POTENTIAL USE OF LOCAL PHYTOPLANKTON STRAINS FOR REARING THREE DIFFERENT BIOTYPES OF THE GENUS Brachionus plicatilis (Müller, 1786) CRYPTIC SPECIES COMPLEX G. Seretidou*, E. Foudoulaki, V. Kostopoulou, A. Economou-Amilli and I. Tzovenis Laboratory of Ecology and Taxonomy, Faculty of Biology, University of Athens, Zografou 15784, Athens, Greece itzoveni@biol.uoa.gr Laboratory of Fish pathology & Nutrition, Institute of Aquaculture & Genetics, Hellenic Center of Marine Research, Ag. Kosmas Ellininkon, Greece
In brief Mediterranean aquaculture still relies on live feeds during the first feeding of marine fish larvae Live food = phytoplankton + rotifers, Artemia etc. Greek hatcheries use imported phytoplanktic and rotifer strains The purpose of this research was to investigate the imported phytoplankton replacement with local phytoplanktic strains isolated from Greek coastal lagoons 12 local Tetraselmis sp. strains were fed to 3 rotifer Brachionus plicatilis complex strains Assessment: productivity and enrichment in essential fatty acids compared with 2 reference strains 2
Intensive rearing of sea bream/bass Common feeding plan Sparus aurata 3 rd -17 th day phytoplankton+ rotifers 17 th -24 th day phytoplankton+ rotifers + Artemia 34 th 45 th day Artemia Common feeding plan Dicentrarchus labrax 3 rd -24 th day phytoplankton+ rotifers + Artemia 28 th 45 th day Artemia + artificial diet 3
EFAs in hatchery food chain Microalgae ARA-EPA-DHA Rotifers (ARA-EPA-DHA) Further enrichment Larvae 4
Experimental design Rotifers: 3 strains(triplicates, 2 ml, 1 amictic female/ml, 25, 22 ) Brachionus plicatilis L3 Brachionus Tiscar SM 2 Brachionus Almenara Phytoplankton: 16 strains(log phase, in excess, 25, 2 ) Tetraselmis suecica * (incertae sedis) Nannochloropsis oculata* (N. gaditana) Selection of strains for FAME (filtration in GF/C, lyophilization, GC-FID) Reference strains Optimum yield strains (*) reference strain 5
Phytoplankton strains Tetraselmis spp 6
Phytoplankton reference strains Nannochloropsis oculata Chlorella sp 7
Rotifer strains B. plicatilis L3 B. Almenara B. Tiscar SM 2 8
Methodology-Culture Phytoplankton Cultivation photoperiod 12:12 h L: D light intensity 1 klux batch cultures Temperature 21 ± 1 C, Harvesting at the exponential phase Salinity 25 Rotifer rearing Initial density 1 ind/ml Culture volumes 2 ml Temperature(21±1 C) Photoperiod 12:12 h L:D Light intensity 1 klux Growth measurements: stereoscope, meander (amictic females, eggs) in daily basis for 25 days/triplicate The total sampling volume didn t overpass the 1 % of the starting culture volume 9
Methodology rotifers Cultures 1
Methodology assessment Specific growth rate: 16 SGR = (lnn n lnn n 1 )/ t 14 Maximum production: 12 1 N tmax = rotifer /ml 8 6 Egg ratio: 4 ER = eggs/amictic females 2 Productivity: -2 productivity (Pmax) = rotifers/ml/day Productivity expressed in terms of biovolume: V tmax = μm 3 /ml/day L3-CHUII B y =,8388x -,8179 R² =,9716 5 1 15 2 Sampling on pre-combusted GF/C filters, freeze-drying Fatty acids were determined after direct transesterification (Lepage & Roy, 1986) 11
Results - SGR 1,6 1,4 Mean Brachionus plicatilis L3 Mean±SD 1,6 1,4 Mean Brachionus "Tiscar" Mean±SD 1,2 1,2 SGR max 1,,8,6 1,,8,6,4,4,2,2,, SGR max SGR max 1,6 1,4 1,2 1,,8,6,4,2 Mean Brachionus "Almenara" Mean±SD, B. Tiscar SM 2 + N. occulata = the highest sgr (1,17 day -1 ) among all the mass cultures. 12
Results - N tmax 18 16 14 Brachionus plicatilis L3 Mean Mean±SD 18 16 14 Brachionus "Tiscar" Mean Mean±SD Nt max 12 1 8 6 4 2 Nt max 12 1 8 6 4 2 Nt max 18 16 14 12 1 8 6 4 2 Brachionus "Almenara" Β. Tiscar SM 2 Mean Mean±SD 14 > Mes 2, R 1-1, R 9-2 > 1 = N. oculata B. plicatilis L3 6 > T. chuii, T3-1, > 4 > reference strains B. Almenara T. chuii +reference strains > local strains 13
Results - P tmax 3 25 Mean Brachionus plicatilis L3 Mean±SD 3 25 Brachionus sp. "Tiscar" Mean Mean±SD rotifers/ml/day 2 15 1 5 bc c a bc bc c c c b bc rotifers/ml/day 2 15 1 5 rotifers/ml/day 3 25 2 15 1 5 Mean Brachionus sp. "Almenara" Mean±SD B. Tiscar SM 2 + Mes 2 maximum P (18 rotifer/ml /day) B. Tiscar SM 2 best performance in total 14
Results - biov tmax productivity μm 3 /ml/day 4E8 3,5E8 3E8 2,5E8 2E8 1,5E8 1E8 Brachionus plicatilis L3 Mean Mean±SD Outliers Extremes productivity μm 3 /ml/day 4E8 3,5E8 3E8 2,5E8 2E8 1,5E8 1E8 Brachionus sp. "Tiscar" Mean Mean±SD Outliers Extremes 5E7 5E7 productivity μm 3 /ml/day 4E8 3,5E8 3E8 2,5E8 2E8 1,5E8 1E8 5E7 Brachionus sp. "Almenara" Mean Mean±SD Outliers Extremes B. Tiscar SM 2 Max T.chuii - B. plicatilis L3 (2,21 1 8 μm 3 ml -1 ) B. Tiscar SM 2, 5 strains > 1 8 μm 3 ml -1 day -1 B. plicatilis L3 3 strains> 1 8 μm 3 ml -1 day -1 B. Almenara All < 1 8 μm 3 ml -1 day -1 15
Results- biov tmax Current effect: F(3, 15)=5,1192, p=, Vertical bars denote,95 confidence intervals 1,8E9 1,6E9 1,4E9 Rotifer spp B.plicatilis L3 Rotifer spp B.tiscar SM 2 Rotifer spp B.almenara 1,2E9 1E9 8E8 6E8 4E8 2E8 Vt max μm 3 /ml ANOVA 2-way rotifer x phyto interaction B. plicatilis L3 max biomass with T. chuii, T 3-1 & B. tiscar SM2 max biomass with Mes 2, R 1-2, R 9-2 B. almenara max with T. chuii, T. suesica maxima of B. plicatilis L3 & Tiscar not different, Almenara low 16
Results-Fatty acids Brachionus plicatilis L3 FAME starvation N. oculata T. suecsica V2-3 T3-1 T. chuii % avg std avg std avg std avg std avg std avg std 16: 2,93 1,36 24,5,64 17,94 4,43 19,98,75 16,76,1 14,95,99 16:1n-9 15,27,65 23,41,12 3,42 2,1 4,9,35 3,23,41 2,84,38 18:1n-9 19,48 1,81 7,61,15 11,8 2,32 1,62,19 9,37 2,98 9,5 3,38 18:2n-6 5,72,63 3,44,16 4,97,32 6,72 1,27 5,11 1,17 5,87,22 18:3n-3 3,22 1,76 1,47,28 13,19 5,41 9,17 1,64 11,55 1,15 1,39,32 2:4n-6 2,14,33 3,59,7,62,88 1,42,18 1,24,14 2,43,38 2:4n-3 1,4 1,52,, 2,71 3,83 8,41,97 7,95,54 7,94 2,27 2:5n-3 4,91,85 14,96,11 6,55,9 6,19 1,78 6,49,46 6,41,99 TFA mg/g 64,3 11,45 53,12 16,79 22,48 7,13 26,76 7,67 27,49 8,19 3,35 17,49 SFA 27,8 1,85 35,21,81 25,86 6,85 27,11,46 23,2,99 21,15,16 MUFA 51,91 3,28 38,59,5 33,7 6,85 29,81 4,17 29,42 1,48 33,18 3,87 n-9 35,54 3,16 34,8,19 21,57 6,52 2,95,49 19,17 1,81 2,31 6,82 n-6 8,44,9 7,92,62 7,7,36 8,87 1,94 7,81,8 1,5,75 n-3 11,7 5,26 17,37,17 33,33 13,13 33,79 7,16 38,84 2,98 34,73 2,88 EPA+DHA 4,91,85 14,96,11 6,55,9 6,19 1,78 6,49,46 6,41,99 Results-Fatty acids Brachionus Tiscar SM 2 FAME starvation Mes2 N. oculata T. suecica R1-2 T3-1 % avg std avg std avg std avg std avg std avg std 16: 33,33 4,26 1,31 14,58 22,81,65 23,9 1,4 22,58 4,62 2,86,2 16:1n-9 14,75,8 6,77,36 15,4,58 3,35,27 3,63 2,23 3,82,57 18:1n-9 16,4 3,94 14,4 9,74 5,32,33 15,24 3,16 15,76 5,65 1,6,84 18:2n-6 3,29 1,18 2,82 3,99 4,11,15 4,9,71 6,25,1 4,46 1,6 18:3n-3 3, 3,49 14,34 7,48 6,87,16 17,12 3,52 12,5 4,76 14,76 1,51 2:4n-6 2,58,3,34,49 3,6,1,7,1,92,55,96,27 2:5n-3 4,63 1,29 3,28 1,62 1,8 2,54 3,58,56 4,79 1,37 5,1,34 22:1n-9,54 1,89 4,68 2,54 14,16,31 3,48 4,45 4,47 2,8 6,62,7 TFA mg/g 49,84 12,82 28,89 13,37 41,43 9,69 62,85 25,98 23,82 11,14 36,47 1,72 SFA 4,84 3,36 32,55 4,9 32,9,4 31,44 6,29 29,98 5,8 27,1,19 MUFA 39,97 3,67 38,11 9,44 44,65,79 34,48,4 37,29 6,13 34,96 1,58 n-9 31,85 4,3 29,61 11,39 36,32,64 23,12 1,24 24,88 5,54 22,32 1,33 n-6 8,59 1,69 3,51 4,97 7,97,8 5,76,58 7,6 1,18 6,49 1,63 n-3 1,8 6,18 22,5 12,87 14,18,52 28,32 6,8 24,95 1,51 31,22,18 EPA+DHA 4,63, 3,28 1,62 1,8 2,54 3,58,56 4,79 1,37 5,1,34 17
Conclusions Rotifer strains showed a preference for food species and differentiated their performance accordingly The rotifer strains with the best performance were the B. "Tiscar" SM 2 and B. plicatilis L3 The phytoplankton strain which promotes growth, reproduction and enrichment in both rotifer biotypes was Tetraselmis T 3.1 B. "Tiscar" SM 2 can be reared efficiently with the local strains T 3-1 and R 1-2 while B. plicatilis L3 with V 2-3 and T 3-1 To obtain dense rotifer populations the suggested phytoplankton strains are the Mes 2, R 1-2, R 9-2 for B. 'Tiscar' SM 2 and the T 3-1 for B. plicatilis L3 & Mes 17 did not promote rotifer growth and they unfit as food for these strains. B. "Almenara" may be suitable for rearing freshwater and brackish fish species as in high salinity it failed to produce dense populations The rotifers reflect the fatty acid profile of their diet The EPA proportions improved greatly in L3 with the enrichment of microalgae especially with Nannochloropsis EPA precursors like 2:4ω3 were significantly elevated when L3 was fed with the other microalgae For producing dense populations of rotifers suitable local strains were identified For enrichment a further investigation on strains with suitable profiles including DHA must be carried out 18
thanks