phosphatidylinositol ratio on malformation in larvae and juvenile gilthead sea bream (Sparus aurata)

Aquaculture 304 (2010) 42–48

Contents lists available at ScienceDirect

Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

The effect of dietary phosphatidylcholine/phosphatidylinositol ratio on malformation in larvae and juvenile gilthead sea bream (Sparus aurata) E. Sandel a,⁎, O. Nixon a, S. Lutzky a, B. Ginsbourg a, A. Tandler a, Z. Uni b, W. Koven a a b

Israel Oceanographic and Limnological Research Institute, The National Center for Mariculture, P.O. Box 1212, Eilat 88112, Israel Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot, Israel, P.O. Box 12, Rehovot, 76100, Israel

a r t i c l e

i n f o

Article history: Received 26 May 2009 Received in revised form 9 March 2010 Accepted 11 March 2010 Keywords: Osteocalcin BGP Phosphatidylcholine Phosphatidylinositol Deformity Larval rearing Juvenile quality

a b s t r a c t Malformation in commercially raised fish, such as cranial, vertebral and gill cover deformities is a major factor reducing their market value. Although these deformities are most apparent in the juvenile and adult stages they may originate from suboptimal nutrition during the critical larval rearing stage. Previous research hypothesized that dietary phosphatidylinostol (PI) was more effective in reducing deformities than the main membrane phospholipid, phosphatidylcholine (PC). Consequently, the aim of this study was to test the effect of different dietary ratios of PC and PI fed to the gilthead sea bream (Sparus aurata) larvae, on developmental performances in juvenile fish in terms of survival, growth and malformation rate. Four microdiet (MD) treatments, that differed in their PC/PI ratio and replaced 75% of the Artemia ration (wt/wt), were fed to 20–34 dph (days post hatching) sea bream larvae. In addition to the high PC/PI or low PI containing MD control, a commercial reference treatment (100% Artemia ration) was also given. At 40 dph, the larvae were graded in all treatments into small (b 1.3 mg dry wt larva−1) and large (N 2.9 mg dry wt larva−1) larvae, in order to test if growth rate influenced treatment effect throughout development to 141 dph. There was no marked (P N 0.05) treatment effect on growth rate in 40 dph larvae. On the other hand in later juvenile development (67 dph), decreasing dietary PC/PI ratio contributed to significantly (P b 0.05) better growth and (P N 0.05) higher survival. Moreover, reducing dietary PI markedly (P b 0.05) increased jaw (cranial) deformity in both size groups at 67 dph which may have adversely affected juvenile feeding on a dry hard starter feed. Conversely, increasing dietary PI (reducing PC/PI ratio) showed a non-significant trend of increased skeletal deformity which was markedly (P b 0.05) higher in faster growing larvae in all MD treatments. Although there was no clear effect of PC/PI ratio on gill cover deformity rate, there was a size dependent susceptibility to this deformity where smaller larvae showed the highest incidence of this malformation. Osteocalcin (BGP) mRNA levels correlated well (R2 = 0.964) with development in the faster growing fry fed the high PI diet. Higher production of BGP may have reduced (P b 0.05) the jaw deformity while tending to cause over-mineralization and deformity of the skeleton. The results suggest an effective dietary PC/PI ratio of 1.28 for sea bream larvae during culture. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The gilthead sea bream (Sparus aurata L.) is a major commercial species in European aquaculture that is widely farmed in countries around the Mediterranean Sea (125,355 T, FAO, 2007). However, skeletal deformity in market size fish continues to significantly plague the industry (Cahu et al., 2003) reaching levels in excess of 30% of production. Malformed fish are sold at considerably lower prices or are manually removed, raising the cost of production (Koumoundourous et al., 1997). Moreover, deformation can affect fish performance in terms of swimming ability, food conversion, growth rate, survival, as well as susceptibility to

⁎ Corresponding author. P.O. Box 12, Rehovot 76100, Israel. Tel.: +972 8 9489575; fax: +972 8 9489577. E-mail address: [email protected] (E. Sandel). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.03.013

stress and pathogens (Andrades et al., 1996; Koumoundourous et al., 1997; Boglione et al., 2001). The onset of deformity frequently occurs during larval development where over 27% of the population can be affected leading to severely reduced survival (5%) in the juvenile and adult stages (Andrades et al., 1996). In fact, it is becoming increasingly clear that nutritional factors during larval rearing can predispose or directly cause deformity (Andrades et al., 1996; Afonso et al., 2000) which ultimately affects market production levels and fish quality. A number of studies have demonstrated that the morphogenesis of marine fish larvae could be altered by inappropriate dietary levels of micro-nutrients such as vitamin A (Villeneuve et al., 2005a,b; Hernandez et al., 2006; Fernández et al., 2008). On the other hand, deficient dietary macronutrients such as lipids can also influence malformation. Cahu et al. (2003) and Zambonino et al. (2005) found that feeding a compound diet containing increasing levels of soybean lecithin (2.7–11.6% DW) to first feeding larval sea bass (Dicentrarchus

E. Sandel et al. / Aquaculture 304 (2010) 42–48

labrax) increased survival and significantly reduced vertebral and jaw malformations. Geurden et al. (1997, 1998) demonstrated in carp (Cyprinus carpio) that different dietary phospholipid classes strongly affect growth and/or larval development. Phosphatidylcholine (PC) was shown to be a growth enhancer while phosphatidylinositol (PI) prevented skeletal deformities. PC stimulates the synthesis and secretion of the apoB48 protein of triacylglycerol-rich lipoproteins (Field and Mathur, 1995) which promotes growth by increasing energy flux from the intestinal mucosa into the blood (Seiliez et al., 2006). On the other hand, excessive levels of dietary PC can lead to decreased survival and a higher malformation rate in carp larvae (Geurden et al., 1998). Apart from PI's structural role in the membrane, where it serves as an anchor for a wide variety of cell surface proteins (Cahu et al., 2003), this phospholipid and its metabolically active derivatives, inositol trisphosphate (IP3) and diacylglycerol (DAG), appear to modulate protein kinase C activity (Bell and Sargent, 1987; Seiliez et al., 2006). As second messengers, they also regulate the calcium entry into the cell from nuclear reservoirs (Cahu et al., 2003). However, how the involvement of PI in these physiological pathways influences deformity remains incompletely understood. A key physiological process that PI may be influencing is the ontogeny of bone synthesis and mineralization which has been addressed recently in gilthead sea bream (Gavaia et al., 2000; Pombinho et al., 2004). In parallel, efforts have been made toward the cloning of cDNAs and genes corresponding to proteins involved in fish bone and cartilage formation. Osteocalcin (bone Gla protein, BGP), a vitamin K-dependent protein routinely used as a marker for bone and cartilage growth in mammalian systems, has been purified, its cDNA and gene cloned, and specific antibodies developed (Pinto et al., 2001, 2003; Simes et al., 2003; Pombinho et al., 2004). Presently, the accepted practice worldwide for the intensive larval rearing of marine fish is the feeding of live prey during the first weeks where the developing fish are then gradually weaned onto a dry diet (Fernandez-Diaz and Yufera, 1997; Kolkovski et al., 1997). Unfortunately, the use of live food as experimental diets is severely limited. Although the fatty acid profiles, total lipid and specific micro-nutrients of rotifers and Artemia can be manipulated, protein quality and level as well as tissue phospholipid class composition are genetically determined and cannot be altered by the zooplankter's diet (Rosenlund et al., 1997; Koven et al., 2001). This means that investigating the effect of various dietary phospholipids on larval performance must be carried out only through the provision of an acceptable microdiet to the larvae. The aim of the present study was to determine, through the use of a soft gelatin micro-encapsulated diet, the effect of dietary PC/PI ratio on the incidence of skeletal deformity, osteocalcin expression and the performance of gilthead sea bream larvae, juveniles and fry. 2. Materials and methods 2.1. Diet preparation Four gelatin based micro-encapsulated diets, identical in their nonlipid fractions and total lipid levels but differing in their phospholipid composition, were prepared at KARMAT coating industries (Kibbutz Ramot Menashe, Israel). The microdiet (MD) was designed to time release specific amino acids as an attractant while encapsulating other free amino acids that stimulate specific digestive hormones in order to improve larval digestion and assimilation. The composition of the MDs is outlined in Table 1. 2.2. Fish rearing Eggs were obtained from natural spawning sea bream brood stock maintained at the Ardag fish farm (Ardag Red Sea Mariculture Ltd., Eilat, Israel) and stocked (100 eggs L−1) in 1500-LV-tanks at the

43

Table 1 Composition of microdiets.a Diet

A

B

C

D

Formulation (g/kg−1) basal dietb De-oiled soybean lecithinc Phospholipon 80 Hd

950 – 50

950 20 30

950 40 10

950 50 –

a

Produced at KARMAT Coating Industries Ltd., Israel. Per kg diet: 559.5 g fish meal; 44 g gelatin; 6 g acacia; 63 g fish oil (Matmor Feeds, Israel); and 27 g EPAX 1050 (EPAX AS, Aalesund, Norway). Coating amino acids: 20 g glycine; 20 g arginine; 20 g alanine and 20 g betaine (Sigma, St. Louis, USA); 0.5 g vitamin C (Stay C, Hoffman LaRoche, Switzerland); 40 g mono calcium phosphate (Fluka, Buchs, Switzerland); 10 g vitamin and mineral premix; and 10 g choline chloride (Sigma, St. Louis, USA). Supplemented amino acids: 10 g valine; 10 g isoleocine; 10 g tryptophan; and 20 g phenylalanine(Sigma, St. Louis, USA). c Deoiled soybean lecithin (Enzymotec, Migdal HaEmeq, Israel): 70.7% total PL; 23.9% phosphatidylcholine; 20.2% phosphatidylinositol; 14.5% phosphatidylethanolamine; 6.2% phosphatidic acid; 5.2%. d Phospholipon 80 H (Phospholipid GmbH, Köln, Germany): hydrogenated PC; 60%, Lyso PC: 10%. b

National Center for Mariculture in Eilat, a branch of Israel Oceanographic and Limnological Research. At 5 days post hatching (dph), rotifers (Brachionus rotundiformis), enriched (0.2 g 1 × 106 rotifers−1) with AlgaMac-2000 (Bio Marine, USA), were fed to the larvae (10 rotifers mL−1) until 16 dph. The rotifers, relaying on a mixed diet of algae (Nannochloropsis spp.) and yeast, were harvested daily from a circular 5-m diameter 32-m3 tank. Then, the larvae were randomly distributed (100 larvae L−1) to twenty-five 400-L conical fiberglass tanks in a temperature and salinity controlled system which was continuously supplied with filtered (10 µm), oxygenated (6 mg L−1) diluted sea water (25‰) with a tank exchange rate of 30% h−1. The larvae were co-fed rotifers with Artemia nauplii (Great Salt Lake, USA, 1 nauplii mL−1), also enriched with AlgaMac-2000, until 20 dph. The temperature was gradually raised from 19 °C to 24 °C. All animal procedures and handling complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1985). Four MD treatments, that differed in their PC/PI ratios and replaced 75% of the normal Artemia ration (mg dry weight, DW), were fed to 20–34 dph larvae using a specially designed speed regulated belt feeder (C-Rose Ltd., Koranit, Israel). This ration level replacement was chosen as the experimental MD has in previous testing successfully replaced up to 75% of the Artemia ration at an MD:Artemia ratio (DW) of 2:1. This means that each of the tanks also received unenriched Artemia at 25% of the DW ration. The highest PC/PI ratio diet (lowest PI) was considered the control (Diet A) while a commercial reference diet (100% enriched Artemia ration — Art treatment) was also tested. The biochemical analysis of the enriched Artemia with AlgaMac-2000 was tested with Trace GC Ultra Gas Chromatography, On-column (Thermo Finnigan, USA) (Table 2). Each of the five diets was tested in replicates of five 400-L tanks (Table 3). For the verification of the PC/PI values from the different diets, a separation of the phospholipids by TLC was carried out essentially as follows. Total lipid extracts were dissolved in chloroform (100 mL) and applied to the origin of a Silica Gel 60 TLC plate (20 cm × 20 cm) (Merck Ltd.). The dried lipid spots were developed with Methyl-acetate/Isopropanol/Chloroform/Methanol/ KCl (25:25:25:10:9, v/v/v/v/v) for 1 h, 40min dehydration and another 1 h in Hexane/Diethyl ether/Acetic acid (80:20:2, v/v/v). Plates were allowed to dry and then sprayed with 0.1% 2′,7′-Dichlorofluoroscein in 97% methanol (Sigma, Israel). Bands corresponding to PC and PI were identified under u.v. illumination and collected by scraping the silica into a glass tube. The particle size of the four diets offered to 20–26 dph larvae was 125–200 µm while a larger particle (200–400 µm) was fed to 27–34 dph larvae. From 35 to 40 dph the larvae, maintained in their respective treatment tanks, were fed with Artemia (1 Artemia mL−1) while weaning onto a dry starter feed (size #0) (Coppens International, Helmond, The Netherlands).

44

E. Sandel et al. / Aquaculture 304 (2010) 42–48

Table 2 Selected fatty acid indices (mg g−1 DW) of Great Salt Lake Artemia after 24 h enrichment on AlgaMac-2000. Fatty acid

Level

Saturates Monounsaturates 14:0 14:1 15:0 16:0 16:1n-7 16:2 16:3 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20:0 20:3n-6 20:4n-6 (ARA) 20:4n-3 20:5n-3 (EPA) 22:6n-3 (DHA) DHA/EPA

20.47 ± 4.57 14.98 ± 2.54 2.19 ± 0.21 0.71 ± 0.18 0.49 ± 0.34 12.46 ± 2.84 3.63 ± 1.16 0.97 ± 0.45 0.51 ± 0.31 5.07 ± 1.23 9.82 ± 1.15 0.82 ± 0.82 8.58 ± 4.35 18.6 ± 4.94 2.75 ± 0.98 0.25 ± 0.18 0.05 ± 0.05 0.54 ± 0.32 0.34 ± 0.24 4.36 ± 0.24 5 ± 0.35 1.16 ± 0.09

Values are expressed as means ± S.E.M.

At 40 dph, the larvae were graded in all treatments into small (b1.3 mg DW larva−1) and large (N2.9 mg DW larva−1) size classes, in order to test the dietary effect on growth rate throughout development. Small and large larvae from each of the five dietary treatments were stocked (200 small and 150 large larvae aquaria−1) in forty 27-L glass aquaria where each size and treatment was tested in replicates of four aquaria. Each aquarium was continuously supplied with ambient seawater (40‰) at 23 °C at a rate of 0.18 L min−1. The larvae were fed from 40 to 67 dph on Artemia (1000 Artemia aquarium−1) and starter feed (150 mg size #1) (Coppens International, Helmond, The Netherlands) 3 times per day. At 67 dph, 100 representative juveniles from each of the 40 size and treatment aquaria were stocked into a corresponding 10-L mesh cage floating in 8000-L tanks for growing out until 141 dph. The remaining fry in each aquarium were taken for dry weight, deformity examination. 2.3. Determination of deformities As the skeleton of young fish has a low bone density, X-raying for deformities was not practical. Alternatively, the 67 dph juveniles were stained for bone and cartilage using a modified method by Potthoff (1984). In brief, this process involved dehydrating specimens in 70– 99% ethanol for 1 to 5 days, staining them for cartilage (Alcian blue in acetic acid and 100% ethanol) for 1 day, neutralizing by washing with sodium borate for 12–48 h and bleaching in a solution of 3% H2O2 and 1% KOH. This was followed by tissue digestion in trypsin (in sodium borate) for 1–14 days, depending on specimen size and staining them for bone (Alizarin red in 1% KOH). The samples were then de-stained Table 3 The dietary PC/PI ratio in the experiment treatments.

(g/100 g dry diet)

Phospholipids Phosphatidylcholine Phosphatidylinositol PC/PI in total diet

A

B

C

D

Art

25% Artemia + 75% MD

25% Artemia + 75% MD

25% Artemia + 75% MD

25% Artemia + 75% MD

100% Artemia

5.7 1.86 3.07

4.54 1.95 2.32

4.42 2.76 1.6

3.88 3.04 1.28

4.42 1.78 2.48

in KOH:glycerol solutions (3:1 to 1:1 to 1:3) and preserved in 100% glycerol. Before staining, gill cover deformities were visually observed and noted. After staining, the juveniles were examined under a binocular microscope and then were sorted for cranial and vertebral deformities. At 141 dph, the remaining fry were manually sorted for phenotypic cranial and gill cover deformities and then, as the bone density had sufficiently increased, were analyzed using the more rapid X-ray approach (Philips, Media 65 CT-H) and the results digitally analyzed using the program; Fujifilm FCR XG-1. 2.4. Molecular marker for bone growth Pinto et al. (2001) has shown no detection of sparus osteocalcin (bone Gla protein; BGP) mRNA by RT-PCR before 37 dph. That is why the mRNA spBGP levels were tested from the experimented juveniles in all treatments throughout development at 40, 50, 62, 73, and 83 dph. Total RNA from whole larva was extracted in four replicates (four juveniles per treatment with Total RNA Isolation Reagent (TRIzole®, Gibco-BRL, Gaithersburg, USA) according to the manufacturer's protocol from 100 mg larvae. The RNA was treated with RNAse free-DNAse I (Promega, Madison, WI) to remove contaminating genomic DNA. A total of 1 μg of the pooled RNA was used to synthesize the first cDNA strand using Superscript™ II RT (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. A specific primer, Sparus BGP (Sigma, St. Louis, USA), was designed based on the Sparus BGP mRNA: 5′-TGGCAGCCATCTGTCTGACTT-3′ (forward); 5′-TCAGTGTCCATCATGTGCTCG-3′ (reverse). cDNA (1 μL) was used as a template for a hot-start RT-PCR in the presence of 1.5 μM degenerate primer, 2 μL of 10× buffer, 1.5 mM MgCl2, 1 μL of dNTPs (final 200 μM each), and 5 U of Taq DNA polymerase (Promega). RT-PCR conditions were 95 °C for 2 min, and 37 cycles of 95 °C for 2 min, 53 °C for 1 min and 68 °C for 2 min followed by 72 °C for 5 min. The RT-PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized by staining with ethidium bromide. As an internal control for the relative amount of RNA used for each sample, Sparus β Actin was also amplified from the same amount of RT-PCR reaction, using two specific primers designed according to the published Sparus β Actin cDNA (GenBank Accession no. X89920); 5′-TTCCTCGGTATGGAGTCC-3′ (forward); 5′-GGACAGGGAGGCCAGGA-3′ (reverse). The abundance of Sparus BGP was normalized to the density of the Sparus β Actin transcripts by densitometry with a high-resolution scanner. Gel-Pro densitometer software (Version 3.0, Media Cybernetics, Silver Spring, MD) was applied to determine the amount of mRNA in each band. 2.5. Statistical analysis Statistical analysis of the results was conducted by SPSS 13 (SPSS, Inc.). Results are given as mean ± SEM. Significance between means were analyzed using one-way ANOVA followed by the Tukey–Kramer Honestly Significant Difference (HSD) multiple range test (P b 0.05). 3. Results 3.1. Larval growth and survival throughout development There was no marked (P N 0.05) treatment effect on growth rate in 40 dph larvae although larvae fed the MDs were significantly (P b 0.05) smaller than the commercial reference diet (Art) larvae (Table 4). On the other hand, in later juvenile development (67 dph), decreasing dietary PC/PI ratio contributed to better growth in the large group juveniles. Respectively, juveniles fed diet D (44.4 mg DW larva−1) had significantly (P b 0.05) better growth compared to juveniles fed diet A (27.5 mg DW larva−1) (Table 4). Both small (b1.3 mg DW larva−1) and large (N2.9 mg DW larva−1) larvae fed the lowest PC/PI MD demonstrated a non-significant

E. Sandel et al. / Aquaculture 304 (2010) 42–48

45

Table 4 The effect of dietary PC/PI ratio, given from 20 to 34 dph on small (b1.3 mg DW larva−1) and large (N 2.9 mg DW larva−1) gilthead sea bream dry weight (at 33, 40 and 67 dph) and survival rate (at 40, 67 and 141 dph). n = 4. Dietary groups (PC/PI ratio) A (3.07) Larval dry weight, mg 33 dph 40 dph 67 dph Small (b1.3 mg DW larva−1) Large (N2.9 mg DW larva−1) Survival rate, % 40 dph 67 dph Small (b1.3 mg DW larva−1) Large (N2.9 mg DW larva−1) 141 dph Small (b1.3 mg DW larva−1) Large (N2.9 mg DW larva−1)

B (2.32)

C (1.6)

D (1.28)

Art (2.48)

0.9 ± 0.1a 1.6 ± 0.1a

0.8 ± 0.1a 1.6 ± 0.1a

0.8 ± 0.1a 2.1 ± 0.5a

0.8 ± 0.1a 1.4 ± 0.1a

26.9 ± 2.6a 27.5 ± 0.5a

16.6 ± 1.1a 35.7 ± 3.5a,b

21.5 ± 3a 34.5 ± 2.4a,b

16.9 ± 2.8a 44.4 ± 2.8b

19.5 ± 2.4a 29.8 ± 3.7a,b

73.1 ± 9.5a

70.9 ± 13.2a

62.5 ± 19.7a

86.7 ± 3.7a

89.7 ± 1.7a

92.3 ± 1.7a 87 ± 0.1a

85.8 ± 2.4a 96.3 ± 1.5a

86.5 ± 9.5a 94.25 ± 3.5a

90.3 ± 3.6a 96 ± 1.0a

95 ± 1.5a 89.3 ± 3.2a

44.3 ± 6.3a 49 ± 1.0a

48 ± 3.5a 51.5 ± 2.7a

51.2 ± 9.2a 44.6 ± 8.9a

55.8 ± 9.7a 56.3 ± 12.4a

68.6 ± 7.4a 47 ± 8.8a

Values are expressed as means ± S.E.M. Different superscript letters

a,b

2 ± 0.2b 4 ± 0.1b

in the same row are significantly different (P b 0.05).

(P N 0.05) but consistently higher survival throughout development than larvae fed the other MD treatments. The commercial reference (100% Artemia ration) larvae showed the best survival overall throughout development in only the small (b1.3 mg DW larva−1) size larvae but not significantly (P N 0.05) (Table 4). 3.2. Deformity rates throughout development Examination of jaw (cranial) deformity in both size groups at 67 dph (Fig. 1) showed a markedly (P b 0.05) PI dose response effect on reduced deformity that was size dependent. Dietary treatment D (PC/PI = 1.28) juveniles demonstrated the lowest level of cranial deformity(b1%) while treatment B (PC/PI = 2.32) juveniles showed the highest incidence of deformity in all the four MD treatments (5.5%). Interestingly, the commercial reference diet (100% Artemia ration) with PC/PI = 2.48 showed the highest cranial deformity rate than the other treatments (14–17%). In contrast to cranial deformity, large juveniles (N2.9 mg DW larva−1) demonstrated a non-significant (PN 0.05) trend of increasing dietary PC/PI ratio (or decreasing PI) with decreasing vertebral deformity (Fig. 2). Dietary treatments C and D (PC/PI=1.6 and 1.28) showed the highest level of vertebral malformations while these deformities were the most reduced in treatment A (PC/PI=3.07) of all four MD treatments. The

Fig. 1. The effect of dietary PC/PI ratio on level (%) of cranial deformities in small (b 1.3 mg DW larva−1) and large (N 2.9 mg DW larva−1) gilthead sea bream 67 dph juveniles. Levels within a size group having different letters were significantly (P b 0.05) different. Results are expressed as mean ± S.E.M. n = 4.

commercial reference treatment (100% Artemia ration) with PC/PI=2.48 showed, as in the cranial deformity, the highest vertebral deformity rate between all treatments. Importantly, the vertebral deformity rate within treatments was significantly (Pb 0.05) higher in the large size (N2.9 mg DW larva−1) juveniles compared to the small size (b1.3 mg DW larva−1) in diets A, B and D (Fig. 2). Although there was no clear effect of PC/PI ratio on gill cover deformity rate, there was a size dependent susceptibility to this deformity where smaller juveniles generally showed the highest incidence of this malformation which was marked (Pb 0.05) at a ratio of 2.32 (Fig. 3). Throughout development during the juvenile and fry stages (67– 141 dph), a general size-independent and non-significant (PN 0.05) trend of increasing incidence of cranial deformity was observed (Fig. 4) suggesting that the cranial deformity was not deleterious for the growing fry. Vertebral deformity, as observed in cranial malformation, generally increased throughout that time of development independently of fry size (Fig. 4) suggesting that this deformity was continually developing and/or was not deleterious. On the contrary, gill cover deformity tended to decrease (Fig. 4) from 67 to 141 dph, suggesting operculum regeneration and/or that the exposure of the gills was deleterious with age. In order to maximize observed treatment effects, juveniles grown from small (b1.3 mg DW larva−1) and large (N2.9 mg DW larva−1)

Fig. 2. The effect of dietary PC/PI ratio on level (%) of vertebral deformities in small (b 1.3 mg DW larva−1) and large (N 2.9 mg DW larva−1) gilthead sea bream 67 dph juveniles. Levels within a size group having different letters were significantly (P b 0.05) different. Levels between size groups fed the same treatments and having different (*) were significantly (P b 0.05) different. Results are expressed as mean ± S.E.M. n = 4.

46

E. Sandel et al. / Aquaculture 304 (2010) 42–48

Fig. 3. The effect of dietary PC/PI ratio on level (%) of gill cover deformities in small (b 1.3 mg DW larva−1) and large (N 2.9 mg DW larva−1) gilthead sea bream 67 dph juveniles. Levels within a size group having different letters were significantly (P b 0.05) different. Levels between size groups fed the same treatments and having different (*) were significantly (P b 0.05) different. Results are expressed as means ± S.E.M. n = 4.

larvae from only the control treatment A (PC/PI = 3.07) and the lowest PC/PI treatment D (PC/PI= 1.28) from different age classes (dph) were analyzed and compared for BGP mRNA levels. After normalizing the Sparus BGP densitometry results to those of the Sparus β Actin, a nonsignificant trend (P N 0.05) was noticeable (Fig. 5) in which the mRNA levels in large treatment D juveniles increased throughout development from 40 to 83 dph (R2 = 0.964 of regression curve). The pattern of mRNA levels in the controlled (A) juveniles was unclear and lower in the older juveniles (R2 = 0.284 of regression curve). 4. Discussion The growth promoting effects of dietary phospholipid for marine fish larvae has been well documented (Cahu et al., 2003). A number of authors have argued that this was primarily due to the contribution of the dominant membranal phospholipid, phosphatidylcholine (PC), to lipoprotein synthesis (Geurden et al., 1995; Coutteau et al., 1997; Fontagne et al., 1998; Tocher et al., 2008) which enhanced the transport of lipids assembled in the intestinal enterocytes to the cellular membranes of the body, a necessary requisite during rapid tissue deposition. On the other hand, Kanazawa et al. (1981) also showed that dietary incorporation of phospholipid reduced malformation, espe-

Fig. 5. (a) A replicate detection in Sparus BGP mRNA by RT-PCR from five developmental stages (40–83 dph) in large (N 2.9 mg DW larva−1) gilthead seabream fry fed dietary PC/PI ratio of 3.07 (A) and 1.28 (D). (b) Relative quantity of Sparus BGP mRNA levels (40–83 dph) in large (N 2.9 mg DW larva−1) juveniles fed diets A and D. The units are arbitrary and relative to Sparus β Actin mRNA. n = 3.

cially twist of jaw and scoliosis, in Ayu larvae, Plecoglossus altivelis. As phospholipids include various phosphoglycerides other than phosphatidylcholine (e.g., phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol), this implies that less dominant phospholipid classes might be critical in reducing deformities in fish larvae. Geurden et al. (1997, 1998) concluded that although PC promoted growth in carp (C. carpio) it was much less effective in reducing skeletal deformities than phosphatidylinositol (PI). This finding was reinforced by Cahu et al. (2003) who found that a diet having a PC/PI ratio of 2.18 (1.6% of DW diet) fed to European sea bass (D. labrax) from first feeding prevented deformities during development. In fact, the various MD PC/PI ratios tested in the present study bracketed the most effective ratio reported by Cahu et al. (2003). Until 40 dph, variable dietary PC/PI ratio did not affect larval growth while the commercial reference (100% Artemia ration) significantly

Fig. 4. The effect of dietary PC/PI ratio on deformity (%) of cranial, vertebral and gill cover in gilthead sea bream throughout development (67–141 dph). n = 4.

E. Sandel et al. / Aquaculture 304 (2010) 42–48

(P b 0.05) demonstrated the best weight gain. This was surprising as preliminary studies carried out at the NCM showed that a 75% MD ration successfully replaced a complete Artemia ration in terms of growth and survival. However, it was later discovered that the pigment astaxanthin (extracted from the algae Hematococcus sp.) was not added in the production of the present MD treatments. Astaxanthin is one of the most important carotenoids in marine organisms, being responsible for the pigmentation of skin and flesh of fish, mainly salmonids, but also influencing survival and growth of fish larvae (Dominguez et al., 2005). This may explain the growth discrepancy between the MDs and the commercial reference diet. On the other hand, there was a PC/PI treatment growth effect from 40 to 67 dph. The faster growing larvae (N2.9 mg DW larva−1) at 40 dph exhibited a significant PI dose dependent growth response at 67 dph, which was not observed with the slower growing larvae (b1.3 mg DW larva−1) graded at 40 dph (Table 4). In fact, the juvenile fish fed the highest PI containing MD (D) during larval rearing grew more rapidly than the commercial reference. The improved growth performance of juveniles originating from faster growing 40 dph larvae fed the highest PI diet may be tied to the significantly lower incidence of jaw (cranial) deformity in these fish compared to their cohorts fed the higher PC/PI ratios. Several authors have reported that larvae with abnormal jaw die because of their difficulty in feeding (Kurokawa et al., 2008). In this experiment, the commercial reference diet (100% Artemia ration) showed the highest cranial deformity rate (14–17%) than the other treatments. This finding is supported by the results of Galeotti et al. (2000) who reported opercula deformities of 17% in S. aurata larvae reared on live food. The overall incidence of jaw deformity was relatively low and not a major factor in larvae feeding on the soft gelatin MD capsule and also tended to increase from 67 to 141 dph implying that it was not deleterious. Nevertheless, jaw deformity may have adversely affected feeding on the hard particulate starter feed later on in juvenile development which, in turn, influenced growth performance. Moreover, the data suggested that the dietary PI effect on jaw deformity was more prominent in faster growing individuals. This follows as the effect of a limiting dietary nutrient on reducing deformity would likely be first expressed in fish with the most rapid muscle and bone deposition. In contrast, there was a non-significant (P N 0.05) trend of increasing vertebral deformity with increased dietary PI or decreasing PC/PI ratio. In fact, faster growing larvae fed the dietary treatments demonstrated markedly higher skeletal deformity (P b 0.05) than their slower growing cohorts in treatments A, B and D. Similarly to jaw deformity, there was a general increase in the population from 67 to 141 dph of vertebral malformation testifying to the non-lethal nature of this deformity and the added cost that would be necessary to remove them from the population. Andrades et al. (1996) supported this finding by reporting that gilthead sea bream larvae displaying lordosis were not hampered in the ability to swim or eat. There was no clear effect of PC/PI ratio on level (%) of gill cover deformities but there was a significant size dependent susceptibility (diet B) in which smaller larvae showed the highest incidence of opercula deformities. This was supported by Verhaegen et al. (2007) who showed that 65 dph gilthead sea bream larvae with opercula deformities were significantly smaller compare to normal specimens. Gill cover malformation generally decreased throughout development (Fig. 4) suggesting that the exposure of the gills was deleterious with age. This might be expected as malformed operculae lower the fish's resistance to oxygen stress and predispose them to bacterial infections of the gills (Paperna et al., 1980; Galeotti et al., 2000). On the other hand, regeneration of the operculae has been shown in preliminary studies in which gilthead sea bream larvae lacking full operculae largely succeeded to regenerate them during metamorphosis (De wolf et al., 2004; Verhaegen et al., 2007). An extensive investigation on genes regulated by nutrients was carried out recently, using micro-arrays including a large number of genes. The data provided crucial information regarding digestive

47

functions, metabolism and skeletal development (Cahu et al., 2009). One of these genes, found to give positive results, is osteocalcin (BGP), a vitamin K requiring protein in bone hyroxyapatite that has a strong affinity for calcium and has been correlated with the mineralization of bone (Szulc et al., 1994; Simes et al., 2008). Nishimoto et al. (2003) found in carp that bone hydroxyapatite binding of BGP is enhanced in the presence of calcium ions. Inside the cell, it is known that PI functions as a precursor of second messengers such as Inositol 3 phosphate (IP3) regulating calcium ions' entry into the cell from the endoplasmatic reticulum (Cahu et al., 2003; Tocher et al., 2008). One possible biological pathway out of many affecting skeletal morphogeneses, relies in the relation between PI and BGP. This study suggests that dietary PI may be increasing the availability of calcium for bone mineralization, which possibly stimulates BGP production. In a study on European sea bass larvae (ORCIS project, 2005), it has been hypothesized that high mRNA levels of BGP may have led to overmineralization of the vertebrae causing spinal malformations. However, the requirement for mineralization in the normal development of jaws and vertebral column may differ during larval growth resulting in high dietary PI reducing jaw deformity while simultaneously over mineralizing the vertebrae. The effect of BGP levels on different skeletal anomalies should be further investigated on gilthead sea bream larvae in future research. In summary, decreasing the dietary PC/PI ratio (5% total phospholipid) during larval rearing significantly increased juvenile growth. This may have resulted from a reduced incidence of jaw deformity which leads to more effective pellet feeding. Moreover, the decreased rate of cranial deformity may be due to PI enhancing osteocalcin synthesis and normal jaw development. The results conclude that a PC/PI ratio of 1.28 or a PI level of 3.04 g 100 g−1 DW diet gave the best larval and fry performance. Acknowledgments This work was supported by the FineFish Collective Research Project (no. 012451) an EU 6th Framework Program. References Afonso, J.M., Montero, D., Robaina, L., Astorga, N., Izquierso, M.S., Ginés, R., 2000. Association of a lordosis–scoliosis–kyphosis deformity in gilthead seabream (Sparus aurata) with family structure. Fish Physiology and Biochemistry 22, 159–163. Andrades, J.A., Becerra, J., Ferrhdez-Llebrez, P., 1996. Skeletal deformities in larval, juvenile and adult stages of cultured gilthead sea bream (Sparus aurata L.). Aquaculture 141, 1–11. Bell, M.V., Sargent, J.R., 1987. Protein kinase C activity in the spleen of trout (Salmo gairdneri) and the rectal gland of dogfish (Scyliorhinus canicula), and the effects of phosphatidylserine and diacylglycerol containing (n-3) polyunsaturated fatty acids. Comparative Biochemistry and Physiology 87B, 875–880. Boglione, C., Gagliardi, F., Scardi, M., Cataudella, S., 2001. Skeletal descriptors and quality assessment in larvae and post-larvae of wild-caught and hatchery-reared gilthead sea bream (Sparus aurata L. 1758). Aquaculture 192, 1–22. Cahu, C., Zambonino Infante, J., Takeuchi, T., 2003. Nutritional components affecting skeletal development in fish larvae. Aquaculture 227, 254–258. Cahu, C., Gisbert, E., Villeneuve, L., Morais, S., Hamza, N., Wold, P.A., Zambonino Infante, J., 2009. Influence of dietary phospholipids on early ontogenesis of fish. Aquaculture Research 40, 989–999. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P., 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155, 149–164. De Wolf, T., Courtens, V., Capiferri, U., Pirone, A., Lenzi, C., Lenzi, F., 2004. The influence of light conditions on the operculum recovery of sea bream (Sparus aurata L.) fry. Book of abstracts. Aquaculture Europe 2004, Biotechnologies for quality, Barcelona, Spain 20–23 October, 2004, p. 292. Dominguez, A., Ferreira, M., Coutinho, P., Fabrega, J., Otero, A., 2005. Delivery of astaxanthin from Haematococcus pluvialis to the aquaculture food chain. Aquaculture 250, 424–430. FAO Fishery and Aquaculture Information and Statistics Service. Aquaculture production 2005. FAO yearbook. Fishery statistics. Aquaculture production. 100/2. Rome, FAO. 2007. 202p. Fernández, I., Hontoria, F., Ortiz-Delgado, J.B., Kotzamanis, Y., Estévez, A., ZamboninoInfante, J.L., Gisbert, E., 2008. Larval performance and skeletal deformities in farmed gilthead sea bream (Sparus aurata) fed with graded levels of vitamin A enriched rotifers (Brachionus plicatilis). Aquaculture 283, 102–115.

48

E. Sandel et al. / Aquaculture 304 (2010) 42–48

Fernandez-Diaz, C., Yufera, M., 1997. Detecting growth in gilthead seabream, Sparus aurata L., larvae fed microcapsules. Aquaculture 153, 93–102. Field, F.J., Mathur, S.N., 1995. Intestinal lipoprotein synthesis and secretion. Pergamon. 34, 185–198. Fontagne, S.p., Geurden, I., Escaffre, A.M., Bergot, P., 1998. Histological changes induced by dietary phospholipids in intestine and liver of common carp (Cyprinus carpio L.) larvae. Aquaculture 161, 213–223. Galeotti, M., Beraldo, P., De Dominis, S., D'Angelo, L., Ballestrazzi, R., Musetti, R., Pizzolito, S., Pinosa, M., 2000. A preliminary histological and ultrastructural study of opercular anomalies in gilthead sea bream larvae (Sparus aurata). Fish Physiology and Biochemistry 22, 151–157. Gavaia, P.J., Sarasquete, C., Cancela, M.L., 2000. Detection of mineralized structures in early stages of development of marine Teleostei using a modified Alcian Blue– Alizarin Red-double staining technique for bone and cartilage. Biotechnic and Histochemistry 75, 79–84. Geurden, I., Radünz-Neto, J., Bergot, P., 1995. Essentiality of dietary phospholipids for carp (Cyprinus carpio L.) larvae. Aquaculture 131, 303–314. Geurden, I., Coutteau, P., Sorgeloos, P., 1997. Effect of a dietary phospholipid supplementation on growth and fatty acid composition of European sea bass (Dicentrarchus labrax L.) and turbot (Scophthalmus maximus L.) juveniles from weaning onwards. Fish Physiology and Biochemistry 16, 259–272. Geurden, I., Marion, D., Charlon, N., Coutteau, P., Bergot, P., 1998. Comparison of different soybean phospholipidic fractions as dietary supplements for common carp, Cyprinus carpio, larvae. Aquaculture 161, 225–235. Hernandez, L.H., Teshima, S., Koshio, S., Ishikawa, M., Gallardo-Cigarroa, F.J., Alam, M.S., Uyan, O., 2006. Effects of vitamin A palmitate, beta-carotene and retinoic acid on the growth and incidence of deformities in larvae of red sea bream Chrysophrys major. Ciencias Marinas 32, 195–204. Kanazawa, A., Teshima, S., Inamori, S., Iwashita, T., Nagao, A., 1981. Effects of phospholipids on growth, survival rate and incidence of malformation in the larval Ayu. Memoirs of Faculty of Fisheries, Kagoshima University 30, 301–309. Kolkovski, S., Tandler, A., Izquierdo, M.S., 1997. Effects of live food and dietary digestive enzymes on the efficiency of microdiets for seabass (Dicentrarchus labrax) larvae. Aquaculture 148, 313–322. Koumoundourous, G., Oran, G., Divanach, P., Stefanakis, S., Kentouri, M., 1997. The opercular complex deformity in intensive gilthead sea bream Sparus aurata L. larviculture. Moment of apparition and description. Aquaculture 149, 215–226. Koven, W., Kolkovski, S., Hadas, E., Gamsiz, K., Tandler, A., 2001. Advances in the development of microdiets for gilthead seabream, Sparus aurata: a review. Aquaculture 194, 107–121. Kurokawa, T., Okamoto, T., Gen, K., Uji, S., Murashita, K., Unuma, T., Nomura, K., Matsubara, H., Kim, S.K., Ohta, H., Tanaka, H., 2008. Influence of water temperature on morphological deformities in cultured larvae of Japanese eel, Anguilla japonica, at completion of yolk resorption. Journal of the World Aquaculture Society 39, 726–735. Nishimoto, S.K., Waite, J.H., Nishimoto, M., Kriwacki, R.W., 2003. Structure, activity, and distribution of fish osteocalcin. Journal of Biological Chemistry 278, 11843–11848.

Paperna, I., Ross, B., Colorni, A., Colorni, B., 1980. Disease of marine fish cultured in Eilat mariculture project. Studies and Review, General Fisheries Council for the Mediterranean 57, 29–32. Pinto, J.P., Ohresser, M., Cancela, M.L., 2001. Cloning of the bone Gla protein gene from the teleost fish Sparus aurata. Evidence for overall conservation in gene organization and bone-specific expression from fish to man. Gene 270, 77–91. Pinto, J.P., Conceicao, N., Gavaia, P.J., Cancela, M.L., 2003. Matrix Gla protein gene expression and protein accumulation co-localize with cartilage distribution during development of the teleost fish Sparus aurata. Bone 32, 201–210. Pombinho, A.R., Laizé, V., Molha, D.M., Marques, S.M.P., Cancela, M.L., 2004. Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type specific expression of matrix Gla protein and osteocalcin. Cell and Tissue Research 315, 393–406. Potthoff, T., 1984. Clearing and staining techniques. In: Moser, H.G. (Ed.), Ontogeny and Systematics of Fishes. : American Society of Ichthyologists and Herpetoligists, Spec. Publ., vol. 1. Allen Press, Lawrence, KS, pp. 35–37. Rosenlund, G., Stoss, J., Talbot, C., 1997. Co-feeding marine fish larvae with inert and live diets. Aquaculture 155, 183–191. Seiliez, I., Bruant, J.S., Zambonino Infante, J.L., Kaushik, S., Bergot, P., 2006. Effect of dietary phospholipid level on the development of gilthead sea bream (Sparus aurata) larvae fed a compound diet. Aquaculture Nutrition 12, 372–378. Simes, D.C., Williamson, M.K., Ortiz-Delgado, J.B., Viegas, C., Price, P.A., Cancela, M.L., 2003. Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish. Journal of Bone and Mineral Research 18, 244–259. Simes, D.C., Viegas, C.B., Williamson, M.K., Price, P.A., Cancela, L., 2008. Purification of matrix Gla protein and osteocalcin from the adriatic sturgeon (Acipencer naccarii), an ancient bony fish with a cartilaginous endoskeleton. Bone 42, S65. Szulc, P., Arlot, M., Chapuy, M.-C., Duboef, F., Meunier, P.J., Delmas, P.D., 1994. Serum undercarboxylated osteocalcin correlates with hip bone density in elderly women. Journal of Bone and Mineral Research 9, 1591–1595. Tocher, D.R., Bendiksen, E., Campbell, P.J., Bell, J.G., 2008. The role of phospholipids in nutrition and metabolism of teleost fish. Aquaculture 280, 21–34. Verhaegen, Y., Adriaens, D., De Wolf, T., Dhert, P., Sorgeloos, P., 2007. Deformities in larval gilthead sea bream (Sparus aurata): a qualitative and quantitative analysis using geometric morphometrics. Aquaculture 268, 156–168. Villeneuve, L., Gisbert, E., Le Delliou, H., Cahu, C.L., Zambonino-Infante, J.L., 2005a. Dietary levels of all-trans retinol affect retinoid nuclear receptor expression and skeletal development in European sea bass larvae. British Journal Nutrition 93, 1–12. Villeneuve, L., Gisbert, E., Zambonino-Infante, J.L., Quazuguel, P., Cahu, C.L., 2005b. Effects of lipids on European sea bass morphogenesis: implication of retinoid receptors. British Journal Nutrition 94, 877–884. Zambonino, J., Cahu, C., Villeneuve, L., Gisbert, E., 2005. Nutrition, Development and Morphogenesis in Fish Larvae: Some Recent Developments, Aqua Feeds: Formulation and Beyond, pp. 3–5.