Latin American Journal of Aquatic Research E-ISSN: 0718-560X [email protected] Pontificia Universidad Católica de Valparaíso Chile Salas-Leiva, Joan; Opazo, Rafael; Remond, Camille; Uribe, Eduardo; Velez, Antonio; Romero, Jaime Characterization of the intestinal microbiota of wild-caught and farmed fine flounder (Paralichthys adspersus) Latin American Journal of Aquatic Research, vol. 45, núm. 2, mayo, 2017, pp. 370-378 Pontificia Universidad Católica de Valparaíso Valparaíso, Chile Available in: http://www.redalyc.org/articulo.oa?id=175051041012 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Lat. Am. J. Aquat. Res., 45(2): 370-378, 2017 Intestinal microbiota of fine flounder 370 1 DOI: 10.3856/vol45-issue2-fulltext-12 Research Article Characterization of the intestinal microbiota of wild-caught and farmed fine flounder (Paralichthys adspersus) Joan Salas-Leiva1,2, Rafael Opazo1,5, Camille Remond1, Eduardo Uribe3,5 Antonio Velez4 & Jaime Romero1,5 1Laboratorio de Biotecnología de los Alimentos, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile 2Doctorado en Acuicultura, Programa Cooperativo Universidad Católica del Norte Pontificia Universidad Católica de Valparaíso, Universidad de Chile 3Departamento de Acuicultura, Universidad Católica del Norte, Coquimbo, Chile 4Fundación Chile, Cultivos Marinos Tongoy, Coquimbo, Chile 5Aquapacífico, Coquimbo, Chile Corresponding author: Jaime Romero ([email protected]) ABSTRACT. A comparative analysis of the cultivable intestinal microbiota of farmed (AF) and wild-caught (WF) Paralichthys adspersus was performed. The 16S rRNA gene was used for taxa identification, and the ITS region for strain differentiation. We detected the presence of Vibrio, Bacillus, Photobacterium, Staphylococcus and Carnobacterium in AF, and Exiguobacterium, Klebsiella, Arthrobacter, Raoultella, Kluyvera, Myroides, Streptococcus, Vagococcus, Staphylococcus, Acinetobacter, Psychrobacter, Lactobacillus, Weissella and Lactococcus in WF. The microbial community was more diverse in WF than in AF. Some bacterial groups were only found in wild-caught fish and may be studied as potential beneficial agents for improving production traits in farmed fish. As the first study of microbiota of P. adspersus, it provides significant information that can potentially help improve farming practices by using strains as species-specific probiotics. Keywords: microbiota, intestinal microbiology, fine flounder, lactic acid bacteria, 16SrRNA. INTRODUCTION netic relationships of the species (Sullam et al., 2012). Comparative analyses using data from fish intestines The flatfish Paralichthys adspersus has great potential and other environments revealed that the microbiota of in Chilean aquaculture. However, its farming at a fish is not a simple reflection of the organisms in their commercial scale is limited by low growth rates (Silva, local habitat, but also the result of the host-specific 2010). Since few studies on digestion and nutrient selective pressures within the intestine (Bevins & metabolism are available, new information regarding Salzman, 2011; Navarrete et al., 2012). the intestinal microbiota that can facilitate or promote The effect of the diet on microbiota is one of the these functions is needed to improve farming practices most documented aspects in farmed fish. Wong et al. of this species. Microbiota plays a major role in (2013) report changes in the composition of the nutrition, growth, health, and survival of the host fish microbiota of Oncorhynchus mykiss when fishmeal is because some bacteria supply exogenous nutrients, replaced for soybean meal. The fine flounder feeds on produce extracellular enzymes, vitamins and fatty acids fish, crustaceans and mollusks in the wild, but is raised (Dhanasiri et al., 2011). For such reason, there is with artificial diets and kept captive in different habitats interest in understanding how bacterial populations in than its natural ones, similarly as other farmed the gastrointestinal tract are structured, and the role organisms. In general, such conditions increase the they play on the fitness of their hosts. interest in knowing what microbiota changes are Studies in mammals reveal associations among the experienced by organisms in captivity with respect to composition of the microbiota and host diet, anatomy those on the wild. There are diversity studies regarding and phylogeny (Ley et al., 2008). In fish, the variation the microbiota in S. salar (Holben et al., 2002), Danio in the composition of the microbiota is strongly rerio (Roeselers et al., 2011), Gadus morhua (Dhanasiri associated with the habitat, trophic level, and phyloge- et al., 2011), Peneaus monodon (Rungrassamee et al., ______________________ Corresponding editor: Sandra Bravo 237 1 Latin American Journal of Aquatic Research 2014). However, in the genus Paralichthys, such infor- 0.1M, EDTA 0.01M, NaCl 0.15M, pH 7.8) was added. mation is restricted to P. olivaceus (Kim & Kim, 2013). The mixture was homogenized in vortex and then serial Colston & Jackson (2016) argue whether work on dilutions were spread and incubated as described captive animals can be used to predict the gut above. Water samples were obtained directly from the microbiomes of animals in the wild. This problem has farm’s water source (water influent); total counts and been suggested before (Amato, 2013), yet there is still viable count were performed from serial dilutions as a substantial lack of studies that have attempted to described above. All the samples were analyzed in characterize the enteric microbial communities in hosts triplicate. within a natural environment. Populations of lactic acid bacteria (LAB) are highly PCR amplification, sequencing and analysis of bac- variable in the intestines of fish and change as the teria aquatic environment does, i.e., farming or wilderness The isolates were grown overnight in Tryptic soy broth (Hagi et al., 2004). LAB is a group widely investigated (TSB) at 37°C and harvested by centrifugation. in animals because it plays an important role on the Extraction of bacterial genomic DNA was carried out health and nutrition of the host (Vázquez et al., 2005; as previously reported (Navarrete et al., 2012). In order Lauzon & Ringø, 2012). Korean researchers observed to select the strains that were going to be sequenced, we that species richness of LAB in P. olivaceus was amplified a section of 16S rRNA for all isolates significantly higher in the intestines of wild fish than in (positions 341 to 907) which was subjected to PCR- farmed specimens (Kim & Kim, 2013). Our study RFLP, using PCR conditions described in (Navarrete et aimed to characterize cultivable bacterial populations al., 2012). PCR products were digested with EcoRI-HF of microbiota of P. adspersus farmed (AF) and wild- ™ and Hpa II (New England BioLabs Inc.), and caught (WF) emphasizing on the detection of LAB. visualized as (Romero et al., 2002). Bacterial strains corresponding to different PCR-RFLP patterns were selected for sequencing (Macrogen, USA). For MATERIALS AND METHODS sequencing, the region 27 to 1492 of the 16S rRNA was amplified as described (Romero et al., 2002). Isolates Fish with identical sequences were subjected to ITS analysis Farmed fine flounder were obtained from aquaculture (González et al., 2003). This information allowed the facilities of Cultivos Marinos Tongoy (AF: n = 15, comparison of microbiota diversity between WF and average weight = 100 g). Wild fine flounder (WF: n = AF. In both cases, PCR-RFLP and ITS profiles were 7, weight = ~300 g) were captured in the Region of assessed with cluster analysis. The sequences were Coquimbo. AF came from the same cohort fed with edited and cleaned up with BIOEDIT software (Hall, commercial pellets, without addition of probiotics, 1999) and compared to those of the public RDP immunostimulants or inhibitors. AF and WF showed no database for identification. apparent deformities or diseases. RESULTS Isolation and counts of intestinal microbiota The intestinal contents of wild-caught and farmed fish The bacterial counts in the intestinal contents of wild were collected and treated as previously described flounder (WF) and aquaculture flounder (AF) and) (Navarrete et al., 2010). Serial dilutions of intestinal were 5.14 ± 0.71 and 5.49 ± 0.59 (Log10 Colony contents were spread over plates of Trypticase soy agar Forming Units, CFU g-1), respectively when TSA was (TSA) for screening heterotrophic bacteria and Man, used. These levels were according to reported bacterial Rogosa and Sharpe Agar (MRS) for screening of lactic load in other flatfish (MacDonald et al., 1986; Eddy & acid bacteria (LAB). Incubation was carried out at 17°C Jones, 2002; Verner-Jeffreys et al., 2003; Martin- for 10 days and colonies were isolated in fresh medium Antonio et al., 2007; Fig. 1). Total counts under for evaluation. The total bacterial counts were assessed epifluorescence microscope averaged 7.25 ± 0.81 and -1 by epifluorescence microscopy using acridine orange 7.30 ± 1.06 (Log10 bacteria g ) of intestinal content AF (Romero & Espejo, 2001). Briefly, serial dilution of and WF respectively; hence, the cultivability in AF and intestinal content were filtered (0,2 µm) and then WF was roughly near to 1%. The total bacterial counts