NOTA CIENTÍFICA

 

First characterization of gastrointestinal culturable bacteria of Patagonian toothfish Dissostichus eleginoides (Nototheniidae)

Primera caracterización de bacterias gastrointestinales cultivables en el bacalao de profundidad Dissostichus eleginoides (Nototheniidae)

 

Rocio Urtubia¹, Pablo Gallardo¹, César A. Cárdenas², Paris Lavin^2,3 and Marcelo González-Aravena^2*

¹Centro de Cultivos Marinos Bahía Laredo, Facultad de Ciencias, Universidad de Magallanes, Avda. Bulnes 01855, Punta Arenas, Chile
²Laboratorio de Biorrecursos Antárticos, Departamento Científico, Instituto Antártico Chileno, Plaza Muñoz Gamero 1055, Punta Arenas, Chile. *mgonzalez@inach.cl
³Present address: Laboratorio de Complejidad Microbiana y Ecología Funcional, Instituto Antofagasta, Universidad de Antofagasta, Chile

---

ABSTRACT

The Patagonian toothfish Dissostichus eleginoides is one of the most important fisheries from the Southern Ocean. The biology of this species is relatively well studied and some nutritionals issues have also been reported; however there is no information about the composition of the bacterial community of the gastrointestinal tract, which is essential to characterize the microbiota of this fish. The bacterial flora of D. eleginoides is here described for the first time using culturable methods. By applying traditional culture-based techniques and 16S rDNA sequencing methods it was possible to characterize the families Vibronaceae and Moraxellaceae, which were mainly represented by Vibrio and Psychrobacter, respectively. This Patagonian fish shows a microbiota very similar to other cold waters fishes.

Key words: Notothenioid, cold-water fish, subantarctic, Bacteria, 16S rRNA

---

INTRODUCTION

Commercial fishing of Patagonian toothfish Dissostichus eleginoides (Smitt, 1898) represents a global challenge over the sustainability in the exploitation of marine resources. This species is particularly vulnerable to over-exploitation because of their slow growth rate, late maturity and low fecundity (Horn 2002, Collins et al. 2010). It is in this scenario that Chile has been developing aquaculture research projects, which allowed to obtain information about the natural diet (Soto 2015), growth and survival rates in captivity (Gallardo 2016). In this process, it is essential to improve our knowledge about the bacterial flora of this fish species, because the gastrointestinal microbiota play a critical role in the physiology, nutrition and health of the host. Microbiome research in other teleost species, especially those important in an aquaculture perspective, has been scarce (Llewellyn et al. 2014). Substantial information about the gastrointestinal microbiota has been produced during the last years, but the focus was put on model and farmed fish species, such as zebra fish, turbot, salmon or trout (Nayak 2010, Ganguly & Prasad 2012, Llewellyn et al. 2014). Few microbiological studies have been conducted on cold-water fishes that have great ecological and economic importance, as halibut or Atlantic cod (Jensen et al. 2002, Ringø et al. 2006). To date there are no reports available of disease outbreaks and mortalities provoked by pathogenic bacteria in the Patagonian toothfish kept under captivity conditions.

The aim of this study was to evaluate the culturable microbiota retrieved from 3 sections of the digestive tract (stomach, middle- and distal intestine) of adult Patagonian toothfish, in order to obtain further information about culturable aerobic heterotrophic bacteria present in the gastrointestinal tract.

 

MATERIALS AND METHODS

Specimens of wild Patagonian toothfish were collected near Diego Ramirez Archipelago (56°29'S, 68°44'W), on board of a factory vessel, at 1500 to 1700 m of depth using long-line fishery. This was performed according the methodology of aquaculture broodstock selection (Gallardo 2016). Caught fishes were then transferred to Centro de Cultivos Marinos Bahía Laredo, located in Laredo Bay (52°58'S, 70°49'W) Punta Arenas, Chile. The fishes were kept for 6 months in 50 m³ fiberglass circular tanks, with flow through system of seawater. The water temperature was 6.4 ± 1.4°C and the fishes were fed 3 times a week, with a sausage that contained a fishmeal mixture rich in highly unsaturated fatty acids, in addition to fish oil, vitamins and immune stimulants. The feeding rate was 0.6 ± 0.25% of body weight per day and the growth rate was 0.06 ± 0.08% per day. Sixty fish were captured, 20% survived and acclimatized to the aquaculture confinement. Of these fishes, one was euthanized due to erratic behavior, hence providing an opportunity to assess the gastrointestinal microbiota of a specimen of D. eleginoides, that was been maintained under controlled conditions.

Isolation and identification of cultivable bacteria

The peritoneal cavity of the fish was aseptically opened with a sterile scalpel and then the gastro intestinal tract (GI) was aseptically excised and sectioned in: stomach, middle and posterior intestine. These sections were rinsed with sterile PBS (Phosphate Buffer Saline) to remove food debris. Tissue samples of intestinal wall used to cultivate bacteria were washed with sterile PBS. The side of the intestinal epithelium was rubbed on plates with solid nutrient media (Rogosa, R2A, Marine Agar and TCBS agar) to recover lactobacilli, heterotrophic bacteria, heterotrophic marine bacteria, and Vibrio spp., respectively. The plates were incubated in duplicate at 10°C for several days until bacterial growth was observed. Colony selection for sequencing processes was made according to morphology and analysis of 16S restriction fragments, as described by Urtubia et al. (2014). PCR amplifications were performed using the same condition as described above for 27F and 1422R primers. 70 isolates (20 from stomach, 22 from middle intestine and 27 from distal intestine) were identified using sequences analyses of 16S rRNA gene.

Data analysis

16S RNA genes sequenced from isolated strains were edited with DNA BASER Sequence Assembler v.4. The phylogenetic assignation was performed based on the sequence similarity using BLAST (Basic Local Alignment Search Tool) program (National Center for Biotechnology Information). The phylogenetic tree was created by means of Bosque software (Ramírez-Flandes & Ulloa 2008). 16S Ribosomal sequence were aligned with 3.6 MUSCLE software (Edgar 2004). The phylogenetic tree was inferred by maximum likelihood method based on the HKY85 model (Hasegawa et al. 1985). The phylogenetic inference corresponded to PhyML (Guindon & Gascuel 2003). Statistical evaluation of tree topologies analysis was performed with 1000 bootstrap repetitions. Sequences of isolated strains were submitted to GenBank database under accession numbers KP259730 _ KP259799 .

 

RESULTS AND DISCUSSION

The characterization and comparison of bacterial flora associated to the digestive tract achieved by cultivable methods is an important step to understand the functional roles that bacteria play and their relationship with the host, as well as the potential use as probiotics in aquaculture (Nayak 2010, Perez-Sanchez et al. 2014). This study describes for the first time, the culturable bacteria associated to the digestive tract of D. eleginoides. Because the Patagonian toothfish used in this study was part of the acclimatization process belonging to a reproduction experiment, no other specimens could be sacrifice to perform further bacterial studies. In the context of the opportunistic sampling, these preliminary results are important as provide a first view of the qualitative bacterial composition.

A total of 113 cultivable isolates were recovered from the 3 sections of the GI tract using different solid media. 53 colonies were obtained from Marine agar, 30 from TCBS and 30 using R2A medium. In contrast, no colonies were obtained in the Rogosa medium. Restriction fragments analysis of these strains allowed to differentiate 70 strains (21 from stomach, 22 from middle intestine and 27 from final intestine). These 70 isolates were purified and identified based on their 16S rRNA gene sequences. The predominant bacteria class was a-Proteobacteria (n= 63), whereas the least abundant classes were Actinobacteria (n= 7) and Firmicutes (n= 1), represented by genera Nesterenkonia (n= 1), Rhodoglobus (n= 4), Frigoribacterium (n= 1), Acinetobacter (n= 1) and Sporosarcina (n= 1), respectively (Fig. 1).

 

Figure 1. Composition of the culturable bacteria associated with the gastrointestinal
tract of the Patagonian toothfish D. eleginoides. The number of strains for each
bacterial genus is shown in parentheses. A) Stomach; B) Middle intestine;
C) Distal intestine
Figura 1. Composición de las bacterias cultivables asociadas al tracto gastrointestinal del
bacalao de profundidad D. eleginoides. Se muestra entre paréntesis el número
de cepas pertenecientes a cada género bacteriano. A) Estómago;
B) Intestino medio; C) Intestino distal

 

Several isolated cultivable bacteria from D. eleginoides belonged to genera described for cold environments were found in this study, such as Psychrobacter, Frigoribacterium and Rhodoglobus. This is consistent with the Southern Ocean environment. In this regard, water temperature may be a key factor affecting not only distribution of D. eleginoides but also bacterial composition along the digestive tract.

Results demonstrated that bacterial strains belonging to the family Vibrionaceae (44.3%) and Moraxellaceae (35.7%) were present in all 3 sections of the GI tract, showing a clear predominance in respect to other bacterial families. Moreover, Micrococcaceae (1.4%), Pseudomonadaceae (1.4%), Microbacteriaceae (8.6%), Sphingomonadaceae (5.7%), Xanthomonadaceae (1.4%) and Planococcaceae (1.4%) registered a low predominance in the intestinal tract sections. The most frequently recovered bacterial genera were Vibrio (n= 26) and Psychrobacter (n= 23), both from Proteobacteria (Fig. 2). The presence of Vibrio species in the microbiota of D. eleginoides is consistent with previous studies performed on several fish species (Nayak 2010). Vibronaceae was mainly represented by Vibrio and Psychrobacter, in all 3 sections of the GI tract. It is evident that cold environments constitute an ecological niche for Psychrobacter bacteria in organisms that inhabit cold waters (Bozal et al. 2003). The genera Vibrio and Psychrobacter were highly represented in stomach (52.3 and 33.3%, respectively) and middle intestine (45.4 and 50.0%, respectively). In contrast, these genera only represented 18.5% of the bacterial isolates from distal intestine. Aliivibrio was detected only in this portion with equal percentage as Vibrio. Of this group, the most predominant isolates corresponded to Vibrio tasmaniensis, Vibrio splendidus and Aliivibrio logei. Consistent with these results, MacCormack & Fraile (1990) obtained mostly Vibrio spp. from the fish Notothenia neglecta, using cultivation-dependent approaches. The authors concluded that Vibrio species constitute the predominant indigenous intestinal microbiota found on this Antarctic notothenioidei fish. Furthermore, intestinal microbiota studies of Notothenia coriiceps and Chaenocephalus aceratus, using non-cultivable methods, showed a relatively low bacterial sequence diversity. The most dominating order in terms of abundance were gamma-Proteobacteria, Photobacterium sp., Allivibrio sp. and Vibrio sp. (Ward et al. 2009). Recently, Sedláèek et al. (2016) described the dominance of Enterobacter isolated from gut contents of several fish species inhabiting Weddell Sea in Antarctica (Notothenia coriiceps, Trematomus bernacchii, Trematomus hansoni, and Trematomus newnesi). Nevertheless, they used a specific media for Enterobacter, evidencing that different results can be obtained depending on the methodological approaches (this includes cultivable and non-cultivable methods). They even suggest that the results may vary depending on the type of sampled tissue (e.g., intestinal contents or GI tract wall). Although, some Vibrio species could be considered as pathogenic bacteria, its presence in Patagonian toothfish intestinal tract seems to be normal. Vibrio has been frequently detected in the intestinal flora of different fish species, for example in halibut, tilapia, Atlantic cod and Atlantic salmon (Nayak 2010, Llewellyn et al. 2014). These results suggest that Vibrio splendidus and Vibrio tasmaniensis are native bacterial flora in D. eleginoides.

 

Figure 2. Phylogenetic tree of 16S ribosomal gene sequences inferred by maximum
likelihood method (using alignment of 1216 positions). Numbers presented are
support values (bootstrap values) of PhyML. Strain sequences are presented with
information by section (ST- stomach, MI- middle intestine and FI- distal intestine);
culture medium (R2A agar, MA- Marine agar and TCBS agar) and
GenBank accession number
Figura 2. Árbol filogenético de las secuencias del gen ribosomal 16S inferidas por
el método de máxima verosimilitud (usando un alineamiento de 1216 posiciones).
Los números presentados corresponden a los valores de soporte (valores
de bootstrap) de PhyML. Secuencias utilizadas son presentadas con la
información por sección (ST-estómago, ML- intestino medio y FI- intestino distal),
medio de cultivo (Agar R2A, MA- Agar marino y Agar TCBS) y número
de acceso en GenBank entre paréntesis

 

The presence in the fish microbiome of several bacterial groups, such as Actinobacteria, may imply that these bacteria could be involved on bacterial control against fish pathogens, like Vibrio spp. (Sanchez et al. 2012). The phylum Actinobacteria was represented by 3 genera: Nesterenkonia sp. (one isolate from stomach), family Micrococcaceae; Frigoribacterium sp. (one isolate from distal intestine) and Rhodoglobus sp. (4 isolates from distal intestine with 99% of 16S rRNA sequence identity), which belong to the Microbacteriaceae family (Fig. 2). Species of the genus Nesterenkonia have been previously reported as halotolerant, being isolated from different saline ecosystems such an arid saline system (El Hidri et al. 2013) or a hypersaline Antarctic lake (Collins et al. 2002). Microbacteriaceae family represented by the genus Rhodoglobus had similar representation to the Vibrionaceae family in the distal intestine, but was only present in this segment. Four isolates showed 99% similarity with a Rhodoglobus sp. isolated from cold environment (Sheridan et al. 2003). This phylum has been reported in carnivorous fishes from marine and freshwater environments (Sullam et al. 2012). Further studies in D. eleginoides using cultivation independent approaches such as denaturing gradient gel electrophoresis (DGGE) or next generation sequencing (NGS) technologies will be necessary to assess with a higher resolution the complexity of bacterial community associated to GI tract. This type of studies will also help to understand how different nutritional and environmental conditions may influence digestive tract bacterial community structure in this species. A better knowledge of the intestinal bacterial community will play a key role in the comprehension of potential fish captivity consequences regarding this emerging species in aquaculture system.

 

ACKNOWLEDGMENTS

Funding was provided by FONDEF-CONICYT DA09i1002 and Master degree thesis supported by INACH code MG_01-12.

 

LITERATURE CITED

Bozal N, MJ Montes, E Tudela & J Guinea. 2003. Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov. International Journal of Systematic and Evolutionary Microbiology 53: 1093-1100.

Collins MA, P Brickle, J Brown & M Belchier. 2010. The Patagonian toothfish: Biology, ecology and fishery. Advances in Marine Biology 58: 227-300.

Collins MD, PA Lawson, M Labrenz, BJ Tindall, N Weiss & P Hirsch. 2002. Nesterenkonia lacusekhoensis sp. nov., isolated from hypersaline Ekho Lake, East Antarctica, and emended description of the genus Nesterenkonia. International Journal of Systematic and Evolutionary Microbiology 52: 1145-1150.

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32(5): 1792-1797.

El Hidri D, A Guesmi, A Najjari, H Cherif, B Ettoumi, C Hamdi, A Boudabous & A Cherif. 2013. Cultivation-dependant assessment, diversity, and ecology of haloalkaliphilic bacteria in arid saline systems of Southern Tunisia. BioMed Research International 2013: 648141.

Gallardo P. 2016. Antecedentes preliminares del cultivo de bacalao de profundidad (Dissostichus eleginoides; Nototheniidae) en la región de Magallanes, Chile. Anales del Instituto de la Patagonia 44(3): 77-84.

Ganguly S & A Prasad. 2012. Microflora in fish digestive tract plays significant role in digestion and metabolism. Reviews in Fish Biology and Fisheries 22(1): 11-16.

Guindon S & O Gascuel. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52(5): 696-704.

Hasegawa M, H Kishino & T Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22(2): 160-174.

Horn PL. 2002. Age and growth of Patagonian toothfish (Dissostichus eleginoides) and Antarctic toothfish (D. mawsoni) in waters from the New Zealand subantarctic to the Ross Sea, Antarctica. Fisheries Research 56(3): 275-287.

Jensen S, Ø Bergh, Ø Enger & B Hjeltnes. 2002. Use of PCR_RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry. Canadian Journal of Microbiology 48(5): 379-386.

Llewellyn MS, S Boutin, S Hossein & N Derome. 2014. Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries. Frontiers in Microbiology 5: 207. <doi: 10.3389/fmicb.2014.00207>

MacCormack WP & ER Fraile. 1990. Bacterial flora of newly caught Antarctic fish Notothenia neglecta. Polar Biology 10(6): 413-417.

Nayak SK. 2010. Role of gastrointestinal microbiota in fish. Aquaculture Research 41: 1553-1573.

Perez-Sanchez T, I Ruiz-Zarzuela, I de Blas & JL Balcaraz. 2014. Probiotics in aquaculture: a current assessment. Reviews in Aquaculture 6(3): 133-146.

Ramírez-Flandes S & O Ulloa. 2008. Bosque: integrated phylogenetic analysis software. Bioinformatics 24(21): 2539-2541.

Ringø E, S Sperstad, R Myklebust, S Refstie & Å Krogdahl. 2006. Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal. Aquaculture 261(3): 829-841.

Sanchez LM, WR Wong, RM Riener, CJ Schulze & RG Linington. 2012. Examining the fish microbiome: Vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products. PLoS ONE 7(5): e35398. <doi:10.1371/journal.pone.0035398>

Sedláèek I, E Staòková & P Švec. 2016. Composition of cultivable enteric bacteria from the intestine of Antarctic fish (family Nototheniidae). Czech Journal of Animal Science 61(3): 127-132.

Sheridan PP, J Loveland-Curtze, VI Miteva & JE Brenchley. 2003. Rhodoglobus vestalii gen. nov., sp. nov., a novel psychrophilic organism isolated from an Antarctic Dry Valley Lake. International Journal of Systematic and Evolutionary Microbiology 53: 985-994.

Soto I. 2015. Determinación e identificación del contenido estomacal de Bacalao de profundidad silvestre (Dissostichus eleginoides, Smitt 1998), como apoyo para su desarrollo acuícola en la Región de Magallanes, Chile. Tesis de Biólogo Marino, Universidad de Magallanes, Puna Arenas, 84 pp.

Sullam KE, SD Essinger, CA Lozupone, MP O'Connor, GL Rosen, R Knight, SS Kilham & JA Russell. 2012. Environmental and ecological factors that shape the gut bacterial communities of fish: A meta-analysis. Molecular Ecology 21(13): 3363-3378.

Urtubia R, P Gallardo, P Lavin, N Brown & M González. 2014. Characterization of culturable bacterial flora in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) with `gaping jaws' syndrome. Latin American Journal of Aquatic Research 42(1): 97-110.

Ward NL, B Steven, K Penn, BA Methé & WH Detrich III. 2009. Characterization of the intestinal microbiota of two Antarctic notothenioid fish species. Extremophiles 13(4): 679-685.

---

Received 21 June 2016 and accepted 6 June 2017

Associate Editor: Pilar Muñoz M.