The sea cucumber, Apostichopus japonicus, is an epibenthic species with a geographical range spanning the northwest Pacific Ocean, including the Bohai Sea, the Yellow Sea, the east coast of Russia, and the coasts of Japan and Korea(Liao, 1997). It is the most economically important holothurian species, and has long been exploited as one of the most valuable seafoods in China, Russia, Japan, and Korea(Sloan, 1984).

The gut microbiota form a symbiotic relationship with the host, playing important roles in nutrition, metabolism, and biological antagonism(Cummings et al., 2004). Reports have shown that the gut microbiota can produce a variety of exogenous digestive enzymes, such as amylase, protease, lipase, cellulase, phytase, tannase, and chitinase(Nelson et al., 1999; Bairagi et al., 2002; Ray et al., 2010; Khan et al., 2011), contributing to food digestion. Furthermore, the gut microorganisms can supply essential nutrients and protect the host from being infected by competing for nutrition and available sites with pathogenic bacteria and producing antibacterial substances(Westerdahl et al., 1991; Hentges, 1992). Because of these important functions the gut microflora should be treated as an organ; however, it has been ignored by researchers for a quite some time(Bocci, 1992).

Traditional culture-dependent and molecular biological methods have been employed to study the gut microflora in the A. japonicus digestive tract(Sun and Chen, 1989; Xiang et al., 2006; Gao et al., 2010; Li et al., 2010; Zhang et al., 2011; Gao et al., 2014). Many A. japonicus gut microorganisms have been isolated and identified to date, such as Vibrio, Pseudomonas, Flavobacterium, Bacillus, Filobacillus, and Saccharomyces(Sun and Chen, 1989; Xiang et al., 2006; Li et al., 2010; Zhang et al., 2011). Bacterial community composition in the A. japonicus digestive tract has been surveyed by polymerase chain reaction/ denaturing gradient gel electrophoresis(PCR-DGGE) and high-throughput 454-pyrosequencing based on 16S rDNA(Gao et al., 2010, 2014). However, few studies on the functions of the microorganisms in the A. japonicus gut, particularly the enzyme-producing bacteria, have been carried out. To our knowledge, only Sun and Chen(1989) and Zhang et al.(2013)found bacteria that could decompose starch and casein in the A. japonicus gut.

Animal gut microbiotas contain allochthonous and autochthonous microbes. The autochthonous microbes can colonize the epithelial surface of the host gut, while the allochthonous microbes are transient(Ringø et al., 2006). Probiotics have been used widely in aquaculture as biological control agents against pathogens and activators of host nutrient intake(Verschuere et al., 2000; Ringø et al., 2010). Successful colonization in the digestive tract is often considered a prerequisite for dietary probiotics(Wu et al., 2013), thus the c and idate strains should preferably come from the host gut autochthonous microbiota.

The aim of the present study was to isolate and identify protease-, amylase-, and cellulase-producing autochthonous strains from the A. japonicus gut. Their extracellular enzyme production abilities were detected to evaluate the possible digestive symbiosis between the gut microbiota and the sea cucumber. Our results will provide c and idate probiotics for use in sea cucumber aquaculture.

Ten sea cucumbers, A. japonicus(212.12±14.56 g), were collected from a bottom enhancement area in Qingdao, Sh and ong, China, in April 2013, while the water temperature was 18°C. The sea cucumbers collected in this area mainly feed on the marine sediments; no medication is used during culturing. The sea cucumbers were transferred to the laboratory immediately and starved for 24 h to clear their digestive tracts before dissection.

The body surface of each sea cucumber was sterilized with 70% ethanol and thoroughly scrubbed with sterile marine water. The sea cucumbers were dissected and the entire gut was removed from the body cavity using sterile instruments. Afterwards, the guts were cleaned with sterile physiological saline solution(pH 7.4). All ten digestive tracts were mixed and cut up with sterile dissecting scissors; 3.0 g gut mixture was homogenized in 30 mL sterile physiological saline solution(PBS, pH 7.4). The gut suspension was used to isolate microorganisms. All operations were carried out under aseptic conditions.

2216E liquid medium(pH 7.6): 37.4 g of 2216E liquid medium(Qingdao Hope Bio-Technology Co. Ltd., Sh and ong Province, China)suspended in 1 000 mL distilled water. Luria-Bertani(LB)liquid medium(pH 7.2): 1.0 g peptone, 0.5 g yeast extract, 1.0 g NaCl, 100 mL filtered marine water. 2# liquid medium(pH 7.0): 4.0 g beef extract, 1.0 g yeast extract, 4.0 g peptone, 10.0 g glucose, 2.5 g NaCl, 1 000 mL filtered marine water. These three mediums were used to separate marine bacteria.

Gause’s Synthetic Broth(GSB)Medium(pH 7.4- 7.6): 20.0 g soluble starch, 0.5 g K₂HPO₄ ·3H₂O, 0.5 g MgSO₄ · 7 H₂O, 0.5 g NaCl, 0.01gFeSO₄ · 7 H₂O, 1.0g KNO₃, 1 000 mL filtered marine water. The GSB medium was used to separate marine-derived actinomycetes.

Potato Dextrose Broth(PDB)medium(pH 7.2): 200.0 g potato, 20.0 g glucose, 1 000 mL filtered marine water. The PDB medium was used to separate marine fungi.

The 2216E agar medium, Luria-Bertani(LB)Agar medium, 2# Agar medium, Gause’s Synthetic Agar(GSA)medium, and Potato Dextrose Agar(PDA)medium were made by adding 1.5% agar to the liquid medium. All of the mediums were sterilized at 121°C for 20 min.

The gut suspension was diluted 1:10 five times and used to isolate the microorganisms(Beveridge et al., 1991). Three replicates(0.1 mL)were taken from each dilution and poured aseptically within a laminar flow on sterilized 2216E agar plates, LB agar plates, 2# agar plates, GSA plates, and PDA plates. The culture plates were incubated at 37°C for 18-24 h. To obtain pure colonies, the well-separated strains with obviously diff erent morphologies were chosen and separated on the corresponding growth medium by the streak plate method The purified colonies were stored in the corresponding agar slant culture-medium at 4°C for further study.

A medium(pH 7.2)containing 1.0 g soluble starch, 0.5 g peptone, 1.0 g yeast extract, 1.5 g agar, and 100 mL filtered marine water was used to screen amylase-producing microorganisms. The plates were incubated at 37°C for 24 h after the colonies had been inoculated with sterile bamboo toothpicks. The amylase-producing colonies exhibited a hydrolyzed zone that did not turn blue after the addition of Lugol’s iodine(Mondal et al., 2010).

A medium(pH 7.2)containing 1.0 g soluble cellulose, 1.0 g NaCl, 1.0 g peptone, 0.5 g yeast extract, 1.5 g agar, and 100 mL filtered marine water was used to screen cellulase-producing microorganisms. The plates were incubated at 37°C for 24 h after the colonies had been inoculated with sterile bamboo toothpicks. The plates were then dyed with 0.5% Congo red for 30 min and soaked in 5% NaCl solution for 1 h. A hydrolyzed circle that did not turn red suggested cellulase activity(Zou et al., 2011).

A medium(pH 7.2)containing 1.0 g casein, 0.1 g yeast extract, 1.5 g agar, and 100 mL filtered marine water was used to screen protease-producing microorganisms. The plates were incubated at 37°C for 24 h after the colonies had been inoculated with sterile bamboo toothpicks. A clear hydrolysis zone around the colony indicated the presence of protease(Zou et al., 2011).

The pure cultured enzyme-producing microorganisms were collected from the fermentation broth culture. The total genomic DNA was extracted using the Hot Water Extraction(HWE)method(Feng et al., 2013) and an Omega E.Z.N.A. TM bacterial DNA kit(Omega Bio-Tek, Norcross, Georgia, United States)according to the manufacturer’s protocol with slight modification. The genomic DNA was detected by electrophoresis on 1% agarose gels with a 2 000-bp DNA ladder.

The 16S ribosomal DNA(16S rDNA)was amplified using the universal primer pairs 27f(5′-AGAGTTTGATCCTGGCTCAG-3′)/ 1492r(5′-TACGGCTACCTTGTTACGACTT-3′)(Zhang et al., 2013). The reaction mixture for PCR amplification was prepared in a total volume of 50 μL with 2 μL template DNA, 1 μL of each primer(20 pmol/mL), 25 μL Ex Taq™ Version 2.0(TaKaRa, Dalian, Liaoning province, China), and 21 μL sterile ultrapure water. The PCR reaction conditions were as follows: 35 cycles of 10 min at 94°C for initial denaturation, 45 s at 94°C for denaturation, 45 s at 55°C for annealing of 16S rDNA, 1 min extension at 72°C, and 10 min final extension. The PCR products were detected by electrophoresis on 1.5% agarose gels with a 2 000-bp DNA ladder.

RFLP analysis was performed on the PCR products from the enzyme-producing colonies isolated. The PCR products were digested with Mbo I, Hinf I, Hae III, Hha I, and Taq I(Haryanti et al., 2003)according to the manufacturer’s protocol with slight modification. The enzyme-digested products were separated on 3% agarose gels with a 100-bp ladder st and ard. The b and s were visualized under ultraviolet transillumination following gel electrophoresis. The similarity cluster diagram of the enzyme-producing strains was constructed based on the 16Sr DNA RFLP restriction patterns.

Based on the RFLP analysis results, purified PCR products from the representative strains were r and omly chosen from each group and sent to Shanghai Hanyu Bio-Lab, China for sequencing. The partial 16S rDNA gene sequences were compared with other sequences in the NCBI database using the BLAST program. Phylogenetic trees were constructed by the neighborjoining method using the software MEGA 5.05(Kumar et al., 2004), and tree topologies were evaluated by bootstrap analysis with 1 000 replicates.

The hydrolysis zone(D) and colony(d)diameters were measured with Vernier calipers after the enzymeproducing strains had been screened. The D/d value was used to compare the enzyme-producing capabilities of all colonies(Gao et al., 2007).

Thirty-nine strains with diff erent colony morphologies were isolated from the A. japonicus gut using isolation mediums and purified by the streak plate method(Table 1). The strains were mainly white, yellow, milky, reddish, and hoary in color, and the diameters ranged from0.42-4.38 mm. Among the 39 isolated strains, ten were isolated from 2216E agar medium, nine from 2# agar medium, nine from LB agar medium, six from GSA medium, and five from PDA medium. According to their morphological characteristics, we conclude that all of the microorganisms isolated were bacteria, no fungus or actinomycetes were isolated.

Table 1 Morphological characteristics of the colonies cultured in the present study

Table Options

All of the 39 strains can produce at least one enzyme(Table 2). The results revealed that 36 strains produce protease, 26 produce amylase, and 14 produce cellulase.

Table 2 Qualitative extracellular enzyme activity of the enzyme-producing strains

Table Options

The 16S rDNA fragments of all the enzyme-producing microorganisms isolated were amplified efficiently. The results also indicated that all of the enzyme-producing microorganisms isolated were bacteria.

The restriction enzymes Mbo I, Hinf I, Hae III, Hha I, and Taq I were used to digest the IBSCL-3 PCR products to identify the one with the most appropriate restriction enzyme pattern in the agarose gel electrophoresis. 16S rDNA PCR products digested with Mbo I produced the most fragments; therefore, this restriction enzyme was selected for the RFLP analysis of all microorganisms.

Mbo I RFLPs diff ered in all of the enzyme-producing strains in terms of fragment number and distribution. Mbo I digestion of the PCR products produced 3-5 fragments(Fig. 1). Based on this restriction enzyme map, the similarity cluster diagram was constructed for all strains and the 39 enzyme-producing strains were divided into 8 operational taxonomic units(OTU)at the 100% similarity level(Fig. 2).

Fig. 1 16Sr DNA macrorestriction map for all of the strains isolated with Mbo I M: 100 bp DNA Ladder; 1 (IBSC2-1); 2 (IBSC2-2); 3 (IBSC2-3); 4 (IBSC2-4); 5 (IBSC2-5); 6 (IBSC2-6); 7 (IBSC2-7); 8 (IBSC2-8); 9 (IBSC2-9); 10 (IBSC2-10); 11 (IBSCL-1); 12 (IBSCL-2); 13 (IBSCL-3); 14 (IBSCL-4); 15 (IBSCL-5); 16 (IBSCL-6); 17 (IBSCL-7); 18 (IBSCL-8); 19 (IBSCL-9); 20 (IBSC2#-1); 21 (IBSC2#-2); 22 (IBSC2#-3); 23 (IBSC2#-4); 24 (IBSC2#-5); 25 (IBSC2#-6); 26 (IBSC2#-7); 27 (IBSC2#-8); 28 (IBSC2#-9); 29 (IBSCG-1); 30 (IBSCG-2); 31 (IBSCG-4); 32 (IBSCG-5); 33 (IBSCG-6); 34 (IBSCG-7); 35 (IBSCP-1); 36 (IBSCP-2); 37 (IBSCP-3); 38 (IBSCP-4); 39 (IBSCP-5).

Figure options

Fig. 2 Similarity cluster diagram of gut enzyme-producing microbiota based on the 16Sr DNA macrorestriction map

Figure options

The 16S rDNA PCR products of IBSC2-1, IBSC2- 10, IBSCL-1, IBSCL-2, IBSCL-9, IBSC2#-3, IBSC2#-4, and IBSCG-6(one strain for each OTU)were chosen for sequencing. Blast analysis revealed that the IBSC2-1, IBSC2-10, IBSCL-1, IBSCL-2, IBSCL-9, IBSC2#-3, IBSC2#-4, and IBSCG-6 16S rDNA sequences exhibited >97% identity with that of Virgibacillus marismortui, Bacillus sp., B. stratosphericus, B. nanhaiensis, B. flexus, B. licheniformis, and B. aryabhattai, respectively. The phylogenic analysis revealed that IBSC2-1 and V. marismortui clustered together, while the other strains were all assigned to the Bacillus(Fig. 3).

Fig. 3 N-J phylogenetic analysis based on the 16Sr DNA sequences

Figure options

The hydrolysis zone(D) and colony(d)diameters were measured after the enzyme-producing strains had been screened; the D/d values are shown in Table 2.

The results show that the protease-producing isolates mainly belonged to the genera Virgibacillus(two strains) and Bacillus(34 strains). All of them exhibited high protease-producing abilities(the D/d values ranged from¹.14-7.91). Of all the strains, IBSC2-8 had the highest protease producing ability(D/d=7.91).

There were 26 amylase-producing strains; all were assigned to the genus Bacillus. IBSC2#-4 exhibited the highest amylase-producing ability(D/d=3.27).

Fourteen strains closely related to the genus Bacillus can produce cellulase. Among them, IBSC2- 5 exhibited the highest cellulase-producing ability(D/d=3.59).

Of all the enzyme-producing strains, six exhibited protease, amylase, and cellulase producing activities; all of these were assigned to the genus Bacillus. The results also revealed that the Bacillus isolated could produce one or more enzymes, but the Virgibacillus isolated could only produce protease.

In the present study, 39 enzyme-producing strains were isolated and screened. Based on the phylogenetic analysis of 16S rDNA sequences they belonged to either Bacillus or Virgibacillus, all strains were cultured under aerobic conditions.

Based on the optimum temperature for growth, microflora can be categorized into five types: psychrophiles, psychrotrophs, mesophiles, thermophiles, and hyperthermophiles, their optimum growth temperatures are 15°C, 20-30°C, 20-45°C, 55-65 ° C, and 80-90°C, respectively. Many studies have isolated mesophiles from the guts of aquatic animals at 28-37°C(Bairagi et al., 2002; Ghosh et al., 2002; Esakkiraj et al., 2009; Roy et al., 2009; Mondal et al., 2010; Peixoto et al., 2011), which is higher than the ambient temperature that the samples were collected at. Mesophilic bacteria can grow in a wide temperature range(minimum¹⁵- 20°C, maximum >45°C), though most grow optimally at 37°C(Roy et al., 2009; Mondal et al., 2010). The purpose of this study was to isolate mesophiles for c and idate probiotics from the sea cucumber gut. Therefore, we isolated the strains at 37°C, which is higher than the sampling environment temperature.

Bacillus strains have been isolated and identified as the enzyme-producing microflora from the gut of many species of aquatic animals, and many of the strains can produce more than one kind of digestive enzyme. Mondal et al.(2010), isolated four bacterial isolates that could produce extracellular digestive enzymes from the fish, Labeo bata ;two strains exhibiting high amylase, cellulase, and protease activity were identified as Bacillus licheniformis and B. subtilis. A Bacillus sp. strain producing at least three digestive enzymes was isolated from the sea cucumber A. japonicus(Yang et al., 2013); toxicity tests showed that the strain posed no threat to its host. In the present study, all of the Bacillus strains could produce protease, amylase, and /or cellulase. The results of this and previous studies indicate that Bacillus sp. are good producers of proteolytic enzymes and can also produce cellulase and amylase in moderate quantities(Mondal et al., 2010; Yang et al., 2013).

Virgibacillus strains that can produce digestive enzymes have rarely been isolated from the guts of aquatic animals. Similar to the results of Zhang et al.(2013), the Virgibacillus marismortui isolated in the present study can only secrete protease, not cellulase or amylase. Moreover, previous studies have reported that some Virgibacillus bacteria exhibited denitrification abilities(Denariaz et al., 1989). Therefore, the Virgibacillus isolated in our study could be used to improve water quality; however, further study is needed to confirm this.

Protease, cellulase, and amylase are the most important digestive enzymes in the gastrointestinal tract(GI)of animals. However, some vertebrate and invertebrate animals cannot secrete digestive enzymes by themselves, particularly cellulase. These animals require the aid of symbiotic microorganisms in their GI tract to digest the food and make the energy in this compound available to the host(Karasov et al., 2007). For example, according to Stickney and Shumay(1974), cellulase in the catfish(Ictalurus punctatus)igestive tract was derived from alimentary tract microbiota rather than from cellulase secreting cells within the fish. A study on the gut microflora of termites revealed that the most of the cellulase activity took place in posterior intestine and was largely derived from protozoan(Tokuda et al., 2002). The silkworm cannot secrete cellulase and the enzymes produced by its gastrointestinal tract microflora are used to digest food(Zou et al., 2011). The present study demonstrates that Bacillus can secrete protease, cellulase, and amylase, and Virgibacillus can secrete protease. The digestive enzymes they produce can help A. japonicus digest the food and provide essential nutrients for growth.

Many digestive enzymes, such as amylase, cellulase, pectinase, alginase, protease, dipeptidase, and esterase have been detected in the A. japonicus gut to date(Ke, 2010). However, the source of these digestive enzymes was not clear. In our study, all of the 39 strains isolated can produce extracellular enzymes. Among them, 36 strains produce protease, 29 produce amylase, and 11 produce cellulase. We propose that the gut microflora is an important source of digestive enzymes for A. japonicus.

Earlier investigations have shown that grampositive bacteria, particularly members of the genus Bacillus, can secrete a wide range of exoenzymes(Moriarty, 1996, 1999) and Bacillus isolated from the gut of aquatic animals have a beneficial eff ect on the digestive process of the host. Furthermore, Bacillus sp., including B. subtilis, B. cereus, B. coagulans, B. clausii, B. megaterium, and B. licheniformis are used as probiotics(Oggioni et al., 2003). Bacillus benefits the host by improving digestive enzyme activity. Wang and Xu(2006)showed that protease, amylase, and lipase in the common carp(Cyprinus carpio)digestive tract improved significantly while using Bacillus sp. as probiotics. Wang(2007)also showed that digestive enzyme activity improved significantly when photosynthetic bacteria and Bacillus sp. were added to shrimp basal diets, and the treatment group clearly exhibited high growth performance. In addition to their eff ect on digestive enzyme activity, Bacillus can also out-compete pathogenic bacteria for nutrients and space, and can exclude them through the production of antibiotics(Moriarty, 1999; Verschuere et al., 2000)enhancing immunity and reducing mortality in the host. Zhao et al.(2012)reported that the growth target and immunological indicators of juvenile A. japonicus improved significantly on a diet containing 10 9 CFU/g B. subtilis T13 isolated from the intestine of A. japonicus, confirming B. subtilis T13 as a potential probiotic for the juvenile sea cucumber. Therefore, the Bacillus strains isolated in the present study could be c and idate probiotics for A. japonicus aquaculture.

As a detritus feeder, the sea cucumber mainly feeds on surface sediment, including microorganisms, meiofauna, decaying organic debris, and inorganic and dissolved organic matter(Yingst, 1976; Moriarty, 1982; Zhang et al., 1995). Previous studies have proven that benthic diatoms and microorganisms are an important nutrient source for the sea cucumber and most of the animals energy requirements come from bacteria(more than 70%)(Sui, 1988).The microbes isolated from the sea cucumber gut in the present study can produce protease, amylase, and cellulase, which might suggest that the enzyme-producing microbes help the sea cucumber to digest the protein, starch, and cellulose stored in food, e.g., microorganisms and algae.

A previous study revealed that pure cultures of approximately 99% of the microorganisms in the natural environment could not be obtained by traditional isolation and culture methods(Mccaig et al., 2001). However, we can still expand the range of cultivable microorganisms by changing the culture conditions, e.g., reducing the concentration of nutrients in the culture medium or prolonging cultivation time. Therefore, the traditional isolation and culture methods are still an important method for investigating unknown microorganisms. Furthermore, combined with molecular biological methods, traditional culture-dependent methods will help us to obtain a better underst and ing of microbes in complex environments and contribute to further studies.

The results of our study suggest that the autochthonous gut microbiota of the adult sea cucumber A. japonicus produces a variety of enzymes including protease, amylase, and cellulase and exhibit strong enzyme-producing capabilities. The high enzyme-producing capability of the isolates suggest that the gut microbiota play an important role in the digestive process of the sea cucumber A. japonicus.