Effects of Pseudoalteromonas sp. BC228 on Digestive Enzyme Activity and Immune Response of Juvenile Sea Cucumber (Apostichopus japonicus)

MA Yuexin, SUN Feixue, ZHANG Congyao, BAO Pengyun, CAO Shuqing, and ZHANG Meiyan

Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea of Ministry of Agriculture, Dalian Ocean University, Dalian 116023, P. R. China

Effects of Pseudoalteromonas sp. BC228 on Digestive Enzyme Activity and Immune Response of Juvenile Sea Cucumber (Apostichopus japonicus)

MA Yuexin*, SUN Feixue, ZHANG Congyao, BAO Pengyun, CAO Shuqing, and ZHANG Meiyan

Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea of Ministry of Agriculture, Dalian Ocean University, Dalian 116023, P. R. China

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0 (control), 105, 107and 109CFU g-1diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107and 109CFU g-1diet of BC228 in comparison with control (P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107CFU g-1diet of BC228 was significantly higher than that of those fed control diet (P < 0.05). In addition, 105and 107CFU g-1diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets (P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

Apostichopus japonicus; Pseudoalteromonas sp. BC228; digestive enzyme activity; immune response

1 Introduction

The sea cucumber, Apostichopus japonicus Selenka, is an economically important and intensively farmed holothurian. Its annual production exceeded 100000 tons in 2010, creating a direct economic benefit of > 20 billion Yuan (http://www.haisheninfo.com). However, its epidemic diseases such as skin ulceration and peristome tumescence have been frequently occurring in last decade, causing serious economic losses and limiting the sustainable development of sea cucumber culturing industry (Ma et al., 2006a, b; Deng et al., 2009; Li et al., 2010). The primary pathogens isolated from sick sea cucumber were Gram-negative bacteria (Ma et al., 2006a, b; Deng et al., 2009; Li et al., 2010). Virus-like particles were also identified with electron microscope in juvenile and adult sea cucumber suffering from acute peristome edema and disease with symptoms of evisceration and skin ulceration (Wang et al., 2007; Deng et al., 2008). In addition, fungi and parasites in culture environment were opportunistic pathogens of sea cucumber (Wang et al., 2005).

In recent years, biological control of diseases with environmentally friendly methods such as probiotics has become an important research subject in aquaculture (Kesarcodi-Watson et al., 2008). The use of probiotics in the culture of aquatic organisms emerged in 1980s in China, and an exponentially growing application has been witnessed in last 10 years (Qi et al., 2009). Probiotics are preparations of microbial cells or cellular components which contain live and/or dead bacteria, conferring a health benefit on host (Salminen et al., 1999). Many probiotics such as Bacillus subtilis (Vaseeharan and Ramasamy, 2003), Carnobacterium inhibens (Joborn et al., 1997), Lactobacillus rhamnosus (Nikoskelainen et al., 2001), Roseobacter sp. (Ruiz-Ponte et al., 1999), Vibrio alginolyticus (Austin et al., 1995), V. fl uvialis (Irianto and Austin, 2002) and Pseudoalteromonas sp. (ten Doeschate and Coyne, 2008) were tentatively used in aquaculture. When Bacillus sp. K-3 and lactic acid bacterium L-2 were added to the diet of A. japonicus, its intestinal protease and amylase activities were improved significantly in 30 days (Zhang et al., 2009). B. subtilis could improve immune response and disease resistance of sea cucumber (Zhang et al., 2010; Zhao et al., 2011; Zhao et al., 2012).The search for new microorganisms that could be used as probiotics for sea cucumber is still ongoing. Previously, the marine bacterium Pseudoalteromonas sp. BC228 was isolated from healthy A. japonicas in our laboratory and found to have the ability of degrading protein, starch and lipid in vitro (Yang et al., 2013). The aims of this study were to assess its effects on the digestive enzyme activity, immune responses and disease resistance of juvenile A. japonicus and determine its potential of being probiotics of sea cucumber.

2 Materials and Methods

2.1 Bacterium

Pseudoalteromonas sp. BC228 was previously isolated by Yang et al. (2013). It was chosen for its ability of degrading protein, starch and lipid. BC228 was grown in tryptone soya broth (TSB) prepared with aged seawater at 25℃ with constant aeration until early stationary phase. The cell suspension was centrifuged at 1500 r min-1at 4℃for 15 min with pellet re-suspended in saline and incurporated into diets. To determine the concentration of the bacterium, serial dilutions (1/10 volume each step) were performed. The dilutions of 10-4to 10-10were spread onto tryptone soya agar (TSA) plates in triplicate after their OD was measured at 600 nm. Colonies were counted when the bacterium was cultured at 25℃ for 16 h. The relationship between CFU and time and CFU and OD were established.

2.2 Feeding Trial

The formulation of control diet was that of Liu et al. (2012). The experimental diets were prepared daily by supplementing 0 (control), 105, 107and 109CFU g-1diet of BC228 to the control diet.

Juvenile sea cucumber individuals were obtained from a commercial farm in Dalian, China. Prior to feeding trial, sea cucumber were acclimated to the rearing condition for two weeks. Healthy sea cucumbers (0.679 g ± 0.193 g) were randomly selected and distributed into 12 plastic tanks (100 L) 50 individuals each and 3 tanks each diet. For 45 days, sea cucumber were fed once (16:00) a day at a rate of 5% body weight and 50% of water was replaced every second day. Feces and any food remains were removed daily by siphoning. Aeration was maintained with low pressure electrical blowers via air stones. Water temperature was maintained between 14℃ and 7℃, salinity was maintained between 33 and 34 and acidity was maintained between pH 7.8 and pH 8.2.

2.3 Measurement of Enzyme Activity

At the end of feeding trial, sea cucumbers were starved for 24 h. Three sea cucumber individuals each tank were randomly sampled for determining various digestive enzyme activities. The animals were dissected immediately and the whole intestines were removed by incising at the esophagus and cloaca. The samples were blot dried with filter paper, weighed and homogenized in nine volumes of ice-cold 0.85% NaCl using a manual glass homogenizer. The homogenates were then centrifuged with supernatants pipetted into clean centrifuge vials and analyzed.

Total protein of the supernatants was determined according to Bradford (1976) using bovine serum albumin (BSA) as standard. Trypsin activity was assayed according to Holm et al. (1988) using commercial kits (Nanjing Jiancheng Bioengineering Institute, China). The reaction was based on trypsin’s ability of hydrolyzing L-arginine ethyl ester, which resulted in an increase in optical density when measured at 253 nm. One unit of trypsin activity was defined as the amount of enzyme causing an increase in absorbance of 0.003 per min per mg protein in intestine materials at 37℃ and pH 8.0. Lipase activity was measured by the simplified turbidmetric assay (Shihabi et al., 1971) using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China). The reaction was based on lipase’s ability of hydrolyzing triglyceride in stabilized emulsion of olive oil, which resulted in a decrease in optical density when measured at 420 nm. One unit of lipase activity was def i ned as 1 μmol substrate consumed per minute per gram protein in intestine materials at 37℃.

2.4 Immunoassays

Five sea cucumber individuals each tank were randomly sampled on day 45. Sea cucumbers were starved for 24 h ahead of assaying. After dissection, the coelomic fl uid was collected immediately and then thoroughly mixed with an equal volume of isotonic aqueous anticoagulant solution containing 0.02 mol L-1EGTA, 0.34 mol L-1NaCl, 0.019 mol L-1KCl and 0.068 mol L-1Tris-HCl (pH 8.0) (Xing et al., 1998). The coelomic fl uid from fi ve sea cucumber individuals each tank was pooled for immunological analysis. Total protein in the coelomic fl uid was determined according to Bradford (1976) using BSA as standard. After an aliquot of the coelomic fl uid was collected for the phagocytic activity test, the remaining was centrifuged at 2500 r min-1and 4℃ for 10 min with supernatant collected and used directly to assaying lysozyme and phenoloxidase activities.

Phagocytic activity of coelomocytes was assessed by measuring the uptake of neutral red stained zymosan particles (from Saccharomyces cerevisiae) with the method of Hannam et al. (2010). Phagocytic uptake of zymosan particles by coelomocytes was determined against a standard curve and expressed as a function of protein content in the 50 μL sample.

Lysozyme activity in coelomic fl uid was estimated as described by Hultmark et al. (1980) with minor modification. In brief, dried Micrococcus lysodeikticus (Worthington Biochemical Corporation, USA) cells were suspended in ice-cold 0.1 mol L-1phosphate buffer (pH 6.4), to give a density of 30 units on a Klett-Summerson colorimeter (A5700.2–0.3). Five μL coelomic fl uid sample was mixed with 300 μL of bacterial suspension in microplate in an ice bath and the absorbance (A0) was measured at 570 nm immediately. The mixture was incu-bated at 37℃ for 30 min. The microplate was then transferred back to ice bath for 10 min and the absorbance (A) was measured at 570 nm. The number of units (U) was estimated following the formula

Phenoloxidase activity was determined spectrophotometrically using L-3, 4-dihydroxyphenylalanine (L-zDOPA) as substrate and trypsin as elicitor following the method described by Smith and S?derh?ll (1991). One unit of enzyme activity was defined as the amount of enzyme causing an increase in absorbance of 0.001 per min per mL coelomic fl uid.

2.5 Challenge Test

After feeding for 45 days, 10 sea cucumber individuals were sampled randomly each diet and challenged with an intra-peritoneal injection of 0.1 mL of V. splendidus suspension (107CFU mL-1) (Ma et al., 2006b). The temperature of seawater was maintained between 8℃ and 7℃. The sea cucumbers were fed with control diet for 30 days disease evaluation as reported previously by Ma et al. (2006b).

2.6 Statistical Analysis

In order to determine significant differences among diets, data were analyzed using one-way analysis of variance (ANOVA), and followed by Duncan’s multiple comparison of means using SPSS 19.0 for Windows. Difference was significant if P < 0.05.

3 Results

3.1Activity of Digestive Enzyme

Sea cucumber fed BC228 containing diets had higher intestinal trypsin activity than those fed control diet (P < 0.01; Fig.1A). The intestinal lipase activity of sea cucumbers were significantly enhanced by 107and 109CFU g-1diet of BC228 (P < 0.01; Fig.1B).

Fig.1 Effects of Pseudoalteromonas sp. BC228 on intestinal trypsin (A) and lipase (B) activities of sea cucumber. Means (n = 3) with different letters are significantly different (P < 0.05).

3.2 Immune Response

Sea cucumber fed the diet containing 107CFU g-1diet of BC228 showed significantly higher phagocytic activity than those fed other diets (P < 0.05; Fig.2).

Lysozyme activity in coelomic fl uid in sea cucumber fed diet supplemented with 105CFU g-1diet of BC228 was significantly higher than those fed with other diets (P < 0.01; Fig.3A). An increase of coelomic fl uid phenoloxidase activity was observed in sea cucumber which was fed with the diet containing 107CFU g-1diet of BC228 (P < 0.01; Fig.3B).

Fig.2 Effect of Pseudoalteromonas sp. BC228 on phagocytic activity in coelomocytes of sea cucumber. Means (n=3) with different letters are significantly different (P < 0.05).

3.3 Challenge Test

The challenge test showed that administration of BC228 enhanced disease resistance of sea cucumbers against V. splendidus injection (Table 1). The cumulative mortality of sea cucumbers fed with the different diets which were supplemented with BC228 was 0–10%, which was lower than that of animals fed with the basal diet (50%).

Fig.3 Effects of Pseudoalteromonas sp. BC228 on lysozyme (A) and phenoloxidase (B) activities in the coelomic fl uid of sea cucumber. Means (n =3) with different letters are significantly different (P < 0.05).

Table 1 Cumulative incidence and mortality of sea cucumber Apostichopus japonicus fed with Pseudoalteromonas sp. BC228 diets for 45 days and challenged with Vibrio splendidus NB13

4 Discussion

4.1 Digestive Enzyme

Bacteria may benef i t the digestive processes of aquatic animals. Of diverse bacterial genera described in introduction section, Pseudoalteromonas sp. C4 was believed to be able increase the amount of alginate lyase of abalone (Haliotis midae) required for the digestion of kelp, thereby making more nutrients readily available for absorption by abalone (ten Doeschate and Coyne, 2008). In this study, intestinal trypsin and lipase activities of sea cucumber were significantly enhanced by dietary BC228. It is not clear whether such increased activities were due to endogenous enzymes synthesized by sea cucumber as a result of BC228 stimulation, and/or due to enzymes synthesized by BC228 as the bacterium is able to secrete extracellular protease and lipase (Yang et al., 2013). Nevertheless, BC228 did not increase intestinal amylase activity of sea cucumber (unpublished data) although it could secrete extracellular amylase (Yang et al., 2013), which suggested that the exogenous enzyme produced by the probiont would contribute at best only a little to the total enzyme activity of the intestine of sea cucumber. Perhaps, the presence of the probiont might stimulate the production of endogenous enzymes by sea cucumber. These findings were in agreement with that of Ziaei-Nejad et al. (2006) for Indian white shrimp (Fenneropenaeus indicus). Therefore, one of the proposed functions of BC228 tested in this study was to improve the activities of digestive enzymes in the intestine of sea cucumber.

4.2 Immune Indices

Coelomocytes are the major cells of defense in echinoderms against infection and injury, which phagocytose, entrap and encapsulate invading microorganisms (Gliński and Jarosz, 2000). Improvement of phagocytic activity in coelomocytes has been reported for sea cucumber fed with B. subtilis T13 (109CFU g-1diet) for 30 days (Zhao et al., 2012) or B. subtilis (107CFU g-1diet) for eight weeks (Zhang et al., 2010). In the present study, significant enhancement of phagocytic activity in coelomocytes was observed in sea cucumber fed diet containing 107CFU g-1diet of BC228 for 45 days. Similar benefit has also been observed on the phagocytic activity of hemocytes in shrimp (Penaeus monodon) fed with Bacillus S11 for 60 days (Rengpipat et al., 2000). Lysozyme is an important humoral innate defense factor, which widely distributes in both invertebrates and vertebrates (Magnadóttir et al., 2005). Lysozyme has an antibacterial activity by catalyzing the hydrolysis of peptidoglycans of cell walls of bacteria, especially the Gram-positive (Jollès and Jollès, 1984). In general, lysozyme exists in coelomocytes and coelomic fl uid of echinoderms (Canicattí and Roch, 1989; Shimizu et al., 1999). In this study, the lysozyme activity in coelomic fl uid showed a significant increase in sea cucumber fed the diet containing 105CFU g-1diet of BC228 for 45 days. The stimulation of serum lysozyme activity has been recognized after feeding with B. subtilis AB1 (Newaj-Fyzul et al., 2007) and Carnobacterium sp. BA211 in rainbow trout (Oncorhynchus mykiss) (Irianto and Austin, 2002) at 107cells g-1diet for 14 days, and B. subtilis and Lactobacillus acidophilus at 107CFU g-1diet in Nile tilapia (Oreochromis niloticus) for one and two months (Aly et al., 2008). The phen- oloxidase system is important in immune defense of invertebrates. Canicatti and Seymour (1991) found that phenoloxidase occurred in the proenzyme form in Holothuria tubulosa coelomocyte lysate and exogenous trypsin could enhance phenol-loxidase activity. The enzyme is responsible for melanin deposition during the processes of foreign-body encapsulation. In this study, greatly elevated phenoloxidase activity in coelomic fl uid was observed in sea cucumber fed the diet containing 107CFU g-1diet of BC228 for 45 days. Similarly, serum phenoloxidase activity in shrimp (Litopenaeus vannamei) fed the diet containing 1010CFU g-1diet of Bacillus OJ for 28 days increased significantly (Li et al., 2009). The present study clearly demonstrated the immunomodulatory effect of BC228 when it was included in the diets of sea cucumber.

4.3 Challenge Test

Alteromonas haloplanktis (Pseudoalteromonas haloplanktis, Gauthier et al., 1995) was capable of reducing the mortality of Argopecten purpuratus larvae when they were exposed to 103CFU mL-1of Vibrio anguillarum (Riquelme et al., 1996). B. subtilis supplementation in diet could increase disease resistance of sea cucumber to pathogenic bacterium V. splendidus (Zhang et al., 2010; Zhao et al., 2012). Our results showed that the dietary administration of BC228 reduced cumulative incidence and mortality of juvenile sea cucumber after being challenged with V. splendidus, which may be due to the stimulation of the innate immune response (Zhang et al., 2010; Zhao et al., 2012). To our knowledge, this is the fi rst study to demonstrate Pseudoalteromonas sp. on enhancing the digestive enzyme activity, the immune response and disease resistance of sea cucumber.

In addition, effects of BC228 on digestive enzymes and immune responses were dose-dependent. The dietary bacterium that transited in the digestive tract might be active to modulate the response of the host, depending on the dose supplied. Similarly, suitable dose B. subtilis T13 in diet could improve the immune response and disease resistance of sea cucumber (Zhao et al., 2012). The effect of B. subtilis E20 on the protease activity in the gut and gut contents of Litopenaeus vannamei was also dosedependent (Liu et al., 2009). Therefore, when digestive enzyme, immune response and disease resistance were all taken into consideration, a 107CFU g-1supplement of BC228 was recommended for A. japonicus according to the results of this study.

In conclusion, BC228 has the potential of being the probiotics of sea cucumber as it improves the digestive enzyme activity and immune response of sea cucumber, and enhances its resistance against V. splendidus.

Acknowledgements

This work was financially supported by the National High Technology Research and Development Program of China (863 Project) of China (2012AA10A412).

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(Edited by Qiu Yantao)

(Received March 26, 2013; revised May 6, 2013; accepted September 23, 2014)

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

* Corresponding author. Tel: 0086-411-84763096

E-mail: mayuexin@dlou.edu.cn