Introduction

Apostichopus japonicus is an important species for aquaculture in China. In northern China, A. japonicus is cultivated on a commercial scale by sea ranching or in shallow ponds, with the latter being more commonly employed in northern China (Choo 2008). Breeding methods for this species have improved in the past decade, and its transcriptome has been sequenced, although extensive studies on the thermal resistance, disease resistance, and growth rates of A. japonicas have been conducted, no effective and practical method to prevent the development of skin ulcerations during its cultivation has been established (Du et al. 2012; Gianasi et al. 2017; Zhao et al. 2017). The skin ulceration usually takes place when water temperature gets low from December to March, and once occurs, it spreads to a whole pond within days and brings a lethal death rate higher than 90%. Besides such potential devastating blow to A. japonicus cultivation, skin ulceration can be frequently found on couples of day basis. A. japonicus only possesses innate immunity, thereby making it relatively vulnerable to harsh cultivation environments. One preventative measure against skin ulcerations is to administer probiotics or prebiotics (Zhang et al. 2010; Ma et al. 2013; Sun et al. 2012; Yang et al. 2014, 2015). However, these lesions continue to occur despite efforts to elucidate the molecular mechanisms underlying its immune system (Xue et al. 2015). Therefore, an efficient approach in identifying the pathogens that cause skin ulcerations during the cultivation of A. japonicus is warranted.

Several studies on the causative pathogens of skin ulcerations have been conducted; however, none have utilized next-generation or third-generation sequencing (Li et al. 2010; Tiruvayipati et al. 2013). The 16S ribosomal RNA (16S rRNA) gene has been employed in profiling bacteria and Archaea since the late 1970s (Woese and Fox 1977). Multiple 16S rRNA databases have accumulated massive amounts of sequence data, and high-throughput sequencing technologies, predominantly the Roche 454 and Illumina sequencing platforms, have facilitated deep microbial community profiling. However, investigations on bacterial composition do not go beyond the family level (Vishnivetskaya et al. 2011; Besemer et al. 2012; Mosher et al. 2012). Recent studies using the Pacific Biosciences (PacBio) RS platform have revealed genus-level discrimination of 16S rRNA amplicons from microbial communities (Mosher et al. 2013). Because fewer resources are necessary due to improvements in phylogenetic reconstruction, we performed a comparative assessment of A. japonicus from a single local breeding spot using the PacBio RS platform to identify candidates responsible for, or linked to, skin ulcerations. To keep skin microbial consortia samples intact, we did not make any effort to exclude host tissues at the sampling stage. Such limitations may also assess the specificity of the polymerase chain reactions (PCRs) and sequencing depth of the PacBio RS platform, as indicated by the generation of good analyzable mega data without contamination.

Materials and methods

Sampling of A. japonicus

We sampled three healthy normal (NM) A. japonica and three individuals with skin ulceration deterioration (UD) in a same cultivating pond (122°15′N, 37°01′E) in Weifang, Shandong Province, China, on May 5th, 2016. Those with UD were identified by appearances of positive skin ulceration spots. The healthy group (HG) consists of three NM samples with an average weight of 69.0 ± 21.2 g and the three UD samples had an average weight of 41.8 ± 13.0 g. All samples were transported on ice and dissected later for a tiny piece (2 g) of skin tissue (for UD samples, ulceration dots were collected) within 4 h. We did not exclude host tissues or only tried to brush off some of the microbial consortia on the skin during sampling and instead utilized whole-skin tissue samples in PacBio RS II sequencing to precisely reflect the actual skin microbial composition.

PCR amplification of 16S rRNA genes

Genomic DNA was extracted following the Earth Microbiome Project (EMP) protocol (http://www.earthmicrobiome.org/protocols-and-standards/dna-extraction-protocol/). The purity and concentration of DNA in the final 40-μL eluent that was, respectively, extracted from the samples was tested on both NanoDrop NC2000 (Thermo Scientific, Wilmington, DE, USA) and TBS380 Fluorometer (Turner BioSystems, Sunnyvale, CA, USA). The primers employed to amplify the V1–V9 region of the 16S rRNA were 27F (3′-AGAGTTTGATCMTGGCTCAG) and 1492R (5′-ACCTTGTTACGACTT). The reaction mixture consisted of 5 μL of a 5× reaction buffer, 5 μL of a 5× GC buffer, 2 μL of each dNTP (2.5 mM), 1 μL of the forward primer (10 μM), 1 μL of the reverse primer (10 μM), 2 μL of the DNA template (4–25 ng μL−1), 8.75 μL of ddH2O, and 0.25 μL of 5U/μL Q5 DNA polymerase. All the samples were subjected to two-step PCR: (1) 25 cycles of the following: initial denaturation at 98 °C for 3 min, denaturation at 98 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 180 s, and a final extension at 72 °C for 5 min; (2) 16 cycles of the following: initial denaturation at 98 °C for 3 min, denaturation at 98 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 180 s, and a final extension at 72 °C for 5 min.

PacBio RS single-molecule real-time (SMRT) sequencing

The PCR products (0.2–0.7 μg) were prepared using Pacific Biosciences Template Prep Kit 1.0 for DNA damage repair and end repair. A total of 50 μL of the damage repair mix was, respectively, incubated at 37 °C for 60 min and cooled at 4 °C. By adding 2 μL of the end-repair eluent, the 52-μL mixture was incubated at 25 °C for 5 min. The reaction mix was cleaned using 0.6× Agencourt AMPure XP beads (Beckman Coulter, Beverly, MA, USA). Then, the mixtures were subjected to a blunt-ligation reaction preparation step at − 25 °C for 1 h, 65 °C for 10 min, and 4 °C overnight. Ligation failure products were removed by adding exonucleases and incubating at 37 °C for 60 min. The resulting 41-μL mixture containing the library was purified using 0.6× Ampure PB beads twice. Once the primers were annealed and DNA polymerase had bound, the SMRT bell-polymerase complexes were sequenced.

Results

PacBio RS II SMRT chip generated a total of 18,195 reads from the V1–V9 amplicons of all six samples. The circular consensus sequencing data were processed through the SMRT pipeline (https://github.com/PacificBiosciences/smrtflow) to make sure that the error rate was < 10% and full passes were > 3. The length distribution of all reads was as expected of typical V1–V9 amplicons of the 16S rRNA gene (Fig. 1).

Fig. 1
figure 1

Length distribution of reads from NM and UD groups using PacBio RS II SMRT chip

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From a total of 16,276 optimized full-length 16S rRNA reads from all six samples, 97% were clustered to the bacterium 16S rRNA sequence of the Greengenes database (Release 13.8, http://greengenes.secondgenome.com/) (DeSantis et al. 2006) using QIIME (Quantitative Insights Into Microbial Ecology, v1.8.0, http://qiime.org/) (Caporaso et al. 2010) up to species level (Table 1).

Table 1 OTU clustering and classification of outcomes
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The rarefaction curve depicted well-presentable sequencing depth for each sample (Fig. 2). All samples reached saturation as indicated by the plateau.

Fig. 2
figure 2

Rarefaction curves of OTU numbers of the samples

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The composition of each sample at the species level shows a three-stage trend in Fig. 3.

Fig. 3
figure 3

Main species composition of the samples

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NM1, 2, and 3 comprised HG A. japonicus, UD1 showed the early stages of skin ulceration symptom, and UD2 and 3 exhibited advanced stages of skin ulceration.

To identify the bacterium that potentially causes skin ulceration, we compared the bacterial communities between HG and UD1 and that between UD1 and UD2 and 3. Then, we filtered the data matrix to identify strains that showed at least a twofold increase from the HG group to UD1 and still existed at average densities in UD 2 and 3 (Fig. 4). Our results showed that all the filtered strains decreased in number as A. japonicus reached the advanced stages of skin ulceration.

Fig. 4
figure 4

Strains showing at least a twofold increase in number and a positive average in the UD 2 and 3 groups

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Figure 4A shows a shift in the bacterial community profile from HG to UD1, and that in B depicts similarities in profiles between UD1 and UD 2 and 3. Eight bacterial species exhibited de novo emergence (DE) as reflected in the microbial composition during the early stage of skin ulceration. The specific number of the DEs in UD1 was as follows: Desulforhopalus singaporensis (0.44‰), Lactobacillus fabifermentans (0.44‰), Ornithinibacillus contaminans (0.44‰), Polaribacter dokdonensis (0.44‰), Bizionia argentinensis (0.87‰), Rubritalea tangerina (0.87‰), Luteolibacter algae (1.31‰), and Lutibacter agarilyticus (4.36‰).

Discussion

D. singaporensis, L. fabifermentans, P. dokdonensis, B. argentinensis, L. agarilyticus, and L. algae from the DE group are associated with various metabolic pathways related to skin ulcerations but have not been reported as actual pathogens (Lie et al. 1999; Yoon et al. 2006; Bercovich et al. 2008; De Bruyne et al. 2009; Lanzarotti et al. 2011; Park et al. 2013; Aran et al. 2014; Campanaro et al. 2014; Treu et al. 2014; Yoon et al. 2017).

Approximately seven species have been validated in the genus Ornithinibacillus (Mayr et al. 2006). Most are free-living bacteria isolated from natural environments, such as marine sediment and saline soil. O. scapharcae TW25T is considered to be the only Ornithinibacillus that has pathogenic potential. O. contaminans DSM22953T, which emerged in this study, was first isolated from a blood sample of a 75-year-old woman. It is Gram-positive, endospore-forming, and rod-shaped. O. contaminans DSM22953T, similar to O. scapharcae TW25T, contains a number of virulence-relevant genes, which indicates that it may also be pathogenic. However, no case reports of disease due to O. contaminans DSM22953T have been reported to date. The present study thus may suggest a case study of its pathogenicity (Kämpfer et al., 2010; Jiang et al., 2016). Further research is due for conclusions.

R. tangerina is Gram-negative, non-motile, facultatively anaerobic, and coccoid or rod-shaped. R. tangerina was isolated from the visceral specimen of an unidentified sea hare collected from Himetsu, Sado, Niigata, Japan, in 2006. Information on this species is limited (Yoon et al. 2007).

Other strains that increased in density during possible different stages of skin ulceration in cultured A. japonicas included the following:

  1. 1.

    Carnobacterium maltaromaticum LMA28 can be found in the wild and in food products, and can grow anaerobically. This species is pathogenic to fish, whereas cases involving humans have not been reported (Leisner et al. 2007).

  2. 2.

    Aerococcus viridans is a member of the bacterial genus Aerococcus. It is a causative agent of gaffkaemia, a disease in lobsters (Greenwood et al. 2005).

  3. 3.

    Bacillus thuringiensis is a Gram-positive, soil-dwelling bacterium that is commonly used as a biological pesticide. It was first discovered in 1901 by Japanese biologist Shigetane Ishiwata (Roh et al. 2007) and then rediscovered in Germany by Ernst Berliner, who reported that it causes a disease called Schlaffsucht in flour moth caterpillars. B. thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, which causes anthrax; the three organisms differ mainly in their plasmids (Økstad and Kolstø 2011). Besides B. thuringiensis, there are a total of 14 members of the genus Bacillus that showed an increase in density in UD 1. Among these, B. thuringiensis and B. pseudomycoides are the only two Bacillus species that were observed on UD 2 and 3 at average levels.

  4. 4.

    Seven members of the Burkholderia appeared during the early stage of skin ulceration, except for Burkholderia vietnamiensis G4, which also emerged in HG. However, none of these species were identified during the advanced stage of skin ulceration. Several members of this genus are considered to be biosecurity threats (Woods and Sokol 2006; Sawana et al. 2014).

The two predominant bacteria of the skin health consortia included B. cereus and Oceanobacillus polygoni. B. cereus averaged 459.93 ± 30.38‰ and O. polygoni 295.24 ± 73.53‰ in the HG group. The population of both species dramatically dropped to 43.80 ± 24.20‰ and 24.70 ± 12.17‰, respectively, during the advanced stage of skin ulceration. B. cereus is a Gram-positive, rod-shaped, aerobic, motile, β-hemolytic bacterium that is commonly found in soil and food. Some strains of B. cereus are harmful, whereas others can be beneficial as probiotics for animals (Charalampopoulos and Rastall 2009). O. polygoni is a Gram-positive, peritrichously flagellated, straight, facultatively alkaliphile, rod-shaped bacterium that was first isolated from indigo fermentation fluid in 2013 (Hirota et al. 2013). A strain of O. polygoni, MBF-HG6, is salt-tolerant and alkaliphilic and produces bioflocculants (Li et al. 2017). The role of O. polygoni in skin ulceration remains unclear.

Saccharicrinis carchari was the predominant bacterium (846.85 ± 5.75‰) during the advanced stage of skin ulceration. However, this bacterium was not observed in all three HG samples and only showed a 1.54-fold increase in percentage from HG to UD1 (0.44‰). S. carchari was the only strain that illustrated a shift in the trend wherein it continuously increased. It is a Gram-negative, facultatively anaerobic, gliding, non-endospore-forming, yellow-pigmented, straight or slightly curved, rod bacterium that was initially isolated from shark gill homogenate. This strain is catalase-positive and oxidase-negative (Liu et al. 2014). Its role in skin ulceration remains unclear.

Conclusions

The present study examined changes in the skin microbial consortia of A. japonicus with skin ulcerations. PacBio sequencing revealed differences in bacterial communities at the species level. O. contaminans and S. carchari were the predominant bacterial species in the skin ulcerations. Our approach of keeping skin samples intact and eliminating unrelated sequences by selective PCR for 16S rRNA sequencing on a PacBio RS platform proved to be sufficient for performing a full-scale, species-discriminating inspection of the skin consortia community. Our technique may thus be potentially employed in future investigations on skin microbial consortia in aquatic invertebrates.