Development of microbound diets for crab larvae

Transcription

1 Development of microound diets for cr lrve C. Zeng, P. Southgte, J. Genodep, H. Bmford nd M.H. Holme School of Mrine Biology nd Aquculture Jmes Cook University, Austrli

2 Introduction The frming of crs for hrd/soft-shell cr productions is emerging sector of quculture. Cr lrve hve their specific feeding hit/feeding mode tht differs from peneid nd fish lrve. Reserch ims t development of microound diets (MBD) for cr lrvl culture nd s vehicle for lrvl nutrition study. Adopting techniques previously used with fish lrve, MBD were leled with 14 C for evluting utiliztion of the formulted diets y lrve.

3 The Cr Species Mud cr Scyll serrt Blue swimmer cr Portunus pelgicus

4 Life Cycle of Scyll serrt (After Nouhiro Ohshiro, 1991)

5 Life Cycle of Portunus pelgicus

6 Htchery culture of mud crs Berried femle Egg & newly htched Zoe-I lrv Meglop Zoe-V lrv First juvenile cr stge (Photos: Dvid Mnn)

7 Htchery culture of lue swimmer cr Berried femle cr Eggs» Juvenile Cr Meglop Zoe-l lrve

8 Two mjor types of formulted lrvl diets Microound diets (MBD): dietry components held within gelled hydrocolloid mtrix or inder. Microencpsulted diets (MED): encpsulting solution, colloid or suspension of dietry ingredients with memrne. MBD MED

9 Why MBD ws the choice? Possily more ttrctive to cr lrve ecuse the sence of cpsule wll, llowing leching of nutrients tht my promote feeding of cr lrve. Ese of preprtion. Cn e produced within nrrow size rnge. Possily more digestile ecuse no cpsule wll to rek down prior to digestion of dietry ingredients.

10 Why 14 C-leling technique ws dopted for detecting lrvl ingestion of MBD? Difficulties in studying lrvl feeding of qutic nimls due to their smll size qutic medium logistics 14 C-lelling technique hs een used successfully in fish lrve gilthed rem Sprus urt (Tndler & Kolkovski 1991; Kolkovski et l. 1993) rrmundi Ltes clcrifer (Prtridge & Southgte 1999)

11 Production of the 14 C -leled MBD 14 C-leling of lge Nnnochloropsis sp. with N 2 [ 14 C]O 3 14 C-leling of rotifers y feeding them with 14 C -leled lge Incorporting 14 C-leled rotifers s one of the ingredients of the MBD

12 SET-UP FOR 14 C-LABELING OF ALGAE AND ROTIFERS Aertion supply tuing Croy with lge KOH trp Mgnetic stirrer

13 Composition of 14 C-leled MBD used for feeding cr lrve Ingredient Squid mel 14C-leled rotifers Fish oil Corn oil Lecithin Cholesterol Disic clcium phosphte Minerl mix Vitmin mix Binder TOTAL % Dry Weight

14 MBD mnufcture Dry Ingredients + Wet Ingredients + Binder Oven Dry Mix Mix Grind Sieve MBD Prticles

15 Cr lrve production for MBD trils Broodstock femles collected from the wild Femle mintined in n outdoor recirculting system until spwn Trnsferred erried crs to indoor tnks for egg incution nd htching Lrve mss rered in severl 300 L indoor tnks fed rotifers nd Artemi nuplii

16 Set-up for the lrvl MBD feeding trils Air Aertion tuing Plstic ottle Glss tuing PVC pipe

17 Mesurement of rdioctivity of lrvl smples fter MBD feeding trils After feeding, lrve wshed to remove uneten food nd killed y quick freezing Trnsferred to scintilltion vil nd dissolved y dding soluilizing fluid (Soluene 350, Pckrd) Heted in n oven to complete soluilistion; dd scintilltion cocktil (Hionic Flour, Pckrd) Rdioctivity of the lrvl smples mesured using et liquid scintilltion counter

18 Modifiction of 14 C-leling leling technique for pplying on cr lrve Two mjor potentil sources of errors: MBD prticles remin dhering to lrve in spite of wshing. Lrvl sorption of wter-solule 14 C leched from MBD. Experimentl design & setup: Tretments: ) Ded lrve hold with MBD prticles ) Live lrve hold in MBD lechte (MBD prticles removed fter incuted for 5h) C) Bckground smple Setup sme s susequent MBD trils Experiment lsted for 5 h 5 independent replictes (100 lrve per replicte) for ech tretment

19 Modifiction of 14 C-leling technique for cr lrve Summry of result: Cr lrve did not sor significnt mount of solule 14 C. The likely source of error comes from MBD dhering to lrve despite thorough wshing - differs from results from fish lrve. Rdioctivity (DPM) Ded lrve control set up for every susequent MBD 14 C leling tril to correct lrvl rdioctivity redings. 0 Ded lrve with MBD prticles Live lrve in 14 C lechte from MBD (contining only solule 14 C) Tretments Bckground rdioctivity

20 Lrvl Utiliztion of Experimentl MBD Questions: 1. Do cr lrve utilize MBD? 2. Is it different t different stges? 3. Does it chnge over time?

21 Mterils nd Methods Helthy lrve of desired stge rndomly selected nd removed from mss culture Lrve strved for h prior to stock culture vessels Lrve llowed 2 h cclimtiztion period in culture vessels Fed 14 C-leled MBD to lrve for desired time period After feeding, ll lrve in replicte smpled for rdioctivity mesurement nd susequent conversion to MBD utiliztion 5 replictes (5x1L culture vessel) nd ded lrve control (for correcting smple rdioctivity redings) for ech tretment Sttistics: ANOVA + Duncn s multiple rnge test

22 Utiliztion of MBD y S. serrt lrve All lrvl stges ingested MBD. Mximl utiliztion ttined within 1h of feeding for erly Zoe I-III lrve. Mximl utiliztion ttined in 2 h for Zoe IV nd V lrve. Lrvl Stge Lrvl Ingestion of MBD (µg lrv -1 ) 1 h 2 h 3 h 4 h 5 h Zoe I * 0.30 ± ± ± ± ±.03 Zoe II * 0.27 ± ± ± ± ±.07 Zoe III * 1.69 ± ± ± ± ±.79 Zoe IV ** 0.90 ± ± ± ± ±.61 Zoe V ** 5.50 ± ± ± ± ± 1.1 Meglop * 30.7 ± ± ± ± ± 2.7

23 Mximl MBD utilized y vrious stges of S. serrt lrve over 5h feeding period MBD utiliztion incresed with lrvl development. Sustntil increse occurred etween Zoe II to III, Zoe IV to V nd Zoe V to meglop. - Ingestion (µg lrv -1 ) Zoe I Zoe II Zoe III Zoe IV Zoe V Meglop Lrvl Stges

24 Utiliztion of MBD y P. pelgicus lrve All lrvl stges ingested MBD. No significnt difference in utiliztion for vrious MBD feeding period exmined (0.5, 1, 2, 3 nd 4 h) for ll instrs. Rnge of MBD utilized incresed with lrvl development. Sustntil increse t meglopl stge. Lrvl Instr MBD utilized (μg per lrv) over 0.5 to 4 h Zoe I Zoe II Zoe III Zoe IV Meglop

25 Mximl MBD utilized y P. pelgicus lrve over 4 h feeding period 9 Microgrms MBD Ingested Per lrv Zoe I Zoe II Zoe III Zoe IV Meglop Lrvl Stge

26 MBD prticle size preference nd optiml feeding rtion for cr lrve Introduction Optiml prticle size nd rtion re likely to vry with the development of cr lrve. Mterils nd Methods: Except lrve fed for I h for ll tretments, sic experimentl protocols similr to previous trils. MBD rtion equivlents to dry weight of live feeds fed to prticulr lrvl stge under stndrd condition designted s 100%.

27 Prticle Size Rnges tested -S. serrt - S. serrt Lrvl Instr Zoe I Zoe II Zoe III Zoe IV Meglop Prticle Size Rnges Tested (μm) <150; <150; ; <150; ; ; ; ; ;

28 Prticle Size Rnges tested -P. pelgicus- P. pelgicus Lrvl Instr Zoe I Zoe II Zoe III Zoe IV Meglop Prticle Size Rnges Tested (μm) ; ; ; ; ; ; ; ; ; ; ;

29 Prticle size preference - summry of results S. serrt: No significnt difference in lrvl utiliztion of MBD for the prticle sizes tested t Zoe I nd III stges. Significntly higher rte of utiliztion of lrger prticles t Zoe V nd Meglopl stge. P. pelgicus: No significnt difference in lrvl utiliztion of MBD for the prticle size rnges tested t Zoe I, II & III. Significntly higher rte of utiliztion for lrger prticles t Zoe IV & Meglopl stge. No significnt difference in meglopl utiliztion of MBD of comintion size rnges ( µm) nd lrger size rnges ( & µm).

30 Prticle size preference results S. serrt Lrvl MBD utiliztion (µg lrv -1 ) < Zoe V Zoe I Zoe III < Meglop MBD Prticle Size Rnge (µm)

31 Prticle size preference results P. pelgicus Zoe III Zoe IV Microgrms Diet Ingested Per Lrv Prticle Size Zoe III μm μm μm Prticle Size Microgrms Diet Ingested Per Lrv Prticle Size Zoe IV μm μm μm Prticle Size Microgrms Diet Ingested Per Lrv Prticle Size Meglop Meglop μm μm μm μm Prticle Size μm

32 MBD rtion tested -S. serrt Lrvl Instr % stndrd live feed rtion & MBD dry weight equivlent (mg) for ech 1 L culture replicte * 300% 200% 100% 50% 25% 15% 12.5% Zoe I Zoe III Zoe V Meglop * Tril 1: fed incresed rtion: 300, 200 nd 100% of stndrd rtion; Tril 2: fed reduced rtion: 100, 50, 25, 15, 12.5% of stndrd rtion.

33 MBD rtion tested -P. pelgicus Lrvl Instr % stndrd live feed rtion & MBD dry weight equivlent (mg) for ech 1 L culture replicte 200% 100% 50% 25% 12.5% Zoe I Zoe II Zoe III Zoe IV Meglop

34 Optiml rtion - summry of results S. serrt: No significnt difference in MBD utiliztion when lrve fed higher (200% & 300%) thn stndrd (100%) rtions for ll lrvl stges. At Zoe I, III & V, no significnt difference in MBD utiliztion when rtion reduced to 50%. However, rtion further reduced to 25% resulted in significnt decrese MBD utiliztion. At meglopl stge, no significnt difference in MBD utiliztion even rtion reduced s low s 12.5%. P. pelgicus: No significnt difference in lrvl MBD utiliztion for ll rtions tested t Zoe I, III nd V stges. Significntly higher MBD utiliztion for 200% rtion t Zoe II stge. MBD utiliztion only significntly difference etween 12.5% nd 100%, 200% levels t meglopl stge.

35 Optiml rtion results S. serrt Lrvl Ingestion (µg lrv -1 ) Zoe I 25% 50% 100% Zoe V Zoe III 25% 50% 100% Meglop 25% 50% 100% 12.50% 25% 50% 100% Rtion

36 Optiml rtion results P. pelgicus Zoe I Zoe II Microgrms Diet Ingested Per Lrv Optiml Rtion Zoe I mg 5.4 mg 2.7 mg Rtion Microgrms Diet Ingested Per Lrv Optiml Rtion Zoe II mg 7.1 mg 3.6 mg Rtion Microgrms Diet Ingested Per Lrv Optiml Rtion Meglop Meglop mg 8.2 mg 4.1 mg 2.1 mg 1.0 mg Rtion

37 Introduction Binder is likely to ffect ttrctiveness nd lrvl utiliztion of MBD. The most suitle inder my vry with the development of lrve. 14 Screening MBD inders for S. serrt lrve 14 C-leled MBD of sme dietry ingredients prepred with 5 inders: gr, lginte, crrgeenn,, geltin nd zein nd fed to vrious stges of S. serrt lrve for 2 h. Leching rtes of the five MBD were lso tested.

38 Results: Zoe I A For ll lrvl stges tested, MBD utiliztion did not differ significntly mong 5 MBD prepred with different inders. Ingestion (µg lrv -1 ) Agr Alginte Crrgeenn Geltin Zein Agr Alginte Crrgeenn Geltin Zein Zoe III Zoe V B C Agr Alginte Crrgeenn Geltin Zein Meglop Agr Alginte Crrgeenn Geltin Zein D Binders Utiliztion of MBD prepred with five inders (gr, lginte, crrgeenn, geltin nd zein) y Scyll serrt lrve

39 MBD leching rte results: Zein-ound MBD consistently showed the lowest leching mong five inders tested. Conclusion: As greter leching did not enhnce ingestion, Zein-ound MBD with miniml leching is more desirle s unnecessry leching wstes dietry nutrients nd deteriortes wter qulity. % Leching Zein Geltin Agr Alginte Crrgeenn c c 30 min 60 min 240 min Immersion Time

40 MBD feeding trils: lrvl survivl nd development Introduction Lrvl rering experiment is the ultimte test of the suitility of MBD. MBD cn e used for totlly replcement /co-feeding with live feed to reduce the costs nd relince on live feed nd for delivering specific nutrients to lrve.

41 MBD feeding tril: S. serrt meglop rered communlly Five tretments: 1) 100%MBD (dry weight equivlent to 5 Artemi/ml); 2)100% Artemi (5/ml); 3) 75%MBD+ 25%Artemi; 4) 50%MBD+ 50%Artemi nd 4) 25%MBD+ 75%Artemi. 2-dy old meglope removed from mss culture for stocking into 2 L conicl culture vessels. 5 replictes for ech tretment, 12 meglope/replicte. Lrve fed twice dily with 100% wter chnge prior to feeding. Tretment 1 Tretment 2 Tretment 3 Tretment 4 Tretment 5 12 lrve 12 lrve 12 lrve 12 lrve 12 lrve

42 Summry of results: Lrve fed 25%MBD+75%Artemi initilly hd significnt high survivl. However, finl survivl to 1 st cr not significntly different mong ll tretment. Overll survivl to 1 st cr stge very low for ll tretments due to cnnilism. % Survivl c c c c c 100 % MBD 75%MBD+25%Artemi 50%MBD+50%Artemi 25%MBD+75%Artemi 100 % Artemi 0 Dy 3 Dy 4 Dy 5 Dy 6 Dy 7 Dy 8 Dy 9 Dy10 Dy11 Dys of Culture

43 MBD feeding tril: S. serrt meglope rered individully To eliminte the effects of cnnilism, 2-dy old meglope were stocked individully into 20 x 150 ml flt ottom culture vessels. Meglope were fed either 100% MBD or 100% Artemi. Lrve fed twice dily with 100% wter chnge prior to feeding.

44 Summry of results: 90%survivl to 1 st cr stge for oth tretment of fed 100% MBD or 100% Artemi. Meglope fed MBD egn moult erlier. Dys of Culture Age of Meglope fed 100% Artemi % Survivl % Fist Stge Crs fed 100% MBD % Survivl % First Stge Crs

45 MBD feeding tril: replcement of live feed component of the sl MBD for S. serrt meglope Six tretments: 1) Rotifer mel MBD; 2) Artemi mel MBD; 3) Squid mel MBD; 4) Fish mel MBD; 5) Unfed control nd 6) Live Artemi control. Newly moulted meglope stocked individully into 250 ml flt ottom culture vessels. Ech tretment hs 15 meglope rered individully. MBD fed twice dy. 100% wter exchnge dily. Tretment 1 Tretment 2 Tretment 3 Tretment 4 Tretment 5 Tretment 6 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve 1 lrve

46 Summry of results: Survivl nd development of meglope fed squid mel nd fish mel MBD ws not inferior to those fed rotifer or Artemi mel MBD. Tretment Unfed Rotifer mel MBD Artemi mel MBD Squid mel MBD Fish mel MBD Live Artemi % survivl Men meglopl durtion (dys)

47 MBD feeding tril with S. serrt zoe III lrve Four tretments: 1) 100% MBD; 2) 50%MBD+50%Artemi; 3) 100% Artemi nd 4) unfed. Zoe II close to moult removed from mss culture in the evening prior to tril, stocked in seprted continers nd strved overnight. Select newly moulted Zoe III in the morning nd stocked to 2 L conicl vessels. 4 replictes, 25 lrve per replicte. Tretment 1 Tretment 2 Tretment 3 Tretment 4 25 lrve 25 lrve 25 lrve 25 lrve MBD fed 4 times dy. 100% wter exchnge dily.

48 Summry of results: Highest survivl to next stge (Zoe IV) with co-feeding of MBD nd Artemi t 50%:50% rtio. Zoe III le to molt successfully into next stge solely on MBD diet while ll unfed lrve died within 6 dys. Tretment 100% MBD 50% MBD + 50% Artemi 100% Artemi % survivl to next stge Men intermolt period (dys) 8.0 c c Unfed

49 Conclusion All lrvl stges of oth S. serrt nd P. pelgicus ingested MBD. Lrvl ingestion of MBD incresed sustntilly with their development. Lter lrve prefer lrger MBD for oth S. serrt nd P. pelgicus. Optiml rtion re species-specific nd lrvl stge-specific. Different inders ppers to hve reltive little effects on lrvl utiliztion of MBD for S. serrt. For S. serrt meglope, Artemi cn e fully replced with MBD. Through nutrition mnipultion, sustntil improvement in lrvl survivl my ecome chievle for the stge tht often suffered high mortlity. For zoel lrve, co-feeding of MBD ppers to e more fesile option.

50 Future directions Exploiting techniques for ssessment of lrvl digestion nd nutrition ssimiltion. Further ssessment of lrvl survivl nd development under vrious feeding schemes (e.g vrious co-feeding schemes for different lrvl stges, feeding frequency etc). Optimizing the physicl properties of MBD (uoyncy, extrusion methods etc). Use MBD s tool for cr lrvl nutritionl requirement study.

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