Ki Wook Lee 1, Hee Sung Kim 1, Pil Youn Kim 2, Hae Seung Jeong 3, June Kim 3, Ah-Yeong Yun 3 and Sung Hwoan Cho 3*

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1 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 RESEARCH ARTICLE Sustitution effet of white rdish (Rphnus stivus L.) y-produt nd tuni of se squirt (Hloynthi rorentzi, von Drshe) for Undri pinntifid in feed of lone (Hliotis disus, Reeve 1846) Ki Wook Lee 1, Hee Sung Kim 1, Pil Youn Kim 2, He Seung Jeong 3, June Kim 3, Ah-Yeong Yun 3 nd Sung Hwon Cho 3* Open Aess Astrt Sustitution effet of white rdish y-produt (WRB) nd tuni of se squirt (TSS) for Undri in feed on growth nd rss omposition of lone ws determined. Eight-hundred forty juveniles were distriuted into 12 ontiners. Three formulted diets were prepred in triplite. A 15% Undri ws inluded in the ontrol diet. The 15% Undri ws sustituted with sme mount of WRB nd TSS, referred to s the DRG nd TSS diets, respetively. Finlly, dry Undri ws lso prepred to ompre effet of the formulted diets on performne of lone. The experimentl diets were fed to lone one dy t stition level with little leftover. The feeding tril lsted for 16 weeks. Crude protein, rude lipid, nd sh ontent of the experimentl diets hnged over ll period of time. Higher rude protein nd lipid nd sh ontents retined in ll formulted diets ompred to the Undri were oserved t 24 nd 48 h fter sewter immersion. Survivl of lone ws not ffeted y the experimentl diets. Weight gin nd speifi growth rte (SGR) of lone fed the TSS diet ws greter thn ll other diets. Weight gin nd SGR of lone fed the ontrol diet ws not different from those of lone fed the WRB diet. Proximtes of the soft ody of lone ws ffeted y the diets. In onlusion, WRB nd TSS re the promising feed ingredients to reple Undri in lone feed; espeilly, TSS is superior to either Undri or WRB. The formulted diets produed improved growth performne of lone over the single Undri. Keywords: Alone Hliotis disus, Sustitution effet, White rdish y-produt (WRB), Tuni of se squirt (TSS), Undri Bkground Sine mrolge (MA) is one of the most expensive (US$3 4/kg) nd highest omponents (15 30% mount) in formulting ommeril lone feed in Estern Asi, espeilly Kore (Jng et l. 2018), development of lterntive soure tht is oth hep nd supply-stle for MA needs to ontinue for effetive lone (Hliotis spp.) ulture. MA used s ommon feed for yer-round lone ulture re either Lminri jponi Areshoug or Undri * Correspondene: hosunh@kmou..kr 3 Division of Mrine Biosiene, Kore Mritime nd Oen University, Busn 49112, Kore Full list of uthor informtion is ville t the end of the rtile pinntifid Hrvey nd ommonly inluded into lone feed depending on their prie nd vilility. As those MA re, however, exlusively hrvested during winter seson in wild in Estern Asi, either dry or slted MA re ommonly used for lone ulture for the rest of other seson. Alone frmers prefer feeding lone on MA over formulted feed due to esy mngement nd onveniene of frm in Kore lthough well-formulted feed produed etter growth performne over MA (Butist-Teruel et l. 2003; Gril-Esquivel nd Felek 2009; Jungetl.2016; Myung et l. 2016; Jng et l. 2018). In ddition, n interntionl mrket prie of MA is lso expeted to inrese due to high demnd for humn onsumption nd expnsion of lone frm in The Author(s) Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 2 of 8 the future. Therefore, development of n lterntive soure for MA in lone feed is highly desirle. Nturl diet of lones is known to onsist of 40 50% rohydrte, nd they hve vrious digestive enzymes ple of hydrolyzing omplex rohydrtes (Fleming et l. 1996). Mi et l. (1995) demonstrted tht lone (H. tuerult nd H. disus hnni) hd high potentil for utilizing rohydrte for energy. Alone utilized rohydrte etter thn lipid s n energy soure (Britz et l. 1994; MonjendVin1998; Thongrodetl.2003), proly explined y the ft tht lones hve high levels of the digestive enzymes of proteses, mylse, lginse, grse, rrgeense, nd lminrinse, ut low levels of lipses (Emerson 1967; Gómez-Pinhetti nd Grí-Rein 1993; Britz et l. 1994; Ersmus et l. 1997; Gri-Esquivel nd Felek 2006). Therefore, unlike fish utilizing rohydrte poorly (Wilson 1994; Stone 2003), lones seem to utilize vriety of plnt or rop s energy soure in feed well. Reyes nd Fermin (2003) reported tht the omined terrestril lef mel (Moring oliefer) nd freshwter quti fern (Azoll pinnt) with niml protein (white fish nd shrimp mels) were the promising feed ingredients for the prtil diet for the frmed lone (H. sinin) in the Philippines. White rdish (Rphnus stivus L.) is widely onsumed vegetle rop in Kore s the trditionl food, Kimhi, omposed of its root nd leves. White rdish y-produt, minly omposed of unsuitle stems nd leves (WRB) for humn onsumption re dumped nd regrded s trsh. Leves of white rdish re known to hve nti-inflmmtory tivity in lipopolyshridestimulted mrophges vi nuler ftor-kpp B intivtion (Prk nd Song 2017). Wter extrts of white rdish seeds hve een used in trditionl mediine to tret digestive inflmmtion, dirrhe, nd domen illness in Est Asi (Kim et l. 2015; Choi et l. 2016). Therefore, WRB hs high potentil s n lterntive soure for MA in lone feed. Kim et l. (2016) lso reported tht Lminri jponi t 20% in lone (H. disus) feed ws ompletely sustituted with rie rn, whih is one of griulturl wstes. Sine tuni of se squirt (Hloynthi roretzi von Drshe) (TSS) is usully dumped to the se fter deshelling for humn onsumption, it is regrded s pollution soure. Annul quulture prodution of se squirt rehed 31,353 tons in 2016 (KOSIS 2017). Kng et l. (1996) demonstrted tht the enzymti hydrolystes of TSS ould e used s the protein nd rohydrte soures for quulture. Both WRB nd TSS seem to hve high potentil s lterntive soure for MA in lone feed. Therefore, in this study, sustitution effet of WRB nd TSS for Undri in feed on growth nd rss omposition of lone (H. disus) ws determined. Methods Preprtion of lone nd rering onditions Juvenile lone were purhsed from privte hthery nd trnsferred to n lone frm (Oen nd Fisheries Reserh Institute, Jeju Speil Self-Governing Provine, Jeju, Kore). Before n initition of the feeding tril, lones were limted to the experimentl onditions for 2 weeks nd fed with the dry Undri one dy t the rtio of % totl iomss. Eight-hundred forty juvenile lones verging 5.4 g were rndomly distriuted into eh of the 12, 70-L plsti retngulr ontiners ( m) (70 per ontiner). Twelve ontiners were pled into 10-ton onrete flow-through rewy systems (wter volume: 2.8 tons) s flow rte of 48.2 L/min/rewy. The snd-filtered sewter t temperture rnging from 17.2 to 19.3 C (men ± SD: 18.4 ± 0.06 C) t 17:00 h ws supplied throughout the feeding tril. Aertion ws supplied into eh rewy nd the photoperiod followed nturl onditions. The experimentl diets were fed to lone one dy (17:00 h) t stition level (out 2 3% iomss) with little leftover. Ded lones were removed dily nd the ottoms of the ontiners were siphon-lened dily. The feeding tril lsted for 16 weeks. At the end of the feeding tril, lone ws hrvested nd olletively weighed from eh ontiner. Preprtion of the experimentl diets Three formulted diets were prepred in triplite (Tle 1). Fish mel, fermented soyen mel nd sein were inluded s the protein soure in the formulted diets. Dextrin nd squid liver nd soyen oils were used s the rohydrte nd lipid soures, respetively, in the formulted diets. A 15% Undri ws inluded in the ontrol diet. The 15% Undri ws sustituted with the sme mount of WRB nd TSS, referred to s the DRG nd TSS diets, respetively. The formulted diets were stisfied for dietry protein nd lipid requirements for lone (Mi et l. 1995, ; Fleming et l. 1996; Butist-Teruel et l. 2003). Finlly, dry Undri ws prepred to ompre effet of the formulted diets on performne of lone. Next, 20% sodium lginte ws dded to three formulted diets. All the ingredients were mehnilly mixed well nd wter dded t rtio of 1:1. A pste ws mde from eh of the diets y using n eletroni mixer nd shped into 0.15-m thik sheets, whih were then ut y hnd into 1-m 2 flkes. The flkes were then dipped into n queous solution of 5% CCl 2 for 1 min.

3 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 3 of 8 Tle 1 Feed ingredients in the experimentl diets (%, dry mtter sis) Experimentl diets Ingredients Control WRB TSS Undri Fish mel Fermented soyen mel Csein Dextrin Undri powder 15 (CP:14.9%, CL:2.7%) White rdish y-produt (WRB) 15 (CP: 21.6%, CL: 1.3%) Tuni of se squirt (TSS) 15 (CP: 28.5%, CL: 0.7%) Squid liver oil Soyen oil Sodium lginte Vitmin premix Minerl premix Nutrient (%) Dry mtter Crude protein Crude lipid Ash Crohydrte Vitmin premix ontined the following mount whih were diluted in ellulose (g/kg mix): exipient, 317; rioflvin, 23.8; pyridoxine, 4.7; niin, 95.2; C-pntothente, 33.3; inositol, 476.9; foli id, 1.5; p-mino enzoi id, 47.6 Minerl premix ontined the following ingredients (g/kg mix): exipient, 45.5; MgSO 4, 140.8; NH 2 PO 4, 92.4; KH 2 PO 4, 246; C(H 2 PO 4 ) 2, 139.5; ZnSO 4, 22.5; C-ltte, 310; AlCl 3, 0.15; KI, 0.15; MnSO 4, 2; CoCl 2,1 Crohydrte ws lulted y the differene (100 % rude protein % rude lipid % sh) The flkes were dried t room temperture for 2 dys nd stored t 20 C until use. Anlytil proedures of the diets nd rss Twenty lones t the strt nd from eh ontiner t the end of the feeding tril were smpled nd frozen for hemil nlysis nd growth mesurement. Prior to exmintion, ll smples were slightly thwed, followed y seprtion of the shell nd soft-ody tissue. Shell length nd shell width were mesured in millimeters with digitl liper (Mitutoyo Corportion, Kwski, Jpn), nd the soft ody weight ws mesured. Speifi growth rte (SGR, %/dy) ws lulted using the formul of Britz (1996): SGR = [(ln(wf) In(Wi))/dys of feeding] 100, where In(Wf) = nturl log of the finl men weight of lone nd In(Wi) = nturl log of the initil men weight of lone. The seprted soft ody tissue from ll lones from eh ontiner ws then homogenized nd used for proximte nlysis. Crude protein ontent ws determined y the Kjeldhl method (Auto Kjeldhl System, Buhi B-324/435/412, Switzerlnd), rude lipid ws determined using n ether-extrtion method, moisture ws determined y oven drying t 105 C for 24 h nd sh ws determined using muffle furne t 550 C for 4 h. All methods were ording to stndrd AOAC (1990) prties. Wter stility of the experimentl diets The three formulted diets nd dry Undri were pled in seprte 70-L plsti retngulr ontiners ( m) without lone in duplite. These ontiners were then pled within 5-ton onrete flow-through rewy indoor tnk t flow rte of 48.2 L/min nd susmpled t 12, 24, nd 48 h to evlute lehing of nutrients in the diets to determine their wter stility. Nutrient levels in the diets were ssessed using the sme proedure desried ove for lone rss. Wter stility of nutrients in the diets ws expressed s the perentge of finl dry ontent to initil dry ontent for eh nutrient sed on Mi et l. (1995) s study. Sttistil nlysis One-wy ANOVA nd Dunn s multiple rnge test (Dunn 1955) were used to determine the signifine of the differenes mong the mens of tretments y using SAS version 9.3 progrm (SAS Institute, Cry, NC, USA). Wter stility of the experimentl diets ws tested y ANOVA with repeted mesurement designs (Cody nd Smith 1991). Perentge dt ws rsinetrnsformed prior to sttistil nlysis. Results Crude protein, rude lipid, nd sh ontent of the experimentl diets were signifintly (P < ) hnged over ll periods of time (Figs. 1, 2, nd 3), nd their signifint (P < ) intertions (experimentl diets time) were lso oserved. After 12 h immersion in sewter, the retention of rude protein ontent in the TSS diet ws signifintly (P < 0.05) higher thn those in the ll other diets nd highest nd lowest for the TSS diet nd Undri, respetively (Fig. 1). The mount of dry mtter ontent retined in the ontrol nd WRB diets ws lso signifintly (P < 0.05) higher thn tht in the Undri fter 12 h immersion in sewter. The retention of rude lipid in the experimentl diets ws not signifintly (P > 0.05) different t 12 h fter sewter immersion, ut the retined perent of rude lipid in the ll formulted diets ws signifintly (P < 0.05) higher thn tht in the Undri t 24 h (Fig. 2). The proportion of sh ontent retined in the ll formulted diets ws signifintly (P < 0.05) higher thn tht in the Undri fter 12 h immersion in sewter nd highest

4 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 4 of ) 80 Control Crude protein ( WRB TSS Undri Elpsed time (h) Fig. 1 Chnges in rude protein ontent (%) of the experimentl diets t 12, 24, nd 48 h fter sewter immersion (mens of duplite ± SE). [ANOVA with repeted design: times (P < ) nd their intertion (experimentl diets time) (P < )]. Different letters in eh time point indites differene etween diets within eh time point nd lowest for the TSS diet nd Undri, respetively (Fig. 3). Survivl of lone rnged from 91.4 to 96.2% ws not ffeted y the experimentl diets (Tle 2). However, weight gin nd speifi growth rte (SGR) of lone fed the TSS diet ws signifintly (P < 0.05) greter thn ll other experimentl diets. Weight gin nd SGR of lone fed the ontrol diet ws lso signifintly (P < 0.05) greter thn those of lone fed the Undri, ut not signifintly (P > 0.05) different from those of lone fed the WRB diet. Shell length of lone fed the TSS diet ws signifintly (P < 0.05) higher thn ll other experimentl diets (Tle 3). Shell width nd height ws not signifintly (P > 0.05) ffeted y the experimentl diets. Soft ody weight of lone fed the TSS diet ws signifintly (P < 0.05) higher thn tht of lone fed the WRB diet nd Undri, ut not signifintly (P > 0.05) different from tht of lone fed the ontrol diet. Moisture ontent of the soft ody of lone fed the ontrol diet ws signifintly (P < 0.05) higher thn tht of lone fed the WRB diet nd Undri, ut not signifintly (P > 0.05) different from tht of lone fed the TSS diet (Tle 4). Crude protein ontent of the soft ody of lone fed the WRB nd TSS diets ws signifintly (P <0.05) higher thn tht of lone fed the ontrol diet nd Undri. Crude protein ontent of the soft ody of lone fed the ontrol diet ws Control ) ( Crude lipid WRB TSS Undri Elpsed time (h) Fig. 2 Chnges in rude lipid ontent (%) of the experimentl diets t 12, 24, nd 48 h fter sewter immersion (mens of duplite ± SE). [ANOVA with repeted design: times (P < ) nd their intertion (experimentl diets time) (P < )]. Different letters in eh time point indites differene etween diets within eh time point

5 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 5 of Control Ash ( ) 60 WRB TSS 40 Undri Elpsed time (h) Fig. 3 Chnges in sh ontent (%) of the experimentl diets t 12, 24, nd 48 h fter sewter immersion (mens of duplite ± SE). [ANOVA with repeted design: times (P < ) nd their intertion (experimentl diets time) (P < )]. Different letters in eh time point indites differene etween diets within eh time point lso signifintly (P < 0.05) higher thn tht of lone fed the Undri. Crude lipid ontent of the soft ody of lone fed the WRB nd TSS diets ws signifintly (P < 0.05) higher thn tht of lone fed the Control diet nd Undri. Ash ontent of the soft ody of lone fed the ontrol nd WRB diets nd Undri ws signifintly (P < 0.05) higher thn tht of lone fed the TSS diet. Higher rude protein nd lipid ontent of the soft ody of lone fed the formulted diets ompred to those of lone fed the Undri ws well refleted from dietry nutrient ontent. Disussion Beuse lones re slow-eters, wter stility of the diet is ritil to determine growth of lone nd severity of wter pollution in frms. Higher rude protein nd lipid nd sh ontents retined in the ll formulted diets (Control, WRB, nd TSS) diets ompred to the Undri t 24 nd 48 h fter sewter immersion indited tht wter stility of the formulted diets ws superior to the Undri. Espeilly, wter stility of the TSS diet seemed to e superior to other diets t 24 nd 48 h fter sewter immersion in this study. Similrly, wter stility of the formulted lone feed ws superior to dry MA (Undri nd Lminri) t 24, 48, nd 72 h fter sewter immersion (Lee et l. 2016). Butist-Teruel et l. (2003) lso reported tht wter stility of the formulted diets ws estimted to e 64% t 24 h when niml nd plnt protein soures ws tested for lone (H. sinin). SGR of lone fed the formulted diets rnged from 0.60 to 0.68%/dy in this study ws omprle to those otined ( nd %/dy for the sme speies of lone with initil weight of 3.3 nd 4.3 g grown t men temperture of 18.0 nd 19.5 C, respetively) in Lee et l. (2016) nd Jng et l. (2018) s studies nd superior to ones otined ( nd %/dy for the sme speies of lone with initil weight of 0.43 nd 1.29 g, respetively, grown t men temperture of 18.4 C) in Kim et l. (2016) nd Jung et l. (2016) s studies. No differene in weight gin nd SGR of lone fed the ontrol nd WRB diets indited tht WRB is promising lterntive soure for Undri in lone feed. This ould e explined y the ft of omprle Tle 2 Survivl (%), weight gin (g/lone) nd speifi growth rte (SGR) of juvenile lone fed the experimentl diets sustituting Undri with the dry white rdish y-produt (WRB) nd tuni of se squirt (TSS) for 16 weeks Experimentl diets Initil weight (g/lone) Finl weight (g/lone) Survivl (%) Weight gin (g/lone) SGR (%/dy) Control 5.4 ± ± ± ± ± WRB 5.4 ± ± ± ± ± TSS 5.4 ± ± ± ± ± Undri 5.4 ± ± ± ± ± Vlues (mens of triplite ± SE) in the sme olumn shring the sme supersript letter re not signifintly different (P > 0.05) Speifi growth rte (SGR, %/dy) = [(Ln(Wf) Ln(Wi))/dys of feeding] 100, where Ln(Wf) = nturl log of the finl men weight of lone nd Ln(Wi) = nturl log of the initil men weight of lone

6 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 6 of 8 Tle 3 Shell length (mm), shell width (mm), shell height (mm), nd soft ody weight (g/individul) of lone fed the experimentl diets sustituting Undri with the dry white rdish y-produt (WRB) nd tuni of se squirt (TSS) for 16 weeks Experimentl diets Shell length (mm) Shell width (mm) Shell height (mm) Soft ody weight (g) Control 45.9 ± ± ± ± 0.07 WRB 45.6 ± ± ± ± 0.04 TSS 47.1 ± ± ± ± 0.06 Undri 45.0 ± ± ± ± 0.01 Vlues (mens of triplite ± SE) in the sme olumn shring the sme supersript letter re not signifintly different (P > 0.05) nutritionl vlues [rude protein (CP): 21.6% nd rude lipid (CL): 1.3%] in WRB with ones [CP: 28.5% nd CL: 0.7%] in TSS (Tle 1). Similrly, the griulturl yprodut, rie rn, ws the good lterntive soure for L. jponi in lone feed (Kim et l. 2016). Reyes nd Fermin (2003) lso showed tht lolly ville yerround terrestril lef mel nd quti fern re the promising feed ingredients for prtil diet for frmed lone (H. sinin) in the Philippines. Sine lone utilize rohydrte etter thn lipid s n energy soure (Britz et l. 1994; Thongrod et l. 2003) nd hve high levels of the digestive enzymes (Emerson 1967; Gómez- Pinhetti nd Grí-Rein 1993; Gri-Esquivel nd Felek 2006), vriety of terrestril leves hs worth of evluting lterntive soure for MA in lone feed to lower feed ost. Fleming et l. (1996) lso reported tht lone hd vrious digestive enzymes ple of hydrolyzing omplex rohydrtes. Fesiility nd suitility of WRB s the lterntive soure for MA in ommeril diet on lone frm, therefore, needs to e proved efore prtil pplition (Tle 3). Improved weight gin nd SGR of lone fed the TSS diet ompred to the ontrol nd WRB diets in this study indited tht TSS is superior lterntive soure to either Undri or WRB for MA in lone feed. MA in lone feed ws ompletely sustituted with TSS without retrdtion of growth of lone (H. disus) nd the mximum weight gin ws otined in lone fed the diet sustituting TSS with 40% MA when TSS ws sustituted with 0, 20, 40, 60, 80, nd 100% MA t 20% in lone feed (Jng et l. 2018). Fesiility of sustitution of TSS with Undri in ommeril lone feed needs to e tested in ommeril-sle frm. Kng et l. (1996) explined tht the enzymti hydrolystes of TSS ould e used s the protein nd rohydrte soures for quulture. Extrts of TSS were suitle feed dditive for quulture use (Lee et l. 1994; Hong et l. 2002). Differene in oth studies [improved weight gin nd SGR in lone fed the TSS diet sustituting 100% MA with TSS in this study vs slightly poor weight gin nd SGR of lone fed diet sustituting 100% MA with TSS in Jng et l. (2018) s study] ould e resulted from differene in mino id lne in the experimentl diets in oth studies: [ssuming no redution, even slightly inrese in rude protein ontent (essentil mino id) due to no redution in the min protein soure in the TSS diet in the former vs redution in some essentil mino id, suh s rginine, histidine, isoleuine, leuine, lysine, phenyllnine, threonine, nd vline due to deresed soyen mel in the diet sustituting 100% MA with TSS in the ltter] (Tle 4). The essentil mino ids, suh s lysine, methionine, nd histidine re the limiting ftors to evlute suitility of lterntive protein soure for fish mel in lone feed (Shipton nd Britz 2001; Butist-Teruel et l. 2003; Cho et l. 2008; Cho 2010). Mi et l. (1994) lso showed tht the essentil mino ids (rginine, methionine, threonine, nd histidine) were the limiting ftors in six speies of MA (Ulv ltu, Chondrus rispus, Plmri plmt, Alri esulent, Lminri digitt, ndl. shrin) for growth of lone (H. tuerult nd H. disus hnni). Shell length nd the soft ody weight of lone were reltively well refleted from growth rte of lone in Tle 4 Chemil omposition (%) of the soft ody of lone fed the experimentl diets sustituting Undri with the dry white rdish y-produt (WRB) nd tuni of se squirt (TSS) for 16 weeks t the end of the 16-week feeding tril Experimentl diets Moisture Crude protein Crude lipid Ash Control 77.9 ± ± ± ± 0.09 WRB 76.9 ± ± ± ± 0.07 TSS 77.5 ± ± ± ± 0.07 Undri 76.8 ± ± ± ± 0.07 Vlues (mens of triplite ± SE) in the sme olumn shring the sme supersript letter re not signifintly different (P > 0.05)

7 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 7 of 8 this study. Similrly, the iologil riteri mesured were oinident with growth rte of lone (Butist- Teruel et l. 2003; Cho 2010). Higher rude protein nd lipid ontent of the soft ody of lone fed the formulted diets ompred to those of lone fed the Undri ws well refleted from dietry nutrient ontent, greeing with other studies showing tht the proximtes of the soft ody of lone ws diretly ffeted y dietry nutrient ontent (Uki et l. 1986; Mi et l. 1995, ; Thongrod et l. 2003; Cho et l. 2008; Gri-Esquivel nd Felek 2009; Cho 2010; Kim et l. 2016; Myung et l. 2016). Conlusions In onlusion, WRB nd TSS re the promising feed ingredients to reple Undri in lone feed; espeilly, TSS is superior to either Undri or WRB. The formulted diets produed improved growth performne of lone over the single Undri. Arevitions MA: Mrolge; SGR: Speifi growth rte; TSS: Tuni of se squirt; WRB: White rdish y-produt Aknowledgements This reserh ws supported y Bsi Siene Reserh Progrm through the Ntionl Reserh Foundtion of Kore (NRF) funded y the Ministry of Siene, ICT & Future Plnning. Funding The design of the study, olletion, nlysis, nd interprettion of the dt nd writing of the mnusript were supported y Bsi Siene Reserh Progrm through the Ntionl Reserh Foundtion of Kore (NRF) funded y the Ministry of Siene, ICT & Future Plnning (2017R1A2B ). Avilility of dt nd mterils All dtsets generted during nd/or nlyzed during the urrent study re ville from the orresponding uthor on resonle request. Authors ontriutions SHC designed the study nd prepred the drft. PYK, HSK, nd HSJ rn the feeding tril. JK nd AY nlyzed the hemil omposition of the diets nd rss of lone. All uthors red nd pproved the finl mnusript. Ethis pprovl All experimentl protools followed the guidelines of the Institutionl Animl Cre nd Use y Committee of the Kore Mritime nd Oen University. Consent for pulition Not pplile. Competing interests The uthors delre tht they hve no ompeting interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Author detils 1 Deprtment of Convergene Study on the Oen Siene nd Tehnology, Kore Mritime nd Oen University, Busn 49112, Kore. 2 Oen nd Fisheries Reserh Institute, Jeju Speil Self-Governing Provine, Jeju 63358, Kore. 3 Division of Mrine Biosiene, Kore Mritime nd Oen University, Busn 49112, Kore. Reeived: 12 Novemer 2017 Aepted: 7 Ferury 2018 Referenes AOAC. Offiil methods of nlysis, 15th edition. Arlington, VA: Assoition of Offiil Anlytil Chemists; Butist-Teruel MN, Fermin AC, Koshio SS. Diet development nd evlution for juvenile lone, Hliotis sinin: niml nd plnt protein soures. Aquulture. 2003;219: Britz PJ. Effet of dietry protein level on growth performne of South Afrin lone, Hliotis mide, fed fishmel-sed semi-purified diets. Aquulture. 1996;140: Britz PJ, Heht T, Knuer I, Dixon MG. The development of n rtifiil feed for lone frming. S Afr J Si. 1994;90:7 8. Cho SH. Effet of fishmel sustitution with vrious niml nd/or plnt protein soures in the diet of the lone Hliotis disus hnni Ino. Aqu Res. 2010;41:e Cho SH, Prk J, Kim C, Yoo J. Effet of sein sustitution with fishmel, soyen mel nd rusten mel in the diet of the lone Hliotis disus hnni Ino. Aqu Nutr. 2008;14:61 6. Choi KC, Cho SW, Kook SH, Chun SR, Bhttri G, Poudel SB, Kim MK, Lee KY, Lee JC. Intestinl nti-inflmmtory tivity of the seeds of Rphnus stivus L. in experimentl ulertive olitis models. J Ethnophrmol. 2016;179: Cody RP, Smith JK. Applied sttistis nd the SAS progrmming lnguge. 3rd ed. Englewood Cliffs, NJ: Prentie-Hll, In.; p Dunn DB. Multiple rnge nd multiple F tests. Biometris. 1955;11:1 42. Emerson DN. 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8 Lee et l. Fisheries nd Aquti Sienes (2018) 21:10 Pge 8 of 8 lone [Hliotis disus (Reeve, 1846)] sujeted to vrious stress onditions. J Shellfish Res. 2016;35: Mi K, Merer JP, Donlon J. Comprtive studies on the nutrition of two speies of lone, Hliotis tuerult L. nd Hliotis disus hnni Ino. II. Amino id omposition of lone nd six speies of mrolge with n ssessment of their nutritionl vlue. Aquulture. 1994;128: Mi K, Merer JP, Donlon J. Comprtive studies on the nutrition of speies of lone, Hliotis tuerult L. nd Hliotis disus hnni Ino. III. Responses of lone to vrious levels of dietry lipid. Aquulture. 1995;134: Mi K, Merer JP, Donlon J. Comprtive studies on the nutrition of two speies of lone, Hliotis tuerult L. nd Hliotis disus hnni Ino. IV. Optimum dietry protein level for growth. Aquulture. 1995;136: Monje H, Vin MT. The effet of ellulose on the growth nd ellulolyti tivity of lone Hliotis fulgens when uses s n ingredient in formulted rtifiil diet. J Shellfish Res. 1998;17: Myung SH, Jung W, Kim HS, Kim YE, Cho SH, Jw MS, Kim PY, Kim MK, Prk M, Kim B. Effets of dietry sustitution of fishmel with the omined dry mirolge, Nnnohloropsis oeni (NO) iomss residue nd sein on growth nd ody omposition of juvenile lone (Hliotis disus). Aqu Res. 2016;47: Prk H, Song M. Leves of Rphnus stivus L. shows nti-inflmmtory tivity in LPS-stimulted mrophges vi suppression of COX-2 nd inos expression. Prev Nutr Food Si. 2017;22:50 5. Reyes OS, Fermin AC. Terrestril lef mels or freshwter quti fern s potentil feed ingredients for frmed lone Hliotis sinin (Linneus 1758). Aqu Res. 2003;34: Shipton TA, Britz PJ. The prtil nd totl replement of fishmel with seleted plnt protein soures in diets for the South Afrin lone, Hliotis mide L. J Shellfish Res. 2001;20: Stone DAJ. Dietry rohydrte utiliztion y fish. Rev Fish Si. 2003;11: Thongrod S, Tmtin M, Chirt C, Boonyrtplin M. Lipid to rohydrte rtio in donkey s er lone (Hliotis sinin, Linne) diets. Aquulture. 2003;225: Uki N, Kemuym A, Wtne T. Requirement of essentil ftty ids in the lone Hliotis disus hnni. Bull Jpn So S Fish. 1986;52: Wilson RP. Utiliztion of dietry rohydrte y fish. Aquulture. 1994;124: Sumit your next mnusript to BioMed Centrl nd we will help you t every step: We ept pre-sumission inquiries Our seletor tool helps you to find the most relevnt journl We provide round the lok ustomer support Convenient online sumission Thorough peer review Inlusion in PuMed nd ll mjor indexing servies Mximum visiility for your reserh Sumit your mnusript t