132 ΒΘΟΥΗΜΘΚΗ ΤΣΑΗ ΚΑΘ ΠΕΡΘΕΚΣΘΚΟΣΗΣΑ Ε ΛΘΠΑΡΑ ΟΞΕΑ ΣΟΤ ΣΡΟΥΟΖΩΟΤ Brachionus Nevada Ε ΥΕΗ ΜΕ ΣΗ ΤΣΑΗ ΣΗ ΣΡΟΦΗ Κωστοπούλοσ Β. 1, Μήλιοσ Ε. 2 *, Βερροιόποσλος Γ. 3 1 Ειιεληθό Κέληξν Θαιαζζίσλ Εξεπλώλ, Θλζηηηνύην Υδαηνθαιιηεξγεηώλ, Ηξάθιεην Κξήηε, 71003 2 Γεσπνληθό Παλεπηζηήκην Αζελώλ, Τκήκα Επηζηήκεο Ζσηθήο Παξαγσγήο θαη Υδαηνθαιιηεξγεηώλ, Εξγαζηήξην Εθεξκνζκέλεο Υδξνβηνινγίαο, Θεξά Οδόο 75, Βνηαληθόο, Αζήλα, 11855 3 Εζληθό & Καπνδηζηξηαθό Παλεπηζηήκην Αζελώλ, Τκήκα Βηνινγίαο, Τνκέαο Ζσνινγίαο θαη Θαιάζζηαο Βηνινγίαο, Παλεπηζηεκηνύπνιε, Αζήλα, 15784 Περίληψη Έλα ζηέιερνο ηνπ ηξνρόδσνπ Brachionus Nevada, ην νπνίν αλήθεη ζην ζύκπιεγκα εηδώλ ηνπ B. plicatilis, εθηξάθεθε κε ην δηαθνπηόκελν ζύζηεκα (batch) ρξεζηκνπνηώληαο δύν ζηηεξέζηα: καγηά Saccharomyces cerevisiae (ρεηξηζκόο 1) θαη Culture Selco (ρεηξηζκόο 2). Οη δύν ηξνθέο δηέθεξαλ σο πξνο ηε βηνρεκηθή ζύζηαζε θαη ηελ πεξηεθηηθόηεηα ζε ιηπαξά νμέα. Η καγηά πεξηείρε ζηαηηζηηθά πςειόηεξα επίπεδα (% ηνπ μεξνύ βάξνπο) πξσηετλώλ, ελώ ην Culture Selco πςειόηεξα επίπεδα DNA, RNA, ιίπνπο θαη n-3 ιηπαξώλ νμέσλ. Οη παξαπάλσ ζηαηηζηηθέο δηαθνξέο αληρλεύζεθαλ θαη ζηα ηξνρόδσα ησλ αληίζηνηρσλ ρεηξηζκώλ. Η πεξηεθηηθόηεηα ησλ ηξνρόδσσλ ζε DNA, RNA θαη ιηπίδηα παξνπζίαζε ρξνληθή δηαθνξνπνίεζε, ζε αληίζεζε κε ηελ πεξηεθηηθόηεηα ζε πξσηεΐλεο θαη ιηπαξά νμέα πνπ παξέκεηλε ζηαζεξή θαη ζηνπο δύν ρεηξηζκνύο. Σηελ πεξίπησζε ηνπ ρεηξηζκνύ 1, κεηαβνιέο παξαηεξήζεθαλ εληόο ηεο εκέξαο, ελώ ζηνλ ρεηξηζκό 2 κεηαμύ ησλ εκεξώλ. Τν παξαπάλσ απνδόζεθε ζηε ζύζηαζε ηεο ηξνθήο θαη ηηο κεηαβνιέο πνπ επέθεξε ζηε δνκή ηνπ πιεζπζκνύ. Η αλάιπζε ησλ ιηπαξώλ νμέσλ θαηέδεημε όηη ηα ηξνρόδσα δηαζέηνπλ κεραληζκνύο, κε ηνπο νπνίνπο ζπλζέηνπλ νξηζκέλα ιηπαξά νμέα απαξαίηεηα γηα ηελ αλάπηπμή ηνπο. Σύκθσλα κε ηα απνηειέζκαηα, ηα ηξνρόδσα πνπ εθηξέθνληαη κε δηαθνξεηηθά ζηηεξέζηα, απαηηνύλ έλα δηαθνξεηηθό πξσηόθνιιν εκπινπηηζκνύ, πξνθεηκέλνπ λα απνθηήζνπλ έλα βηνρεκηθό πξνθίι θαηάιιειν γηα ηε δηαηξνθή ησλ ηρζπδίσλ. Λέξεις κλειδιά: καδηθή εθηροθή, ητζσογελλεηηθοί ζηαζκοί, ηροτόδωα. *Σπγγξαθέαο επηθνηλσλίαο: Μήιηνπ Ε. (elenmi@aua.gr) BIOCHEMICAL COMPOSITION AND FATTY ACID CONTENT IN THE ROTIFER brachionus Nevada IN RELATION TO FEED COMPOSITION Kostopoulou V. 1, Miliou H. 2 *, Verriopoulos G. 3 1 Hellenic Center for Marine Research, Institute of Aquaculture, Gournes Pediados, Heraklion, Crete, 71003, Greece 2 Agricultural University of Athens, Department of Animal Science and Aquaculture, Laboratory of Applied Hydrobiology, Iera Odos 75, Votanikos, Athens, 11855, Greece 3 National & Kapodistrian University of Athens, School of Sceince, Faculty of Biology, Department of Zoology and Marine Biology, Panepistimioupoli, Athens 15784, Greece Abstract A strain of the rotifer Brachionus Nevada, which belongs to the B. plicatilis species complex, was batch cultured using two feeding regimes: baker s yeast Saccharomyces cerevisiae (treatment 1) and Culture Selco (treatment 2). The two feeds differed in biochemical composition and fatty acid content. Baker s yeast had significantly higher protein levels (% of dry weight), whereas Culture Selco showed significantly higher levels of DNA, RNA, lipids and n-3 HUFAs. The abovementioned differences were also detected in the corresponding rotifers. Rotifer DNA, RNA and lipid content showed temporal variation, in contrast to protein and fatty acid content, which remained constant in both treatments. The temporal variation was diurnal in treatment 1 and daily in treatment 2. This was attributed to feed composition and concomitant changes it brought to population structure. Fatty acid analysis showed that
133 rotifers are able to synthesize certain fatty acids that are necessary for their development. According to this study, rotifers fed on different regimes require a different enrichment protocol in order to reach a satisfactory biochemical profile for fish larvae. Keywords: mass culture, hatcheries, rotifers. *Corresponding author: Miliou Helen (elenmi@aua.gr) 1. Introduction The rotifer Brachionus plicatilis is commonly used in aquaculture as first live feed of fish larvae. In this study, the biochemical composition and fatty acid content of a strain of B. Nevada, one of the lineages grouped with B. plicatilis s.s., is presented. 2. Materials and methods Sampling took place in an aquaculture farm in Greece (Octapus S.A.). Rotifers were batch-cultured in tanks (2.5 m 3 capacity) for 4 days. All cultures were performed at 25 o C and 35 g L -1 salinity under constant illumination and aeration. A preexperimental culture of B. Nevada was maintained under the abovementioned conditions using Tetraselmis suecica as food. For the main culture phase, two routinely used feeding regimes were chosen: baker s yeast Saccharomyces cerevisiae and Culture Selco (CS) (INVE N.V., Belgium). These two diets differ in their biochemical composition (Kostopoulou et al. 2009) and fatty acid profile (Table 1). Rotifer samples were collected twice daily at 8:00 and 16:00 for biochemical assays and fatty acid analyses (500 ml). The sampling of rotifers started on day 0 at 16:00 (0 h) and ended on day 4 at 8:00 (88 h). The intermediate samples were taken at 16 h, 24 h, 40 h, 48 h, 64 h and 72 h. A more analytical description of the experimental design can be found in Kostopoulou et al. (2006) as Experiment 1. DNA, RNA, protein and lipid analyses were assayed according to the method of Holland and Hannant (1973). Fatty acid methyl esters were separated by gas liquid chromatography (GLC) on a Perkin Elmer Gas Chromatograph. The data of the fatty acid content of the two diets had to be square root transformed. Comparisons between and within treatments, and significance of regressions were tested by ANOVA (STATGRAPHICS, v.5). Untransformed mean values ± standard error (S.E.) are presented. 3. Results DNA, RNA and lipid content was significantly higher in treatment 2 (0.20±0.02, 3.16±0.16 and 11.78±0.52% of dry weight, respectively) compared to treatment 1 (0.15±0.01, 2.62±0.13 and 9.25±0.48% of dry weight, respectively). Protein content was significantly higher in treatment 1 than in treatment 2 (55.65±0.99% and 52.23±0.76% of dry weight, respectively). Regarding the temporal variation of the biochemical parameters, DNA and RNA showed a significant diurnal variation in treatment 1, whereas in treatment 2 significantly higher values were observed within the first 40 hours of sampling, decreasing thereafter to reach higher values again towards the end of the sampling period (88 hrs). Lipids showed the opposite picture. Proteins showed no significant temporal variation. DNA and RNA were positively related (P<0.001) to ovigerous females (abundance %) in both treatments. Lipids were positively related (P<0.001) to immature rotifers in treatment 1.
134 Rotifer fatty acid composition (% total fatty acid) was influenced by the diet in the case of: 16:4n-1, 20:5n-3 (EPA), 22:2n-6, 22:5n-3, 22:6n-3 (DHA) and 18:2n-6. The content of these fatty acids was statistically higher in CS and CS-fed rotifers, except for the last one, which was higher in yeast and yeast-fed rotifers (Tables 1 and 2). There were other fatty acids whose difference in feed did not influence rotifer composition, which was similar in the two treatments (14:0, 16:0, 16:1, 18:0, 18:1, 18:4n-3, 20:1n-9, 20:4n-6 (ARA), 22:1n-11, 21:5n-3 and 24:1n-9). Another category was that of fatty acids that were absent or present in lower amounts in feeds than in rotifers (14:1, 15:0, 15:1, 16:2n-4, 17:0, 18:3n-3, 18:3n-6, 18:4n-1, 20:0, 20:2n-6, 20:3n-6, 20:3n-3, 20:4n- 3, 22:1n-9, 22:4n-6, 22:5n-6). Table 1. Selected 1 fatty acid composition (% of total fatty acids) of the two feeding regimes (Diet 1: yeast; Diet 2: Culture Selco ) used in rotifer cultures. Mean ± standard error are presented. Food Diet 1 Diet 2 P Fatty acids n = 3 n = 3 14:0 0.23 ± 0.02 1.47 ± 0.31 ** 16:0+16:1 34.09 ± 0.94 19.02 ± 2.95 * 16:4n-1-0.33 ± 0.12 ** 18:0+18:1 39.53 ± 0.65 28.69 ± 2.04 * 18:2n-6 23.17 ± 0.85 6.62 ± 0.91 *** 18:3n-3-1.20 ± 0.17 *** 18:4n-3-1.48 ± 0.14 *** 20:1n-9 0.35 ± 0.02 1.82 ± 0.38 ** 20:4n-6 (ARA) - 0.97 ± 0.27 *** 20:5n-3 (EPA) 0.12 ± 0.04 11.21 ± 0.91 *** 22:1n-11 0.58 ± 0.07 3.15 ± 1.12 * 22:2n-6 0.21 ± 0.03 0.95 ± 0.13 ** 21:5n-3 0.01 ± 0.01 1.42 ± 0.51 ** 22:5n-3 0.17 ± 0.04 3.08 ± 0.92 ** 22:6n-3 (DHA) 0.94 ± 0.23 13.42 ± 0.34 *** 24:1n-9 0.27 ± 0.05 2.92 ± 1.26 * n-3 1.24 ± 0.31 31.94 ± 1.51 *** n-6 23.38 ± 0.88 9.18 ± 0.57 *** n-3/n-6 0.05 ± 0.01 3.50 ± 0.26 *** DHA/EPA 7.68 ± 0.60 1.21 ± 0.08 *** *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: non significant - : not detectable 1 Fatty acids of less than 0.8 (% of total fatty acids), showing no significant differences between treatments, are not listed. 4. Discussion The increased lipid levels in rotifers fed on CS (treatment 2) can be attributed to the increased lipid level of this diet. The higher lipid levels of these rotifers were constant throughout the experimental period, in contrast to the lipid-poor population of yeast-fed rotifers. Such temporal decrease in rotifer lipid content is due to the lower lipid input of the food (yeast). Rotifers supply their offspring with the necessary energy, which decreases thereafter as it is not compensated by the food, as shown by the positive relation of lipid content to immature rotifers in treatment 1. The significantly
135 higher levels of nucleic acids in CS-fed rotifers could be attributed to the increased proportion of ovigerous females present in these populations (Kostopoulou et al. 2006). The correlation of nucleic acids with ovigerous females could reflect the active embryonic development during that phase of the cycle. Table 2. Selected 1 fatty acid composition (% of total fatty acids) of rotifers fed yeast (Treatment 1) or Culture Selco (Treatment 2). Initial refers to the pre-experimental rotifer population fed on phytoplankton. Mean ± standard error are presented. Rotifers Initial Treatment 1 Treatment 2 P Fatty acids n = 4 n = 14 n = 14 16:4n-1 0.09 ± 0.04 a 0.08 ± 0.02 a 0.23 ± 0.02 b *** 18:2n-6 12.63 ± 0.74 b 11.65 ± 0.67 b 9.91 ± 0.51 a * 18:3n-3 14.36 ± 1.96 b 14.40 ± 0.96 b 9.43 ± 0.58 a *** 20:2n-6 0.29 ± 0.03 ab 0.39 ± 0.03 b 0.24 ± 0.04 a * 20:3n-6 0.33 ± 0.01 ab 0.38 ± 0.02 b 0.27 ± 0.03 a ** 20:3n-3 0.17 ± 0.04 ab 0.27 ± 0.03 b 0.07 ± 0.03 a *** 20:4n-3 1.69 ± 0.19 ab 2.16 ± 0.10 b 1.57 ± 0.13 a ** 20:5n-3 (EPA) 3.58 ± 0.33 a 2.90 ± 0.28 a 5.83 ± 0.26 b *** 22:2n-6 0.59 ± 0.08 a 0.85 ± 0.08 a 1.08 ± 0.09 b * 22:5n-3 0.72 ± 0.06 b 0.44 ± 0.05 a 1.15 ± 0.03 c *** 22:6n-3 (DHA) 2.25 ± 0.33 a 2.10 ± 0.29 a 4.52 ± 0.46 b *** n-3 23.42 ± 1.97 23.03 ± 0.97 23.20 ± 1.21 ns n-6 15.08 ± 0.76 14.77 ± 0.72 12.88 ± 0.45 ns n-3/n-6 1.55 ± 0.08 1.62 ± 0.13 1.84 ± 0.14 ns DHA/EPA 0.63 ± 0.08 0.85 ± 0.18 0.76 ± 0.06 ns *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: non significant 1 Fatty acids showing no significant differences between treatments are not listed. The fatty acid profile of rotifers was influenced by the two feeds in a varied way. The saturated and monounsaturated fatty acids were similar in the rotifers of the two treatments, although the respective diets showed significant differences. Such stable rotifer composition, regardless of feed, can be linked to a need for these acids during growth and development. The fatty acids that have received most attention are 20:5n-3, 20:4n-6 and 22:6n3, because of their important role in fish larval growth and development. In this study, rotifers were enriched in those two fatty acids when cultured with CS, in contrast to yeast, which seems to be nutritionally deficient. The higher content of HUFAs in CS-fed rotifers, together with their higher lipid content can be linked to their population structure. Based on Kostopoulou et al. (2006), CS- fed rotifers were characterized by increased proportion of ovigerous females. A link appears to exist between lipid content and reproductive output. The results of the fatty acid analysis point towards activation of specific pathways in yeast-fed rotifers. This is evidenced by the presence of a number of fatty acids in yeast-fed rotifers, which were detected in lower levels or not at all in the corresponding feed (yeast) and which, in some cases, even exceeded the levels of CSfed rotifers. These fatty acids act as precursor molecules in HUFAs synthesis (e.g. 18:3n-3, 18:4n-3, 20:3n-3 and 20:4n-3 for biosynthesis of 20:5-n and 22:6n-3 or 20:2n- 6 and 20:3n-6 for biosynthesis of 20:4n-6). When the feed is deficient in HUFAs
136 (yeast), the rotifers activate specific pathways that lead to the production of those HUFAs. In the opposite case (CS), activation does not need to occur. Overall, CS-fed rotifers showed a better fatty acid profile than yeast-fed ones along with a constant lipid content throughout the batch culture and an increased proportion of ovigerous females. References Holland D.L., Hannant P.J. (1973) Addendum to a micro-analytical scheme for the biochemical analysis of marine invertebrate larvae. Journal of the Marine Biological Association of the UK, 53: 833-836. Kostopoulou V., Miliou H., Katis G., Verriopoulos G. (2006) Changes in the population structure of the lineage Nevada belonging to the Brachionus plicatilis species complex, batch-cultured under different feeding regimes. Aquaculture International, 14: 451-466. Kostopoulou V., Miliou H., Verriopoulos G. (2009) Morphometric changes in a strain of the lineage Nevada belonging to the Brachionus plicatilis (Rotifera) complex. Aquaculture Research, 40: 938-949.