Synbiotic Effect of Bacillus Mycoides and Organic Selenium on Immunity and Growth of Marron, Cherax Cainii (Austin, 2002)

Aquaculture Research, 2016, 1–12 doi:10.1111/are.13105

Synbiotic effect of Bacillus mycoides and organic selenium on immunity and growth of marron, Cherax cainii (Austin, 2002)

Irfan Ambas1,2, Ravi Fotedar2 & Nicky Buller3 1Department of Fishery, Faculty of Marine Science and Fishery, Hasanuddin University, Makassar, Indonesia 2Sustainable Aquatic Resources and Biotechnology, Department of Environment and Agriculture, Curtin University, Bentley, WA, Australia 3Animal Health Laboratories, Department of Agriculture and Food Western Australia, South Perth, WA, Australia

Correspondence: I Ambas, Department of Fishery, Faculty of Marine Science and Fishery, Hasanuddin University, JL. P Kemerdekaan km. 10, Makassar 90245, Indonesia. E-mail: [email protected]

B. mycoides may improve a particular immune Abstract parameters of marron and to a lesser extent their The present feeding trial examined the effect of growth. synbiotic use of Bacillus mycoides and organic selenium (OS) as Sel-Plex on marron immunity, Keywords: marron, synbiotic, Bacillus mycoides, growth and survival. The marron were cultured in organic selenium, immunity and growth recirculated tanks and fed test diets consisting of a basal diet; basal diet supplemented with Introduction B. mycoides (108 CFU g1 of feed); basal diet supplemented with OS (Sel-Plex) (0.2 g kg1 of feed) Prebiotics and probiotics have been extensively used and basal diet supplemented with synbiotic in aquaculture (Burr & Gatlin 2005; Denev, Staykov, (B. mycoides at 108 CFU g1 and OS 0.2 g kg1 Moutafchieva & Beev 2009; Ganguly, Paul & feed) diet, in triplicate. The effect of the prebiotic Mukhopadhayay 2010; Dimitroglou, Merrifield, OS (Sel-Plex) on the growth rate of B. mycoides Carnevali, Picchietti, Avella, Daniels, Guroy€ & Davies was also studied in vitro. The results suggested 2011; Kristiansen, Merrifield, Vecino, Myklebust & that synbiotic use of B. mycoides and OS signifi- Ringø 2011; Merrifield & Zhou 2011). Prebiotic is a cantly improved some immune parameters of mar- non-digestible food ingredient that beneficially affects ron, particularly the glutathione peroxidase, and the host by selectively stimulating the growth and/or to some extent total haemocyte counts. However, the activity of one or a limited number of bacteria in the synbiotic feed did not synergistically improve the colon and thus improves host health (Gibson & marron growth, in fact the use of B. mycoides-sup- Roberfroid 1995; Ringø, Olsen, Gifstad, Dalmo, plemented diet alone demonstrated significantly Amlund, Hemre & Bakke 2010). Prebiotics have higher growth in marron compared with the shown beneficial effects on numerous aquatic ani- growth of marron fed on other test diets. mals (Zhou, Buentello & Gatlin 2010), and reviews Supplementation of the basal diet with host origin of their potential use in aquaculture have been docu- B. mycoides significantly increased the intestinal mented (Merrifield, Dimitroglou, Foey, Davies, Baker, bacterial population (3.399 825 CFU g1 of Bøgwald, Castex & Ringø 2010; Ringø et al. 2010; gut) in marron compared with other diets. Organic Merrifield & Zhou 2011). The prebiotics commonly selenium as Sel-Plex in Trypticase Soya Broth also used and evaluated in aquatic animals to date confirmed that OS did not increase the amount of include inulin, fructooligosaccharides, mannano- growth of B. mycoides and resulted in a lower ligosaccharides (MOS), galactooligosaccharides, intestinal bacterial population in the synbiotic xylooligo-saccharides, arabinoxylooligosaccharides, diet-fed marron. In conclusion, synbiotic of OS and isomaltooligosaccharides and GroBiotic-A (Ringø

© 2016 John Wiley & Sons Ltd 1 Synbiotic effect of B. mycoides & OS on marron I Ambas et al. Aquaculture Research, 2016, 1–12 et al. 2010), chitosan oligosaccharides (COS) and Arnold 2010), sea cucumber, Apostichopus organic selenium (OS). In marron Cherax cainii japonicus (Zhang, Ma, Mai, Zhang, Liufu & Xu (Austin 2002), the prebiotics evaluated were MOS 2010), yellow croaker Larimichthys crocea (Ai, Xu, (Sang, Ky & Fotedar 2009; Sang & Fotedar 2010) Mai, Xu & Wang 2011), cobia Rachycentron and OS (Nugroho & Fotedar 2013b). canadum (Geng, Dong, Tan, Yang & Chi 2011) and Probiotics, defined as live microorganisms when Zebrafish Danio rerio (Nekoubin, Javaheri & Iman- administered in adequate amounts confer a health pour 2012). A review of synbiotic use of probiotics benefit on the host (FAO/WHO, 2002), have been and prebiotics for fish in aquaculture has also been recommended as alternatives for use as growth documented (Cerezuela et al. 2011). promoters, diseases control and for the safety of Sel-plex, an organic form of selenium derived sustainable aquaculture (For reviews see; Ringø, from yeast produced by Alltech, showed beneficial Strøm & Tabacheck 1995; Ringø & Gatesoupe effects in marron when administered at 0.2 g kg1 1998; Gatesoupe 1999; Verschuere, Rombaut, of feed (Nugroho & Fotedar 2013b). Bacillus Sorgeloos & Verstraete 2000; Irianto & Austin mycoides, a strain isolated from the gastrointestinal 2002; Austin 2006; Balcazar, Blas, Ruiz-Zarzuela, tract of marron, demonstrated favourable probiotic Cunningham, Vendrell & Mu0zquiz 2006; Farzanfar properties such as growth inhibition of Vibrio mim- 2006; Das, Ward & Burke 2008; Sahu, Swarnaku- icus and Vibrio cholerae non-01 (Ambas, Buller & mar, Sivakumar, Thangaradjou & Kannan 2008; Fotedar 2015), and improvement in marron Denev et al. 2009; Ninawe & Selvin 2009; health and immunity (Ambas, Suriawan & Fotedar Qi, Zhang, Boon & Bossier 2009; Merrifield 2013; Ambas, Fotedar & Buller 2015). The pre- et al. 2010; Nayak 2010; Prado, Romalde & Barja sent study examined the synbiotic effect of the pro- 2010; Cerezuela, Meseguer & Esteban 2011; biotic B. mycoides and the prebiotic OS (Sel-Plex) Kolndadacha, Adikwu, Okaeme, Atiribom & on growth, health condition and immunity as Mohammed 2011; Lara-Flores 2011; Martınez tested by total haemocyte count (THC), DHC, bac- Cruz, Ibanez, Monroy Hermosillo & Ramırez Saad teraemia, clotting time and glutathione peroxidase 2012; Ibrahem 2013; Lakshmi, Viswanath & Gopal (GPx) enzyme activity of marron. 2013; Pandiyan, Balaraman, Thirunavukkarasu, George, Subaramaniyan, Manikkam & Sadayappan Materials and methods 2013) and more recently (Lazado, Marlowe & Caipang 2014; Michael, Amos & Hussaini 2014; Animal and feed preparation Newaj-Fyzul, Al-Harbi & Austin 2014; Ghanbari, Kneifel & Domig 2015; Hai 2015). Marron, Cherax cainii (Austin, 2002) Probiotics and prebiotics generally have been 10.83 0.28 g were supplied by Marron Growers studied separately (Li, Tan & Mai 2009; Cerezuela, Association of Western Australia located in North- Fumanal, Tapia-Paniagua, Meseguer, Morinigo~ & cliffe and Manjimup. The marron were acclimated Esteban 2013). Synbiotics as a combination of pro- to experimental tanks and feed for 2 weeks before biotics and prebiotics have not been intensively the experiment commenced. During the acclima- explored (Li et al. 2009). To date, the synbiotic tion, marron were fed a basal diet at 1.5% of total studies on aquatic animals is limited to gilthead biomass per tank once a day at 17:00 hours. sea bream Sparus aurata L. (Tapia-Paniagua, Commercial feed was purchased from Glenn Reyes-Becerril, Ascencio-Valle, Esteban, Clavijo, Forrest, (Glenn Forrest, Australia). with a composi- Balebona & Morinigo~ 2011; Cerezuela, Guardiola, tion of 26% crude protein, 9% crude fat and 5% Meseguer & Esteban 2012; Cerezuela et al. 2013), crude ash. The commercial marron feed was rainbow trout Oncorhynchus mykiss (Rodriguez- homogenized using a blender to obtain a desirable Estrada, Satoh, Haga, Fushimi & Sweetman 2009), pellet size for marron and for inclusion of B. my- Japanese flounder Paralichthys olivaceus (Ye, Wang, coides, OS (Sel-Plex) and synbiotic of B. mycoides Li & Sun 2011), shrimp Penaeus japonicus (Zhang, and OS (Sel-Plex) before storing at 4°C. Bacillus Tan, Mai, Zhang, Ma, Ai, Wang & Liufu 2011), mycoides was added at 108 CFU g1 of feed follow- shrimp Litopenaeus vannamei (Li et al. 2009; ing the previous study (Ambas et al. 2013), Thompson, Gregory, Plummer, Shields & Rowley whereas OS (Sel-Plex) was supplemented at 2010), European lobster Homarus gammarus L. 0.2 g kg1 of feed (Nugroho & Fotedar 2013b). (Daniels, Merrifield, Boothroyd, Davies, Factor & The marron were fed the experimental diets for

10 weeks at 1.5% of total biomass per tank, once DHC ð%Þ¼ðNumber of haemocytes cell per day in the late afternoon (Jussila 1997). type/total haemocytes cells countedÞ100 Experimental set up

The experimental tanks were 250-L cylindrical Haemolymph bacteria (Bacteraemia) plastic tanks (80-cm diameter and 50-cm high) filled with freshwater and supplied with constant Assessment of bacteraemia was performed follow- aeration. The tank was also equipped with a sub- ing the established procedure described by Fotedar, mersible thermostat set to 24°C. Water in each Tsvetnenko and Evans (2001) with a minor modi- tank ran continuously at a rate of approximately fication. The haemolymph was withdrawn into a 3 L min1 using recirculating biological filtration. sterile syringe and placed onto a sterile glass slide Sufficient PVC pipes of appropriate diameters were from which a 0.05-mL aliquot was smeared onto placed into each tank as marron shelters. Follow- a blood agar (BA) plate. This technique was effec- ing the acclimation, the marron were distributed tive in removing bubbles of the haemolymph so equally into 12 experimental culture tanks at the that an accurate volume was smeared onto the density of 12 marron tank1, where each group plates. Subsequently, the plates were placed in a was in triplicate. sterilized container before overnight incubation at 25°C. The total colony-forming units (CFU) for 1 Data collection each plate and CFU mL were calculated on the basis of a total volume of 0.05 mL1 plate. Total and differential haemocyte count Haemolymph clotting time The THC and differential haemocyte count DHC (proportion of hyaline cell) of marron were mea- Determination of haemolymph clotting time fol- sured on the final day of the feeding trial. Haemo- lowed the established method (Fotedar et al. 2001; cyte sample preparation and calculation were Jussila, McBride, Jago & Evans 2001). Briefly, the done using an established method (Jussila, Jago, haemolymph of marron was withdrawn using a Tsvetnenko, Dunstan & Evans 1997; Fotedar sterile syringe and dispensed into an Eppendorf 1998). Briefly, 0.5 mL of haemolymph withdrawn tube. A 30-lL aliquot was quickly transferred and from the second last ventral segment of marron drawn into a capillary tube (Chase, Scientific was placed into a haemocytometer (Neaubauer, Glass, Rockwood, TN, USA), then the tube was Munich, Germany) immediately viewed under a repeatedly inverted until the haemolymph stopped camera equipped with a microscope, images were moving, which was noted as haemolymph clotting then taken for THC and DHC counts. For each time (seconds). treatment group, the procedure was repeated ten Glutathione peroxidase times using a different animal. The THC was calculated as THC = (cells counted 9 dilution fac- Sample preparation and determination of the GPx tor 9 1000)/volume of grid (0.1 mm3). activity in marron tissue followed the established The proportion of hyaline cells (DHC) was also method (Rotruck, Pope, Ganther, Swanson, Hafe- prepared using established methods (Bancroft & man & Hoekstra 1973). The marron tissue was Stevens 1977; Hai & Fotedar 2009). One drop of diluted with physiological saline at a ratio of 1:1 the mixture of anticoagulant and haemolymph and stored at 4°C until used. The GPx activity was was smeared onto a glass microscope slide and expressed as micrograms of GSH consumed per air-dried before fixing in 70% methanol for minute per milligram of protein. 10 min. The slides were stained in May–Grunwald Intestinal bacterial density and Giemsa for 10 min each. Identification of the haemocyte cell groups followed the criteria Bacterial density in marron intestine was deter- described by Bauchau (1981) and Johansson, mined following the established protocol (Hai & Keyser, Sritunyalucksana and Soderhall (2000). Fotedar 2009). In brief, five marron of equal size On each slide, a total of 200 cells were counted from each treatment group were selected and and the percentage of a haemocyte group was rinsed in distilled water. Subsequently, the shells calculated using the equation: were cleaned with 70% alcohol then rinsed again

in distilled water to remove the external bacteria. measurement (t) and at the commencement (W0), Following dissection, the intestine of each marron where t is experimental period (day). was removed and weighed, then homogenized using a sterilized pestle and mortar. Subsequently, Survival rate (%) the homogenates were diluted serially with sterilized normal saline then lawn inoculated to nutri- At the end of the experimental period, the number ent agar (NA) plates prior to incubation for 24 h of marron in each tank was counted and survival at 25°C. The total colony-forming units (CFU) for rate was calculated using the following formula; each plate and CFU mL1 were calculated on the 1 basis of a total volume of 0.05 mL plate (Buller SR ¼ðNt=N0Þ100 2004). where SR is the survival rate (%); Nt is the num-

ber of marron at time t and N0 is the number of Bacillus mycoides Effect of OS on marron at the commencement (0) respectively. Sel-Plex (Alltech) does not dissolve in water, but does dissolve in NaOH. The recommended dose as Water quality a feed additive is 0.2 g kg1. To determine whether OS (Sel-Plex) dispersed in water, or dis- The water quality in each tank was kept at opti- solved in 0.08% sodium hydroxide would affect mum conditions for marron by performing water – the growth of B. mycoides, an experiment was per- exchange at a rate of 10 15% of the total water formed by adding the Sel-Plex and solvent to volume twice a week, after siphoning out the fae- media as treatments in triplicate as follows; (1) ces and uneaten feed. Several water quality B. mycoides in TSB only, (2) B. mycoides in TSB parameters were monitored weekly including total with 0.2 g kg1 OS (Sel-Plex), and (3) B. mycoides ammonia and nitrite, which were measured using in sodium hydroxide added to TSB. To achieve the Calorimeter PR 1890, USA; temperature and pH lowest concentration of sodium hydroxide which using a digital pH/mV/C meter, Cyberscan pH300; dissolved Sel-Plex 0.2 g kg1 OS (Sel-Plex), a Eutech instruments, Singapore, Singapore; and dis- working solution of OS (Sel-Plex) in sodium solved oxygen using a digital DO meter SM600, hydroxide was prepared by a serial dilution to the Milwaukee, Romania. desired concentration. Prior to the experiment, pure B. mycoides was Data analysis grown on BA plates and incubated overnight at 24°C, then emulsified in sterile normal saline to be The data were analysed using SPSS statistical pack- used as an inoculum. Subsequently, 100 lL of the age version 22.0 for Windows and Microsoft Excel. inoculum was added to each media of the three Significant differences among treatment means treatments then incubated 24 h at 25°C. Determi- were determined using one way analysis of vari- nation of B. mycoides density in each treatment ance (ANOVA). All significant tests were performed < was obtained by total bacterial count on BA plates at P 0.05 level. The results were presented as (Buller 2004). means SE (standard error).

Speciﬁc growth rate Results

The specific growth rate (SGR) of marron was Immune competence determined by measuring the average weight of 15 marron from each treatment group at the Immune competences selected in this study (THC, beginning and end of the feeding trial. The specific DHC, bacteraemia, clotting time and GPx activity) growth rate was calculated as follows: are presented in Table 1 and Fig. 1. All immune parameters observed were significantly higher 1 (P < 0.05) in supplement-fed marron compared SGR ð%Þ¼100 ðln Wt ln W0Þt with basal diet-fed marron. where SGR is the specific growth rate in weight (% Synbiotic use of B. mycoides and OS synergisti- 1 g day ), Wt is the weight of marron at cally improve immunity particularly the GPx

Table 1 Immune competence (mean SE) of marron fed Bacillus mycoides, organic selenium (OS) and synbiotic diets

Parameters Control B. mycoides OS B. mycoides + OS

THC (106 mL1) 2.858 1.1c 9.471 0.8a 4.162 1.0b 8.129 1.4a Hyaline cell (%) 24.4 3.2c 40.8 4.2a 32.5 3.1b 38.3 2.9a Bacteraemia (CFU mL1) 4020 1.686c 668 433a 2584 832b 2576 470b Clotting time (s) 71.7 6.9c 36.4 6.8a 56.9 5.6b 53.7 12.8b

B. mycoides was added at 108 CFU g1 of feed, whereas OS was supplemented at 0.2 ppm kg1 of feed. Data in the same row having different superscripts indicates signiﬁcantly different at 0.05 (n = 10; P < 0.05).

300 d 250 c b 200 a

150

Figure 1 Glutathione peroxidase 100 activity (lg of GSH consumed per minute per milligram of protein) of min-1 mg protein-1) 50 marron muscle fed basal, Bacillus mycoides, organic selenium and GPx acvity (μg GSH consumed 0 synbiotic diets (n = 6). Basal diet B. mycoides OS B.mycoides+OS enzyme activity of marron and to some extent inhibit B. mycoides growth, as the total colony THC and DHC (hyaline cell). In general, the synbi- count in sodium hydroxide prepared with TSB otic feed improved marron immunity significantly media was similar (366.6 9 106 CFU mL1)to higher compared with OS and basal diet-fed the total colony of B. mycoides in TSB only. marron but comparable to the single use of Specific growth rate B. mycoides. Growth of marron fed various supplemented diets Intestinal bacteria density at the end of the feeding trial is presented in At the termination of the feeding trial, the intesti- Table 2. The highest specific growth rate was nal bacterial density (million g1 of gut) was sig- detected in marron fed B. mycoides-supplemented nificantly different (P < 0.05). Marron fed diet. Although the lowest growth rate was B. mycoides showed the highest intestinal bacterial observed in marron fed the basal diet, the growth density (3.399 825 CFU g1 of gut) compared rate was not significantly different to OS or synbi- with basal diet-fed marron (519 176 CFU g1 otic-fed marron. of gut) (Fig. 2). Survival rate (%)

The survival rate of marron fed various supple- Effect of OS on Bacillus mycoides mented diets at the termination of the feeding trial The growth (number of colonies) of B. mycoides is presented in Fig. 3. No mortality was observed was measured by total bacterial count after 24 h in tanks of marron fed B. mycoides, OS (Sel-Plex) of incubation and indicated that dissolved OS (Sel- or a combination of both, but deaths occurred in Plex) in TSB media did not improve the growth of basal diet-fed marron. B. mycoides. In fact, the number of B. mycoides colonies was signiﬁcantly lower (186.6 9 Water quality 106 CFU mL1) in TSB media containing OS compared with a count of 382.6 9 106 CFU mL1 in The water quality in this study was maintained TSB media without OS. In addition, 0.08% of within the optimum range for marron growth, as sodium hydroxide used as an OS solvent did not each tank was equipped with a bio-ﬁlter

4500 a 4000 3500 3000

2500 b b 2000 1500 (milliong-1 of gut) (milliong-1 of 1000 c Intestinal bacteria density 500 Figure 2 Intestinal bacterial pop- 0 ulation (106 CFU g1of gut) of Control B.mycoides OS synbiotic marron fed various diets (n = 5).

Table 2 Speciﬁc growth rate (% day1) of marron fed Bacillus mycoides, organic selenium (OS) and synbiotic diets

Parameters Basal diet B. mycoides OS B. mycoides + OS

Initial weight 10.83 0.28 10.83 0.28 10.83 0.28 10.83 0.28 Final weight 13.06 + 1.52 14.29 1.55 13.21 1,57 13.71 1.63 SGR 0.16 0.05a 0.21 0.07b 0.17 0.06a 0.18 0.04a

B. mycoides was added at 108 CFU g1 of feed, whereas OS was supplemented at 0.2 ppm kg1 of feed. Data in the same row having different superscripts indicate signiﬁcantly different at 0.05 (n = 15; P < 0.05).

105 aaa 100

95 b 90

Survival rate (%) 85 Figure 3 Survival rate (%) of 80 marron fed different diets at the Control B.mycoides OS synbioc termination of the feeding trial. recirculation system and regular water exchange time (Jussila et al. 1997; Sang et al. 2009), was conducted. No significant difference was glutathione-S-transferase (GST) and GPx (Nugroho observed for the water quality in tanks among the & Fotedar 2013a) have been successfully used as treatments (Table 3). tools to evaluate immunity and health status in marron studies. The present study demonstrated that supplementation with either B. mycoides,OS Discussion (Sel-Plex), or their combination as a synbiotic sig- Probiotics and prebiotics have been proven to nificantly improved marron immune parameters, improve immunity and physiological aspects in particularly GPx enzyme activity and THC. The various aquaculture species; hence studies on feed significant increase in the GPx and THC in the supplements are now focused on synbiotics to synbiotic-fed marron compared with use of OS explore further synergetic effects in aquatic ani- (Sel-Plex) alone or the basal diet-fed marron indi- mals. cated a synergetic effect of the synbiotic on mar- Total haemocyte count, proportion of hyaline ron immunity. Previous studies using OS (Sel-Plex) cell (DHC), bacteraemia and haemolymph clotting also demonstrated improved marron THC

Table 3 Nitrite (ppm), pH, temperature (oC) and dissolved oxygen (ppm) in tanks during trial

Parameters Control B. mycoides OS B. mycoides + OS

Nitrite 0.045 0.01a 0.034 0.01a 0.035 0.01a 0.033 0.01a pH 7.60 0.05a 7.62 0.07a 7.58 0.05a 7.65 0.03a Temperature 23.27 0.81a 23.47 0.62a 23.65 0.84a 23.53 0.43a Dissolved oxygen 6.21 .020a 6.25 0.18a 6.24 0.42a 6.21 0.23a

Data in the same row having different superscripts indicate signiﬁcantly different at 0.05 (n = 3; P < 0.05). OS, organic selenium.

(Nugroho & Fotedar 2013a, 2015), and the use of bacteraemia in marron. Our previous study also B. mycoides (Ambas et al. 2013). Increased THC confirmed a significant bacteraemia reduction in and improved immunity of aquatic animals fed a marron fed B. mycoides at 48 and 96 h post injec- synbiotic diet has also been observed in shrimps tion challenge test with the pathogen V. mimicus P. japonicus (Zhang et al. 2011) and L. vannamei (Ambas et al. 2013). Chisholm and Smith (1995) (Li et al. 2009), rainbow trout O. mykiss (Rodri- evaluated antibacterial activity of four species of guez-Estrada et al. 2009; Mehrabi, Firouzbakhsh & crustaceans and found that their haemocytes con- Jafarpour 2012), Japanese flounder P. olivaceus (Ye tained factors able to reduce the viable count of et al. 2011), sea cucumber Apostichopus japonicas injected bacteria within 4 h. (Zhang et al. 2010) and koi Cyprinus carpio koi The antioxidant enzymes, including GPx, which (Lin, Mao, Guan, Luo, Luo & Pan 2012). However, is a very potent antioxidant, protect the body from synbiotic use of b-glucan and Bacillus subtilis or damage from oxidation by free radicals (Chiu, Pediococcus acidilactici did not improve THC of Hsieh, Yeh, Jian, Cheng & Liu 2010), which can shrimp, L. vannamei (Wongsasak, Chaijamrus, cause cellular damage and oxidative stress Kumkhong & Boonanuntanasarn 2015). (Parrilla-Taylor & Zenteno-Savın 2011). In this The haemocyte is responsible for destroying study, GPx was significantly higher in the muscle invasive microorganisms (bacteraemia) and foreign of synbiotic-fed marron (Fig. 1) compared with particles, thus greater THC contributes to better other test diets. Supplementation with OS (Sel- cellular immunity (Sang, Fotedar & Filer 2011). In Plex) also significantly improved the antioxidant Australia, bacteraemia has been associated with enzymes, GST and GPx, in marron haemolymph mortalities in crayfish (Eaves & Ketterer 1994; (Nugroho & Fotedar 2013a). In contrast, synbiotic Edgerton & Owens 1999), as a result of exposure use of b-glucan and B. subtilis in feed resulted in to environmental stressors (Jussila et al. 1997; significantly lower superoxide dismutase (SOD) Evans & Edgerton 2002; Sang et al. 2009). Once activity in shrimp L. vannamei, compared with pathogens or foreign particles enter the haemo- b-glucan alone (Wongsasak et al. 2015). coel, the haemocyte initiates phagocytosis (Li, Yeh The SGR of marron improved significantly when & Chen 2010), whereas in crayfish the phagocyto- fed B. mycoides, OS (Sel-Plex) and their synbiotics sis is chiefly executed by hyaline cells (Johansson in supplemented feeds compared with basal diet- et al. 2000). In the present study, bacteraemia of fed marron. However, marron fed synbiotic feed probiotic-, OS- and synbiotic-fed marron was sig- was not significantly different to singular supple- nificantly lower compared with basal diet-fed mentation of either B. mycoides or OS (Sel-Plex). marron indicating that probiotic, OS and their Improved growth using synbiotic feed has been synbiotic effectively reduces foreign particles in the demonstrated in rainbow trout O. mykiss finger- haemolymph as a result of higher THC and lings (Rodriguez-Estrada et al. 2009; Mehrabi et al. proportion of hyaline cells. 2012), European lobster H. gammarus (Daniels Chronic bacteraemia results in immunosuppres- et al. 2010), Japanese flounder Paralichthys (Ye sion, reduced growth rates in Cherax quadricarina- et al. 2011) and Nile tilapia Oreochromis niloticus tus (Edgerton & Owens 1999) and mortality, thus (Hassaan, Soltan & Ghonemy 2014). clearance of the bacterial pathogens in the circu- The main purpose of using both prebiotics and lating haemolymph is essential to maintain animal probiotics in aquatic animals is to improve health, health. Sang et al. (2009) and Nugroho and Fote- immunity and growth by stimulating commensal dar (2015) demonstrated that greater THC and a intestinal bacteria of the host. The feed supplemen- higher proportion of hyaline cells reduce tation of B. mycoides or OS (Sel-Plex) alone

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–12 7 Synbiotic effect of B. mycoides & OS on marron I Ambas et al. Aquaculture Research, 2016, 1–12 significantly improved intestinal bacterial density avoid any adverse effects of the synbiotic feeds on compared with the basal diet-fed marron; how- the hosts as was found in sea bream, S. aurata L., ever, the synbiotic use did not further improve the where the combined administration of inulin and total bacterial population compared with single B. subtilis increased susceptibility to infection (Cer- supplementation. The highest intestinal bacterial ezuela et al. 2012). load of B. mycoides-fed marron compared with Members of the Bacillus genus represent the basal diet and other probiotic candidates was also majority of bacteria used as probiotics due to their demonstrated in the previous study using various physical and biological characteristics (Wang, Li & probiotic sources (Ambas et al. 2013). Zhang et al. Lin 2008). The most widely used in synbiotic stud- (2011) observed a significant increase in total bac- ies include B. coagulans (Lin et al. 2012), B. subtilis terial counts of shrimp P. japonicus fed a synbiotic (Zhang et al. 2010, 2011; Geng et al. 2011; Cere- diet. Microbiota of GIT can be considered as a zuela et al. 2012, 2013; Zhang, Yu, Tong, Tong, metabolically active organ (Gaggıa, Mattarelli & Dong, Xu & Wang 2014; Wongsasak et al. 2015), Biavati 2010), which provides an initial barrier to B. licheniformis (Zhang et al. 2011; Hassaan et al. pathogen entry (Sugita, Tsunohara, Ohkashi & 2014), B. megeterium (Li et al. 2009) and B. clausii Deguchi 1988; Verschuere et al. 2000; Ramirez & (Ye et al. 2011). In the present study, B. mycoides Dixon 2003; Ringø, Olsen, Mayhew & Myklebustd was selected as it was of host origin and possessed 2003), thus its stability (density and diversity) is a number of probiotic properties (Ambas, Buller essential for the health of an organism (Rollo, Sul- et al. 2015) such as improved marron immunity pizio, Nardy, Silvi, Orpianesi, Caggiano, Cresci & against V. mimicus (Ambas et al. 2013) and Carnevali 2006; Denev et al. 2009). improved GIT health status (Ambas, Fotedar et al. The effect of the Sel-Plex, yeast-derived OS, on 2015). Overall, the present study suggested that B. mycoides evaluated in this study demonstrated B. mycoides and OS (Sel-Plex) contribute a syner- that density of B. mycoides was higher in OS-free getic effect on marron immunity particularly on TSB media compared with B. mycoides in oxidative enzyme activity (GPx) and the THC, but TSB + OS media indicating that OS (Sel-Plex) did to a lesser extent on growth rate. In addition, an not improve the growth of B. mycoides. Cerezuela in vitro test revealed that dissolved OS (Sel-Plex) in et al. (2011) reviewed synbiotic studies and con- TSB media did not improve B. mycoides growth. cluded that dietary synbiotic effect is most likely dependent on the fish species, feeding dose and Acknowledgments duration, and the type of prebiotics and probiotics synbiotic combinations. In shrimp, L. vannamei,a The authors thank the Government of Indonesia combination of b-glucan and P. acidilactici was particularly The Directorate General of Higher better than b-glucan and B. subtilis in terms of Education (DIKTI) and The Department of Fishery, SOD activity of the animal (Wongsasak et al. Faculty of Marine Science and Fishery, Hasanud- 2015). din University of Makassar Indonesia which finan- Synergetic effect of B. mycoides and OS was cially supported this study. The authors also observed on marron immunity (GPx and THC), thank Mr Ardiansyah for technical assistance in but not on the growth rate. A similar result was GPx samples preparation and analysis and observed in koi, C. carpio koi, using dietary COS Mr Suleman for his assistance in analysing the and B. coagulansi, which synergistically improved data. innate immunity and resistance, but did not improve the growth (Lin et al. 2012). Presumably References this was due to the fact that the probiotic B. mycoides did not grow well on OS-added substrate Ai Q., Xu H., Mai K., Xu W. & Wang J. (2011) Effects of dietary supplementation of Bacillus subtilis and fruc- and resulted in a lower intestinal bacterial popula- tooligosaccharide on growth performance, survival, tion (Fig. 2), as each strain has substrate prefer- non-specific immune response and disease resistance of ence (Rastall & Maitin 2002). No synbiotic studies juvenile large yellow croaker, Larimichthys crocea. have examined in vitro the effect of pure prebiotics Aquaculture 17, 155–161. solely on the selected probiotics to determine Ambas I., Suriawan A. & Fotedar R. (2013) Immunologi- whether the prebiotics improve or inhibit the cal responses of customised probiotics-fed marron, growth of the probiotics. This test is essential to Cherax tenuimanus, (Smith 1912) when challenged

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