THE EFFECT OF DIFFERENT SEAWEED DIETS ON GUT REGENERATION OF THE SEA CUCUMBER (Apostichopus japonicus) Pham Truong Giang1,2, Yong-Ki Hong3 Research Institute for Aquaculture No 3, Nha Trang, Vietnam KOICA-PKNU International Graduate Program of Fisheries Science, Korea Email: truonggiangria3@gmail.com Department of Biotechnology, Pukyong National University, Nam-gu, Busan 608737, Republic of Korea Email: ykhong@pknu.ac.kr ABSTRACT Apostichopus japonicus is one of the economically highly valued species with important nutritional value, medicinal purposes and beneficial physiological properties for consumers Among the different edible body parts of sea cucumber, the intestine (Konowata) is well known for nourishment Therefore, we aimed to evaluate the effect of various seaweed diets on rate of gut regeneration and continuous commercial production of sea cucumber intestine After days of recovery from evisceration, sea cucumber’s of a size of 29.1 ±4.0 gram were fed with powder of 14 different seaweed species for 14 days For choosing the best method to release gut, we applied a chemical injection method with KCl, NaCl, urea, methanol, ethanol, and chloroform Also physical stimuli including sonication, shaking, electric shocks and pressing by forceps were applied After 21 days of culturing, body weight, gut weight growth rate were evaluated in each group Among the chemical and physical stimuli applied, injection with 0.7 M KCl was found to be the best method to release the gut (100% released and survived) Sea cucumber gut regeneration from E cava, U pinnatifida and G amansii, respectively was higher than other groups which consisted of L catenata, H pannosa, and U pertusa This study shows the potential of various seaweeds used as sea cucumber feed for effective regeneration of gut Key words: sea cucumber, seaweed, gut regeneration, feed I INTRODUCTION The ability of echinoderms to ‘repair’ injured or autotomized body parts is well known (Hyman, 1955; Candia, 2006) For example, they can completely discard most of their internal organs and then rapidly regrow them (Mashanov and Garcia-arraras., 2011) This may occur when they face physical, chemical or environmental stress, or are under threat of predation (Smith and Olds., 2011) Sea cucumbers, or holothurians, are exclusively marine invertebrates classified in the phylum Echinodermata; class Holothuroidea (Mashanov and Garcia-arraras., 2011) Holothurians are characterized by a relatively long life span, estimated to about four to ten years (Storelli et al., 2001) They continuously renew cells in their adult tissues, including the digestive tube (Mashanov et al., 2004) and regenerate the same structure multiple times over their lifetime (Mashanov et al., 2010) Visceral regeneration in sea cucumbers provides a promising system for fundamental questions of regenerative biology (Mashanov and Garciaarraras., 2011) Gut regeneration in sea cucumbers points to a connection between development and regeneration (Smith and Olds., 2011) Furthermore, the sea cucumber is one of the ‘sea treasures’ which has been used not only as luxury food in certain countries but also in medicines (Pangkey et al., 2012) Gut content analysis and observations on live sea cucumbers show that their diet comprises mainly inorganic compounds, organic detritus of macroalgae, decaying animals, and microorganisms (Dar and Ahmad., 2006) Powdered macroalgae and sea mud have been used as the main components of formulated feeds in semi-intensive sea cucumber culture systems (Liu et al., 2010) In northern China, G lemaneiformis, S thunbergii, S polycystum, Z marina, U lactuca and L japonica, are the most common seaweeds used in feeding cultured sea cucumbers (Liu et al., 2010; Xia et al., 2012) Apostichopus japonicus is the most commonly cultured species in China, mainly due to its high quality meat and the success of the culturing method applied for the species (Huizeng., 2001) While knowledge on feeding larval and juvenile sea cucumbers is well established, the feeding during rearing (grow-out) of sea cucumbers should be studied more intensively (Pangkey et al., 2012) Therefore, the present study aims to evaluate the effect of several seaweeds on the rate of gut regeneration and potential significance in the future culture of sea cucumber products II MATERIALS AND METHODS Stimulation methods experiment Fresh, living sea cucumbers of approximately the same size (29.0±4.0 g) were bought from a seafood market at Busan, South Korea Several physical and chemical stimulation methods were applied to eviscerate their gut safely, and for use in the feeding experiments In case of chemical methods, potassium chloride (0.7 M KCl), sodium chloride (NaCl), urea, methanol, ethanol, or, chloroform was injected in the sea cucumbers’ body at different concentrations via the coelom with 4% body weight In case of physical methods, stimulation such as sonication, shaking, electric shocks and pressing with forceps were used (Figure 1) The holothurians were then transferred to glass aquarium (55×30×35 cm, water volume of 45 L) and survival rate was observed Seaweed materials Seaweeds and sand were collected from Gijang-Gun beach The sand was washed and sieved to grain size between 150 and 300 μm, while seaweed was dried and then ground into a fine powder of 250 and 400 μm Sand and seaweed were mixed at equal volume at 1:1 ratio before adding sea water and thoroughly mixing into a thick paste to avoid size stratification of grains The paste was then made into pellets (2cm various shapes) before rapid freezing to −20 °C Sea cucumber gut regeneration experiment Sea cucumber gut was eviscerated by 0.7M KCl 4% of body weight (the result of stimulation experiment above) they were randomly distributed into rectangular boxes with individuals per box, in a total of 43 tanks including a control tank The animals were then left for seven days of wound was recovery (Mashanov and Garcia-arrara., 2011) The pelletized seaweed feed was fed directly to the animals after the seven days recovery period, and then every 48 hours with feed to 3% of body weight Any feed left-overs were collected, and feaces as well as debris were removed hour before each new feeding round Rearing condition and feeding The aquarium conditionwas were maintained at salinity and pH ranging 30 - 32 ‰ and 7.8 - 8.2, respectively, at 14±0.4°C water temperature, light at 10hrs and dark at 14 hrs and a dissolved oxygen level >6ppm The ammonia concentration was less than 0.3 mg/L The water exchange rate (recirculating) in the aquarium was 3L/min Sea cucumber Chemical methods Physical methods Evaluation Figure Sea cucumber evisceration methods Figure.2 Procedures gut regeneration of the sea cucumber Apostichopus japonicus Figure.3 Procedures used for seaweed feeds for feeding experiment Statistical analysis GWR = GW ì BW-1 ã GWR: Gut weight ratio (gram) • GW : Gut weight (gram) • BW : Body weight (gram) GGR = GWRa ì GWbf-1 ã GGR: Gut gowth rate • GWRa: Gut weight ratio after regeneration (gram) • GWRbf: Gut weight ratio before experiment (gram) Statistics was performed using software SPSS16.0 with possible differences among treatments being tested by one-way ANOVA Microsoft’s excel package 2010© was used III RESULTS Stimulation methods The results of both chemical and physical stimulation methods to induce gut releasing in sea cucumber are shown in Table Table.1 Inducing evisceration by different stimulation methods in sea cucumber Stimuli Percentage released Percentage survival KCl 100 100 NaCl 11 44.4 Methanol 14.3 57 Ethanol 42.86 Chloroform 28.6 Sonication (5mins) 58 58 Forceps pinch (gentle-strong) 100 Electric shock (4.5-12V, 5mins) 33.3 Shaker (247rpm/1hour) 100 Effect of Seaweed diets in the gut regeneration Table.2 Gut growth rate (Mean±SE) relative to seaweed species after 21 days of experiment Seaweed species Gut growth rate (Mean±SE) Gellidium amansii (GA) 0.0512 ± 0.005 Scytosiphon lomentaria (SL) 0.0476 ± 0.01 Ulva pertusa (UPS) 0.0165 ± 0.004 Laminaria japonica (LJ) 0.0234 ± 0.005 Sargassum honoreri (SH) 0.0239 ± 0.005 Ecklonia cava (EC) 0.1085 ± 0.013 Sargassum hemiphyllum (SH) 0.0475 ± 0.014 Undaria pinnatifida (UP) 0.063 ± 0.007 Sargassum thumbergii (ST) 0.0476 ± 0.01 Carpopeltis comea (CC) 0.0219 ± 0.006 Lomentaria catenata (LC) 0.0121 ± 0.003 Enteromorpha linza (EL) 0.0244 ± 0.003 Chondrus ocellatus (CO) 0.0268 ± 0.005 Hypnea pannosa (HP) 0.0156 ± 0.003 Before feeding (BF) 0.101 ± 0.005 Control group CNTR (without feeding) 0.0148 ± 0.002 Sea cucumber gut regeneration in E cava, U pinnatifida and G amansii showed a mean value of 0.1085 ± 0.01 gram/gram, 0.063 ± 0.01 gram/gram and 0.0512 ± 0.001 gram/gram, respectively Meanwhile, the least gut regeneration values were found to be 0.012 ± 0.01 gram/gram, 0.0156 ± 0.003 gram/gram and 0.0165 ± 0.004 gram/gram in L catenata, H pannosa, U pertusa, respectively (Figure and Table 2) Fig.4 Gut regeneration of sea cucumber in different seaweed diets after 21 days (Letter above bars refers no significant differences between of them) IV DISCUSSION Holothurians are known for gut evisceration when facing different kind of disturbances (Vandenspiegel et al., 2000; Garcia-arraras and Greenberg., 2001; Mashanov et al., 2010) This study demonstrates the effects of various seaweed diets on gut growth However, actual gut evisceration methods were considered the most important aspect before feeding seaweed, for instance gut evisceration induced by chemical and physical means The criteria used for effectiveness of the induction were the evisceration rate, intensity of stimuli and survival of the animal after evisceration Rapid evisceration after inducing stimulus made collection of time taken to release, identification of the intensity of stimulus and distinguishing the harmlessness of the stimulus toward the animals after keeping them for at least one week In this studies, KCl at 0.4M showed similar results (Byrne, 1986; Tan et al., 2008; Zang et al., 2012) where KCl shows good performance with 100% (Table 1) evisceration as well as well being of the animals after more than four days compared with other sources of induction, narcosis effect of KCl might be the reason of its effectiveness while NaCl showed similar results as Byrne, 1986 with no evisceration, and chloroform, methanol and ethanol led no or less evisceration with survival rate between 0-57% (Table 1) In contrast, physical induction by sonication showed better performance in terms of evisceration rate and survival rate afterwards, which represents a kind of environmental disturbance as discussed in Vandenspiegel et al., 2000 especially Pinching by forceps was far different which no evisceration (Table 1) In electric shocks case, sea cucumber no evisceration probably due to weak voltage (4,5- 12V) compare with Byrne (1986), when voltage increased from 30 to100V but sea cucumber die all in third day Shaking failed to induce evisceration, probably due to weak revolution per minutes (rpm) used which might not be enough to induce evisceration (Table 1) All successful stimuli led to evisceration of all parts of the gut, excepting the cloaca, as has been been observed through test -stressing animals after evisceration by dissecting as explained previously (Byrne, 2001 and Mashanov, 2010) In the present study KCl and sonication were considered as effective and safe for animal survival after gut eviscerations hence for the feeding experiments KCl was used since have more favourable criteria than sonications The effects of seaweed in sea cucumber have been successfully studied previously (Xia et al., 2012) In this study, the main target was gut weight regeneration after feeding different seaweed powder diets The regeneration and autotomy of sea cucumber body parts have been studied for years and 21days confirmed to be sufficient to fully regenerate the gut in sea cucumber (Mashanov and Garcia-arraras., 2011) while 20 days was sufficient for bioactive compound accumulation (Bangoura, 2012) and vary in marine invertebrates (Pawlik, 1990) In this study seaweeds have been shown to vary in a statistically significant way for variation in gut regeneration Ecklonia cava, Undaria pinnatifida and Gellidium amansii showed the best results of 0.1085 ± 0.01 gram/gram, 0.063 ± 0.01 gram/gram and 0.0512 ± 0.01 gram/gram, respectively Lomentaria catenata, Hypnea pannosa and Ulva pertusa, showed the lowest values of 0.0121± 0.003 gram/gram, 0.0156 ± 0.003 gram/gram and 0.0165 ± 0.004 gram/gram, respectively (Table 3), while the remaining eight species ranged in between These results could be due to the presence of bioactive compounds in particular seaweed species has been absorb by sea cucumber (Guerard et al 2011) V CONCLUSSION Using seaweed powder for regeneration gut of sea cucumber is a promising idea in the sea cucumber industry with wide field apply such as medicinal, pharmaceutical and biotechnological and food for human as well The final result show that: For evisceration of sea cucumber, KCl 0.4M is the best method with 100% eviseation and survival rate as well In this study, E cavar is the best seaweed which can be used for gut regenaration of sea cucumber Apostichopus japonica, resulting within 21 days in a growth of 0.1085 ± 0.01 gram/gram References Bangoura Issa., 2012 Feeding of brown Seaweed Ecklonia cava, Ecklonia stolonifera and Eisenia bicyclis to Abalone for accumulation of phlorotannin PhD thesis 86-103pp Byrne M., 1986 Induction of evisceration in the holothurian Eupentacta quinquesemita and evidence for the presence of an endogenous evisceration factor J Exp Biol 120:25–39pp Byrne M., 2001 The morphology of autotomy structures in the sea cucumber Eupentacta quinqesemita before and during evisceration J Exp Biol 204:849–863pp Candia C M., 2006 Regeneration in echinoderms: repair, regrowth, cloning ISJ 3:64-76 Dar M.A., Ahmad H.O., 2006 The feeding 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ratio after regeneration (gram) • GWRbf: Gut weight... aquarium was 3L/min Sea cucumber Chemical methods Physical methods Evaluation Figure Sea cucumber evisceration methods Figure.2 Procedures gut regeneration of the sea cucumber Apostichopus japonicus... into pellets (2cm various shapes) before rapid freezing to −20 °C Sea cucumber gut regeneration experiment Sea cucumber gut was eviscerated by 0.7M KCl 4% of body weight (the result of stimulation