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Characterization of the intestinal microbiota of wild-caught and farmed fine flounder (Paralichthys adspersus).

INTRODUCTION

The flatfish Paralichthys adspersus has great potential in Chilean aquaculture. However, its farming at a commercial scale is limited by low growth rates (Silva, 2010). Since few studies on digestion and nutrient metabolism are available, new information regarding the intestinal microbiota that can facilitate or promote these functions is needed to improve farming practices of this species. Microbiota plays a major role in nutrition, growth, health, and survival of the host fish because some bacteria supply exogenous nutrients, produce extracellular enzymes, vitamins and fatty acids (Dhanasiri et al., 2011). For such reason, there is interest in understanding how bacterial populations in the gastrointestinal tract are structured, and the role they play on the fitness of their hosts.

Studies in mammals reveal associations among the composition of the microbiota and host diet, anatomy and phylogeny (Ley et al., 2008). In fish, the variation in the composition of the microbiota is strongly associated with the habitat, trophic level, and phylogenetic relationships of the species (Sullam et al., 2012). Comparative analyses using data from fish intestines and other environments revealed that the microbiota of fish is not a simple reflection of the organisms in their local habitat, but also the result of the host-specific selective pressures within the intestine (Bevins & Salzman, 2011; Navarrete et al., 2012).

The effect of the diet on microbiota is one of the most documented aspects in farmed fish. Wong et al. (2013) report changes in the composition of the microbiota of Oncorhynchus mykiss when fishmeal is replaced for soybean meal. The fine flounder feeds on fish, crustaceans and mollusks in the wild, but is raised with artificial diets and kept captive in different habitats than its natural ones, similarly as other farmed organisms. In general, such conditions increase the interest in knowing what microbiota changes are experienced by organisms in captivity with respect to those on the wild. There are diversity studies regarding the microbiota in S. salar (Holben et al., 2002), Danio rerio (Roeselers et al., 2011), Gadus morhua (Dhanasiri et al., 2011), Peneaus monodon (Rungrassamee et al., 2014). However, in the genus Paralichthys, such information is restricted to P. olivaceus (Kim & Kim, 2013). Colston & Jackson (2016) argue whether work on captive animals can be used to predict the gut microbiomes of animals in the wild. This problem has been suggested before (Amato, 2013), yet there is still a substantial lack of studies that have attempted to characterize the enteric microbial communities in hosts within a natural environment.

Populations of lactic acid bacteria (LAB) are highly variable in the intestines of fish and change as the aquatic environment does, i.e., farming or wilderness (Hagi et al., 2004). LAB is a group widely investigated in animals because it plays an important role on the health and nutrition of the host (Vazquez et al., 2005; Lauzon & Ringo, 2012). Korean researchers observed that species richness of LAB in P. olivaceus was significantly higher in the intestines of wild fish than in farmed specimens (Kim & Kim, 2013). Our study aimed to characterize cultivable bacterial populations of microbiota of P. adspersus farmed (AF) and wildcaught (WF) emphasizing on the detection of LAB.

MATERIALS AND METHODS

Fish

Farmed fine flounder were obtained from aquaculture facilities of Cultivos Marinos Tongoy (AF: n = 15, average weight = 100 g). Wild fine flounder (WF: n = 7, weight = ~300 g) were captured in the Region of Coquimbo. AF came from the same cohort fed with commercial pellets, without addition of probiotics, immunostimulants or inhibitors. AF and WF showed no apparent deformities or diseases.

Isolation and counts of intestinal microbiota

The intestinal contents of wild-caught and farmed fish were collected and treated as previously described (Navarrete et al., 2010). Serial dilutions of intestinal contents were spread over plates of Trypticase soy agar (TSA) for screening heterotrophic bacteria and Man, Rogosa and Sharpe Agar (MRS) for screening of lactic acid bacteria (LAB). Incubation was carried out at 17[degrees]C for 10 days and colonies were isolated in fresh medium for evaluation. The total bacterial counts were assessed by epifluorescence microscopy using acridine orange (Romero & Espejo, 2001). Briefly, serial dilution of intestinal content were filtered (0,2 gm) and then stained. Total counts were calculated after counting 10 fields for each sample, using 100x of a epifluorescence microscope. Analysis of feed was done following (Romero & Navarrete, 2006); briefly, pellets were weighed and an equal weight of sterile TE buffer (Tris 0.1M, EDTA 0.01M, NaCl 0.15M, pH 7.8) was added. The mixture was homogenized in vortex and then serial dilutions were spread and incubated as described above. Water samples were obtained directly from the farm's water source (water influent); total counts and viable count were performed from serial dilutions as described above. All the samples were analyzed in triplicate.

PCR amplification, sequencing and analysis of bacteria

The isolates were grown overnight in Tryptic soy broth (TSB) at 37[degrees]C and harvested by centrifugation. Extraction of bacterial genomic DNA was carried out as previously reported (Navarrete et al., 2012). In order to select the strains that were going to be sequenced, we amplified a section of 16S rRNA for all isolates (positions 341 to 907) which was subjected to PCRRFLP, using PCR conditions described in (Navarrete et al., 2012). PCR products were digested with EcoRI-HF [TM] and Hpa II (New England BioLabs Inc.), and visualized as (Romero et al., 2002). Bacterial strains corresponding to different PCR-RFLP patterns were selected for sequencing (Macrogen, USA). For sequencing, the region 27 to 1492 of the 16S rRNA was amplified as described (Romero et al., 2002). Isolates with identical sequences were subjected to ITS analysis (Gonzalez et al., 2003). This information allowed the comparison of microbiota diversity between WF and

AF. In both cases, PCR-RFLP and ITS profiles were assessed with cluster analysis. The sequences were edited and cleaned up with BIOEDIT software (Hall, 1999) and compared to those of the public RDP database for identification.

RESULTS

The bacterial counts in the intestinal contents of wild flounder (WF) and aquaculture flounder (AF) and) were 5.14 [+ or -] 0.71 and 5.49 [+ or -] 0.59 (Log10 Colony Forming Units, CFU [g.sup.-1]), respectively when TSA was used. These levels were according to reported bacterial load in other flatfish (MacDonald et al., 1986; Eddy & Jones, 2002; Verner-Jeffreys et al., 2003; MartinAntonio et al., 2007; Fig. 1). Total counts under epifluorescence microscope averaged 7.25 [+ or -] 0.81 and 7.30 [+ or -] 1.06 (Log 10 bacteria g) of intestinal content AF and WF respectively; hence, the cultivability in AF and WF was roughly near to 1%. The total bacterial counts in the water of the farm showed an average of 3.3 [+ or -] 0.47 (Log10 bacteria m[L.sup.-1]), whereas the viable count in TSA showed an average of 1.67 [+ or -] 0.35 (Log10 CFU m[L.sup.-1]). Simultaneously, feed samples were examined, showing a viable count of 2.99 [+ or -] 0.55 (Log10 CFU [g.sup.-1]). Lactic acid bacteria were examined using MRS, however, only samples from intestinal contents of WF showed colonies at level of 3.51 [+ or -] 0.60 (Log10 CFU [g.sup.-1]).

A total of 100 bacterial isolates were included in this study. A set of 73 isolates were analyzed by 16SrRNA sequencing and 16SrRNA RFLP, and 47 sequences of partial 16SrRNA were deposited in GenBak under the Accession Numbers: KP453988-KP453997; KP731 550-KP731586. Examination of these sequences in RDPii database allowed the taxonomic identification of the isolates.

The phylum Proteobacteria was the dominant bacterial group in WF, and ranked second after Firmicutes in AF (Fig. 2). Proteobacteria is the dominant phylum in the microbiota of other flatfish, such as, Solea senegalensis (Martin-Antonio et al, 2007; TapiaPaniagua et al., 2010), P. olivaceus (Sugita et al., 2002; Kim & Kim, 2013) and Scophthalmus maximus (Xing et al., 2013). This bacterial group may contribute to the digestive process by providing a variety of enzymes (Neulinger et al., 2008; Smriga et al., 2010). In our research, the lower representation of Proteobacteria in AF with respect to WF requires further study to establish if this is affecting the nutrient metabolism in the fish resulting in low growth rates. The two most representative Gammaproteobacteria in AF were Vibrio and Photobacterium both belonging to Vibrionaceae, whereas in WF, Psychrobacter and Acinetobacter were the most common isolates (Fig. 2).

Isolates from the phylum Firmicutes correspond to 60% of bacteria in AF. In flatfish, they have been reported as the second most abundant phylum (Sugita et al., 2002; Martin-Antonio et al., 2007; Tapia-Paniagua et al., 2010; Kim & Kim, 2013; Xing et al., 2013). The most representative Firmicutes were Bacillus and Carnobacterium in AF, and Staphylococcus and Lactobacillus in WF (Fig. 2). Bacillus is frequently isolated from the microbiota of several marine species including flatfish (Hovda et al., 2007; Sun et al., 2010; Kim & Kim, 2013).

The examination of the microbiota at the genus level reveals that WF showed 11 genera, representing a higher diversity than AF with only 5 genera. In WF, Psychrobacter and Acinetobacter were the dominant bacteria summing a 52% of relative abundance. In contrast, in AF, Vibrio and Bacillus were the dominant genera (Fig. 2). Interestingly, Bacillus was the most abundant genus in the water of the farm and in the feed, 23% y 50%, respectively. Furthermore, the composition of the water of the farm showed two bacterial genera (Staphylococcus and Bacillus) in coincidence to the microbiota of AF and the genus Bacillus was detected in the feed and in the microbiota of AF (Figs. 2-3). Bacillus, Lysinibacillus and Pseudomonas were common component between water and feed (Fig. 3). Interesting bacterial groups such as lactic acid bacteria (LAB) showed also differential distribution, Carnobacterium was isolated from AF, whereas Lactobacillus, Weisella, Lactococcus, Streptococcus and Vagococcus were retrieved from WF. Figure 4 represents the clustering and molecular diversity of the bacterial isolates including LAB retrieved from the different samples examined, based on RFLP of 16S rRNA gene and some of them by sequencing. In this figure, the RFLP profile of isolates from different sources can be compared; in a roughly distribution, Gammaproteobacteria grouped separatedly from Bacilli, independently of the origin.

DISCUSSION

Low levels of cultivability observed in this work are attributed to the lack of knowledge of suitable conditions for culture to recover certain bacterial populations. However, this is not a limitation to study microbiota composition, because some abundant genus obtained by culture methods can be also retrieved from DNA analysis (Romero & Navarrete, 2006; Navarrrete et al., 2010). Reduced cultivability P. adspersus may be due to the fact that microbiota is formed of species capable of forming colonies on agar plates but with low efficiency, or is composed of unknown species that do not grow on common microbiological media.

Vibrio has been reported as a typical genus in the intestine of the farmed P. olivaceus (Tanasomwang & Muraga, 1988; Sugita & Ito, 2006), P. dentatus (Eddy & Jones, 2002), S. senegalensis (Tapia-Paniagua et al., 2010), H. hippoglossus (Verner-Jeffreys et al., 2003) and S. maximus (Montes et al., 2003). Intriguingly, in our study, Vibrio was isolated from AF but not from WF. One explanation is based on the use of artificial diets that may increase the load of Vibrio in P. adspersus, as it was previously observed in S. senegalensis and P. olivaceus (Tanasomwang & Muroga, 1988; Martin-Antonio et al., 2007). However, it is necessary to consider that the absence of Vibrio in WF may be due to (i) low dominance of this genus in the intestinal community, caused by the presence of bacteria with antagonistic effect against vibrios (Sun et al., 2009) or (ii) Vibrio spp. are viable but noncultivable on media (Silva et al., 2011). Some species of Vibrio and Photobacterium have been classified as opportunistic pathogens in farmed flatfish including P. adspersus (Miranda & Rojas, 1996; Sugita et al., 2002; Villamil et al., 2003; Vazquez et al., 2005; MartinAntonio et al., 2007) and in some species like Epinephelus coioides, Vibrio spp. are part of the microbiota of fish with low growth rates (Sun et al., 2009). Despite its negative connotation, Vibrio spp. has a wide enzymatic activity (i.e., amylase, protease, lipase, chitinase) (MacDonald et al., 1986; Sugita & Ito, 2006); and further studies are needed to establish its positive or negative role in AF.

Psychrobacter and Acinetobacter have been isolated from marine fish (MacDonald et al., 1986; Tanasomwang & Muroga, 1988; Eddy & Jones, 2002; Sugita et al., 2002; Ringo et al., 2006; Sun et al., 2009). Our research constitutes the first report of Psychrobacter in Paralichthys species. The occurrence of Psychrobacter in WF is remarkable because it is associated with positive physiological effects on growth rates and improved health of the host (Sun et al., 2009, 2011). Sun et al. (2009) found that Psychrobacter only occurred and predominated the microbiota of E. coioides with fast growth rates. It has also been suggested that dietary management of E. coioides with Psychrobacter sp. inhibits the growth of pathogenic bacteria (Vibrio spp.), and set conditions in the gut bacteria promoting the establishment and colonization of other types of bacteria in the intestinal tract of fish.

In our study, Firmicutes corresponded to a abundant bacterial population in AF. In flatfish, they have been reported as the second most abundant phylum (Sugita et al., 2002; Martin-Antonio et al., 2007; TapiaPaniagua et al., 2010; Xing et al., 2013; Kim & Kim, 2013). LAB, part of this phylum, are interesting bacteria because they have been used as a probiotic to improve health and growth rates of several flatfish (Villamil et al., 2002). The diversity and load of LAB of fish is affected by nutritional and environmental factors. According to Kim & Kim (2013) the food commonly used in aquaculture of P. olivaceus is an unsuitable substrate for the colonization of LAB in the gut of this fish. For this reason, LAB populations are low or non-present in farmed P. olivaceus. The presence of a single genus of LAB in AF (Carnobacterium) is consistent with expectations in fish fed with artificial diets. The presence of this genus was detected in TSA instead of MRS, because it has been previously described that Carnobacterium is inhibited by components of this medium such as acetate (Leisner et al., 2007). Other interesting genus is Weissella, it has been isolated from a broad variety of animals and specific Weissella strains are also receiving attention as potential probiotics, acting by inhibition of pathogens, and also some strains are known to produce copius amounts of non-digestible polysaccharides, with potential application as prebiotics (Fusco et al., 2015). Recently, the species named Weissella ceti species was associated with diseased fish (Figueiredo et al., 2014). Natural diets provide various nutritional factors such as amino acids, B vitamins, among others, that facilitate growth and colonization of LAB in the intestinal tract (Madigan et al., 2010). The feed supplied to AF did not have any B vitamins supplemented, whereas small amounts of anchovy eaten by WF may contain tiamin (B1) and riboflavin (B2) that could help support LAB in WF (Reyes et al., 2009).

Probiotic bacteria isolated from a particular host or their environment are more beneficial to the host itself or related species than bacteria isolated from other sources. This is due in large part because there is specificity in colonization by the strain-host complex or vice versa. Ying et al. (2007) observed that Lactobacillus adhesion to the surface of the intestinal tract of P. olivaceus depends on the specificity of the host strain. Given the above, we suggest that the microbial community of wild P. adspersus provides a suitable environment for the adhesion and colonization of this type of bacteria, and that the LAB isolated in this study could be studied as a specific probiotic to improve production traits in AF.

The phyla Actinobacteria and Bacteroidetes were underrepresented in WF and absent in AF (Fig. 1) similarly to the observed in farmed and wild P. olivaceus (Kim & Kim, 2013). Sullam et al. (2012) showed that in S. senegalensis these phyla are approximately 10% of the total microbiota. In our study, 13% of the microbiota of WF were represented by these phyla. Actinobacteria represents one of the largest taxonomic units among the 18 major lineages currently recognized within the domain Bacteria; members of this phylum exhibit diverse physiological and metabolic properties, such as the production of extracellular enzymes and the formation of a wide variety of secondary metabolites, however, its role in the microbiota of fish is not well documented (Ventura et al., 2007).

Studies in mammals report that certain Firmicutes are linked to the intake of nutrients, and extraction and regulation of energy homeostasis from their diet (Krajmalnik-Brown et al., 2012). However, in rodents an increase of Firmicutes may be caused by the increase in the intake of carbohydrates and/or polysaccharides (Turnbaugh et al., 2008). The artificial food of AF containing an 11% of carbohydrates could explain the higher proportion of Firmicutes in AF with respect to WF. WF fed mainly on anchovy Engraulis ringens, a carbohydrate-free food when eaten fresh (Reyes et al., 2009).

The load and diversity of fish microbiota are influenced by many intrinsic and extrinsic factors (Nayak, 2010). As it has been illustrated in Fig. 1, the load of bacterial microbiota in the intestinal contents of flatfish is roughly similar, in different species and different origin (wild or reared). Food strongly influences the composition of the microbiota in fish. As example, Dhanasiri et al. (2011) evidenced a reduction of microbiota diversity when wild fish was fed with artificial diets (i.e., microbiota diversity was reduced more than 60% and Vibrionaceae and Clostridiaceae increased over Ignavibacteriaceae and Erysipelotrichaceae). Similar results are reported for S. senegalensis (Martin-Antonio et al., 2007) and P. olivaceus (Kim & Kim, 2013). Our results for WF also showed greater bacterial diversity than AF (Fig. 2). However, our findings are not consistent with those for wild salmon (Salmo salar) and sturgeon (Acipenser ruthenus) indicating lower diversity of the microbiota in wild than in farmed fish (Holben et al., 2002; Bacanu & Oprea, 2013).

DOI: 10.3856/vol45-issue2-fulltext-12

Received: 7 December 2016; Accepted: 10 January 2017

ACKNOWLEDGEMENTS

J. Salas-Leiva received a scholarship from CONICYTChile. This work was funded by Fondecyt 1140734/1171129 from CONICYT and Centro Aquapacifico 15PCTI-46284 from Corfo. The authors thank Victoria Urzua and Mauricio Valdes for technical assistance. Authors declare no conflicts of interest regarding the publication of this manuscript.

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Joan Salas-Leiva (1,2), Rafael Opazo (1,5), Camille Remond (1), Eduardo Uribe (3,5) Antonio Velez (4) & Jaime Romero (1,5)

(1) Laboratorio de Biotecnologia de los Alimentos, Instituto de Nutricion y Tecnologia de los Alimentos, Universidad de Chile, Santiago, Chile

(2) Doctorado en Acuicultura, Programa Cooperativo Universidad Catolica del Norte Pontificia Universidad Catolica de Valparaiso, Universidad de Chile

(3) Departamento de Acuicultura, Universidad Catolica del Norte, Coquimbo, Chile

(4) Fundacion Chile, Cultivos Marinos Tongoy, Coquimbo, Chile

(5) Aquapacifico, Coquimbo, Chile

Corresponding author: Jaime Romero (jromero@inta.uchile.cl)

Corresponding editor: Sandra Bravo

Caption: Figure 1. Viable counts retrieved in the intestinal contents collected from aquaculture (AF) and wild flounder (WF), obtained in this study and compared with the data available for other flatfish of aquaculture origin, in scientific literature.

Caption: Figure 2. Composition of the intestinal microbiota. a) Wild-caught flounder, b) farmed flounder. Inner circle correspond to phylum, first ring correspond to genus of bacteria isolated in Tripticase Soy Agar (TSA); second ring correspond to Lactic acid bacteria (LAB) isolated in MRS; only wild-caught, showed this outer ring. All percentages are presented in relative abundances.

Caption: Figure 3. Bacterial populations present in a) water (aquaculture facility) and b) feed. Inner circle correspond to phylum, ring correspond to genus of bacteria isolated in TSA. All percentages are presented in relative abundances.
Figure 4. Dendrogram of 16S rRNA gene RFLP of bacterial isolates
retrieved from microbiota of fine flounder. For some of the
isolates the 16S rRNA gene was sequenced and the identification
was included as phylum and genus.

Isolate   Source.   Taxonomy                                  Media

LC6       AF        Gammaproteobacteria.    Vibrio             TSA
LC7                                                            TSA
LC9                                                            TSA
LC12a                                                          TSA
LC12b                                                          TSA
F2a       Feed                                                 TSA
A5c       Water     Gammaproteobacteria.    Pseudomonas        TSA
A10b                                                           TSA
A4                  Gammaoroteobacterio.    Psvchrobacter      TSA
A5b                 Gammaproteobacteria.    Psychrobacter      TSA
LS12      WF        Gammaproteobacteria.    Psychrobacter      TSA
LS13a                                                          TSA
LS10a                                                          TSA
LS16a
LSlOb                                                          TSA
LS11a               Gammaproteobacteria.    Raoultella         TSA
A9        Water     Gammaproteobacteria.    Psychorobacter     TSA
LS9                 Gammaproteobacteria.    Kluyvera           TSA
LS6a                Gammaproteobacteria.    Acinetobacter      TSA
LS17a                                                          TSA
LS18                                                           TSA
A6        Water                                                TSA
LS1       WF                                                   TSA
LC11      AF        Gammaproteobacteria.    Acinetobacter      TSA
A11       Water                                                TSA
LS13b     WF                                                   TSA
LS14a     WF        Gammaproteobacteria.    Psychorobacter     TSA
A7        Water                                                TSA
LS25b     WF                                                   TSA
LS22      WF                                                   TSA
F2b       Feed                                                 TSA
LS6       WF                                                   TSA
LS6a                                                           TSA
LS6b                                                           TSA
LS24a               Gammaproteobacteria.    Acinetobacter
LS3a                                                           TSA
LS14b                                                          TSA
LS20e                                                          TSA
F3b       Feed                                                 TSA
A3        Water                                                TSA
LS11b     WF        Gammaproteobacteria.    Stenotrophomonas   TSA
F3b       Feed                                                 TSA
LS16b     WF                                                   TSA
LS13      AF                                                   TSA
F3c       Feed                                                 TSA
LS25a     WF                                                   MRS
LS29      WF                                                   MRS
FF        Feed                                                 TSA
A12a      Water                                                TSA
F2c       Feed                                                 TSA
LS15a     WF        Bacilli.                Exiguobacterium    TSA
F1d       Feed                                                 TSA
F1a       Food                                                 TSA
AS        Water                                                TSA
LC1       AF        Bacilli.                Carnobactorium     TSA
LC2       AF        Bacilli.                Carnobactorium     TSA
LS4b      WF                                                   TSA
LC5b      AF        Bacilli.                Bacillus           TSA
A1        Water                                                TSA
LC4b      AF                                                   TSA
LC4a                                                           TSA
LC10                                                           TSA
LC3                                                            TSA
LS5       WF        Bacilli.                Klebsiella
LS3b      WF                                                   TSA
F1c       Feed                                                 TSA
A10a      Water                                                TSA
LS4a      WF        Flavobactoria           Myroides           TSA
LS21a               Bacilli.                Lactobacillus      TSA
LS23a               Bacilli.                Lactococcus        MRS
LS24b                                                          MRS
LS20d               Bacilli.                Weisdla            MRS
A5a       Water                                                TSA
LS20b     WF        Bacilli.                Weisella           MRS
LS2a                Bacilli.                Streptococcus      TSA
LS24a               Bacilli.                Weisella           MRS
LS21c               Bacilli.                Lactobacillus      MRS
LS2b                                                           MRS
LS21b                                                          MRS
LS23b                                                          MRS
LS26a               Bacilli.                Lactobacillus      MRS
LS26b                                                          MRS
LS24c                                                          MRS
LS24e                                                          MRS
LS7a                Flavobactoria           Myroides           TSA
LS27                                                           MRS
LS241                                                          MRS
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Title Annotation:Research Article
Author:Salas-Leiva, Joan; Opazo, Rafael; Remond, Camille; Uribe, Eduardo; Velez, Antonio; Romero, Jaime
Publication:Latin American Journal of Aquatic Research
Article Type:Ensayo
Date:May 1, 2017
Words:5274
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