C. Lobo 1 M.V. Martín 2 X. Moreno-Ventas 3 S.T. Tapia-Paniagua 4 C. Rodríguez 5 M.A. Moriñigo 4 I. García de la Banda 1. Abstract 1 INTRODUCTION

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1 Received: 13 September 2016 Accepted: 21 March 2017 DOI: /anu ORIGINAL ARTICLE Shewanella putrefaciens Pdp11 probiotic supplementation as enhancer of Artemia n- 3 HUFA contents and growth performance in Senegalese sole larviculture C. Lobo 1 M.V. Martín 2 X. Moreno-Ventas 3 S.T. Tapia-Paniagua 4 C. Rodríguez 5 M.A. Moriñigo 4 I. García de la Banda 1 1 Oceanographic Center of Santander, Spanish Institute of Oceanography, Santander, Spain 2 Oceanographic Center of Tenerife, Spanish Institute of Oceanography, Tenerife, Spain 3 Ecological Area of Water and Environmental Sciences and Technics, University of Cantabria, Santander, Spain 4 Department of Microbiology, Faculty of Sciences, University of Málaga, Málaga, Spain 5 Departamento de Biología Animal, Edafología y Geología, Facultad de Ciencias, Universidad de La Laguna, La Laguna, Spain Correspondence Inés García de la Banda, Oceanographic Center of Santander, Spanish Institute of Oceanography, Santander, Spain. ines.gbanda@st.ieo.es Funding information Spanish National Development Plan for Sole farming (JACUMAR), Grant/Award Number: LENGUADO III. REDESOLEA.; Ministry of Science and Technology, Grant/Award Number: AGL C03-02 Abstract Adequate enrichment of live prey like Artemia, naturally deficient of essential highly unsaturated fatty acids (HUFA), such as docosahexaenoic acid (22:6n- 3, DHA), is critical for the rapidly developing tissues, survival, normal development and production of good- quality fingerlings. The aim of the study was to evaluate the effects of a pulse (10 30 dah) of Shewanella putrefaciens Pdp11 (2.5*10 7 cfu/ml) using Artemia metanauplii as live vector, on its proper lipid profiles and resultant Solea senegalensis body composition and performance. Probiotic administration significantly increased total lipids and specifically n- 3 HUFA levels in Pdp11- enriched Artemia. The live prey lipid modulation was also reflected in the total lipid contents and fatty acid profiles of Pdp11 sole specimens, which achieved a higher growth performance. A fatty acid multivariate principal component analysis confirmed a neat separation of two groups corresponding to Control and probiotic fish for each age sampled (23, 56, 87 and 119 dah). In addition, a further SIMPER analysis highlighted that the Pdp11 Artemia effect on sole lipid profile was different for each fatty acid and was gradually diluted with age. Results suggest an ability of Pdp11 strain to produce n- 3 HUFA as an effective tool for fish marine larviculture optimization. KEYWORDS Artemia, fatty acid profile, larviculture, probiotic, Shewanella, Solea senegalensis 1 INTRODUCTION Senegalese sole (Solea senegalensis, Kaup 1858) is considered a species with high interest for aquaculture diversification in the south of Europe whose production has shown an important increase in recent years (APROMAR, 2015). Significant research has been recently performed to improve quality production of sole juveniles (Morais et al., 2016). A key factor to improve S. senegalensis fry quality should connect nutrient requirements to feeding protocols at first larval stages. In this way, S. senegalensis larvae are usually cultured with live prey in co- feeding with inert diets, where rotifers are frequently enriched with Isochrysis galbana, due to its interesting contents of DHA (Li, Xu, Chen, Zhou, & Yan, 2014) and different commercial products are usually utilized for Artemia metanaupli enrichment (Boglino et al., 2012). Live prey is also of great influence at first feeding as the composition of gut larval microbiota is closely related to that present in the diet (Suzer et al., 2008). In this context, probiotic application is increasing in marine aquaculture based on the beneficial effects obtained in promoting nutritive specimen condition and immunity (Newaj- Fyzul, Al- Harbi, & Austin, 2014). Moreover, the UE has recently approved the first probiotic (Pediococcus acidilactici MA 18/5M; Bactocell, Lallemand Animal Nutrition, Wisconsin, USA) to be used for salmonids and shrimps (E1712/4d.1712). Most utilized probiotics are lactic acid bacteria (LAB) of terrestrial origin although there is increasing interest to isolate probiotics from aquatic environment (Lauzon et al., 2010). In John Wiley & Sons Ltd wileyonlinelibrary.com/journal/anu Aquaculture Nutrition. 2018;24:

2 LOBO et al. 549 this way, Shewanella putrefaciens Pdp11, isolated from the skin mucus of healthy cultured gilthead seabream Sparus aurata (Chabrillón, Rico, Balebona, & Moriñigo, 2005), has demonstrated in vivo beneficial effects on S. senegalensis ongrowing (Tapia- Paniagua et al., 2012). These authors also reported that the probiotic strain was able to colonize sole gut, to modulate intestinal microbiota and to enhance growth performance, enzymatic digestive activities and body composition on S. senegalensis juveniles. Regarding to S. putrefaciens Pdp11 application in sole larviculture, a modulation of gut microbiota and an improvement of growth and body composition were obtained after bioencapsulation in Artemia metanauplii (10 86 days after hatching- dah- ) by Lobo, Moreno- Ventas et al. (2014). Furthermore, a shorter pulse of S. putrefaciens Pdp11 (10 30 dah), using Artemia metanauplii as live vector, enhanced S. senegalensis larval and fry growth and total protein and lipid contents in relation to a digestive enzyme stimulation promoted by a suitable intestinal microbiota modulation (Lobo, Tapia- Paniagua et al., 2014). In spite of these findings, little attention has been focused in studying S. putrefaciens Pdp11 effects on fish body lipid profiles. This aspect is particularly interesting for Senegalese sole larviculture to ensure that the daily intake of energetic and structural essential fatty acids is adequate to achieve larval requirements for rapidly developing tissues such as brain and eye (Padrós, Villalta, Gisbert, & Estevez, 2011). Probiotic supply has been reported as enhancing growth performance in relation to an increase in lipid body composition in haddock Melanogrammus aeglefinus (Plante et al., 2007) and in the cyprinid Catla catla (Bandyopadhyay & Das Mohapatra, 2009). Similarly, Tapia- Paniagua et al. (2014) have recently demonstrated a positive correlation between the digestive microbiota of sole juveniles receiving S. putrefaciens Pdp11 and some fatty acid levels in liver (arachidonic acid 20:4n- 6, ARA, linolenic 18:3n- 3, ALA- and linoleic 18:2n- 6, LA- ) and muscle (ARA and eicosapentaenoic acid 20:5n- 3, EPA). In this context, lipid and fatty acid fortification in rotifers and Artemia related to some microorganisms supplied to live prey has been reported by some authors (Jeeja, Joseph, & Raj, 2011; Sorgeloos, Dhert, & Candreva, 2001) and the lipid composition of several invertebrate species is known to be influenced by their microbial environment (Plante et al., 2007). In fact, some strains of fish intestinal microbiota have been described as possible PUFA producers (Ringo, Sinclair, Birkbeck, & Barbour, 1992; Yano, Nakayama, & Yoshida, 1997). For this reason, the aim of the present study was to evaluate the effect of S. putrefaciens Pdp11 (10 30 dah), using Artemia metanauplii as live vector on Artemia proper fatty acid profiles as well as on S. senegalensis larval and fry growth performance and lipid and fatty acid contents. 2 MATERIAL AND METHODS 2.1 Microorganisms Shewanella putrefaciens Pdp11 was grown in 5 ml of tryptone soya broth (Oxoid Ltd., Basingstoke, UK) supplemented with 15 g/kg NaCl (TSBs) for 18 hr at 22 C, with continuous shaking. Aliquots of 0.1 ml of the culture were spread onto plates of tryptone soya agar (Difco, Sparks, USA), supplemented with 15 g/kg NaCl (TSAs) and incubated daily at 22 C. Bacterial suspensions were prepared daily by scraping the cells from the plates and suspending them in sterile phosphatebuffered saline (PBS, ph 7.2). After 10 min of shaking, the number of bacterial cells per ml was measured at 600 nm using a Hach DR/2500 Laboratory Spectrophotometer (Loveland, Colorado, USA). Probiotic cells were supplied in a dose ( cfu/ml) to 12 hr Origreen (Skretting, Burgos, Spain) pre- enriched Artemia metanauplii. This dose has been previously reported as suitable by Lobo, Moreno- Ventas et al. (2014) and is in the range of other probiotics used in larviculture (Dias et al. 2011). No probiotic cells were supplied to the pre- enriched Artemia metanauplii used as Control. 2.2 Rearing conditions Embryos were obtained from natural spawning of captive S. senegalensis brood stock kept at the Spanish Institute of Oceanography (Santander, Spain). Embryos were incubated at 19.0 ± 0.5 C in 70- L cylinder- conical incubating tanks with gentle aeration and a continuous water flow of 0.5 L/min. Newly hatched larvae (40 individuals per L) were distributed into 250- L circular polyester tanks by triplicate, with a constant aeration and sea water renewal. Temperature varied between 17.8 ± 0.8 C (1 56 day after hatching, dah), 18.9 ± 0.4 C (57 85 dah) and 19.6 ± 1.1 C ( dah). Salinity was 35.4 g/l throughout the trial. Illumination (1,000 lux on surface water) was provided with Sylvania mini- lynx fast start lamps. It was continuous until 10 dah, and a 12:12 L:D cycle was established until 21 dah. Afterwards, postlarvae were reared in semidarkness (<20 lux on surface). Continuous water inflow was maintained to provide suitable oxygen and nitrite sea water levels for larval and postlarval culture (Lund et al., 2008; Parra & Yúfera, 1999). Once the larvae metamorphosed and became benthic, the experimental tanks were emptied, and survival checked. Fish were then randomly redistributed relative to both dietary assayed treatments and stocked at a density of 3,000 individuals per m 2 (3 replicates per dietary treatment). Due to the high stocking density, on 41 dah, postlarvae from each replicate were taken out to adjust density to 2,500 individuals per m 2. Finally, after the weaning period (on 87 dah), the tanks were emptied and fish were randomly redistributed and stocked by triplicate at a density of 1,250 individuals per m 2 until the end of the feeding trial. The feeding regime, based on Cañavate and Fernández- Díaz (1999), is shown in Table 1. From 2 to 9 dah, I. galbana-enriched rotifers were added to the tanks twice a day to maintain a rotifer density of 20 individuals per ml. Microalgae (Nannochloropsis gaditana, cells per ml and I. galbana, cells per ml) were also supplied to larval rearing tanks during this period to ensure a good rotifer quality. From 10 to 86 dah, co- feeding was carried out with Artemia and four sequential commercial pellets (diets A, B, C and D) in both experimental diets. In this way, Artemia nauplii (AF strain INVE Aquaculture, Ghent, Belgium) was supplied from 10 to 12 dah and Artemia metanauplii (EG strain INVE Aquaculture, Ghent, Belgium) thereafter. Artemia metanauplii ( per ml) were enriched with Origreen (fresh protein 430 g/kg, total lipids 300 g/kg, n- 3 HUFA 105 g/kg,

3 550 LOBO et al. dah Rotifer (ind/ml) Artemia (ind/ml) Morning Afternoon Morning Afternoon Inert diet (eight times per day) TABLE 1 Feeding regime of S. senegalensis larvae, postlarvae and fry as a function of age (dah) a 3 a Diet A a 16 a Diet B a 20 a Diet C a 10 a Diet D a Mean value of Artemia metanauplii added twice on each period of the day. DHA/EPA 5/1; Skretting, Burgos, Spain) in 45- L incubators provided with sea water (23 C). The enrichment was performed in two steps, an initial pre- enrichment (12 hr) and a second one (3 9 hr) prior to its administration to sole larvae. Then, Artemia metanauplii was supplied to the larval tanks four times a day, whereas dry feed was added eight times a day. Weaning started at 57 dah with diets C and D, and from 86 dah, postlarvae were fed exclusively with dry feed (diet E). The amount of inert feed was gradually increased from 57 dah (39 g/m 2, 69.2% of total feed) to 86 dah (117 g/m 2 ), while Artemia doses were progressively reduced from 20 metanauplii per ml. At the end of the trial, fry were fed at 11.3% of total tank biomass. Proximate composition, total content of fatty acids (FA) and fatty acid profile (g/kg of dry weight) of experimental diets supplied along larval and fry culture are shown in Table Dietary treatments Two live feeding regimes were compared by triplicate: Pdp11 and Control. Pdp11 regime consisted of S. putrefaciens Pdp11 bacterial strain bioencapsulated in Origreen pre- enriched Artemia, meanwhile Control regime was based on enriched Artemia without any probiotic supply. Probiotic diet was supplied to sole (10 30 dah), being all fish groups further fed with Control Artemia metanauplii (30 86 dah). S. putrefaciens Pdp11 concentration in Artemia was in the range of 10 4 cfu in Artemia per metanauplii along the bioencapsulation period (3 9 hr), accounting at least for 50% of total bacteria. These values are similar to those reported by other authors (Villamil, Figueras, Planas, & Novoa, 2003). No mortalities of S. putrefaciens Pdp11 nor of Artemia metanauplii (90% 92% survival) were registered during the bioencapsulation period. Artemia from both incubators (45 L) were maintained at 23 C, being collected and rinsed with 1- μm filtered sea water for 5 min prior to its administration to sole to eliminate oily residues and to reduce bacterial load. 2.4 Growth and survival For growth studies, thirty specimens from each replicate were weekly and randomly sampled after moderate anaesthesia. Specimens were rinsed with distilled water, put onto preweighed glass fibre filters and dried at 60 C for 48 hr, in order to obtain fish dry weight. Survival was checked counting dead specimens on a daily basis along the experiment. 2.5 Analysis of body composition Three samples from Control and S. putrefaciens Pdp11 Artemia metanauplii were collected, washed several times with distilled water and immediately frozen and stored at 80 C till analysis. For the study of body composition, samples were randomly collected from each rearing tank on four sampling dates: 23 (n = 5), 56 (n = 6), 87 (n = 6) and 119 (n = 5). Each of those samples was a pool of larvae with six to 75 individuals per sample according to larval size: 75 (23 dah), 15 (56 dah) and six (87 and 119 dah). Fish samples were washed several times with distilled water prior to be frozen at 80 C. All samples were subjected to total soluble protein, lipid and fatty acid content analysis. Total soluble protein was determined following the method of Bradford (Bradford, 1976). Total lipid content was assessed by extraction with chloroform methanol 2:1 as described by Blight and Dyer (1959) modified by Fernández- Reiriz et al. (1989) and gravimetrically determined after centrifugation. After transesterification of total lipids (Lepage & Roy, 1986), fatty acid profiles were analysed by gas chromatography fitted with a flame ionization detector at 260 C (8700 PerkinElmer, Beaconsfield, UK). A capillary fused- silica column SPTM (30 m length, 0.25 mm internal diameter and 0.2 μm film thicknesses) was used for fatty acid methyl ester (FAME) separation. After holding at 140 C for 5 min, the temperature was raised to 177 C (1 C per min), to 180 C (0.50 C per min) and to 210 C (2 C per min). Finally, the injector was maintained at 275 C for 7 min. Injection procedures were made in a split ratio mode (10:1), and the area of the internal standard 19:0 was utilized for quantification. 2.6 Statistical analysis All data of growth and biochemical composition are presented as means ± SEM. The specific growth rates (SGR %) of the two experimental groups were calculated as follows, SGR % = ln (Wf) ln (Wi)/t * 100, where Wf and Wi are the mean value of dry weight at the end and at the beginning of each analysed period, respectively, and t stands for the total of days of such period. After testing for normality (Kolmogorov Smirnov test), a one- way ANOVA was performed to detect significant differences in growth parameters and lipid composition between treatments (p <.05). In those

4 LOBO et al. 551 TABLE 2 Proximate composition, total content of fatty acids (FA) and main fatty acid profiles (g/kg dry weight) of inert experimental diets. (Mean; n = 3) Inert diet Diet A Diet B Diet C Diet D Diet E Dry matter Protein Lipids Total content FA Fatty acids 14: : : : : SAFA : :1 a : :1 b :1 c :1 b MUFA :2n : :2n :3n :4n :4n :4n :5n :5n :6n PUFA n n n n- 3 HUFA n- 3/n DHA/EPA EPA/ARA DHA/EPA/ARA MUFA/n- 3 HUFA a Include n- 5, n- 7, n- 9 and n- 11 isomers. b include n- 7 and n- 9 isomers. c include n- 7, n- 9 and n- 11 isomers. cases where significant differences were found, a Tukey or Games Howell post hoc comparison tests (at α =.05) were applied according to variance homogeneity (Levene s test). The effect of probiotic supplementation (S. putrefaciens Pdp11 Artemia versus. Control Artemia) and the effect of fish age (23, 56, 87 and 119 dah) on fatty acid composition of specimens were also assessed by a two- way ANOVA (Zar, 1999). If variances were heterogeneous, a Welch s test was applied (Zar, 1999). Fish fatty acid values were chemometrically analysed by principal components analysis (PCA). Factor scores were subsequently analysed

5 552 LOBO et al. by a one- way ANOVA followed by Tukey multiple comparison tests (Zar, 1999). All the above- mentioned analyses were performed using the SPSS v19 software (SPSS Inc, Chicago, USA). Finally, a SIMPER (similarity percentage procedure analysis) was executed to assess the contribution of each fatty acid to the dissimilarity detected in Control and Pdp11 specimens along the experimental period (23, 56, 87 and 119 dah). Based on the analysis of Bray Curtis (dis) similarity, the strongly contributing matrices from the detected fatty acids were quantified and ranked according to Clarke (1993). In this way, the average contribution of each fatty acid of Control fish is compared in turn with each one of SpPdp11 specimens. SIMPER analysis allowed to quantify the average contribution, by fatty acids, to the measure of dissimilarity between treatments (Clarke & Warwick, 1994). This statistical analysis was performed using PAST software (Hammer, Harper, & Ryan, 2001). 3 RESULTS Growth performance was enhanced by Pdp11- enriched Artemia metanauplii supply as it is shown in Table 3. In this context, Pdp11 fish growth (DW) resulted significantly higher (p <.05) at the end of metamorphosis (23 dah). Moreover, specific growth rates (SGR%) were also significantly higher for Pdp11 postmetamorphic larvae until weaning, TABLE 3 Dry weight (mg), specific growth rate (SGR%) and survival rate (%) of S. senegalensis fed Control Artemia and S. putrefaciens Pdp11- enriched Artemia (10 30 dah). (Mean ± SEM; n = 30) dah Control Pdp Dry weight 0.04 ± ± 0.00 Dry weight 1.51 ± 0.01 b 1.66 ± 0.02 a SGR (%) 17.3 ± 0.1 b 17.7 ± 0.2 a Survival (%) 97.5 ± ± 0.9 Dry weight 37.6 ± 0.9 b 50.2 ± 1.0 a SGR (%) 9.74 ± 0.24 b ± 0.06 a Survival (%) 97.4 ± ± 1.0 Dry weight 78.7 ± ± 2.5 SGR (%) 2.42 ± 0.08 a 1.58 ± 0.12 b 119 Survival (%) 97.0 ± ± 1.0 Dry weight ± 9.5 b ± 10.7 a SGR (%) 3.16 ± ± 0.14 Survival (%) 89.8 ± ± 1.3 Different letters in the same line denote significant differences (p <.05) among treatments. where a marked decrease was observed thereafter in both experimental groups, especially in probiotic fish, along this period (56 87 dah). Once weaning had overcome (119 dah), a slight growth recovery was reached by both experimental groups, although Pdp11 fish growth continued significantly higher than Control (Table 3). In addition, final survival rates were similar for both fish groups (88.1% 89.8%). Shewanella putrefaciens Pdp11 and Artemia metanauplii proximate composition and fatty acid profile are shown in Table 4. As reflected, S. putrefaciens Pdp11 strain contains moderate amount of lipids with more than 90% of fatty acids being saturates and monoenes being the odd- chain fatty acids 15:0 and 17:1 among the most abundant ones. Among PUFA, 16:2n- 4 and particularly EPA seem to also have some relevance in these microorganisms. As a result, probiotic bioencapsulation further displayed differences in Control and S. putrefaciens Pdp11 Artemia metanauplii. Specifically, lipid contents as well as total fatty acid contents (g/kg of dry weight) were higher (p <.05) in S. putrefaciens Pdp11- enriched Artemia metanauplii compared to Control ( versus g/kg and versus g/kg) (Table 4). S. putrefaciens Pdp11 significantly increased total saturated fatty acids (SAFA) in Pdp11- enriched Artemia metanauplii, and specifically, 16:0 and 18:0 were higher (p <.05) compared to Control. A tendency for higher contents of 18:1n- 9 and 20:1n- 9 was also registered in S. putrefaciens Pdp11- enriched Artemia metanauplii in comparison with Control, not being significantly for total monounsaturated fatty acids (MUFA). Remarkable is that 18:3n- 3 and 18:4n- 3 decreased but total n- 3 HUFA increased in S. putrefaciens Pdp11- enriched Artemia metanauplii because of the higher contents of 20:5n- 3 and 22:6n- 3. Accordingly, the relative ratios of n- 3/n- 6 and MUFA/n- 3 HUFA decreased and those of EPA/ ARA increased in S. putrefaciens Pdp11- enriched Artemia metanauplii with respect to Control. Evolution of Control and Pdp11 fish body composition in terms of protein and lipid contents along the experimental period is shown in Table 5. Several differences in lipid and protein levels were detected between both experimental groups throughout fish development. For instance, at 23 dah, Pdp11 postmetamorphic larvae contained more lipids than Control, unlike the protein contents, which were lower at this age. From 23 to 87 dah, it was observed a marked decrease in lipid content in both postmetamorphic larval groups coinciding with a phase of high growth although at 87 dah, again Pdp11 specimens contained more lipids than those of Control. Finally, lipid levels were restored in both experimental groups at 119 dah to reach similar levels as those present before weaning (56 dah). It is also noticeable that Pdp11 fish displayed an earlier decrease of lipids ( to g/kg DW) (Table 5) coinciding with an also earlier or faster growth (Table 3). As a contrary, total lipid drop off in Control specimens lasted longer (until 87 dah) and correlated well with a delayed growth displayed by these specimens. Concerning to fatty acid profile (g/kg of dry weight) of S. senegalensis specimens at 23, 56, 87 and 119 dah fed Control and S. putrefaciens Pdp11- enriched Artemia metanauplii, results are shown in Table 5. A positive correlation between the most abundant fatty acids of S. putrefaciens Pdp11- enriched Artemia and those present in Pdp11

6 LOBO et al. 553 TABLE 4 Proximate composition, total content of fatty acids (FA) and fatty acid profile (g/kg dry weight) of S. putrefaciens Pdp11 at 23 C, Control Artemia and S. putrefaciens Pdp11- enriched Artemia (Mean ± SEM; n = 3) S. putrefaciens Pdp11 Control Artemia S. putrefaciens Pdp11 Artemia Dry matter ± ± ± 9.81 Protein ± ± ± Lipids ± ± 1.86 b ± 1.93 a Total content FA ± ± 0.55 b ± 0.65 a Fatty acids 14: ± ± ± : ± ± 0.02 a 0.37 ± 0.02 b 16: ± ± 0.03 b ± 0.36 a 17: ± ± ± : ± ± 0.07 b 5.99 ± 0.04 a SAFA 6.27 ± ± 0.40 b ± 0.43 a 14: ± ± ± :1 a 6.24 ± ± ± : ± ± ± :1 b 0.80 ± ± 0.17 b ± 0.01 a 20:1 c 0.03 ± ± 0.02 b 1.47 ± 0.08 a 22:1 c 0.05 ± ± ± 0.10 MUFA ± ± ± :2n ± ± ± : ± ± ± :2n ± ± 0.11 b ± 0.12 a 18:3n ± ± 0.48 a ± 0.18 b 18:4n ± ± 0.04 a 1.46 ± 0.04 b 20:4n ± ± ± :4n ± ± 0.00 a 0.54 ± 0.01 b 20:5n ± ± 0.00 b 5.76 ± 0.03 a 22:5n ± ± ± :6n ± ± 0.15 b ± 0.37 a PUFA 1.70 ± ± 0.34 b ± 0.08 a n ± ± ± 0.12 n ± ± 0.20 b ± 0.14 a n ± ± 0.27 b ± 0.03 a n- 3 HUFA 0.99 ± ± 0.13 b ± 0.35 a n- 3/n ± ± 0.03 a 2.26 ± 0.03 b DHA/EPA 0.08 ± ± ± 0.08 EPA/ARA ± ± 0.05 b 2.01 ± 0.00 a DHA/EPA/ARA 5.04 ± ± ± 0.03 MUFA/n- 3 HUFA ± ± 0.07 a 1.58 ± 0.04 b Different letters in the same line denote significant difference (p <.05) among treatments. a Include n- 5, n- 7, n- 9 and n- 11 isomers. b Include n- 7 and n- 9 isomers. c Include n- 7, n- 9 and n- 11 isomers. postmetamorphic larvae was observed at 23 dah. In fact, EPA and DHA contents tended to be higher, although not at a significant level, in 23- day- old Pdp11 postmetamorphic larvae than in Control fish, as they also were in S. putrefaciens Pdp11 live prey. Nevertheless, in terms of fatty acid composition, the effect of probiotic- enriched Artemia metanauplii was probably getting diluted by inert diet incorporation since weaning on. At this stage, both experimental groups presented a similar fatty acid profile which reflected

7 554 LOBO et al. TABLE 5 Proximal composition, total content and main fatty acid (FA) profiles (g/kg of dry weight) of 23, 56, 87 and 119 dah S. senegalensis larvae fed Control and S. putrefaciens Pdp11- enriched diets. (Mean ± SEM; n = 5 for 23 and 87 dah or n = 6 for 56 and 119 dah) 23 dah 56 dah 87 dah 119 dah Control Pdp11 Control Pdp11 Control Pdp11 Control Pdp11 Dry matter ± ± ± ± ± ± ± ± 4.06 Protein ± 1.77 aa ± 0.85 ba ± 2.65 aab ± 3.71 ba ± 8.01 AB ± AB ± B ± 5.94 B Lipids ± 0.42 ba ± 0.20 aa ± 1.59 B ± 2.12 B ± 2.98 bc ± 1.14 ab ± 3.00 B ± 1.91 B Total content FA ± 0.43 ba ± 0.38 aa ± 0.66 B ± 1.25 B ± 1.65 bb ± 3.64 aab ± 1.98 A ± 3.92 AB Fatty acids 14: ± 0.01 C 0.81 ± 0.00 C 0.93 ± 0.01 BC 0.94 ± 0.02 B 1.72 ± 0.12 ab 3.36 ± 0.10 ba 4.52 ± 0.18 A 3.75 ± 0.20 A 16: ± 0.02 bb ± 0.03 ab 8.24 ± 0.08 B 9.35 ± 0.16 B 7.99 ± 0.32 bb ± 0.34 aa ± 0.53 A ± 0.55 A 17: ± 0.00 A 0.92 ± 0.00 A 0.70 ± 0.01 bb 0.86 ± 0.02 ab 0.52 ± 0.02 bc 0.74 ± 0.03 ac 0.80 ± 0.02 aab 0.69 ± 0.01 bc 18: ± 0.01 ba 6.24 ± 0.01 aa 4.14 ± 0.04 B 4.65 ± 0.08 B 3.05 ± 0.05 bc 3.87 ± 0.11 ac 4.03 ± 0.09 B 3.76 ± 0.10 C SAFA ± 0.04 bb ± 0.04 ab ± 0.13 B ± 0.29 BC ± 0.49 bb ± 0.59 aa ± 0.84 A ± 0.84 A 16:1 a 3.96 ± 0.02 BC 3.95 ± 0.03 B 4.14 ± 0.05 B 4.56 ± 0.08 B 2.91 ± 0.18 bc 5.77 ± 0.16 aa 7.13 ± 0.29 A 6.03 ± 0.31 AB 18:1 b ± 0.04 ba ± 0.05 aa ± 0.11 B ± 0.31 AB 8.53 ± 0.26 bc ± 0.40 ab ± 0.48 AB ± 0.58 B 20:1 c 1.11 ± 0.01 BC 1.12 ± 0.01 B 0.76 ± 0.02 C 0.96 ± 0.04 B 1.21 ± 0.07 bb 2.12 ± 0.08 aa 1.93 ± 0.09 A 1.70 ± 0.10 A 22:1 c 0.12 ± 0.00 B 0.18 ± 0.00 B 0.24 ± 0.02 B 0.17 ± 0.01 B 1.13 ± 0.08 ba 2.03 ± 0.08 A 1.73 ± 0.07 A 1.47 ± 0.09 A MUFA ± bab ± 0.08 a ± 0.17 bb ± 0.40 a ± 0.61 bc ± 0.74 a ± 0.97 A ± :2n ± 0.00 B 0.61 ± 0.00 AB 0.48 ± 0.01 B 0.53 ± 0.01 B 0.59 ± 0.02 B 0.74 ± 0.04 AB 0.98 ± 0.03 A 0.90 ± 0.04 A 18:2n ± 0.04 A 8.20 ± 0.03 A 3.25 ± 0.13 B 3.79 ± 0.19 B 3.99 ± 0.15 B 5.80 ± 0.41 AB 7.21 ± 0.15 A 5.98 ± 0.40 AB 18:3n ± 0.15 A ± 0.10 A 4.04 ± 0.27 B 4.99 ± 0.35 B 2.35 ± 0.10 B 3.85 ± 0.49 B 2.35 ± 0.30 B 2.35 ± 0.34 B 18:4n ± 0.03 A 2.03 ± 0.02 A 0.45 ± 0.03 C 0.65 ± 0.05 B 0.58 ± 0.03 BC 0.85 ± 0.12 B 1.05 ± 0.04 B 0.98 ± 0.11 B 20:4n ± 0.03 A 3.29 ± 0.02 A 1.19 ± 0.04 B 1.41 ± 0.06 B 1.40 ± 0.05 B 1.61 ± 0.16 B 1.51 ± 0.03 B 1.37 ± 0.07 B 20:4n ± 0.01 A 1.01 ± 0.01 A 0.22 ± 0.02 C 0.37 ± 0.03 B 0.31 ± 0.01 BC 0.49 ± 0.04 B 0.57 ± 0.02 B 0.47 ± 0.05 B 20:5n ± 0.03 B 2.26 ± 0.02 A 0.53 ± 0.03 C 0.63 ± 0.03 B 2.16 ± 0.11 B 2.64 ± 0.34 AB 5.14 ± 0.24 A 4.51 ± 0.44 AB 22:5n ± 0.02 B 1.73 ± 0.02 A 0.45 ± 0.03 C 0.51 ± 0.03 B 1.93 ± 0.09 B 2.35 ± 0.30 AB 4.27 ± 0.16 A 3.82 ± 0.31 A 22:6n ± 0.10 B 7.94 ± 0.10 A 1.83 ± 0.10 C 2.28 ± 0.17 B 6.04 ± 0.27 B 5.87 ± 0.71 AB ± 0.43 A ± 0.80 A PUFA ± 0.40 A ± 0.30 A ± 0.63 B ± 0.85 C ± 0.70 B ± 2.56 BC ± 0.78 A ± 2.42 AB n ± 0.33 A ± 0.25 A 7.53 ± 0.47 B 9.43 ± 0.62 C ± 0.54 B ± 1.96 BC ± 0.74 A ± 1.92 A n ± 0.07 A ± 0.05 A 4.44 ± 0.17 B 5.20 ± 0.24 B 5.39 ± 0.17 B 7.42 ± 0.56 B 8.72 ± 0.16 A 7.35 ± 0.46 B n ± ± 0.06 A ± 0.11 b ± 0.27 aab 8.84 ± 0.33 b ± 0.42 aab ± ± 0.64 B n- 3 HUFA 9.78 ± 0.15 B ± 0.14 A 3.03 ± 0.18 C 3.79 ± 0.26 B ± 0.43 B ± 1.37 A ± 0.83 A ± 1.59 A n- 3/n ± 0.02 A 2.57 ± 0.01 A 1.63 ± 0.05 B 1.78 ± 0.05 B 2.48 ± 0.06 A 2.04 ± 0.12 AB 2.79 ± 0.08 A 3.04 ± 0.08 AB (Continues)

8 LOBO et al. 555 TABLE 5 (Continued) 23 dah 56 dah 87 dah 119 dah Control Pdp11 Control Pdp11 Control Pdp11 Control Pdp11 DHA/EPA 3.22 ± 0.01 A 3.49 ± 0.01 A ± 0.06 A 3.51 ± 0.09 A 2.85 ± 0.14 A 2.27 ± 0.06 B 2.12 ± 0.02 B 2.42 ± 0.07 B EPA/ARA 0.63 ± 0.00 C 0.68 ± 0.00 C 0.43 ± 0.01 C 0.45 ± 0.01 D 1.56 ± 0.07 B 1.56 ± 0.08 B 3.38 ± 0.11 A 3.19 ± 0.16 A DHA/EPA/ARA 1.23 ± 0.01 C 1.07 ± 0.01 B 3.09 ± 0.13 A 2.62 ± 0.11 A 2.04 ± 0.09 AB 1.68 ± 0.20 AB 1.41 ± 0.04 C 1.84 ± 0.12 AB MUFA/n- 3 HUFA 2.69 ± 0.05 B 2.09 ± 0.02 B 7.93 ± 0.46 A 7.36 ± 0.45 A 1.38 ± 0.06 B 2.73 ± 0.36 B 1.28 ± 0.09 B 1.24 ± 0.11 B Different lower case letters denote significant differences between treatments at the same day (p <.05). Different capital letters denote significant differences between days in the same treatment (p <.05). a Include n- 5, n- 7, n- 9 and n- 11 isomers. b Include n- 7 and n- 9 isomers. c Include n- 7, n- 9 and n- 11 isomers. the influence of the common inert diet over each experimental Artemia metanauplii composition. A very significant decrease in n- 3 HUFA and, specifically DHA and EPA at 56 dah, was observed for both fish groups although it was sharper for the Control group. The contents of these fatty acids were interestingly recovered at the end of weaning (87 dah) and continued to increase until 119 dah. Noteworthy, total fatty acid contents were considerably lower in Control compared to Pdp11 fish group at 87 dah and, specifically, 16:0, 18:0, 16:1 and 18:1 (16:1n- 7 and 18:1n- 9 being 15% 17% and 44% 47% of MUFA, respectively). Afterwards, at 119 dah, the differences in fatty acid composition between the two fish groups were vanished, being remarkable the dramatic increment of EPA/ARA ratio for both experimental fish groups at 119 dah and which particularly reflects the high EPA levels supplied by the inert diet (see Table 2). The results of the two- way ANOVA used to test for significance of the effects of S. putrefaciens Pdp11- enriched diet and stages of development on fatty acid composition of S. senegalensis are shown in Table 6. A significant effect of age for all fatty acids and no effect of S. putrefaciens Pdp11 were found except for n- 3 HUFA fatty acids. However, interactions between these two factors were observed for 14:0, 18:0, 22:1n- 9, n- 3 HUFA and DHA/EPA. The results of the PCA used to examine the multivariate structure of the data sets of fatty acids for 23, 56, 87 and 119 dah sole specimens are shown in Figure 1. The two components of PCA accounted for 80% and 96% of variation of this data set, although more than 50% of variation was explained by principal component 1 (PC1) for all stages of development (Figure 1a, c, e, g). The PC1 component clearly separated fatty acids that predominate in Pdp11 specimens (on the right) from those characteristic of Control (on the left) reflecting the differences between Control and S. putrefaciens Pdp11- enriched Artemia metanauplii. The principal component 2 (PC2) accounted for a smaller percentage of the variability and showed a high positive weighting for SAFA and MUFA (above the zero line) against a high negative weighting for PUFA (below the zero line). Two clusters were significantly separated (p <.05) in the first factor score (Figure 1b, d, f, h) corresponding to Control and probiotic specimens for all stages of development (23, 56, 87 and 119 dah). In order to break down the contribution of each fatty acid to the observed dissimilarity between S. putrefaciens Pdp11 and Control specimens, a dissimilarity percentage analysis (SIMPER) was carried out and results were summarized in Table 7. Most important fatty acids in creating the observed pattern of dissimilarity attributed to probiotic supply were identified as n- 3 PUFA (22:6n- 3 and 18:3- n3) besides 18:2n- 6, 18:1n- 9 and 16:0. Differences in their contribution dissimilarity varied along the experimental period (23, 56, 87 and 119 dah). In this context, differences in DHA were more acute at the end of metamorphosis (18.38%) and the end of weaning (12.89%). On the contrary, the most intense differences linked to 18:1n- 9 were observed along weaning (17.78% 14.15%). In addition, differences in 18:3n- 3 were sharper at the start of weaning (21.32% 17.55%), while those related to 16:0 were observed since the end of weaning (18.76% 16.80%). Moreover, differences linked to 18:2n- 6 were

9 556 LOBO et al. TABLE 6 Significance of probiotic and age effect on fatty acid (FA) larval contents (g/kg of tissue) after a two- way ANOVA. (n = 5 for 23 and 87 dah or n = 6 for 56 and 119 dah) Probiotic Age Interaction F value Sig F value Sig F value Sig Total content 2.68 ns * 6.76 * Fatty acid 14: ns * 3.06 * 16: ns * 1.41 ns 17: ns * 1.97 ns 18: ns * 3.08 * SAFA 0.96 ns * 1.32 ns 16:1n ns * 2.07 ns 16:1n ns * 5.02 * 16:1n ns * 0.99 ns 18:1n ns * 1.13 ns 18:1n ns * 1.86 ns 20:1n ns * 2.55 ns 20:1n ns 0.51 ns 1.32 ns 22:1n ns * 3.28 * MUFA 1.42 ns * 1.96 ns 16:2n ns * 3.43 * 18:2n ns * 1.65 ns 18:3n ns * 0.21 ns 18:4n ns * 1.11 ns 20:4n ns * 2.00 ns 20:4n ns * 2.16 ns 20:5n ns * 1.71 ns 22:5n ns * 1.10 ns 22:6n ns * 2.50 ns PUFA 1.39 ns * 1.56 ns n ns * 1.44 ns n ns * 1.73 ns n ns * 1.62 ns n- 3 HUFA 6.04 * * 4.25 * n- 3/n ns 6.64 * 1.03 ns DHA/EPA 0.01 ns * 3.27 * EPA/ARA 1.05 ns * 1.64 ns DHA/EPA/ ARA MUFA/n- 3 HUFA 2.03 ns 8.20 * 0.92 ns 1.87 ns * 1.73 ns * denotes significant differences (p <.05). ns denotes non-significant differences (p <.05). registered along all the experimental period (9.83% 6.62%). In this context, SIMPER analysis highlighted how the Pdp11 Artemia metanauplii effect on sole lipid profile was different for each fatty acid and was gradually diluted with inert diet supply and subsequent larval age increment. 4 DISCUSSION Senegalese sole production of high- quality juveniles is still a bottleneck, although recent advances in management and technical improvements have brought relevant progress in productivity (Morais et al., 2016). Proper nutrition at first feeding is a key factor for successful larval and juvenile rearing. In this regard, probiotic supplementation at early stages of fish development may contribute to improve larval growth performance by a beneficial effect at digestive level (Tinh, Dierckens, Sorgeloos, & Bossier, 2008; Zacarías- Soto, Lazo, & Viana, 2011) linked to gut microbiota modulation and enhancement of enzymatic activities (Mandiki et al., 2011; Sun, Yang, Huang, Ye, & Chun- Xiao, 2013). In this way, several strains may provide a supplementary source of nutrients and microbial activity in the host digestive tract (Balcázar et al., 2006; Ramírez & Dixon, 2003). For instance, it has been reported that Bacteroides and Clostridium sp. may contribute to the host s nutrition, especially by supplying fatty acids and vitamins (Sakata, 1990). In addition, some microorganisms such as Agrobacterium sp., Pseudomonas sp., Brevibacterium sp., Microbacterium sp. and Staphylococcus sp. may also enrich nutritional processes (Ringo, Strom, & Tabachek, 1995). This study shows that the administration of S. putrefaciens Pdp11- enriched Artemia metanauplii (10 30 dah) to pre-, pro- and postmetamorphic S. senegalensis larvae provides an advantageous growth when compared to Control fish group. The present results confirmed those obtained by Lobo, Moreno- Ventas et al. (2014) with a longer pulse (10 89 dah) of the same probiotic strain and agree with those obtained for several fish species such as Dover sole (Avella et al., 2011), turbot (Dagá et al., 2013), gilthead sea bream (Avella et al., 2010) and sea bass (Mahdhi, Kamoun, Messina, Santulli, & Bakhrouf, 2012), after different probiotics supply. Moreover, S. putrefaciens Pdp11 had been shown not only to enhance growth but also to activate digestive enzyme activity and to improve intestinal epithelium integrity, modulating body composition administrated in Senegalese sole juveniles (Tapia- Paniagua et al., 2012). The lipid composition of S. putrefaciens Pdp11- enriched Artemia metanauplii revealed important and unexpected differences compared to Control Artemia metanauplii. Probiotic administration seems to significantly increase total lipid and total fatty acid contents, and specifically, n- 3 HUFA levels in S. putrefaciens Pdp11- enriched Artemia metanauplii, which could be discussed from several points of view. S. putrefaciens Pdp11 may modulate the absorptive capacity of Artemia metanauplii enhancing total lipid contents as previously reported by Lobo, Moreno- Ventas et al. (2014), and specifically, the Artemia n- 3 HUFA enrichment capability through a more efficient incorporation of these fatty acids from the enrichment commercial product. A stimulation of digestive enzyme activity after the probiotic use could occur as it has already been described for fish larvae (Suzer et al., 2008). Accordingly, Semova et al. (2012) showed the microbiota ability to induce accumulation of enterocyte lipid drops and to increase the absorption of dietary fatty acids in zebrafish Danio rerio. The possible ability of Artemia metanauplii for elongation/desaturation of the C18 fatty acid precursors even in the presence of

10 LOBO et al. 557 (a) (b) (c) (d) (e) (f) (g) (h) FIGURE 1 Component plots (a, c, e, g) and factor score plots (b, d, f, h) of the PCA for fatty acid composition of 23, 56, 87 and 119 dah S. senegalensis larvae fed Control or S. putrefaciens Pdp11- enriched treatment. The axes of component plots (a, c, e, g) show the first two principal components, with the fraction of explained variance in the parentheses. Circles stand for different clusters in the factor score 1 S. putrefaciens Pdp11 should be ruled out since studies conducted by Navarro et al. (1999) when enriching Artemia metanauplii with radiolabelled fatty acid ethyl esters showed that this capacity is very limited. Moreover, recent studies supplying C 14 DHA to Artemia metanauplii described a lower DHA incorporation compared to other fatty acids (Reis et al., 2013), possibly due to its natural tendency to retroconvert DHA into EPA (Bell, McEvoy, Estévez, Shields, & Sargent, 2003; Navarro et al., 1999). However, it is known that some species of bacteria possess the metabolic capacity to synthesize their own EPA, DHA and ARA (Bell & Tocher, 2009; Bergé & Barnathan, 2005). In this context, bacteria from Flavobacterium Cytophaga group and Vibrionaceae as Pseudomonas and even Shewanella have been reported as PUFA producers (Ivanova et al., 2004; Ringo et al., 1992). Specifically, Shewanella has been described as a very interesting genus in relation to PUFA production (Nikoopour & Griffiths, 2008; Satomi, Oikawa, & Yano, 2003), where S. putrefaciens, S. báltica and S. algae present different PUFA contents linked to environmental factors (Amiri- Jami, Wang, Kakuda, & Griffiths, 2006; Nichols, Olley, Garda,

11 558 LOBO et al. TABLE 7 Dissimilarity index (%) in fatty acid content (g/kg of tissue) of 23, 56, 87 and 119 dah S.senegalensis specimens fed Control and Pdp11 Artemia after SIMPER analysis a. (n = 5 for 23 and 87 dah or n = 6 for 56 and 119 dah) Fatty acid 23 dah 56 dah 87 dah 119 dah 14: : : : :1n :1n :1n :1n :1n :1n :1n :1n :2n :3n :4n :4n :4n :5n :5n :6n a Only showing dissimilarities higher than 1%. Brenner, & McMeekin, 2000). EPA production by S. putrefaciens has also been pointed out by several authors (Bergé & Barnathan, 2005; Nichols, McMeekin, & Nichols, 1994). Although as far as we know, no studies of PUFA incorporation from Shewanella into Artemia metanauplii have been performed, several studies have demonstrated the trophic transfer of both EPA and DHA from bacteria to the rotifer Brachionus plicatilis reaching values of EPA as high as 9.4% of total fatty acids (Nichols, Hart, Nichols, & McMeekin, 1996) and contributing to 0.1% 1.2% of the total dry weight (Lewis, Nichols, Hart, Nichols, & McMeekin, 1998). To support this hypothesis of the probiotic ability to produce n- 3 HUFA, it is remarkable the existing correlation among the significant decrements of 18C n- 3 fatty acid precursors such as 18:3n- 3 and 18:4n- 3 and the increments of 20:5n- 3 and 22:6n- 3 in the Pdp11 Artemia metanauplii. However, further in vitro and in vivo studies might be necessary to confirm the probiotic desaturase expression or the ability of Shewanella putrefaciens to transform C 14 radiolabelled fatty acid precursors into EPA and DHA (see Morais, Mourente, Martínez, Gras, & Tocher, 2015; Reis et al., 2013). On the basis of the above- mentioned studies and present results, it could be concluded that Artemia metanauplii can be enriched with DHA and EPA from S. putrefaciens Pdp11. Finally this study cannot be ruled out neither confirmed if S. putrefaciens Pdp11 is also able to somehow enhance the recently known ability of sole specimens to endogenously generate DHA from EPA along the Δ4 metabolic pathway or even from C18 precursors as it has been described by Morais et al. (2015). Shewanella putrefaciens Pdp11- enriched Artemia metanauplii supplied a higher lipid content and specifically more n- 3 HUFA, to sole larvae compared to that of Control Artemia. This fact is critical especially at the end of metamorphosis (23 dah) where n- 3 HUFA as EPA and DHA are involved in many physiological processes as structural components of membrane phospholipids and as precursors of biologically active eicosanoids (Sargent, Tocher, & Bell, 2002). It is well known that the lack of HUFA and particularly DHA affects growth and development at first stages, playing a crucial role in the formation and functioning of the brain and retina. Recent studies have shown deleterious effects on Senegalese sole juveniles when reared on non- enriched Artemia during the whole larval and postlarval stages (Dámaso- Rodrigues et al., 2010). Furthermore, Navarro- Guillén et al. (2014) have recently confirmed that DHA positively affects growth and survival of Senegalese sole larvae when supplied to vegetable oils to achieve a correct balance between dietary energy and EFA. At the start of weaning (56 dah), both fish groups slowed their growth rate and significantly diminished both the total lipid and total fatty acid contents. Among fatty acids, n- 3 HUFA and, specifically, DHA and EPA dramatically decreased in all fry groups although at higher rate in the Control. It is possible that the necessary adaptation to inert diet after weaning might produce these decrements in fish growth rates and n- 3 HUFA contents. Several studies indicate that fish larvae loose important lipid content and, specifically, n- 3 fatty acids during weaning compromising larval growth and performance (Hamre et al., 2013). This reduction in total fatty acid contents and n- 3 HUFA at early weaning has also been reported in sole larvae by Dámaso- Rodrigues et al. (2010). The fatty acid profile and total lipid contents of fish at the end of weaning (87 dah) also indicate the existence of significant differences related to dietary treatments. While in Pdp11 specimens total lipid levels were similar and total fatty acid contents increased in relation to those corresponding to the start of weaning, total lipid contents decreased and total fatty acids were only maintained in Control fish. Despite these last differences, a considerable increase in n- 3 HUFA was detected in both fish groups likely reflecting the commercial pellet composition. In this context, the higher percentage of DHA and the lower level of 18:2n- 6 of the commercial diets were clearly reflected in the evolution of these fatty acid contents on weaning (56 87 dah) in both experimental groups. At 119 dah, the lipid content and fatty acid profiles of both groups were similar and closely resembled the composition of feed as it has been observed in other sole larvae studies (Boglino et al., 2012; Villalta, Estévez, Bransden, & Bell, 2005). Under a PCA multivariate approach, a neat separation of two major supplementation- related clusters corresponding to Control and probiotic fish for each age persisted along the different stages of development (23, 56, 87 and 119 dah). These results lead us to conclude that an early S. putrefaciens Pdp11 pulse through Artemia metanauplii induces changes that modify larval and fry performance, contributing to an advanced sole development. A similar effect has been also

12 LOBO et al. 559 reported in sole larviculture by Avella et al. (2011) and Lobo, Moreno- Ventas et al. (2014). Differences in DHA were more acute at the end of metamorphosis (23 dah) and weaning (87 dah) remarking a better condition for Pdp11 fish after stress processes. In addition, differences in 18:1n- 9, specially observed at weaning, and 16:0, since weaning, probably showed enhanced metabolic processes for Pdp11 specimens remarking the higher energetic level observed for probiotic fry. In summary, the results of the present study show that S. putrefaciens Pdp11 increases n- 3 HUFA content in Artemia metanauplii. This apparent ability of S. putrefaciens Pdp11 strain to produce n- 3 HUFA, previously reported for Shewanella sp, improved S. senegalensis larval and fry growth and generated changes in total lipid contents and fatty acid profiles persisting along the first stages (23 87 dah) of development. In this context, S. putrefaciens Pdp11 might be used as an effective tool for fish marine larviculture optimization in terms of growth and body composition. ACKNOWLEDGEMENTS This work was supported by the Spanish National Development Plan for Sole farming (JACUMAR) and the Ministry of Science and Technology (AGL C03-02). The authors wish to acknowledge the assistance of the Spanish Oceanography Institute staff. We are also grateful to the EULEN/FERROSER employees and especially to Javier Revilla, Mar Oria and Mar Díaz for their assistance. Covadonga Rodríguez is a member of the Institute of Biomedical Technologies (ITB) of the Canary Islands. COMPLIANCE WITH ETHICAL STANDARDS There were not potential conflict of interests (Financial or not financial). There were not human participants involved in the research. 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