Bacterial Diversity of Gut Content in Sea Cucumber (Apostichopus japonicus) and Its Habitat Surface Sediment

GAO Fei, TAN Jie, SUN Huiling, and YAN Jingping

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, P. R. China

Bacterial Diversity of Gut Content in Sea Cucumber (Apostichopus japonicus) and Its Habitat Surface Sediment

GAO Fei, TAN Jie, SUN Huiling*, and YAN Jingping

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, P. R. China

This study investigated the bacterial diversity of gut content of sea cucumber (Apostichopus japonicus) and its habitat surface sediment in a bottom enhancement area using PCR-based denaturing gradient gel electrophoresis (DGGE) technique. Bacterial diversity evaluation showed that the value of the Shannon-Wiener index of gut content in different intestinal segments of A. japonicus varied between 2.88 and 3.00, lower than that of the surrounding sediment (3.23). Phylogenetic analysis showed that bacterial phylotypes in gut content and the surrounding sediment of A. japonicus were closely related to Proteobacteria includingγ-, α-, δand ε-proteobacteria, Bacteroidetes, Firmicute, and Actinobacteria, of which γ-proteobacteria were predominant. These results suggested that the sea cucumber A. japonicus was capable of feeding selectively, and PCR-DGGE was applicable for characterizing the bacterial community composition in gut content and the surrounding sediment of sea cucumber. Further investigation targeting longer 16S rDNA gene fragments and/or functional genes was recommended for obtaining more information of the diversity and function of bacterial community in the gut content of sea cucumber.

sea cucumber; Apostichopus japonicus; bacterium; PCR-DGGE; gut content; sediment

1 Introduction

The sea cucumber, Apostichopus japonicus (Selenka), has long been exploited as an important economic resource in China, Japan, Russia, and North and South Korea (Sloan, 1984). It is one of the most economically important holothurian species in coastal aquaculture and stock enhancement in China. Culture of sea cucumbers has developed fast in recent years and substantially contributed to the Chinese aquaculture industry with the total production over 100000 tons (wet weight) in 2009.

A. japonicus is a deposit feeding species with a simple, tubular digestive tract that passes a large amount of sediment through the gut and assimilates available organic materials. Associated organic materials are mainly from bacteria, diatoms, detritus of macroalgae, and protozoa in the ingested sediment (Hauksson, 1979; Zhang et al., 1995). Previous studies proved that bacteria in the digestive tract of many animal species play an important role in nutrient digestion and absorption, immunization, and biological antagonism of the hosts (Amaro et al., 2009; Amaro et al., 2012; Hess et al., 2011; Warnecke et al., 2007; Ray et al., 2012). Due to the poorly nutritional food ingested by sea cucumber, this animal species is likely benefited from bacteria in the gut content, especially for nutrient digestion. Research by fatty acid biomarker shows that bacterial components are an important food source of A. japonicus (Gao et al., 2010b), which supplies more than 70% of the energy demands in A. japonicus (Sui, 1988). However, little is known regarding the bacterial diversity in the digestive tract and the habitat of A. japonicus.

Previously, Sun and Chen (1989) investigated the bacterial community composition in digestive tract of A. japonicus using conventional cultivation method. Because most of the bacteria in the nature are non-culturable under laboratory conditions, Sun and Chen’s results only revealed the diversity characteristics of limited cultivable bacterial members, a very small proportion of the total bacterial community in gut content of the sea cucumber. Thus, it is needed to apply culture-independent molecular biological methods in order to improve the understanding of total microbiota in the digestive tract and the habitat of sea cucumber. Bacterial DNA extracted from a microbial community can be used to identify the genetic diversity of dominant bacterial populations with the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) technique (Muyzer et al., 1993). Using 16S rRNA gene fragments, one can describe both cultivable and uncultivable bacteria by phylogenetic relationships and examine temporal and/or spatial variations in bacterial community compositions of environmental samples (Hovda et al., 2012; Li et al., 2008; Thompson et al., 2008). Till now, PCR-DGGE technique has been used toinvestigating the bacterial community compositions in various ecosystems of soil, sediment, seawater, lake, and gut of animals (Lau et al., 2002; Lim et al., 2011; Liu et al., 2008; Uthicke and McGuire, 2007).

Research of gastrointestinal micro-ecology has examined the bacterial diversity in some fish and shrimp species using PCR-DGGE. Hovda et al. (2007) used conventional culture-based techniques combined with molecular phylogenetic analysis of 16S rDNA gene fragments to examine the diversity of intestinal microbiota in farmed Atlantic salmon (Salmo salar L.) and demonstrated that PCR-DGGE is an alternative way of studying the intestinal microbiota in fish. Kim et al. (2007) used conventional microbiological techniques and PCR-DGGE method to study the microbial community composition of intestinal contents and mucosal layer of rainbow trout (Oncorhynchus mykiss) and found many novel sequences that had not been previously recognized as part of the intestinal flora in rainbow trout. Zhou et al. (2009) employed the PCR-DGGE technique to investigate the community compositions of autochthonous microbiota in different parts of gastrointestinal tract, including stomach, pyloric caeca, proximal intestine, mid-intestine, and distal intestine of the adult yellow grouper (Epinephelus awoara) cultured in cages. In addition, Luo et al. (2006, 2009) used PCR-based molecular techniques to study the bacterial diversity in the digestive tract and the surrounding environment of shrimp (Litopenaeus vannamei) cultured in seawater and brackish water. However, so far, little studies have been performed to determine the gut bacterial community of holuthurians by PCR-DGGE technique.

In China, the main culture modes of A. japonicus are pond culture, pen culture and bottom culture (sea ranching). Of these, bottom culture is the most predominant and accounts for 75% of total culture area (Chen, 2004). In the bottom culture mode, sea cucumbers feed on natural diets only, with no use of medicine during the culture course. Thus, the growth conditions of sea cucumbers are very close to those in the wild environment. The purpose of this work was to use 16S rRNA-based PCR-DGGE and sequencing technique to describe the bacterial diversity of different gut contents in the anterior, middle, and posterior intestines of A. japonicus cultured in a bottom enhancement area. In addition, we compared the bacterial community compositions among different gut contents and the surrounding sediment of sea cucumber in order to evaluate the changes in associated bacterial diversity. The results will provide valuable data for sea cucumber feeding strategy and the function of bacteria ingested from food.

2 Materials and Methods

2.1 Sample Collection

In December 2009, sea cucumber (122.8 g ± 12.09 g) and their habitat surface sediment (0–2 cm, n = 5) were collected from a bottom enhancement area in Qingdao, Shandong, China. The samples were stored on ice and transported to the laboratory within one hour after collection. After the surface skin of sea cucumbers was sterilized with 70% ethanol, the ventral surface was dissected with a sterile scalpel to expose the body cavity. Thereafter, the digestive tract of sea cucumbers was divided into three segments: the anterior, the middle and the posterior. The content of each intestinal segment was carefully squeezed out into a sterile freezing tube. All gut content and sediment samples were stored at −80℃ prior to anal ysis.

2.2 DNA Extraction and PCR Amplification

The gut contents of three sea cucumbers and five sediment samples were thawed and then centrifuged at 10000 r min−1and 4 for 3℃ min. After removing the supernatant, the sediment samples were mixed into a composite sample. DNA extraction was done using a SoilMaster TM Extraction Kit (EPICENTRE Biotechnologies, USA) following the manufacturer’s protocol.

The variable V3-region of 16S rDNA gene was amplified from the DNA extracts of sea cucumber gut contents and sediment using the universal bacterial primers GC-341f (CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG) and 534r (ATT ACC GCG GCT GCT GG) (Li et al., 2008; Liu et al., 2008; Muyzer et al., 1993). A touch-down PCR with some modifications was performed on all samples to reduce nonspecific priming (Kim et al., 2007). The 50-μL PCR contained 10 × PCR buffer (10 mmol L−1Tris-HCl (pH 8.3), 50 mmol L−1KCl, 1.5 mmol L−1MgCl2), 150 μmol L−1dNTP, 400 nmol L−1each primer, 30 ng μL−1bovine serum albumin (Yu and Morrison, 2005), and 0.04 UμL−1Taq DNA polymerase (Takara). PCR amplification was carried out under the following conditions: initial denaturation at 94 for 5℃ min, followed by 18 cycles of denaturation at 94℃ for 45s, annealing at 65℃(touchtown 0.8℃ per cycle) for 45s, extension at 72℃for 45 s, and 12 cycles of denaturation at 94℃ for 45s, annealing at 55℃ for 45s, and extension at 72℃ for 30s, and final extra extension at 72℃ for 10min. The PCR products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide (Gao et al., 2010a).

2.3 DGGE Analysis

DGGE was performed to separate the PCR products collected above using a D-Code universal mutation detection system (Bio-Rad, USA). Similarly sized and equal amounts of PCR product of each sample was loaded directly onto 8% polyacrylamide gels (16 cm × 16 cm × 1 mm) in 1 × TAE buffer (40 mmol L−1Tris base, 20 mmol L−1glacial acetate and 1 mmol L−1EDTA) with a denaturing gradient of 35%–50%. Electrophoresis was conducted with a constant voltage of 140 V at 60℃ for about 4h. Gels were stained with SYBR Green I for 20 min, washed with deionized water, and then photographed with a digital imaging system.

2.4 Cloning and Sequencing of DNA Fragments from DGGE Bands

DNA bands of interest were carefully excised from the DGGE gel with a clean pipet tip. The excised bands wereplaced in 1.5 mL microcentrifuge tubes containing 100 μL of sterile water. After incubation at room temperature for 10 min, the tube was centrifuged at 10000 r min−1for 5 min and the supernatant was removed. Then, 30 μL sterile water was added to the tube followed by 4℃ incubation overnight. For constructing bacterial clone libraries, the eluted DNA was amplified with the primers 341F and 534R. The PCR products were cloned into pMD18-T vector (Takara) with TOP10 chemically competent cells (Takara) as the host. After cultured overnight, a portion of each culture was directly used as PCR template with the primer set M13F and M13R. The positive clones were chosen and sent to Sangon Biotech, Shanghai, China for sequencing. The obtained sequences (169–194 bp) were deposited in GenBank with the accession Nos. HM776288-HM776308.

2.5 Diversity and Phylogenetic Analysis

Unweighted pair group method with arithmetic mean (UPGMA) tree was used to determine the similarities among samples, based on the presence or absence of each band in DGGE profiles. To further estimate the bacterial community structure revealed via DGGE, the intensity of each DNA band on the DGGE gel was measured and then analyzed using Quantity One Program (Bio-rad, USA). Sequences retrieved from DGGE bands were compared in the GenBank database to identify their close relatives using BLAST program. Sequences representing distinct phylotypes and their closest relatives were aligned with CLUSTALW (Li et al., 2008; Thompson et al., 1994). Evolutionary distances were calculated using the Kimura’s two-parameter correction method. A rooted phylogenetic tree were constructed using MEGA 5.05 with the bootstrap value of 1000. The Shannon index (H) of diversity is given as follows:

where pi is the proportion of phylotypes i, that is,

Values were presented as means ± SD, analyzed using the SPSS 13.0 statistical software package.

3 Results

3.1 Bacterial Community Structure of Sea Cucumber Gut Content and the Surrounding Sediment PCR-DGGE fingerprinting showed that the bacterial diversity substantially varied among different gut contents and the surrounding sediment of sea cucumber (Fig.1). The number of dominant DNA bands ranged from 17 to 26 each lane, and a total of 32 bands were observed at different positions in the gel (Fig.1). The sediment sample had the maximum band number, and the gut content of middle intestine of one sea cucumber had the least number of bands. Overall, 59.4% of the bands commonly exist in each lane, while the remaining 13 bands represent specific genotypes. Of the specific bands, 10 were from the sediment sample, 2 from the intestinal content of the posterior segment, and 1 from the intestinal content of the anterior segment of sea cucumber.

Fig.1 DGGE fingerprinting profiles of bacterial communities in gut contents and the surrounding sediment of Apostichopus japonicus. A: anterior intestine; M: middle intestine; P: posterior intestine; and S: surrounding sediment. The numbers (1, 2 and 3) in front of A, M, and P represent the three sampled sea cucumbers.

UPGMA cluster analysis showed that the gut contents and the surrounding sediment of sea cucumber shared a low similarity of bacterial community structure (39%) (Fig.2). High similarities of the bacterial community structures (>80%) were observed between different gut contents of the same sea cucumber, i.e., 82% for 1M and 1P, 84% for 2M and 2P, 87% for 3A and 3M (Fig.2).

Fig.2 UPGMA tree showing the similarity of bacterial community structures of different gut contents and the surrounding sediment of Apostichopus japonicus. A: anterior intestine; M: middle intestine; P: posterior intestine; S: surrounding sediment.

3.2 Bacterial Diversity of Sea Cucumber Gut Contents and the Surrounding Sediment

The H index of diversity was the highest (3.23) in the sediment (Fig.3). As for various gut contents of the sea cucumber, the H index of diversity was the highest in the anterior intestine (3.00 ± 0.02), followed by the posterior intestine (2.95 ± 0.05) and the middle intestine (2.88 ± 0.07).

Fig.3 H diversity index values of different gut contents and the surrounding sediment of Apostichopus japonicus. A: anterior intestine; M: middle intestine; P: posterior intestine; and S: surrounding sediment.

3.3 Phylogenetic Relationships of Bacterial Components in Sea Cucumber Gut Contents and the Surrounding Sediment

A total of 24 partial 16S rDNA sequences (169–194 bp) were retrieved from the DNA bands on a DGGE gel (Table 1). Blast analysis revealed that the closest relatives of the predominant bacteria in gut contents and the surrounding sediment of sea cucumber were those inhabiting marine environments, such as marine sediments, sea water, coral tissues, sponge tissues, and the digestive gland of oysters. Fig.4 shows the phylogenetic relationship of bacterial members in the gut contents and the surrounding sediment of sea cucumber retrieved from DGGE bands and their close relatives retrieved from the GenBank database. The 24 sequences can be divided into 8 groups: γ-proteobacteria (10), δ-proteobacteria (3), ε-proteobacteria (1), α-proteobacteria (1), Bacteroidetes (2), Actinobacteria (1), Firmicute (1), and unidentified bacteria (5).

3.4 Comparison of Bacterial Community Composition among Sea Cucumber Gut Contents and the Surrounding Sediments

The bacterial community compositions among different gut contents and the surrounding sediment of sea cucumbers werer compared based on the percentage of different bacterial groups indicated by the corresponding DGGE band density (Fig.5). Members of γ-proteobacteria, δproteobacteria, and Bacteroidetes were present in all samples, while members of Actinobacteria and α-proteobacteria were only found in the content of the posterior intestine of one sea cucumber and the sediment sample, respectively. The proportion of γ-proteobacteria was relatively high in all samples (36.1%–51.5%), indicating that γ-proteobacteria were the predominant bacterial group in gut contents and the surrounding sediments of sea cucumber. The proportions of unidentified bacteria were also relatively high in the samples, 22.6%–41.0%.

Table1 Bacterial 16S rDNA V3 sequences retrieved from sea cucumber gut contents and the surrounding sediment

Fig.4 Phylogenetic tree of partial bacterial 16S rDNA V3 gene fragments retrieved from sea cucumber gut contents and the surrounding sediment. Bootstrap test was based on 1000 replicates and the bootstrap values less than 50% were discarded.

4 Discussion

A limited number of studies have investigated the diversity of microbiota in the digestive tract of sea cucumbers such as Holothuria atra and A. japonicus using conventional cultivation methods combined with or without 16S rDNA-based molecular biological methods (Ward-Rainey et al., 1996; Sun and Chen, 1989). As 85%–99% of the total bacterial populations in nature are non-culturable (Kuske et al., 1997; Zhang et al., 2008), it is difficult to disclose the complexity of the sea cucumber gut ecosystem using culture-dependent methods, and knowledge remains lacking regarding the microbiota in the digestive tract of holothurians. In the present study, we employed the PCR-DGGE technique to examine the bacterial diversity in gut contents and the surrounding sediments of A. japonicus in a bottom enhancement area. The results provide new insights to the allochthonous bacterial community in the digestive tract and the habitat of the sea cucumber in a managed aquaculture ecosystem.

H index of diversity is widely used for evaluating the biodiversity of various ecosystems. In the recent years, this index has been used to evaluate the microbial diversity of microbiota in environmental samples with the PCR-DGGE method (Chen et al., 2009; Li et al., 2008; Liu, 2010). In the present study, H index of diversity was used to describe the bacterial diversity of sea cucumber gut contents and the surrounding sediment. In a study based on conventional cultivation method, Sun and Chen (1989) proposed that the sea cucumber A. japonicus selectively ingests the diets. It was found that Bacillus was abundant in the surrounding sediment but barely detectable in the gut contents of A. japonicus, and that the abundance of bacteria capable of decomposing sodium alginate, chitin and sodium alginate were higher in the gut contents than in the surrounding sediment (Sun and Chen, 1989). In the present study, we found the H index values lower in the content of anterior intestine than in the surrounding sediment (Fig.3). This observation indicates the selective feeding characteristic of the sea cucumber, as the intestinal bacterial components mainly come from the ingested sediments (Hammond, 1982; Sun and Chen, 1989; Slater et al., 2011).

UPGMA analysis shows that the content of anterior intestine and the surrounding sediment of A. japonicus share a low similarity of bacterial community structure (Fig.2). This could be related to previous finding by Amaro et al. (2009) that the surrounding sediment bacterial community is signif i cantly different from that in the oesophagous of the holothurians Molpadia musculus. The contents in the anterior gut or oesophagous were just ingested from the surrounding sediment by the depositfeeding holothurians. Thus, different bacterial community compositions between the anterior gut or oesophagous and the surrounding sediment suggest that holothurians can selectively feed.

Members of γ-proteobacteria are the predominant bacterial group in the gut contents and surrounding sediment of the sampled sea cucumbers, accounting for 36.1%–51.5% of the total bacterial populations (Fig.5). Previously, unculturable γ-proteobacteria were found predominant in gut contents of sea cucumbers cultured under the pond culture mode (Gao et al., 2010a), while culturable γ-proteobacteria (Vibiro, Pseudomonas) were found dominant in the digestive tract of A. japonicus (92.7%; Sun and Chen, 1989). In addition, the predominant cultural bacteria of γ-proteobacteria have been found in the guts of sea cucumber (H. atra) and shrimp (Litopenaeus vannamei, Marsupenaeus japonicus, and Penaeus merguiensis) (Li, 2007; Ward-Rainey et al., 1996; Oxley et al., 2002; Yang et al., 2006). In fact, research shows that γ-proteobacteria are dominant in sea water and marine sediments in vast areas along the Shandong coast, east China (Liu, 2010; Zhang, 2008).

In the present study, Actinobacteria were detected in the posterior intestine of one sea cucumber, despite their absence in gut contents of sea cucumbers cultured in a pond reported by a previous study (Gao et al., 2010a).Actinobacteria have been isolated from the hindgut contents of the sea cucumber H. atra (Ward-Rainey et al., 1996) and the sediment in a prawn pond (Ma et al., 2009). A large number of Actinobacteria have been reported in sediments of Jiaozhou Bay in Shandong Province, China (Liu, 2010; Zhang, 2008). Actinobacteria are small but signif i cant and ubiquitous members in marine microbial communities (Venter et al., 2004). The function of Actinobacteria in the sea cucumber A. japonicus needs to be further studied. In the present study, Bacteroidetes were found in the sea cucumber gut contents and the surrounding sediments (Fig.5), with closest relatives from salt marsh sediments and sponge tissues (Lydell et al., 2004; Mohamed et al., 2008). Previously, Bacteroidetes were detected in the gut contents of A. japonicus cultured in a pond (Gao et al., 2010a) and proven dominant in the surface sediment of a M. japonicus pond and Jiaozhou Bay in Shandong Province, east China (Li, 2007; Liu, 2010).

In this study, the percentage of unidentified bacteria was 22.6%–41.0%. These microorganisms may provide a novel bacterial subdivision with special function(s) in the gut and surrounding sediment of A. japonicus. All the closest relatives of sequences obtained in this study shared similar background: marine environments such as marine sediments, sea water, or marine organisms. Some of the sequences have recently been reported, while the others are never published before, leading to a lack of detailed description on relevant organisms.

This study investigated the bacterial community structure in gut contents and the habitat surface sediment of A. japonicus cultured in a bottom enhancement area. The results showed the changes in the bacterial community composition and diversity index among the sediment and different gut contents of the sea cucumber A. japonicus. Phylogenetic analysis showed that the bacteria mainly belong to Proteobacteria (γ-, α-, δ-, and ε-proteobacteria), Bacteroidetes, Firmicute and Actinobacteria, of which γ-proteobacteria were the predominant. The present study verified that PCR-DGGE is applicable to characterize the bacterial community structure in the gut contents and the surrounding sediment of the sea cucumber A. japonicus. As the retrieved 16S rDNA V3 fragments were about 190 bp in length and merely represent dominant groups, our phylogenetic analysis was limited by the relatively short gene fragment and the bacterial diversity in sea cucumber gut contents and the surrounding sediment was likely underestimated. Therefore, other functional genes and/or longer 16S rDNA gene fragments should be used to acquire more information of the diversity and function of the bacterial community in gut contents of the sea cucumber.

Acknowledgements

This work was supported by the National Key Technology Research and Development Program (Nos. 2011 BAD13B02, 2010BAC68B01), the National High Technology Research and Development Program of China (863 program, No. 2012AA10A412), the Science and Technology Development Projects Funds of Shinan District in Qingdao, Shandong Province (2011-5-023-QT), and the National Science Foundation of China (No.41106145).

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(Edited by Qiu Yantao)

(Received June 20, 2012; revised August 9, 2012; accepted July 17, 2013)

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-532-85819199

E-mail: sunhl@ysfri.ac.cn