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Short Communication Diversity and composition of the bacterial community in Amphioxus feces Minming Pan1, Dongjuan Yuan1,2, Shangwu Chen1 and Anlong Xu1,3 1

2 3

Department of Biochemistry, College of Life Sciences, State Key Laboratory of Biocontrol, National Engineering Research Center of South China Sea Marine Biotechnology, Sun Yat-sen University, Guangzhou, P. R. China Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China Beijing University of Chinese Medicine, 11 Bei San Huan Dong Road, Chao-yang District, Beijing 100029, P. R. China

Amphioxus is a typical filter feeder animal and is confronted with a complex bacterial community in the seawater of its habitat. It has evolved a strong innate immune system to cope with the external bacterial stimulation, however, the ecological system of the bacterial community in Amphioxus remains unknown. Through massive parallel 16S rRNA gene tag pyrosequencing, the investigation indicated that the composition of wild and lab-cultured Amphioxus fecal bacteria was complex with more than 85,000 sequence tags being assigned to 12/13 phyla. The bacterial diversity between the two fecal samples was similar according to OTU richness of V4 tag, Chao1 index, Shannon index and Rarefaction curves, however, the most prominent bacteria in wild feces were genera Pseudoalteromonas (gamma Proteobacteria) and Arcobacter (epsilon Proteobacteria); the highly abundant bacteria in lab-cultured feces were other groups, including Leisingera,Phaeobacter (alpha Proteobacteria), and Vibrio (gamma Proteobacteria). Such difference indicates the complex fecal bacteria with the potential for multi-stability. The bacteria of habitat with 28 assigned phyla had the higher bacterial diversity and species richness than both fecal bacteria. Shared bacteria between wild feces and its habitat reached to approximately 90% (153/169 genera) and 28% (153/548 genera), respectively. As speculative, the less diversity of both fecal bacteria compared to its habitat partly because Amphioxus lives buried and the feces will ultimately end up in the sediment. Therefore, our study comprehensively investigates the complex bacterial community of Amphioxus and provides evidence for understanding the relationship of this basal chordate with the environment.

: Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: 16S rRNA gene / Bacterial community / Environment / Feces / Gut microbe Received: May 26, 2015; accepted: June 23, 2015 DOI 10.1002/jobm.201500124

Introduction The complex ecological system of the bacterial community in animals includes both the transient bacteria that will pass through the digestive tract with the bolus and the resident bacteria that will colonize in the digestive tract. Minming Pan and Dongjuan Yuan contributed equally to this work. Correspondence: Anlong Xu, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China E-mail: [email protected] Phone: 86-20-39332990 Fax: 86-20-39332950 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

This complex community is an integral component of the host and may affect the host biology [1, 2]. However, the gut microbiota is confronted with the different pressures from environmental and endogenous factors, such as diet, age, host genetics, and physiological state [3]. Thus, research aimed to the complex ecological system of the bacterial community in feces and its habitat will aid in the understanding of the process of host defense in response to external bacterial stimulation. The composition and features of the microbe differ greatly from invertebrates to vertebrates. For terrestrial vertebrates, the predominant bacteria present are

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Firmicutes and Bacteroidetes. The mammalian gastrointestinal tract is comprised of ten times as many bacteria as the animal’s own cells and includes hundreds of bacterial phylotypes, which constitute a complex bacterial ecological system [4, 5]. In aquatic vertebrates, the Proteobacteria predominates in the digestive tract [6, 7]. A meta-analysis of 25 bacterial 16S rRNA gene sequence libraries derived from the intestines of different fish species showed that variation in gut microbiota composition in fishes strongly correlated with species habitat salinity, trophic level and possibly taxonomy [8]. Invertebrates possess a different composition of bacterial flora. In the terrestrial invertebrate, the drosophila has a simple gut bacterial community, which is associated with only four to eight species of Firmicutes and Proteobacteria [9, 10]. In marine species, the microbial community of the Pacific white shrimp (Litopenaeus vannamei) includes the genera Pseudoalteromonas and Vibrio (gamma Proteobacteria) [11]. Shrimp (Rimicaris exoculata) have a gut bacteria that is comprised of Deferribacteres, Mollicutes, epsilon- and gamma Proteobacteria [12]. The emptied midgut of the shrimp Pestarella tyrrhena (Decapoda: Thalassinidea) has the microbiota of genera Vibrio (gamma Proteobacteria), Spirochaetes, Entomoplasmatales, Bacteroidetes, and Actinobacteria [13]. The hepatopancreas of Porcellio scaber (Crustacea: Isopoda) consists of six phylogenetic clades of the Mollicutes: the Asteroleplasma group, the Acholeplasma-Anaeroplasma group, the Mycoplasma hominis group, the Mycoplasma pneumoniae group, and the Spiroplasma group and a new lineage called “Candidatus Hepatoplasma crinochetorum” [14]. Amphioxus, the closest extant relative of vertebrates, is an ideal model to study on the origin and evolution of vertebrate immunity [15, 16]. Lymphocyte-like cells and phagocytes have been identified in the pharynx gill slits and the mucosal surfaces of the gastrointestinal tract [17, 18]. Amphioxus is a typical filter feeder animal without the ability to select food and lives buried in the sediment of the coast. Thus, Amphioxus is confronted with a complex bacterial community in seawater. Our previous study showed that Amphioxus has evolved a strong innate immune system to cope with the external stimulation of bacteria [17, 19]. Thus, we hypothesize that the ecological system of the bacterial community of Amphioxus will provide insights into the features of the host immunity and biology. The objective of the present study was to explore the composition of the bacterial community in Amphioxus feces and the sediment of its habitat. Using a massive parallel bacterial 16S rRNA gene tag pyrosequencing approach, the microbe of the ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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Amphioxus feces and the sediment of its marine habitat were investigated.

Materials and methods Animals and sample acquisition Six female and male wild amphioxuses were captured on the coast of Zhanjiang in China (latitude, 20° 530 N; longitude, 110° 320 E). The amphioxuses were placed in 50 ml of the sterile seawater. Then, feces sample was collected in the sterile seawater from the six female and male amphioxuses after 1 h. The habitat sample was composed of the sediment and seawater of the captured locus of Amphioxus because Amphioxus is buried in the sediment of the habitat for most of its life. The collected feces and sediment samples were immediately places on ice for further experiments. In the laboratory, adult Chinese amphioxus were held in aerated seawater with the sediment from its habitat and fed microalgae daily for more than three months, and the fecal samples were collected from a mixture of six female and male amphioxus that were lab-cultured. DNA extraction, amplification, quality control, and pyrosequencing The AxyPrep Bacteria Genomic DNA Miniprep Kit (AxyGen Biosciences) was used for the extraction of bacterial DNA, and PCR amplification of the 16S rDNA (primer loci at 5-1492) gene of habitat and feces samples was performed with 10 ng template DNA in 50 ml reactions according to the manufacturer’s instructions (Roche). Fusion primers adapted for the general sequencing kit of the GS FLX Titanium pyrosequencing platform (Roche) were A-14921470R reverse primer (proximal primer) or B-5-24F forward primer (distal primer). The primer sequences are 50 -AGAGTTTGATCCTGGCTCAG-30 , and 50 -TACGGCTACCTTGTTACGACTT-30 . The PCR amplicon libraries were generated for each DNA sample. The amplification reaction system (total 50 ml) contained 36 ml ddH2O, 5 ml 10 buffer, 2 ml dNTPs; 2 ml of each primer (0.5 mM); 2 ml DNA; and 1 ml Taq (5 U/ml). Cycling conditions were as follows: initial denaturation at 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a final 10 min extension at 72 °C. For quality control, the PCR Amplicon Libraries of each DNA sample encoding for 16S rDNA were inserted into the pGEM-T Easy Vector and then transformed into E. coli DH5a cells. A total of 50 clones were selected for sequencing by the ABI 3730 Analyzer. PCR Amplicon Libraries were generated for each DNA sample, gel purified, and then it was broken into the segment of approximately 300 bp. The segment was

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further quantified using LightCycler 480 (Roche) and the Agilent Bioanalyzer 2100 (Agilent), pooled into equal parts by mass, and pyrosequenced by 454 Genome sequencer (Roche) according to the manufacturer’s protocols. The Amphioxus 16S rDNA sequences were deposited into the NCBI SRA database under the accession number SRX685736. 16S rDNA pyrosequencing data analysis Approximately 5 mg of the 3 PCR amplicon libraries for the two feces samples and habitat sample were sequenced by separate runs on a 454 Genome sequencer (Roche) according to the manufacturer’s protocol. The sequencer generated 282,542 raw pyrosequencing reads. The reads for the marine habitat library, the wild feces library, and the lab feces library were 91,587, 95,488, and 95,467 reads along with the average sequence length of 197, 236, and 147 bp, respectively. Operational taxonomic units (OTUs) by aligning unique reads was created at a 3% genetic distance threshold using the FastGroupII (http://fastgroup. sdsu.edu/fg_tools.htm) to search for a set of nonredundant sequences to be clustered [20]. About 14,729 pyrosequencing reads of fewer than 100 bp were omitted from the analysis to minimize pyrosequencing errors. Bacterial annotation was conducted using the Ribosomal Database Project Classifier (RDP) (http://rdp.cme.msu.edu) Naive Bayesian rRNA Classifier Version 2.2 with 50% confidence threshold [21]. V4 segments of the 16S rRNA were used to perform rarefactions, calculate diversity, and create rarefaction curves through calculating diversity between samples by FastGroupII [20].

Table 2. refOTUs and diversity of bacterial V4 tags of 16S rRNA in amphioxus feces and its habitat (>100 bp). Subject Reads Observed refOTUs Chao1 Shannon

Marine habitat

Wild feces

Lab feces

2,955 721 1586.38 5.03

3,591 171 309.02 2.43

3,275 176 332.74 2.11

province. The bacterial community in wild and labcultured Amphioxus feces and its habitat was examined through PCR for 16S rDNA sequences. A total of 267,813 reads (>100 bp) with average lengths of 246, 256, and 212 bp from the feces and habitat samples were generated by pyrosequencing after the Amphioxus DNA was filtered out (Table 1). The sequence quality of the samples was evaluated by Good’s coverage. The quality was good ranging from 95.71 to 99.83% for each individual sample (Table 1). For the quantitative analysis, 2,955, 3,591, and 3,275 reads of the V4 tag in the habitat, wild and lab-cultured Amphioxus feces were clustered into 721, 171, and 176 OTUs (Table 2). The complexity of the microbiota in the sediment of the habitat of Amphioxus was significantly higher than in the wild and lab-cultured fecal samples based on bacterial richness data (Fig. 1). The diversity of the bacterial community in the surrounding habitat was also higher than those in both of the fecal bacteria samples according to the community evenness (Shannon’s), species richness (Chao1), and rarefaction curves (Table 2 and Fig. 1), but both fecal samples had the similar diversity of bacterial

Results and discussion Diversity of microbe in Amphioxus feces and the sediment of its habitat Amphioxus, Branchiostoma belcheri, was collected from the sandy habitat between Donghai Island and Naozhou Island of the Zhanjiang coastal line of the Guangdong

Table 1. The classification, coverage, and diversity of the amphioxus feces and its habitat by Pyrosequencing reads of 16S rRNA (>100 bp). Subject Reads Average length (bp) Known bacteria Unclassified Good’s coverage Phyla

Marine habitat

Wild feces

Lab feces

86,754 246 84,726 1,284 0.9852 28

93,902 256 93,732 163 0.9983 12

87,157 212 80,855 3,742 0.9571 13

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Figure 1. The microbiota in Amphioxus feces. (A) Amphioxus feces. (B) Rarefaction curves at 3% difference for bacterial V6 tags of the three samples (habitat and both Amphioxus fecal samples).

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community (Table 2 and Fig. 1). The taxonomical analysis showed that the sequence tags from the habitat were assigned to 28 bacterial phyla, and both of the fecal samples contained bacteria from only 12 and 13 phyla (Table 1). The taxonomical result indicated that the diversity of the microbiota samples in the habitat was also significantly higher than both of the fecal samples. Amphioxus is a filter feeder animal without the ability to select its food. The compositions of the bacterial community may be mainly determined by the bacteria found in the seawater. Furthermore, since the wild and lab-cultured Amphioxus lives buried in the sediment, the feces will ultimately end up in the sediment. However, the bacterial composition in

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the sediment of habitat will also be influenced by the bacteria in seawater. Thus, the bacterial composition in the sediment of habitat will be determined by the fecal bacteria and seawater. This could partly explain the fact of less diversity of bacteria in the Amphioxus feces by comparison of its marine environment. Disparity of the bacterial community in wild and labcultured feces Detailed taxonomic analyses from the phylum to genus were conducted to understand the bacterial community in Amphioxus feces. No significant difference in the taxonomic distribution was observed at the phylum level

Figure 2. Taxonomic breakdown of the bacterial tags from Amphioxus and marine habitat. The composition of the bacterial community in the marine habitat and the Amphioxus fecal flora. (A) The bar indicates the classification of bacteria at the Phylum level. (B) The bar indicates the Phylum–Class distribution for taxonomically assigned tags that occurred in the phylum Proteobacteria. (C) The bar indicates the genera distributions for taxonomically assigned tags (note only shows 1.0% in the genus, others in supplementary) for taxonomically assigned tags. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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between the wild and lab-cultured fecal bacteria. The Proteobacteria (87.77%) made up the most abundant phylum, followed by the Bacteroidetes (9.47%) and Fusobacteria (0.24%) in the wild fecal bacteria. In the labcultured feces, the Proteobacteria (51.78%) made up the most abundant phylum, followed by the Bacteroidetes (20.25%) and Fusobacteria (0.05%) (Fig. 2A). A significant difference between the two fecal bacteria samples was observed at the class level in the phylum Proteobacteria. In wild fecal bacteria, the prominent class was gamma Proteobacteria and epsilon Proteobacteria, contributing to 40–50% of the total bacteria, respectively (Fig. 2B). In lab-cultured fecal bacteria, the classes of alpha and gamma Proteobacteria made up more than 95% of the total bacteria (Fig. 2B). A comparison of the bacterial community structure of both fecal samples was further investigated by assessing the abundance ratio of the wild to the lab-cultured Amphioxus feces in the sequence tags (uniformation to 100,000 tags) at the genus level (Supporting Information Table S1). The ratio of the wild to lab-cultured Amphioxus

feces that were greater than 50 were of the genera Pseudoalteromonas, Arcobacter, Marinomonas, and Marinifilum (Fig. 3). In these four genera, the prominent bacteria were of the genera Arcobacter and Pseudoalteromonas, contributing to approximately 66.00 and 53.56% of bacteria in the wild fecal bacteria, respectively, but only 1.30 and 3.07% in the lab-cultured fecal bacteria, respectively (Fig. 2B). Although bacteria from Arcobacter are the typical environmental bacterium [22], Arcobacter species are also known as the common commensal bacteria and are widely spread in animal feces [23]. Pseudoalteromonas sp. has been reported as the commensal bacteria of octocoral [24]. As for other two genera, genus Marinifilum was specific to wild Amphioxus feces and contributes 79.60% of the phylum Bacteroidetes. Genus Weeksella, gram-negative bacteria, has been reported the commensal bacteria of the mucous membranes in humans and other warm-blooded animals [25]. Thus, the features of these prominent bacteria suggest that Pseudoalteromonas, Arcobacter, and Weeksella might also be involved in constructing the bacteria of the Amphioxus digestive tract.

Figure 3. Comparison of the Amphioxus feces bacterial communities of the principal 116 genera in the Amphioxus habitat and the fecal flora. (A) The heatmap shows the 116 genera in Amphioxus habitat and fecal flora. (B) The histogram shows the genera with a ratio that is greater than 50. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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The ratio of the lab-cultured to wild fecal bacteria in the sequence tags that was greater than 50 was of the genera Pyrococcus, Serratia, Weeksella, Polaribacter, Gaetbulibacter, Sulfitobacter, Shimia, Croceibacter, Marinicella, Leisingera, Litoreibacter, Donghicola, Enterobacter, Mariniflexile, Cohaesibacter, and Paracoccus (Fig. 3A and B). The bacterial formation of the lab-cultured fecal bacteria was mostly driven by genera Leisingera and Phaeobacter from alpha Proteobacteria (Fig. 2). Thus, genera Leisingera might be a specific genus in lab-cultured amphioxus. It proposed that further functional study on this genus in Amphioxus might provide some informations to understand the relationship of the bacteria and host biology. Comparison of the shared bacteria in both of the Amphioxus feces samples From a total of 630 genera, 120 genera were shared between the wild and lab-cultured Amphioxus feces samples, and 116 genera were shared by the three samples tested (Fig. 4 and Supporting Information Table S1). Four of the genera that were shared in both of the fecal bacteria samples were found only at the very low levels among the sequence tags in the sediment of the Amphioxus habitat (Fig. 4 and Supporting Information Table S1). A much refined taxonomic analysis of the common bacteria in the feces was conducted at the genus level. The most abundant genera in the bacterial community of the wild Amphioxus feces were Pseudoalteromonas and Vibrio (gamma proteobacteria) and the genus Arcobater (epsilon proteobacteria), whereas the lab-cultured fecal bacteria had higher abundances from the genera Phaeobacter, Shimia, and Leisingera (alpha Proteobacteria) and the genera Vibrio and Serratia (gamma Proteobacteria), and Bacteriodetes. The genera Pseudoalteromonas and Vibrio form the microbial community of the digestive tract in shrimp [11]. The most abundant bacteria found in both fecal samples could represent the composition of bacteria in the digestive tract to some extent. The fluctuating bacterial compositions in

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both of the Amphioxus feces samples indicate that the intestine microbiota is plastic and can respond to environmental stress in order to maintain the mutualistic relationship with the host. The Amphioxus microbiota in feces is a complex system with the potential for multistability that could be determined by the changes of the environment. Comparison of bacteria in feces and the sediment of Amphioxus habitat The detailed analysis of the composition in the habitat sediment showed that the Proteobacteria (72.11%) made up the most abundant phylum, followed by the Bacteroidetes (12.68%), Planctomycetes (1.74%), and Fusobacteria (0.46%). Members of the gamma Proteobacteria made up 63.04% of the Proteobacteria diversity and members of the alpha and epsilon Proteobacteria made up 3.80 and 2.01%, respectively. More than 90% (153/169 genera) of the wild fecal bacteria were similar to its habitat and only 12 of 169 genera were specific to the wild fecal bacteria (Fig. 4). About 77% (238/308 genera) of labcultured fecal bacteria were similar to its habitat and only 66 of the 308 genera were specific to the lab-cultured fecal bacteria (Fig. 4). The shared genera between the Amphioxus feces and the habitat sediment consisted mostly of harmless environmental bacteria from the coastal seawater and a few other potential pathogenic species from the genera Serratia and Enterobacter [26, 27]. Thus, the sediment of its habitat contains a majority of bacteria of both Amphioxus feces at the genus level. As speculative, since Amphioxus lives buried in the sediment of its habitat, and thus, the bacterial compositions in the sediment of its habitat were partly retrieved from its feces. Moreover, the sediment contains the specific bacteria reached to approximately 50% (273/548 genus), indicating the higher diversity and species richness of bacteria in the sediment of habitat than feces.

Conclusions

Figure 4. Distribution of genera in the bacteria communities of Amphioxus from the 16S rDNA sequencing. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

In short, our study comprehensively investigated the high complexity of the ecological system of the bacterial community in Amphioxus feces and explored the structure of the fecal bacteria. These findings pave the way for a comprehensive investigation of the ecological system of the bacteria in the Amphioxus digestive tract. Based on our previous systemic study on the sophisticated and expanded innate immune system in amphioxus, we determined that fecal bacteria were a valid resource for understanding the mucosal immune responses to external bacterial stimulation.

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vannamei: digestive tract microbial community of pacific white shrimp (Litopenaeus vannamei). Springerplus, 3, 280.

Acknowledgments This study was supported in part by the project 2011CB946101 and 2013CB835304 of the National Basic Research Program (973) and project 2012AA092201 of the State High-Tech Development Project (863), key project 91231206, 30730089, and project 31270018 of the National Nature Science Foundation of China, project (GD2012-D-002) was supported by Administration of Ocean and Fisheries of Guangdong Province.

Conflict of interests The authors declare that they have no competing interests.

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J. Basic Microbiol. 2015, 55, 1336–1342

Diversity and composition of the bacterial community in Amphioxus feces.

Amphioxus is a typical filter feeder animal and is confronted with a complex bacterial community in the seawater of its habitat. It has evolved a stro...
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