 1). The pellets of 1 mm diameter were prepared by mixing the feed ingredients through a mince mixer and then drying them in the direct sunlight for 6 h. The dried pellets were then allowed to cool at room temperature for 30 min prior to packaging and storing in a dark room.
2.4.1. Challenge with bacteria – trial 1 Total 9 culture tanks were used for this trial. Each tank was stocked with 15 marron (10.44 0.20 g initial mean weight). Each of three test diets was randomly assigned to three tanks, giving three replicates per diet. The marron were provided with the food every two days at the initially feeding rate of 5% of the body weight which was thereafter adjusted for each tank according to feeding response. Uneaten food and faeces were removed before every feeding. Preparation of bacterial stock suspension: Stock suspension of Vibrio mimicus (isolated from blisters of dead yabbies, Cherax albidus) was obtained from the Department of Agriculture, Western Australia. The concentration of stock suspension was approximately 0.53 106 cfu/mL. Challenge test was initiated on day 30 of the feeding trial. Six marron from each test diet-tank were injected through the base of the fifth thoracic leg with 20 mL bacteria stock suspension. The injected marron were marked before releasing back into their original tanks to avoid repeat-sampling. The infected marron were monitored for survival, total haemocyte count, differential haemocyte count, haemolymph clotting time, bacteraemia and lysosomal membrane stability after 24, 48 and 96 h of injection.
H.M. Sang et al. / Fish & Shellfish Immunology 27 (2009) 341–348
2.4.2. Exposure to air during simulated transport – trial 2 Juvenile marron (4.44 0.20 g total weight) after routine acclimation were distributed to 9 independent tanks at a density of 15 marron per tank. Each random block of three tanks was provided with one diet so that each test diet was represented in triplicate. After 112 days of culture, the marron were subjected to a simulated live transport. From each dietary group, twelve marron (4 from each replicate) were tagged with nail polish and put in a polystyrene box (60 40 30 cm) for 36 h. The box had two ice-gel bags covered by a foam layer. Marron were placed on this foam and then covered by another layer of foam. The box was then covered by a lid and sealed. After 36 h in the box, marron were placed back into their culture medium for 6 h and then examined for total haemocyte count, granular cell and haemolymph clotting time. 2.4.3. Exposed to NH3 – trial 3 Marron (94 2.17 g total weight) were stocked into 6 tanks at a density of 8 marron per tank. Three tanks were fed with the BioMosÒ-free diet and the three remaining tanks were fed with the 0.4% Bio-MosÒ supplemented diet for 42 days. The marron in each tank were then exposed to NH3 by adding NH4Cl into the culture media to obtain NHþ 4 concentration of 20 ppm which is equal to a concentration of 1.37 ppm of NH3 at the temperature of 16.8 C and the pH of 8.4 [33]. Survival, total haemocyte count, differential haemocyte count, haemolymph clotting time, bacteraemia and osmoregulatory capacity after 1 day, 3 days and 7 days of NH3-exposure was measured. 2.5. Data collection 2.5.1. Survival Survival rate of marron in all tanks was measured using the following calculation: S [ 1003ðnt =no Þ Where: S is the survival rate; nt is the number of marron at time t and no is the number of marron at the commencement of the challenge. 2.5.2. Total haemocyte count (THC) and differential haemocyte count (DHC) Total and differential haemocyte counts were conducted as per the established procedure for rock lobster with some modification [29]. The base of the fifth thoracic leg of each marron from each culture tank was cleaned with 70% alcohol. A 0.2 mL aliquot of haemolymph was withdrawn into a 1-mL sterile syringe containing 0.2 mL of anticoagulant (1% glutaraldehyde in 0.2 M sodium cacodylate) and dispensed into an Eppendorf tube kept on ice. Total haemocyte counts for individual marron were estimated with a haemocytometer (Neubauer, Germany) under 100-fold magnification, from the anticoagulant/haemolymph mixture. Cells were counted in both grids, and the mean was used as the haemocyte count. The total haemocyte count was calculated as THC ¼ (cells counted dilution factor 1000)/volume of grid (0.1 mm3). For differential haemocyte counts, one drop of haemolymph/ anticoagulant mixture was placed on a slide and smeared. The smear was air dried and fixed in 70% methanol for 10 min. Fixed smears were stained with May-Grunwald and Giemsa (10 min in each) (Bancroft and Stevens, 1977) and mounted with a coverslip. A total of approximately 200 cells were counted on each slide. The granular cells were distinguished on the basis of the larger cell size, smaller pale nucleus, and larger number of eosinophilic granules in the cytoplasm, and their proportion to overall haemocytes was calculated. Hyaline cells had round shape, big nucleus and little or no cytoplasm; while semi-granular cells had a longer shape, big nucleus and little or no cytoplasm.
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2.5.3. Bacteraemia assessment Bacteraemia assessment was performed based on the established procedure [29] with some modification. An 0.05 mL aliquot of haemolymph was withdrawn into a sterile syringe and quickly placed as small drops and smeared on a nutrient agar (NA) plate [34]; the plate was carefully inverted before putting into the incubator chamber at temperature of 25 C for 24 h. Colony forming units (CFU) were counted for each plate and CFU/mL calculated for each sample on the basis of a total volume of 0.05 mL/plate. The CFU/mL were ranked 1 (1–250 CFU/mL) to 12 (2751–3000 CFU/mL), and a final rank of 13 was assigned to those samples in which the colonies were too numerous for an accurate count. 2.5.4. Haemolymph clotting time Haemolymph clotting time was determined following the protocol described by Fotedar et al. [29] with some modification. Haemolymph withdrawn into a sterile syringe was dispensed into an Eppendorf tube. A 30 mL aliquot was quickly transferred into another tube and drawn into a capillary tube. The tube was repeatedly inverted until the haemolymph stopped moving, and the time was noted as haemolymph clotting time. 2.5.5. Neutral red retention assay The lysosomal membrane stability in haemocytes of marron was assessed using neutral red retention assay based on an established protocol [35] with some modification. A stock solution of neutral red was produced by dissolving 10 mg of neutral red powder in 1 mL of dimethyl sulphoxide. The working solution (dye concentration 0.02 mg/mL) was prepared by diluting 10 mL of the stock solution with 5 mL of artificial saline water. A 0.2 mL aliquot of marron haemolymph sample was transferred into an eppendorf tube containing 0.2 mL artificial saline water and gently mixed. The mixture of haemolymph sample and artificial saline water was placed onto a microscope slide treated with a poly-L-lysine solution to enhance cell adhesion. The slide was immediately placed in a 10 C incubator for 15 min to allow the haemocytes to attach to the slide. The slide was removed from the incubator and the excess haemolymph was removed. 40 mL of neural red working solution was added to the slide and the sample was covered with a coverslip. The slide was then returned to the incubator. Every 15 min the slide was taken out and the sample was examined using a microscope. The time at which 50% of the haemocytes had started to lose dye from their lysosomes was recorded as the neutral red retention time of the marron lysosomal membrane. 2.6. Data analysis A multiple comparison test – Least significant different (LSD) and an Independent-sample T test were used to examine the significant difference among treatments using the SPSS version 15.0. Percent data were normalized using an arcsine transformation before analysis. For significant difference, it was required that P < 0.05. One way ANOVA was analyzed and results were expressed as mean standard error in the figures and tables. 3. Results 3.1. Trial 1 – challenge with bacteria Survival: Bio-MosÒ supplemented diets resulted in lower (P < 0.05) marron mortalities when injected with V. mimicus (Fig. 1) compared to marron fed the control diet. 3.1.1. Total and differential haemocyte count There was no significant difference (P > 0.05) in mean THCs of marron fed any of the diets before challenging with the V. mimicus.
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100
Survival (%)
90
b b
80 70
a 60 50
0
24
48
96
Time after infection (h) Control
0.2% BioMos
0.4% BioMos
Fig. 1. Mean SE survival of marron challenged with Vibrio mimicus. Different letters indicate significantly different means at each time at P < 0.05 (n ¼ 18).
24 h after the challenge, the mean THCs of the marron fed the control diet was significantly reduced (P < 0.05) while mean THCs of the marron fed 0.2% Bio-MosÒ and 0.2% Bio-MosÒ diets remained unchanged. After 24 h of bacterial challenge, mean THCs was significantly lower (P < 0.05) in the marron fed the control diet than in the marron fed the Bio-MosÒ diets. However, at 48 h after the challenge, there was no significant difference (P > 0.05) in mean THCs of the marron fed any of the test diets. After 96 h of the challenge, the highest THC was in marron fed 0.4% Bio-MosÒ diet (Table 2). Before the V. mimicus injection, mean proportion of granular cells was significantly lower (P < 0.05) in the marron fed the control
diet than in the marron fed the Bio-MosÒ diets. At 24 h after the injection, there were decreases in mean proportions of granular cells of marron in all treatments but the significant decline (P < 0.05) was only found in marron fed the control diet resulting in the significantly lower proportion of granular cells in the marron fed the control diet. Semi-granular cells were not significantly different between the marron fed the control and the Bio-MosÒ diets and the proportion of these cells remained unchanged during the V. mimicus challenge. However, the proportion of hyaline cells showed the opposite trend to granular cells during the challenge (Table 2). 3.1.2. Haemolymph clotting time There was no significant difference (P > 0.05) in mean haemolymph clotting time of the marron fed any of the diets before and after the V. mimicus injection (Table 2). 3.1.3. Bacteraemia Vibrio spp. in the haemolymph of marron fed the control diets significantly increased (P < 0.05) after 24 and 48 h of V. mimicus injection while there was no significant difference in haemolymph Vibrio spp. of marron fed the Bio-MosÒ diets before and after the injection. The significant difference (P < 0.05) in the Vibrio spp. numbers in the haemolymph of marron fed the control and the Bio-MosÒ diets was observed after 48 h of infection (Table 2). 3.1.4. Neutral red retention time Before V. mimicus injection, NRR times of marron lysosomal membranes in all treatment groups were more than 120 min. 24 h after the injection, the mean NRR times of lysosomal membranes in all the injected marron sharply decreased to less than 30 min. At 48 h after the injection, while the mean NRR time of the marron fed
Table 2 Mean SE immune parameters of marron challenged with Vibrio mimicus. Parameters
Time after infection (h)
Test diets
THCs (million cells/mL)
0* 24 48 96
21.73 122.85 122.30
0.47a 0.04a 0.62a 0.54a
13.66
0* 24 48 96
135.40
0.57a 0.33a 0.60a 1.0a
114.64
Control
Granular cells (%)
Semi-granular cells (%)
Hyaline cells (%)
Vibrio rank
Haemolymph clotting time (s)
NRRT (mins)
0* 24 48 96 0* 24 48 96 0* 24 48 0* 24 48 96 0* 24 48 96
13.26
36.86 123.73 21.86
18.80
110.73 17.16 16.47
0.2% Bio-Mos
0.16a 2.63a 2.0a 2.51a
14.56 13.70 12.91
110.78 114.18 113.11
17.38
110.81 17.05
113.80
0.4% Bio-Mos
0.04a 0.44b 1.08a 0.41a
13.33 13.90
0.81b 3.32b 0.69b 2.54b
1214.05 211.70
13.77
13.67
120.09
1213.35
215.04 15.92
2.19a 2.57a 1.73a 0.38a
2.48ab 0.25b 1.94ab 0.74b
169.96
4.37b 3.90b 1.13b 2.16b
129.95 14.88
1277.97
11.00
0.00a 0.33a a 310.00 0.00
11.00
11.00
22.33
11.33
11.00
3.51a 5.17a 9.06a 13.2a
172.33
182.4 1289.1
291.66
174.00
172.67 165.33
166.33
>120 a 123.33 3.33 a 115.00 0.00 a 240.00 5.00
278.4 278.75 173.08
0.00a 0.33a b 11.00 0.00 158.67 166.33 157.33
2.20c 2.93b 1.34c 1.78b
1.74a 3.09a 2.58a 3.28a
0.73a 2.70a 1.68a 2.26a
1285.79
0.2a 0.45b 0.26a 0.11b
21.53a 6.06a 10.68a 2.96a
>120 a 126.67 3.33 b 255.00 7.64 b 3111.67 4.4
170.90 282.37 281.76
0.00a 0.00a b 11.67 0.67
172.67
144.67 153.33 159.67
10.27a 1.76a 4.98a 3.28a
>120 a 126.67 3.33 b 253.33 1.67 b 393.33 8.82
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. 0* is the time before infection.
H.M. Sang et al. / Fish & Shellfish Immunology 27 (2009) 341–348
100 90
Survival (%)
the control diet still declined, the lysosomal membrane stability of the marron fed the Bio-MosÒ started recovering, especially the mean NRR time of the marron fed the Bio-MosÒ diets increased significantly (P < 0.05). At 96 h after the injection, the lysosomal membrane stability of the marron fed the control diet began to recover but the mean NRR time of the lysosomal membrane was significantly shorter (P < 0.05) than the mean NRR time of the marron fed the Bio-MosÒ diets. Lysosomal membrane stability of the marron fed the 0.2%-Bio-MosÒ diet recovered significantly faster (P < 0.05) than that of the marron fed the control diet.
345
80
a
70 60
b
50 40 30
3.2. Trial 2 – exposure to air during simulated transport
0
1
3
7
Exposure time (day) 3.2.1. Survival After 36 h of simulated transport, no mortalities were observed in any group. 3.2.2. Total and differential haemocyte count THCs of marron fed control diets significantly declined (P < 0.05) after 36 h of transportation while it remained unchanged in the marron fed Bio-MosÒ diets. Before subjection to simulated transportation stress, granular cells proportion was lower in the marron fed the control than the Bio-MosÒ diets and it did not change after the exposure to the transport stressor in marron fed any of the diets (Table 3). 3.2.3. Haemolymph clotting time The haemolymph clotting time of marron fed the control and 0.2% Bio-MosÒ diets significantly reduced after the simulated transportation and it was unchanged in the marron fed 0.4% BioMosÒ diet (Table 3). 3.3. Trial 3 – exposure to NH3 3.3.1. Survival After 7 days of exposure to NH3, survival of marron fed 0.4% BioMosÒ diet was significantly higher (P < 0.05) than that of marron fed the control diets (Fig. 2). 3.3.2. Total and differential haemocyte count THCs and DHC of marron fed the control and 0.4% Bio-MosÒ diets were not significantly different before exposure to NH3. After 1, 3 and 7 days, exposure, THCs of marron fed 0.4% Bio-MosÒ diet was higher (P < 0.05) than marron fed the control diets whereas there was no difference (P > 0.05) among the proportions of granular cells, semi-granular and hyaline cells when marron were fed different diets. THCs of marron fed either the control or Bio-MosÒ supplemented diets significantly reduced after 1, 3 and 7 days of exposure while the proportion of different cells remained unchanged until
Table 3 Mean SE immune parameters of marron challenged after subjected to simulated live transportation. Parameters
Time
13.39
0.2% Bio-Mos a
day 7 when the proportion of granular cells decreased and the proportion of hyaline cells increased (Table 4). 3.3.3. Haemolymph clotting time When exposure to NH3, haemolymph clotting time of marron fed either the control or 0.4% Bio-MosÒ diets significantly increased. After 3 days of exposure, haemolymph clotting time of marron fed 0.4% Bio-MosÒ diet was shorter than marron fed the control diet (Table 4). 3.3.4. Bacteraemia Bacteraemia of marron fed control diets significantly increased (P < 0.05) after 1 day exposure to NH3 whereas there was no change in bacteraemia of marron fed 0.4% Bio-MosÒ diets during 7 days of NH3 exposure. Table 4 Mean SE immune parameters of marron exposed to NH3. Parameters
Exposure time (day)
THCs (million cells/mL)
0* 1 3 7
24.42 31.67 31.11
0* 1 3 7
124.91 213.60
0* 1 3 7
10.58 22.06
0* 1 3 7
174.50 184.33
Granular cells (%)
Semi-granular cells (%)
Hyaline cells (%)
0.16 0.24a
14.43
1.25a 0.98a
122.19
THCs (million cells/mL)
BF AT
Granular cells (%)
BF AT
116.46
BF AT
2.08a 69.00 1.00a 2
22.56
115.79 146.00
13.57
121.12
0.18 0.42b 0.53b 0.34b
1.73b 43.00 1.66b 2 136.00
Vibrio rank
0.4% Bio-Mos b
14.06 14.11 123.41 122.27
b
0.24 0.42b 0.25b 1.42b
1.15b 35.33 3.18c 1
138.00
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. BF: before and AT: after transportation.
0.4% Bio-Mos®
Fig. 2. Mean SE survival of marron challenged with NH3. Different letters indicate significantly different means at each time at P < 0.05 (n ¼ 21).
Test diets Control
Haemolymph clotting time (min)
Control
Haemolymph clotting time (s)
0* 1 3 7 0* 1 3 7
Diets Control 19.87
121.27
121.59
10.39
10.38
0.4% Bio-Mos 0.91a 0.42a 0.24a 0.06a
111.10
1.03a 0.87a 6.62a 2.75a
125.66
0.39a 0.11a 0.35a 0.47a
10.42
32.87 32.22 26.27
129.13 212.90
120.54
10.47 10.81 10.47
0.64a 0.94a 6.33a 2.91a
170.38 286.28
27.66 210.33
1.00a 1.52a 0.66a 1.45a
14.00 16.00
141.00
9.02a 11.62a 6.87a 24.31a
178.33
178.02
13.00
210.00
286.33 2101.17
1280.50
173.93
171.98
12.33
12.33
1
39.17 1261.66 272.50 1255.17
1.74a 0.20b 0.20b 0.03b 3.93a 2.65a 5.42a 0.43a 0.20a 0.25a 0.36a 0.32a 4.04a 2.53a 5.23a 0.75a 0.88a 1.33b 1.52a 1.00b 4.47a 7.33a 2.31b 7.40a
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. 0 *is the time before exposure.
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4. Discussion The results of the current study indicate that application of dietary Bio-MosÒ can significantly improve the marron’s ability to resist stressors such as bacterial infection, exposure to NH3 and live transport indicated by the higher survival and positive immune responses. Similar effects have been described for sea bass [12] and cobia larvae [36] when they were fed dietary Bio-MosÒ. Improved survival of marron when exposed to stressors may be a direct consequence of enhanced health status and positive immune responses of marron as it has been shown in other aquaculture species [15]. The similar outcome is also suggested by improvement in the health of the digestive system of animals fed Bio-MosÒ [11,13,16,36]. Our unpublished data has further confirmed that a better condition of the digestive system, improved immune indicators for better health status of the marron is achieved when fed 0.2%–0.4% Bio-MosÒ supplemented diets. Bio-MosÒ supplementation in the diets has also been previously reported to promote resistance to bacterial infection and stressors in common carp (C. carpio) [13], rainbow trout (S. gairdneri irideus G.) [14], channel catfish (I. punctatus) [15], rainbow trout (O. mykiss) [16] and sea bass (D. labrax) [12]. Total number of haemocytes, proportion of haemocyte types, bacteraemia and haemolymph clotting time have been used as indicators of health of crustacea [29,37–44]. Recently, neutral red retention (NRR) assay has also been used to assess the immune status of shellfish by measuring the dye retention time of lysosomal membranes [45]. Neutral red is a cytotoxic weak base [46] and has been successfully used in the neutral red retention (NRR) assay as a biomarker to test the stability of lysosomal membranes in aquatic animals [35,47–51]. In decapod crustacea, life cycle, food intake, disease outbreaks, pollutants and environmental stress affect the circulating haemocyte count both in quantity and quality [52–54]. In the present study, all marron tested were in the intermolt stage, thus considered to have similar physiological and/or immunological status. Our unpublished data have shown that Bio-MosÒ supplemented diets have improved the immune response of marron when it was applied for the duration of 112 days. In addition, dietary Bio-MosÒ has been approved to have effects on immune indicators of rainbow trout (O. mykiss) after 42 days [16] and sea bass (D. labrax) after 72 days [12] application. The changes in the THCs and DHCs of the marron after exposure to stressors in the current trials imply that bacterial infection, NH3 and air exposure during simulated live transportation could influence the circulating haemocyte counts of marron. After the V. mimicus injection, THCs and proportion of granular cells of marron fed the control diet dropped significantly. The same observations have been reported in black tiger prawn (Penaeus monodon) [55] Chinese shrimp (Fenneropenaeus chinensis) [45] when they are injected with Vibrio anguillarum. The decline in THC after V. mimicus injection could be associated with haemolymph locomotion to the injection site and haemocyte lysis as a result of defense activities [55,56]. However, THC of marron fed the Bio-MosÒ diets did not decrease after the Vibrio injection. Sequeira, Tavares & Arala-Chaves [57] found that Penaeus japonicus haemocytes have the capacity to proliferate, and the proliferation rate can be increased three times when the shrimp are injected with an immunostimulant, lipopolysaccharide. Our previous study (unpublished) also showed that THC in marron increased significantly after feeding with Bio-MosÒ. In the present study, the incorporation of Bio-MosÒ in diets may stimulate and increase the proliferation rate of marron haemocytes to compensate for the loss of haemocytes due to the V. mimicus infection, resulting in the THC proportions to remain constant. The present study also reveals that exposure to NH3 at the concentration of 1.37 mg L1 reduces the THCs of marron.
Circulating haemocytes of several species of crustacean can be affected by extrinsic factors like temperature, pH, salinity, dissolved oxygen and ammonia [58–60]. The decrease in THC is frequently reported in crustacea exposed to stress conditions. Litopenaeus stylirostris following exposure to ammonia at 3.0 mg L1 decreased its THC by 30% [52], Chinese mitten-handed crab (Eriocheir sinensis) reduced the THC indicating suppressed resistance when exposure to ammonia [61]. These reductions in THC relate to the accumulation of haemolymph ammonia and urea, and causes the catabolism of haemocyanin and protein to amino acids [62]. However, the marron fed Bio-MosÒ diets were healthier as shown by lowered reduction rate (80%) of THC compared to the marron fed the control diet (88.75%) after 7 days of exposure to the stressor. Air exposure has been demonstrated to reduce the health status of rock lobster (Panucilus lygnus) [29]. Trial 2 showed that the marron fed BioMosÒ diets are healthier than marron not fed Bio-MosÒ as indicated by the higher THC after 36 h of simulated live transport. It is also inferred from current results that the significant reduction in the THCs of marron fed the diets without Bio-MosÒ supplementation when challenged with bacteria and NH3 exposure has negatively affected the ability to combat the bacterial infection [63,64], thus, resulting in the higher bacteraemia. Crustacean haemolymph clotting involves aggregation of the circulating haemocytes and the gelation of the plasma by factors released from the haemocytes [65]. Haemolymph clotting time has been reported to be changed by the presence of bacteria [66,67], environmental pollutants [68] or holding period in storage tanks and live transportation [39]. In the current experiment, haemolymph clotting time of marron was not affected by V. mimicus injection. Although Durliat & Vranckx [37] found a relationship between hemolymph clotting time and number of haemocytes in lobsters, the authors stated that haemocyte numbers could not be explained by the changes in hemolymph clotting time. To date, little is known about the mechanism by which the changes in crustacean haemolymph clotting time are induced. However, challenging with NH3 and air exposure has affected the haemolymph clotting time of marron. Thus, further research needs to be conducted to define the mechanism in which pollutants, nitrogen metabolites and air exposure stressors affect the clotting time of marron. Marron fed the Bio-MosÒ diet showed the shortest clotting time after 3 days of NH3 exposure and 36 h of air-exposure indicating the ability of Bio-MosÒ to improve initial immune defense activity of marron under these stress conditions. Neutral red is a cytotoxic weak base [46] and has been successfully used in the neutral red retention (NRR) assay as a sensitive indicator of lipid membrane integrity in cases of bacterial infection and immunostimulant toxicity in shellfish. [35,47–51]. Lysosomes found within the semi-granular and granular haemocytes are polymorphic, hydrolytic enzyme-containing organelles that have the capacity to take up and retain neutral red dye. The dye disappears when lysosomal membranes are damaged [46] and unhealthy cells lose neutral red dye at a faster rate than healthy cells due to a decrease in cellular membrane stability [69,70]. In this study, NRR assay was successfully developed for the detection of lysosomes of haemocytes in the marron. Without any stimulation, the NRR time of marron lysosomal membrane in the current experiment was more than 120 min. The injection of V. mimicus altered the stability of the lysosomal membrane of marron as it showed a rapid decline in NRR time. Similarly, NRR time of Chinese shrimp was significantly shortened after Vibrio injection [45]. In marron fed the diet without Bio-MosÒ, the decrease in lysosomal membrane stability is related with the decrease in THC, granular cell count (GC) and semi-granular cell count (SGC) after the injection. A similar phenomenon was found in freshwater prawn (Macrobrachium rosenbergii) [71], oysters (Ostrea edulis L. and Crassostrea gigas (Thunberg)) and the scallop
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(Pecten maxim’s) [47] suggesting that GC and SGC might degranulate at first, followed by lysis of the lysosomes [45]. After 48 h of the injection, the lysosomal membrane stability recovery was significantly faster in marron fed the Bio-MosÒ diets than in those fed the control diet, indicating that Bio-MosÒ as supplementation in diets could have improved the resistance of marron against V. mimicus by hastening the recover rate of lysosomal membrane stability. In conclusion, immunological responses to dietary Bio-Mos supplementation have shown that Bio-MosÒ improves the survival of the marron, improves the health status and increases marron’s defense ability to bacterial infection and other stress conditions. Further research needs to be conducted to clarify the mechanisms of Bio-MosÒ action(s) in the health and immunity improvements of marron. Acknowledgement We thank Alltech Inc, USA for the financial support. We also thank Rocı´o Gonza´lez Lo´pez for assistance with the experimentation. References [1] Scholz U, Garcia Diaz G, Ricque D, Cruz Suarez LE, Vargas Albores F, Latchford J. Enhancement of vibriosis resistance in juvenile Penaeus vannamei by supplementation of diets with different yeast products. Aquaculture 1999;176: 271–83. [2] Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, et al. Disease and health management in Asian aquaculture. Vet Para 2005;132:249–72. [3] Vadstein O. The use of immunostimulation in marine larviculture: possibilities and challenges. Aquaculture 1997;155:401–17. [4] Gatlin III DM. Nutrient and fish health. In: Halver JE, Hardy RW, editors. Fish nutrient. San Diego, CA: Academic Express; 2002. p. 671–702. [5] Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 2008;274:1–14. [6] Capone DG, Weston DP, Miller V, Shoemaker C. Antibacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 1996;145:55–75. [7] Bachere E. Anti-infectious immune effectors in marine invertebrates: potential tools for disease control in larviculture. Aquaculture 2003;227:427–38. [8] Campa-Co´rdova AI, Herna´ndez-Saavedra NY, De Philippis R, Ascencio F. Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to [beta]-glucan and sulphated polysaccharide. Fish Shellfish Immunol 2002;12:353–66. [9] Soltanian S, Thai TQ, Dhont J, Sorgeloos B, Bossier P. The protective effect against Vibrio campbellii in Artemia nauplii by pure b-glucan and isogenic yeast cells differing in b-glucan and chitin content operated with a source-dependent time lag. Fish Shellfish Immunol 2007;23:1003–11. [10] Patterson JA, Burkholder KM. Application of prebiotics and probiotics in poultry production. Poult Sci 2003;82:627–31. [11] Genc MA, Aktas M, Genc E, Yilmaz E. Effects of dietary mannan oligosaccharide on growth, body composition and hepatopancreas histology of Penaeus semisulcatus (de Haan 1844). Aquacult Nut 2007;13:156–61. [12] Torrecillas S, Makol A, Caballero MJ, Montero D, Robaina L, Real F, et al. Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol 2007;23:969–81. [13] Staykov Y. The influence of Bio-Mos on growth rate and immune status of common carp (Cyprinus carpio). In: Alltech’s second annual aquaculture meeting Dunboyne, Co Meath November, oral communication, 2004. [14] Staykov Y, Spring P, Denev S. Influence of dietary Bio-MosÒ on growth, survival and immune status of rainbow trout (Salmo gairdneri irideus G.) and common carp (Cyprinus carpio L.). Bulgaria: Trakia University; 2006. [15] Welker TL, Lim C, Yildirim-Aksoy M, Shelby R, Klesius PH. Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catfish, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. J World Aqua Soc 2007;38:24–35. [16] Staykov Y, Spring P, Denev ES, Sweetman EJ. Effect of a mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquacult Inter 2007;15:153–61. [17] Morrissy NM. Culturing the marvellous marron in Western Australia: 1967–1997. Freshw Crayfish 2000;13:13–38. [18] Rouse DB, Kartamulia I. Influence of salinity and temperature on molting and survival of the Australian freshwater crayfish (Cherax tenuimanus). Aquaculture 1992;105:47–52. [19] Morrissy NM, Evans L, Huner JV. Australian freshwater crayfish: aquaculture species. J World Aquacult Soc 1990;21:113–22.
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Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors Huynh Minh Sang*, Le Trung Ky, Ravi Fotedar Aquatic Sciences, Agriculture and Environment, Curtin University of Technology, 1 Turner Avenue, Technology Park, Bentley, Western Australia 6102, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2009 Received in revised form 1 June 2009 Accepted 4 June 2009 Available online 16 June 2009
Three trials were conducted to determine the effects of mannan oligosaccharide (Bio-MosÒ) on the immune responses of marron. In the first trial marron were challenged with Vibrio mimicus infection, in the second with NH3 exposure and in the third, the marron were exposed to air during a simulated live transportation trial. For V. mimicus infection and live transportation trials, marron (10.44 0.20 g and 4.44 0.20 g initial weights, respectively) were fed three different diets containing 0% (control diet), 0.2% and 0.4% Bio-MosÒ for 30 days and 112 days respectively before challenge, whereas for the NH3 exposure trial, marron (94 2.17 g initial weight) were reared with the control diet and 0.4% Bio-MosÒ diet for 42 days before exposure to NH3. Marron were examined for survival and total haemocyte count (THC), differential haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability as indicators of immune responses during the course of the challenge. Survival of marron infected with bacteria and exposed to NH3, were significantly improved when fed Bio-MosÒ. THCs were significantly reduced in marron fed the control diet when they were infected with bacteria and subjected to live transportation while it remained unchanged in the marron fed the Bio-MosÒ supplemented diets. THCs of marron fed any of the diets were reduced when they were exposed to NH3 but the THCs were higher (P < 0.05) in marron fed Bio-MosÒ diets. Vibrio spp. in haemolymph of marron fed the control diet significantly increased when they were infected with V. mimicus and challenged with NH3 but it remained unchanged in the marron fed the Bio-MosÒ diets. Haemolymph clotting time was higher in marron fed the control diets when subjected to live transportation and 3 days of exposure to NH3. After 96 h infection marron fed the Bio-MosÒ diets had longer NRR time than those fed the control diet. All the findings demonstrated the ability of Bio-MosÒ to improve the survival, health status and immunity of marron under the bacterial infection and stress conditions caused by air and NH3 exposures. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords: Bio-MosÒ Immune response Mannan oligosaccharide Marron Prebiotic
1. Introduction Bacterial and viral diseases are known to be the major constraints in the aquaculture industry in recent years [1] and are impeding the development and sustainability of the industry throughout the world [2]. Thus, the need to control the loss due to disease outbreaks in aquaculture is becoming increasingly important. In most cases, water is disinfected before use and antibiotics are used to protect cultured species from microbial infections [3]. Such practices have led to promote the spread of antibiotic-resistant pathogens in both cultured species and in the environment * Corresponding author. Aquatic Science Research Unit, Curtin University of Technology, Brodie Hall Building, 1 Turner Avenue, Technology Park, Bentley, Western Australia 6102, Australia. Tel.: þ61 92664508; fax: þ61 92664422. E-mail address: [email protected] (H.M. Sang).
[4,5]. In addition, the application of such practices is expensive and undesirable due to having harmful effects to the environment and human health [6]. In addition, the global demand for safe food has prompted the search for natural, alternative growth promoters to be used in aquatic animal feeds. Recently, immunostimulants such as probiotics and prebiotics have shown promise as preventive and environmentallyfriendly alternatives to antibiotics in aquaculture, especially for fishes and crustacea [7–9]. However, the application of prebiotics, classified as non-digestible food ingredients that beneficially affect the host by stimulating growth and/or activity of a limited number of bacteria in the intestine, is in its infancy with crustacea, compared with the progress that has been made in the development of prebiotics for poultry [10]. Thus, there is an increased attention on the use of prebiotics as a key strategy to maintain and expand the aquaculture industries in recent years [11].
1050-4648/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.06.003
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As a common prebiotic, Mannan oligosaccharide (MOS) derived from the cell wall of Saccharomyces cerevisiae, has recently been used in poultry husbandry and in aquaculture as a diet supplement [12]. This compound has been shown to affect gut health by improved adsorption and immune modulation in the target species. In finfish aquaculture, incorporation of MOS in the diet has promoted resistance to bacterial infection and stressors of common carp (Cyprinus carpio) [13], rainbow trout (Salmo gairdneri irideus G.) [14], channel catfish (Ictalurus punctatus) [15], rainbow trout (Oncorhynchus mykiss) [16] and sea bass (Dicentrarchus labrax) [12]. In crustacean aquaculture the use of MOS has been studied by Genc et al. [11] on Penaeus semisulcatus. Marron (Cherax tenuimanus Smith) is one of the world’s largest freshwater crayfish found in Australia and capable of reaching 2 kg in weight [17]. Native to Western Australia, it has been recognised as a potential aquaculture species due to its attractive attributes [18]. Recently, marron was introduced into South Africa, Zimbabwe, Japan, USA, China and the Caribbean as a commercial aquaculture species [19]. As a result of the national and international interest in marron farming, its distribution has been widely extended [20] and research is in progress related to its aquaculture potential. The area of nutrient manipulations in the formulated diets in order to improve marron productivity has attracted many researchers [21–27]. Despite the available information on the macronutrient requirements of marron, the use of supplemented elements in the diet to improve the health status and immunity of marron is still unknown and there is no record on the use of MOS in the diet of marron. In addition, air exposure has been used as a common stressor to evaluate the health of crab (Cancer pagurus) [28], lobster (Panulirus cygnus) [29], western king prawn (Penaeus latisulcatus) and brown tiger shrimp (Penaeus esculentus) [30]. NH3 at toxic level has been proved to alter the physiological and immunological status of different crustacea such as mud crabs (Scylla serrata) [31], blue swimmer crabs (Portunus pelagicus) [32], etc. However, the effects of this stressor on marron have not been investigated. The present paper aims to evaluate the effectiveness of MOS in enhancing the survival and immunity of marron when exposed to bacteria, NH3 and air. Total haemocyte count (THC), different haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability of haemocyte were used as tools to evaluate the immune responses of marron.
Table 1 Ingredients of the test diets. Ingredients
Test diets Control (%)
0.2% Bio-MosÒ (%)
0.4% Bio-MosÒ (%)
Fish oil Soybean meal Fishmeal Wheat flour Ascorbic acid Betaine Calcium ascobate Premix Cholesterol Wheat starch Bio-MosÒ Total
3.20 10.15 33.78 49.35 0.05 1.20 0.02 0.15 0.25 1.85 0.00 100
3.23 10.14 33.83 49.08 0.05 1.20 0.02 0.15 0.25 1.85 0.20 100
3.23 10.14 33.86 48.85 0.05 1.20 0.02 0.15 0.25 1.85 0.40 100
2.2. Experimental animals Marron were supplied by Aquatic Resource Management Pty Ltd, Western Australia and shipped to the Curtin Aquatic Research Laboratory (CARL). The marron were stocked in tanks provided with aerated, recirculating filtered freshwater and were then acclimated to the culture conditions for 2 weeks. During the acclimation period, the marron were fed with a commercial diet supplied by Enviroplus, Australia (26% protein, 47–50% carbohydrate, 9% fats and 8.9% ash) at a rate of 3% total biomass per two days. 2.3. Culture system Plastic cylindrical tanks (800 mm diameter, 500 mm high, and 250 L capacity) were used for the trials. Sufficient PVC pipes and oyster net of appropriate sizes were placed in each tank to provide shelters for the marron. The tank was supplied with aerated recirculating freshwater at a rate of approximately 3 L min1. The water in each tank was filtered through both mechanical and biological filters. 2.4. Feeding and challenge of marron with various stressors A total of 3 independent trials were conducted one after the other. Each trial involved the exposure of the selected stressor to marron under laboratory conditions.
2. Materials and methods 2.1. Test diets Mannan oligosaccharide (Bio-MosÒ, Alltech Inc, USA) which is derived from the cell wall of S. cerevisiae, containing minimum of 30% protein, 1.4% crude fat and maximum of 13% crude fiber was supplemented at two levels of 0.2% and 0.4% to a basal control diet (0% Bio-MosÒ). The concentrations of Bio-MosÒ at 0.2% and 0.4% are the most effective on performance of marron (unpublished data). All test diets contained approximately 34% crude protein, 8% crude lipid, 6% ash, which is considered nutritionally adequate for the growth of juvenile marron [23]. The proximate compositions of the ingredients (supplied by Specialty Feeds Pty Ltd, Western Australia) and Bio-MosÒ (supplied by Alltech Inc, USA) were used as a basis to form the required diets using the software FeedLIVE version 1.52 (Table 1). The pellets of 1 mm diameter were prepared by mixing the feed ingredients through a mince mixer and then drying them in the direct sunlight for 6 h. The dried pellets were then allowed to cool at room temperature for 30 min prior to packaging and storing in a dark room.
2.4.1. Challenge with bacteria – trial 1 Total 9 culture tanks were used for this trial. Each tank was stocked with 15 marron (10.44 0.20 g initial mean weight). Each of three test diets was randomly assigned to three tanks, giving three replicates per diet. The marron were provided with the food every two days at the initially feeding rate of 5% of the body weight which was thereafter adjusted for each tank according to feeding response. Uneaten food and faeces were removed before every feeding. Preparation of bacterial stock suspension: Stock suspension of Vibrio mimicus (isolated from blisters of dead yabbies, Cherax albidus) was obtained from the Department of Agriculture, Western Australia. The concentration of stock suspension was approximately 0.53 106 cfu/mL. Challenge test was initiated on day 30 of the feeding trial. Six marron from each test diet-tank were injected through the base of the fifth thoracic leg with 20 mL bacteria stock suspension. The injected marron were marked before releasing back into their original tanks to avoid repeat-sampling. The infected marron were monitored for survival, total haemocyte count, differential haemocyte count, haemolymph clotting time, bacteraemia and lysosomal membrane stability after 24, 48 and 96 h of injection.
H.M. Sang et al. / Fish & Shellfish Immunology 27 (2009) 341–348
2.4.2. Exposure to air during simulated transport – trial 2 Juvenile marron (4.44 0.20 g total weight) after routine acclimation were distributed to 9 independent tanks at a density of 15 marron per tank. Each random block of three tanks was provided with one diet so that each test diet was represented in triplicate. After 112 days of culture, the marron were subjected to a simulated live transport. From each dietary group, twelve marron (4 from each replicate) were tagged with nail polish and put in a polystyrene box (60 40 30 cm) for 36 h. The box had two ice-gel bags covered by a foam layer. Marron were placed on this foam and then covered by another layer of foam. The box was then covered by a lid and sealed. After 36 h in the box, marron were placed back into their culture medium for 6 h and then examined for total haemocyte count, granular cell and haemolymph clotting time. 2.4.3. Exposed to NH3 – trial 3 Marron (94 2.17 g total weight) were stocked into 6 tanks at a density of 8 marron per tank. Three tanks were fed with the BioMosÒ-free diet and the three remaining tanks were fed with the 0.4% Bio-MosÒ supplemented diet for 42 days. The marron in each tank were then exposed to NH3 by adding NH4Cl into the culture media to obtain NHþ 4 concentration of 20 ppm which is equal to a concentration of 1.37 ppm of NH3 at the temperature of 16.8 C and the pH of 8.4 [33]. Survival, total haemocyte count, differential haemocyte count, haemolymph clotting time, bacteraemia and osmoregulatory capacity after 1 day, 3 days and 7 days of NH3-exposure was measured. 2.5. Data collection 2.5.1. Survival Survival rate of marron in all tanks was measured using the following calculation: S [ 1003ðnt =no Þ Where: S is the survival rate; nt is the number of marron at time t and no is the number of marron at the commencement of the challenge. 2.5.2. Total haemocyte count (THC) and differential haemocyte count (DHC) Total and differential haemocyte counts were conducted as per the established procedure for rock lobster with some modification [29]. The base of the fifth thoracic leg of each marron from each culture tank was cleaned with 70% alcohol. A 0.2 mL aliquot of haemolymph was withdrawn into a 1-mL sterile syringe containing 0.2 mL of anticoagulant (1% glutaraldehyde in 0.2 M sodium cacodylate) and dispensed into an Eppendorf tube kept on ice. Total haemocyte counts for individual marron were estimated with a haemocytometer (Neubauer, Germany) under 100-fold magnification, from the anticoagulant/haemolymph mixture. Cells were counted in both grids, and the mean was used as the haemocyte count. The total haemocyte count was calculated as THC ¼ (cells counted dilution factor 1000)/volume of grid (0.1 mm3). For differential haemocyte counts, one drop of haemolymph/ anticoagulant mixture was placed on a slide and smeared. The smear was air dried and fixed in 70% methanol for 10 min. Fixed smears were stained with May-Grunwald and Giemsa (10 min in each) (Bancroft and Stevens, 1977) and mounted with a coverslip. A total of approximately 200 cells were counted on each slide. The granular cells were distinguished on the basis of the larger cell size, smaller pale nucleus, and larger number of eosinophilic granules in the cytoplasm, and their proportion to overall haemocytes was calculated. Hyaline cells had round shape, big nucleus and little or no cytoplasm; while semi-granular cells had a longer shape, big nucleus and little or no cytoplasm.
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2.5.3. Bacteraemia assessment Bacteraemia assessment was performed based on the established procedure [29] with some modification. An 0.05 mL aliquot of haemolymph was withdrawn into a sterile syringe and quickly placed as small drops and smeared on a nutrient agar (NA) plate [34]; the plate was carefully inverted before putting into the incubator chamber at temperature of 25 C for 24 h. Colony forming units (CFU) were counted for each plate and CFU/mL calculated for each sample on the basis of a total volume of 0.05 mL/plate. The CFU/mL were ranked 1 (1–250 CFU/mL) to 12 (2751–3000 CFU/mL), and a final rank of 13 was assigned to those samples in which the colonies were too numerous for an accurate count. 2.5.4. Haemolymph clotting time Haemolymph clotting time was determined following the protocol described by Fotedar et al. [29] with some modification. Haemolymph withdrawn into a sterile syringe was dispensed into an Eppendorf tube. A 30 mL aliquot was quickly transferred into another tube and drawn into a capillary tube. The tube was repeatedly inverted until the haemolymph stopped moving, and the time was noted as haemolymph clotting time. 2.5.5. Neutral red retention assay The lysosomal membrane stability in haemocytes of marron was assessed using neutral red retention assay based on an established protocol [35] with some modification. A stock solution of neutral red was produced by dissolving 10 mg of neutral red powder in 1 mL of dimethyl sulphoxide. The working solution (dye concentration 0.02 mg/mL) was prepared by diluting 10 mL of the stock solution with 5 mL of artificial saline water. A 0.2 mL aliquot of marron haemolymph sample was transferred into an eppendorf tube containing 0.2 mL artificial saline water and gently mixed. The mixture of haemolymph sample and artificial saline water was placed onto a microscope slide treated with a poly-L-lysine solution to enhance cell adhesion. The slide was immediately placed in a 10 C incubator for 15 min to allow the haemocytes to attach to the slide. The slide was removed from the incubator and the excess haemolymph was removed. 40 mL of neural red working solution was added to the slide and the sample was covered with a coverslip. The slide was then returned to the incubator. Every 15 min the slide was taken out and the sample was examined using a microscope. The time at which 50% of the haemocytes had started to lose dye from their lysosomes was recorded as the neutral red retention time of the marron lysosomal membrane. 2.6. Data analysis A multiple comparison test – Least significant different (LSD) and an Independent-sample T test were used to examine the significant difference among treatments using the SPSS version 15.0. Percent data were normalized using an arcsine transformation before analysis. For significant difference, it was required that P < 0.05. One way ANOVA was analyzed and results were expressed as mean standard error in the figures and tables. 3. Results 3.1. Trial 1 – challenge with bacteria Survival: Bio-MosÒ supplemented diets resulted in lower (P < 0.05) marron mortalities when injected with V. mimicus (Fig. 1) compared to marron fed the control diet. 3.1.1. Total and differential haemocyte count There was no significant difference (P > 0.05) in mean THCs of marron fed any of the diets before challenging with the V. mimicus.
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100
Survival (%)
90
b b
80 70
a 60 50
0
24
48
96
Time after infection (h) Control
0.2% BioMos
0.4% BioMos
Fig. 1. Mean SE survival of marron challenged with Vibrio mimicus. Different letters indicate significantly different means at each time at P < 0.05 (n ¼ 18).
24 h after the challenge, the mean THCs of the marron fed the control diet was significantly reduced (P < 0.05) while mean THCs of the marron fed 0.2% Bio-MosÒ and 0.2% Bio-MosÒ diets remained unchanged. After 24 h of bacterial challenge, mean THCs was significantly lower (P < 0.05) in the marron fed the control diet than in the marron fed the Bio-MosÒ diets. However, at 48 h after the challenge, there was no significant difference (P > 0.05) in mean THCs of the marron fed any of the test diets. After 96 h of the challenge, the highest THC was in marron fed 0.4% Bio-MosÒ diet (Table 2). Before the V. mimicus injection, mean proportion of granular cells was significantly lower (P < 0.05) in the marron fed the control
diet than in the marron fed the Bio-MosÒ diets. At 24 h after the injection, there were decreases in mean proportions of granular cells of marron in all treatments but the significant decline (P < 0.05) was only found in marron fed the control diet resulting in the significantly lower proportion of granular cells in the marron fed the control diet. Semi-granular cells were not significantly different between the marron fed the control and the Bio-MosÒ diets and the proportion of these cells remained unchanged during the V. mimicus challenge. However, the proportion of hyaline cells showed the opposite trend to granular cells during the challenge (Table 2). 3.1.2. Haemolymph clotting time There was no significant difference (P > 0.05) in mean haemolymph clotting time of the marron fed any of the diets before and after the V. mimicus injection (Table 2). 3.1.3. Bacteraemia Vibrio spp. in the haemolymph of marron fed the control diets significantly increased (P < 0.05) after 24 and 48 h of V. mimicus injection while there was no significant difference in haemolymph Vibrio spp. of marron fed the Bio-MosÒ diets before and after the injection. The significant difference (P < 0.05) in the Vibrio spp. numbers in the haemolymph of marron fed the control and the Bio-MosÒ diets was observed after 48 h of infection (Table 2). 3.1.4. Neutral red retention time Before V. mimicus injection, NRR times of marron lysosomal membranes in all treatment groups were more than 120 min. 24 h after the injection, the mean NRR times of lysosomal membranes in all the injected marron sharply decreased to less than 30 min. At 48 h after the injection, while the mean NRR time of the marron fed
Table 2 Mean SE immune parameters of marron challenged with Vibrio mimicus. Parameters
Time after infection (h)
Test diets
THCs (million cells/mL)
0* 24 48 96
21.73 122.85 122.30
0.47a 0.04a 0.62a 0.54a
13.66
0* 24 48 96
135.40
0.57a 0.33a 0.60a 1.0a
114.64
Control
Granular cells (%)
Semi-granular cells (%)
Hyaline cells (%)
Vibrio rank
Haemolymph clotting time (s)
NRRT (mins)
0* 24 48 96 0* 24 48 96 0* 24 48 0* 24 48 96 0* 24 48 96
13.26
36.86 123.73 21.86
18.80
110.73 17.16 16.47
0.2% Bio-Mos
0.16a 2.63a 2.0a 2.51a
14.56 13.70 12.91
110.78 114.18 113.11
17.38
110.81 17.05
113.80
0.4% Bio-Mos
0.04a 0.44b 1.08a 0.41a
13.33 13.90
0.81b 3.32b 0.69b 2.54b
1214.05 211.70
13.77
13.67
120.09
1213.35
215.04 15.92
2.19a 2.57a 1.73a 0.38a
2.48ab 0.25b 1.94ab 0.74b
169.96
4.37b 3.90b 1.13b 2.16b
129.95 14.88
1277.97
11.00
0.00a 0.33a a 310.00 0.00
11.00
11.00
22.33
11.33
11.00
3.51a 5.17a 9.06a 13.2a
172.33
182.4 1289.1
291.66
174.00
172.67 165.33
166.33
>120 a 123.33 3.33 a 115.00 0.00 a 240.00 5.00
278.4 278.75 173.08
0.00a 0.33a b 11.00 0.00 158.67 166.33 157.33
2.20c 2.93b 1.34c 1.78b
1.74a 3.09a 2.58a 3.28a
0.73a 2.70a 1.68a 2.26a
1285.79
0.2a 0.45b 0.26a 0.11b
21.53a 6.06a 10.68a 2.96a
>120 a 126.67 3.33 b 255.00 7.64 b 3111.67 4.4
170.90 282.37 281.76
0.00a 0.00a b 11.67 0.67
172.67
144.67 153.33 159.67
10.27a 1.76a 4.98a 3.28a
>120 a 126.67 3.33 b 253.33 1.67 b 393.33 8.82
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. 0* is the time before infection.
H.M. Sang et al. / Fish & Shellfish Immunology 27 (2009) 341–348
100 90
Survival (%)
the control diet still declined, the lysosomal membrane stability of the marron fed the Bio-MosÒ started recovering, especially the mean NRR time of the marron fed the Bio-MosÒ diets increased significantly (P < 0.05). At 96 h after the injection, the lysosomal membrane stability of the marron fed the control diet began to recover but the mean NRR time of the lysosomal membrane was significantly shorter (P < 0.05) than the mean NRR time of the marron fed the Bio-MosÒ diets. Lysosomal membrane stability of the marron fed the 0.2%-Bio-MosÒ diet recovered significantly faster (P < 0.05) than that of the marron fed the control diet.
345
80
a
70 60
b
50 40 30
3.2. Trial 2 – exposure to air during simulated transport
0
1
3
7
Exposure time (day) 3.2.1. Survival After 36 h of simulated transport, no mortalities were observed in any group. 3.2.2. Total and differential haemocyte count THCs of marron fed control diets significantly declined (P < 0.05) after 36 h of transportation while it remained unchanged in the marron fed Bio-MosÒ diets. Before subjection to simulated transportation stress, granular cells proportion was lower in the marron fed the control than the Bio-MosÒ diets and it did not change after the exposure to the transport stressor in marron fed any of the diets (Table 3). 3.2.3. Haemolymph clotting time The haemolymph clotting time of marron fed the control and 0.2% Bio-MosÒ diets significantly reduced after the simulated transportation and it was unchanged in the marron fed 0.4% BioMosÒ diet (Table 3). 3.3. Trial 3 – exposure to NH3 3.3.1. Survival After 7 days of exposure to NH3, survival of marron fed 0.4% BioMosÒ diet was significantly higher (P < 0.05) than that of marron fed the control diets (Fig. 2). 3.3.2. Total and differential haemocyte count THCs and DHC of marron fed the control and 0.4% Bio-MosÒ diets were not significantly different before exposure to NH3. After 1, 3 and 7 days, exposure, THCs of marron fed 0.4% Bio-MosÒ diet was higher (P < 0.05) than marron fed the control diets whereas there was no difference (P > 0.05) among the proportions of granular cells, semi-granular and hyaline cells when marron were fed different diets. THCs of marron fed either the control or Bio-MosÒ supplemented diets significantly reduced after 1, 3 and 7 days of exposure while the proportion of different cells remained unchanged until
Table 3 Mean SE immune parameters of marron challenged after subjected to simulated live transportation. Parameters
Time
13.39
0.2% Bio-Mos a
day 7 when the proportion of granular cells decreased and the proportion of hyaline cells increased (Table 4). 3.3.3. Haemolymph clotting time When exposure to NH3, haemolymph clotting time of marron fed either the control or 0.4% Bio-MosÒ diets significantly increased. After 3 days of exposure, haemolymph clotting time of marron fed 0.4% Bio-MosÒ diet was shorter than marron fed the control diet (Table 4). 3.3.4. Bacteraemia Bacteraemia of marron fed control diets significantly increased (P < 0.05) after 1 day exposure to NH3 whereas there was no change in bacteraemia of marron fed 0.4% Bio-MosÒ diets during 7 days of NH3 exposure. Table 4 Mean SE immune parameters of marron exposed to NH3. Parameters
Exposure time (day)
THCs (million cells/mL)
0* 1 3 7
24.42 31.67 31.11
0* 1 3 7
124.91 213.60
0* 1 3 7
10.58 22.06
0* 1 3 7
174.50 184.33
Granular cells (%)
Semi-granular cells (%)
Hyaline cells (%)
0.16 0.24a
14.43
1.25a 0.98a
122.19
THCs (million cells/mL)
BF AT
Granular cells (%)
BF AT
116.46
BF AT
2.08a 69.00 1.00a 2
22.56
115.79 146.00
13.57
121.12
0.18 0.42b 0.53b 0.34b
1.73b 43.00 1.66b 2 136.00
Vibrio rank
0.4% Bio-Mos b
14.06 14.11 123.41 122.27
b
0.24 0.42b 0.25b 1.42b
1.15b 35.33 3.18c 1
138.00
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. BF: before and AT: after transportation.
0.4% Bio-Mos®
Fig. 2. Mean SE survival of marron challenged with NH3. Different letters indicate significantly different means at each time at P < 0.05 (n ¼ 21).
Test diets Control
Haemolymph clotting time (min)
Control
Haemolymph clotting time (s)
0* 1 3 7 0* 1 3 7
Diets Control 19.87
121.27
121.59
10.39
10.38
0.4% Bio-Mos 0.91a 0.42a 0.24a 0.06a
111.10
1.03a 0.87a 6.62a 2.75a
125.66
0.39a 0.11a 0.35a 0.47a
10.42
32.87 32.22 26.27
129.13 212.90
120.54
10.47 10.81 10.47
0.64a 0.94a 6.33a 2.91a
170.38 286.28
27.66 210.33
1.00a 1.52a 0.66a 1.45a
14.00 16.00
141.00
9.02a 11.62a 6.87a 24.31a
178.33
178.02
13.00
210.00
286.33 2101.17
1280.50
173.93
171.98
12.33
12.33
1
39.17 1261.66 272.50 1255.17
1.74a 0.20b 0.20b 0.03b 3.93a 2.65a 5.42a 0.43a 0.20a 0.25a 0.36a 0.32a 4.04a 2.53a 5.23a 0.75a 0.88a 1.33b 1.52a 1.00b 4.47a 7.33a 2.31b 7.40a
Different superscript letters in the same row indicate significantly different means at P < 0.05. Means for each immune parameter in any one column not preceded by the same subscript numbers are significantly different at P < 0.05. 0 *is the time before exposure.
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4. Discussion The results of the current study indicate that application of dietary Bio-MosÒ can significantly improve the marron’s ability to resist stressors such as bacterial infection, exposure to NH3 and live transport indicated by the higher survival and positive immune responses. Similar effects have been described for sea bass [12] and cobia larvae [36] when they were fed dietary Bio-MosÒ. Improved survival of marron when exposed to stressors may be a direct consequence of enhanced health status and positive immune responses of marron as it has been shown in other aquaculture species [15]. The similar outcome is also suggested by improvement in the health of the digestive system of animals fed Bio-MosÒ [11,13,16,36]. Our unpublished data has further confirmed that a better condition of the digestive system, improved immune indicators for better health status of the marron is achieved when fed 0.2%–0.4% Bio-MosÒ supplemented diets. Bio-MosÒ supplementation in the diets has also been previously reported to promote resistance to bacterial infection and stressors in common carp (C. carpio) [13], rainbow trout (S. gairdneri irideus G.) [14], channel catfish (I. punctatus) [15], rainbow trout (O. mykiss) [16] and sea bass (D. labrax) [12]. Total number of haemocytes, proportion of haemocyte types, bacteraemia and haemolymph clotting time have been used as indicators of health of crustacea [29,37–44]. Recently, neutral red retention (NRR) assay has also been used to assess the immune status of shellfish by measuring the dye retention time of lysosomal membranes [45]. Neutral red is a cytotoxic weak base [46] and has been successfully used in the neutral red retention (NRR) assay as a biomarker to test the stability of lysosomal membranes in aquatic animals [35,47–51]. In decapod crustacea, life cycle, food intake, disease outbreaks, pollutants and environmental stress affect the circulating haemocyte count both in quantity and quality [52–54]. In the present study, all marron tested were in the intermolt stage, thus considered to have similar physiological and/or immunological status. Our unpublished data have shown that Bio-MosÒ supplemented diets have improved the immune response of marron when it was applied for the duration of 112 days. In addition, dietary Bio-MosÒ has been approved to have effects on immune indicators of rainbow trout (O. mykiss) after 42 days [16] and sea bass (D. labrax) after 72 days [12] application. The changes in the THCs and DHCs of the marron after exposure to stressors in the current trials imply that bacterial infection, NH3 and air exposure during simulated live transportation could influence the circulating haemocyte counts of marron. After the V. mimicus injection, THCs and proportion of granular cells of marron fed the control diet dropped significantly. The same observations have been reported in black tiger prawn (Penaeus monodon) [55] Chinese shrimp (Fenneropenaeus chinensis) [45] when they are injected with Vibrio anguillarum. The decline in THC after V. mimicus injection could be associated with haemolymph locomotion to the injection site and haemocyte lysis as a result of defense activities [55,56]. However, THC of marron fed the Bio-MosÒ diets did not decrease after the Vibrio injection. Sequeira, Tavares & Arala-Chaves [57] found that Penaeus japonicus haemocytes have the capacity to proliferate, and the proliferation rate can be increased three times when the shrimp are injected with an immunostimulant, lipopolysaccharide. Our previous study (unpublished) also showed that THC in marron increased significantly after feeding with Bio-MosÒ. In the present study, the incorporation of Bio-MosÒ in diets may stimulate and increase the proliferation rate of marron haemocytes to compensate for the loss of haemocytes due to the V. mimicus infection, resulting in the THC proportions to remain constant. The present study also reveals that exposure to NH3 at the concentration of 1.37 mg L1 reduces the THCs of marron.
Circulating haemocytes of several species of crustacean can be affected by extrinsic factors like temperature, pH, salinity, dissolved oxygen and ammonia [58–60]. The decrease in THC is frequently reported in crustacea exposed to stress conditions. Litopenaeus stylirostris following exposure to ammonia at 3.0 mg L1 decreased its THC by 30% [52], Chinese mitten-handed crab (Eriocheir sinensis) reduced the THC indicating suppressed resistance when exposure to ammonia [61]. These reductions in THC relate to the accumulation of haemolymph ammonia and urea, and causes the catabolism of haemocyanin and protein to amino acids [62]. However, the marron fed Bio-MosÒ diets were healthier as shown by lowered reduction rate (80%) of THC compared to the marron fed the control diet (88.75%) after 7 days of exposure to the stressor. Air exposure has been demonstrated to reduce the health status of rock lobster (Panucilus lygnus) [29]. Trial 2 showed that the marron fed BioMosÒ diets are healthier than marron not fed Bio-MosÒ as indicated by the higher THC after 36 h of simulated live transport. It is also inferred from current results that the significant reduction in the THCs of marron fed the diets without Bio-MosÒ supplementation when challenged with bacteria and NH3 exposure has negatively affected the ability to combat the bacterial infection [63,64], thus, resulting in the higher bacteraemia. Crustacean haemolymph clotting involves aggregation of the circulating haemocytes and the gelation of the plasma by factors released from the haemocytes [65]. Haemolymph clotting time has been reported to be changed by the presence of bacteria [66,67], environmental pollutants [68] or holding period in storage tanks and live transportation [39]. In the current experiment, haemolymph clotting time of marron was not affected by V. mimicus injection. Although Durliat & Vranckx [37] found a relationship between hemolymph clotting time and number of haemocytes in lobsters, the authors stated that haemocyte numbers could not be explained by the changes in hemolymph clotting time. To date, little is known about the mechanism by which the changes in crustacean haemolymph clotting time are induced. However, challenging with NH3 and air exposure has affected the haemolymph clotting time of marron. Thus, further research needs to be conducted to define the mechanism in which pollutants, nitrogen metabolites and air exposure stressors affect the clotting time of marron. Marron fed the Bio-MosÒ diet showed the shortest clotting time after 3 days of NH3 exposure and 36 h of air-exposure indicating the ability of Bio-MosÒ to improve initial immune defense activity of marron under these stress conditions. Neutral red is a cytotoxic weak base [46] and has been successfully used in the neutral red retention (NRR) assay as a sensitive indicator of lipid membrane integrity in cases of bacterial infection and immunostimulant toxicity in shellfish. [35,47–51]. Lysosomes found within the semi-granular and granular haemocytes are polymorphic, hydrolytic enzyme-containing organelles that have the capacity to take up and retain neutral red dye. The dye disappears when lysosomal membranes are damaged [46] and unhealthy cells lose neutral red dye at a faster rate than healthy cells due to a decrease in cellular membrane stability [69,70]. In this study, NRR assay was successfully developed for the detection of lysosomes of haemocytes in the marron. Without any stimulation, the NRR time of marron lysosomal membrane in the current experiment was more than 120 min. The injection of V. mimicus altered the stability of the lysosomal membrane of marron as it showed a rapid decline in NRR time. Similarly, NRR time of Chinese shrimp was significantly shortened after Vibrio injection [45]. In marron fed the diet without Bio-MosÒ, the decrease in lysosomal membrane stability is related with the decrease in THC, granular cell count (GC) and semi-granular cell count (SGC) after the injection. A similar phenomenon was found in freshwater prawn (Macrobrachium rosenbergii) [71], oysters (Ostrea edulis L. and Crassostrea gigas (Thunberg)) and the scallop
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(Pecten maxim’s) [47] suggesting that GC and SGC might degranulate at first, followed by lysis of the lysosomes [45]. After 48 h of the injection, the lysosomal membrane stability recovery was significantly faster in marron fed the Bio-MosÒ diets than in those fed the control diet, indicating that Bio-MosÒ as supplementation in diets could have improved the resistance of marron against V. mimicus by hastening the recover rate of lysosomal membrane stability. In conclusion, immunological responses to dietary Bio-Mos supplementation have shown that Bio-MosÒ improves the survival of the marron, improves the health status and increases marron’s defense ability to bacterial infection and other stress conditions. Further research needs to be conducted to clarify the mechanisms of Bio-MosÒ action(s) in the health and immunity improvements of marron. Acknowledgement We thank Alltech Inc, USA for the financial support. We also thank Rocı´o Gonza´lez Lo´pez for assistance with the experimentation. References [1] Scholz U, Garcia Diaz G, Ricque D, Cruz Suarez LE, Vargas Albores F, Latchford J. Enhancement of vibriosis resistance in juvenile Penaeus vannamei by supplementation of diets with different yeast products. Aquaculture 1999;176: 271–83. [2] Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, et al. Disease and health management in Asian aquaculture. Vet Para 2005;132:249–72. [3] Vadstein O. The use of immunostimulation in marine larviculture: possibilities and challenges. Aquaculture 1997;155:401–17. [4] Gatlin III DM. Nutrient and fish health. In: Halver JE, Hardy RW, editors. Fish nutrient. San Diego, CA: Academic Express; 2002. p. 671–702. [5] Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 2008;274:1–14. [6] Capone DG, Weston DP, Miller V, Shoemaker C. Antibacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 1996;145:55–75. [7] Bachere E. Anti-infectious immune effectors in marine invertebrates: potential tools for disease control in larviculture. Aquaculture 2003;227:427–38. [8] Campa-Co´rdova AI, Herna´ndez-Saavedra NY, De Philippis R, Ascencio F. Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to [beta]-glucan and sulphated polysaccharide. Fish Shellfish Immunol 2002;12:353–66. [9] Soltanian S, Thai TQ, Dhont J, Sorgeloos B, Bossier P. The protective effect against Vibrio campbellii in Artemia nauplii by pure b-glucan and isogenic yeast cells differing in b-glucan and chitin content operated with a source-dependent time lag. Fish Shellfish Immunol 2007;23:1003–11. [10] Patterson JA, Burkholder KM. Application of prebiotics and probiotics in poultry production. Poult Sci 2003;82:627–31. [11] Genc MA, Aktas M, Genc E, Yilmaz E. Effects of dietary mannan oligosaccharide on growth, body composition and hepatopancreas histology of Penaeus semisulcatus (de Haan 1844). Aquacult Nut 2007;13:156–61. [12] Torrecillas S, Makol A, Caballero MJ, Montero D, Robaina L, Real F, et al. Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol 2007;23:969–81. [13] Staykov Y. The influence of Bio-Mos on growth rate and immune status of common carp (Cyprinus carpio). In: Alltech’s second annual aquaculture meeting Dunboyne, Co Meath November, oral communication, 2004. [14] Staykov Y, Spring P, Denev S. Influence of dietary Bio-MosÒ on growth, survival and immune status of rainbow trout (Salmo gairdneri irideus G.) and common carp (Cyprinus carpio L.). Bulgaria: Trakia University; 2006. [15] Welker TL, Lim C, Yildirim-Aksoy M, Shelby R, Klesius PH. Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catfish, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. J World Aqua Soc 2007;38:24–35. [16] Staykov Y, Spring P, Denev ES, Sweetman EJ. Effect of a mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquacult Inter 2007;15:153–61. [17] Morrissy NM. Culturing the marvellous marron in Western Australia: 1967–1997. Freshw Crayfish 2000;13:13–38. [18] Rouse DB, Kartamulia I. Influence of salinity and temperature on molting and survival of the Australian freshwater crayfish (Cherax tenuimanus). Aquaculture 1992;105:47–52. [19] Morrissy NM, Evans L, Huner JV. Australian freshwater crayfish: aquaculture species. J World Aquacult Soc 1990;21:113–22.
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