*Manuscript Click here to view linked References 1 2 Monitoring of the bioencapsulation of a probiotic Phaeobacter strain in the rotifer 3 Brachionus plicatilis using denaturing gradient gel electrophoresis 4 5 José Pintado1*, María Pérez-Lorenzo1,2, Antonio Luna-González1,3, Carmen G. Sotelo1, 6 María J. Prol1 and Miquel Planas1 7 8 1Instituto de Investigacións Mariñas (CSIC), Eduardo Cabello nº 6, 36208 Vigo, 9 Galicia, Spain. 10 2 Present address: Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, 11 36310 Vigo, Galicia, Spain. 12 3 Present address: Centro Interdisciplinario de Investigación para el Desarrollo Integral 13 Regional-Instituto Politécnico Nacional, Unidad Sinaloa. Boulevard Juan de Dios Bátiz 14 Paredes 250, Guasave, Sinaloa 81101, México 15 16 17 *Corresponding author: Phone +34986231930. Fax +34986292762. E-mail address: 18 [email protected]. 19 20 Abstract 21 22 The bioencapsulation of the probiotic bacteria Phaeobacter 27-4 in the rotifer 23 Brachionus plicatilis was monitored by culture methods and denaturing gradient gel 24 electrophoresis (DGGE) of PCR-amplified 16S rDNA. 25 In a first experiment, the permanence of the probiotic bacteria in clear water and green 26 water was studied. Phaeobacter 27-4 added to the water of the tanks (107 CFU ml-1) 27 remained at levels around 106 CFU ml-1 for 72 h and was not affected by the presence of 28 the algae added (Isochrysis galbana, 105 cells ml-1). The DGGE fingerprints showed a 29 temporal predominance of the probiont in the water and the presence of bacteria 30 belonging to the Flavobacteria, -proteobacteria, and Sphingobacteria groups. A 31 Tenacibaculum strain became predominant when Phaeobacter 27-4 decline, and at the 32 end of the experiment, bacterial profiles became similar to the initial ones with 33 predominance of bacteria belonging to the Oceanospirillaceae family. 1 1 Three different ways of bioencapsulation of the probiont in the rotifer were assayed: 2 E24, addition of Phaeobacter 27-4 for 24 h during the enrichment with I. galbana; E3, 3 addition of Phaeobacter 27-4 during the last 3 h of the enrichment with I. galbana and 4 E3+, with the bioencapsulation done in a separated step, after the 24 h enrichment with 5 I. galbana, being the rotifers filtered, washed and transferred into tanks containing 6 Phaeobacter 27- 4 in seawater, and maintained for 3 h. 7 The result showed that the presence of the algae was not determinant in the 8 effectiveness of the bioencapsulation and the probiont was bioencapsulated in all cases 9 in the first 3 h to a level of 102 cfu rotifer-1. When the rotifers with the bacteria 10 bioencapsulated were transferred to green-water tanks and kept in the conditions used in 11 turbot larvae rearing, Phaeobacter 27-4 maintained in levels close to 102 CFU rotifer-1 12 for 48 h in the case of E24 and E3, and for 24 h in the case of E3+, a period of time 13 sufficient to the larvae to graze on them and to incorporate the probiotic. The E24 14 protocol was selected for the simplicity of the procedure. DGGE fingerprints showed 15 the incorporation of the probiotic and a temporal colonization of the rotifers. 16 Predominant bands identified in the rotifers correspond to -proteobacteria as 17 Pseudoalteromonas. 18 19 Keywords: Probiotic; Phaeobacter 27-4; Bioencapsulation; Rotifer; Larval rearing 20 21 2 1 2 1. Introduction 3 4 In the last years, efforts have been made to develop strategies for microbial control, to 5 decrease the use of therapeutic chemicals and antibiotics (Cabello, 2006), towards a 6 more environmentally friendly and sustainable aquaculture. Probiotics, defined by 7 FAO/WHO (2001) as “live microorganisms which, when administered in adequate 8 amounts, confer a health benefit on the host”, constitute a potential tool in the reduction 9 of mortalities in the rearing of aquatic organisms (Gatesoupe, 1999; Verschuere et al., 10 2000; Vine et al., 2006; Kesarcodi-Watson et al., 2008). 11 12 In turbot larvae (Psetta maxima), the use of probiotics has been studied using 13 commercially available or selected terrestrial lactic acid bacteria (Gatesoupe, 1991; 14 Planas et al., 2004). A better strategy, that avoids the introduction of exotic bacteria to 15 the system, is to select probiotic candidates among isolated strains from healthy turbot 16 (Westerdahl et al., 1991) or hatchery facilities (Huys et al., 2001; Hjelm et al., 2004a). 17 Generally, selection is based on the antagonistic effect to pathogenic bacteria which are 18 responsible of high mortalities in turbot larvae such as Listonella (Vibrio) anguillarum 19 or Vibrio splendidus (Toranzo et al., 1994; Thomson et al., 2005). 20 21 Members of the Roseobacter cluster (-Proteobacteria) produce tropodithietic acid 22 (TDA), compound that inhibits to different and -Proteobacteria (Martens et al., 23 2007). The strain Phaeobacter 27-4, used in this study, was isolated from turbot larval 24 rearing units by Hjelm et al. (2004a) and sequence analysis of 16S rRNA gene showed 25 99.1 % alignment with Phaeobacter gallaeciensis (Ruiz-Ponte et al., 1998). 26 Phaeobacter 27-4 showed antagonism against L. anguillarum and V. splendidus (Hjelm 27 et al., 2004a, b). Furthermore, Planas et al. (2006) demonstrated, in challenge trials with 28 L. anguillarum, a probiotic in vivo effect of Phaeobacter 27-4, being not harmful to 29 turbot larvae. 30 31 Intestinal microbiota of turbot larvae is strongly dependent on the bacteria present in 32 live prey and, to a lesser extent, in the rearing water (Nicolas et al., 1989; Munro et al., 33 1994; Blanch et al., 1997; Reitan et al., 1998). The rotifer Brachionus plicatilis is 34 widely used as live prey in turbot hatcheries and mass culture of rotifers conducts to a 3 1 high load and a variable bacterial microbiota (Verdonck et al., 1994) in their external 2 surface (Munro et al., 1993) and digestive tract (Skjermo and Vadstein, 1993). This 3 microbiota is dominated by strains with a low degree of specialization and high growth 4 rates (Salvesen et al., 1999) that can be detrimental to turbot larvae (Pérez-Benavente 5 and Gatesoupe, 1988; Verdonck et al., 1997). This fact was demonstrated by the 6 increment in the survival of larvae fed with axenic rotifers (Munro et al., 1995). 7 8 So, the control of bacterial microbiota in live feed is an important issue (Planas and 9 Cunha, 1999; Dhert et al., 2001; Skjermo and Vadstein, 1999) and treatments based on 10 disinfection of rotifer eggs for the production of axenic cultures, treatment with 11 hydrogen peroxide or ultraviolet radiation for partial decontamination. can be useful 12 tools (Dhert et al., 2001). However, the elimination of bacteria from live prey implies 13 the loss of a stable microbial balance, predominated by K-strategists, and may favour a 14 more rapid colonization by opportunistic bacteria with high growth rates (r-strategists), 15 once the rotifers are introduced into the larval rearing system. Replacement of the 16 opportunistic bacteria by a preventive colonization with other non-aggressive bacteria 17 with persistence in water or live food can be a good strategy to provide protection to the 18 larvae (Makridis et al., 2000a; Martínez-Díaz et al., 2003). 19 20 Delivery of probiotic bacteria to live prey can not only serve as control agent of 21 opportunistic or pathogenic bacteria but also be a vehicle for introducing probiotics to 22 fish larvae (bioencapsulation) (Gatesoupe, 1994, 1999; Skjermo and Vadstein, 1999; 23 Ringø and Birkbeck, 1999; Makridis et al., 2000b). Feeding larvae with rotifers 24 enriched with Phaeobacter 27-4, parallel to fish pathogen Listonella anguillarum 25 infection, in an experimental challenge model (Planas et al., 2005), brought the 26 accumulated mortality to the level of control, demonstrating the effectiveness of 27 bioencapsulation of the probiotic in rotifers (Planas et al., 2006). 28 29 In a normal practice, before delivery to fish larvae, rotifers are enriched with essential 30 fatty acids, such as polyinsaturated fatty acids (PUFAs), feeding them with microalgae 31 (e.g. Tetraselmis sp., Isochrysis sp., Rhinomonas sp., Rhodomonas sp.) (Dhert et al., 32 2001), The “green water” technique, which consists in the addition of microalgae to the 33 rearing tanks, reduces the proliferation of opportunistic bacteria in the surface of turbot 34 larvae (Salvesen et al., 1999). Algal cultures associate a specific bacterial population 4 1 (Schulze et al., 2006), which might influence bacteria number and composition in 2 rotifers and fish larvae, and members of the Roseobacter clade have been frequently 3 found associated to microalgae cultures (Sandaa et al., 2003; Hjelm et al, 2004b; 4 Nicolas et al., 2004). Algae can also be a factor influencing the grazing of bacteria by 5 rotifers (Nicolas et al., 1989). Consequently, interactions bacteria-microalgae must be 6 considered in the maintenance of the probiont in the rearing tanks and in the 7 bioencapsultion process. 8 9 Monitoring of bacteria introduced in the rearing systems and studying the modification 10 of the associated bacterial microbiota, is an important aspect to understand the way of 11 action of probiotic bacteria. Generally, introduced strains are analysed by culture- 12 dependent methods (e.g. Martínez-Díaz et al., 2003; Planas et al., 2006) and only a few 13 studies use complementary culture-independent techniques to screen the introduced 14 strains, such as inmunocolony-blot (Makridis et al., 2000a, b), ELISA (Makridis et al., 15 2000a, b) or in situ hybridization (Macey and Coyne, 2005). Denaturing Gradient Gel 16 Electrophoresis (DGGE) of 16S rDNA is a very useful technique for genetic 17 fingerprinting of the bacterial community and to monitor changes in its composition 18 (Muyzer, 1997). Furthermore, the excision, re-amplification and sequencing of the 19 bands from the DGGE gels makes possible to identify the bacteria present.