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 sele- nium (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 gÀ1 of feed); basal diet sup- plemented with OS (Sel-Plex) (0.2 g kgÀ1 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 gÀ1 and OS 0.2 g kgÀ1 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 Æ 825 CFU gÀ1 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 kgÀ1 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 Æ 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 gÀ1 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 kgÀ1 of feed (Nugroho & Fotedar 2013b). (Daniels, Merrifield, Boothroyd, Davies, Factor & The marron were fed the experimental diets for 2 © 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–12 Aquaculture Research, 2016, 1–12 Synbiotic effect of B. mycoides & OS on marron I Ambas et al. 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 minÀ1 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