Aquaculture nutrition, tập 18, số 6, 2012

Aquaculture Nutrition 2012 18; 581–588 1 doi: 10.1111/j.1365-2095.2012.00936.x 1,2 CINVESTAV IPN Unidad Me´rida, Laboratorio de Biologı´a y Cultivo de Moluscos, Me´rida, Yucata´n, Me´xico; des Sciences, Ancien logement des maıˆtres, La Rosie`re, Lamentin, Guadeloupe, FWI, France One of the bottlenecks for the queen conch, Strombus gigas, aquaculture is the lack of well-adapted formulated food for optimal growth The goals of this study were to analyse the digestive gland structure of conchs fed with different diets using histochemical techniques and to evaluate the growth and survival of S gigas juveniles with nine formulated diets (protein from 190 to 380 g kgÀ1 and lipids from 26 to 82 g kgÀ1) Proteoglycan granules and acidophilic granules were detected in the digestive cells The abundance of both granule types was variable, according to the nutritional state of the animals The granular content of the digestive cells of conchs fed with artificial diets was scarce when compared with conchs fed on natural food Of the nine formulated feeds, the diet with 365 and 45 g kgÀ1 of protein and lipids, respectively, gave the best growth in weight (0.20 g dayÀ1) and was also associated with digestive cells in the best condition as determined histologically Histochemical analysis of the digestive gland differentiated with Alcian blue staining determines the nutritional status much better than a simple growth index and is therefore more useful in assessing adjustments to the feed formulation to meet the real needs of conchs KEY WORDS: aquaculture, Caribbean, diets, feeding, molluscs, physiology Received 29 January 2011; accepted 14 October 2011 Correspondence: D.A Aranda, CINVESTAV IPN Unidad Me´rida, Laboratorio de Biologı´a y Cultivo de Moluscos, Km antigua carretera a Progreso CP 97310 Me´rida, Yucata´n, Me´xico E-mail: daldana@ mda.cinvestav.mx ª 2012 Blackwell Publishing Ltd Archipel The queen conch, Strombus gigas, (Linnaeus, 1754) is a marine resource of ecological and economic importance in the Caribbean (FAO 2007) Since pre-Columbian time, queen conchs have been an important source of food for the inhabitants of Caribbean coasts and islands (Wing 2001) However, queen conch meat that was a popular staple food is now mostly consumed as a tourist delicacy It is an important source of income in several exporting countries and is an overexploited fishery (Theile 2001) As populations have been declining for several decades, much of the current research focuses on aquaculture, restocking and transplanting techniques to help replenish wild conch populations Queen conch aquaculture has been developed in the Turks and Caicos (to expand conch production farm and to license grow-out farms throughout the Caribbean) and in Florida Harbor Branch Oceanographic Institution (Davis 2000; Shawl et al 2008) Even though queen conch aquaculture is a success in terms of hatchery spat production, growth still depends on the use of large areas of wild environment (Davis 2000) One of the bottlenecks for intensive conch farming is the lack of formulated food for optimal growth in hatchery at a reasonable price (Shawl & Davis 2006; Shawl et al 2008) There is a need to improve husbandry techniques for the grow-out of juveniles with diets that allow a growth rate equal to or higher than that of wild juveniles Moreover, the use of prepared feeds can be very practical as formulations can be manipulated to obtain an optimum nutritional value Furthermore, they are available on demand and if properly prepared may be stored for a longer time period (Bautista-Theurel & Millamena 1999) Aldana Aranda et al (1996) reported a growth rate of 0.31 mm dayÀ1 with Frippak No (480 g kgÀ1 of protein) Glazer et al (1997) fed conchs with Koi fish food and macroalgae, obtaining a growth rate of 0.34–0.35 mm dayÀ1, respectively Rathier (1987), Iversen & Jory (1997) and Aldana Aranda et al (2005) obtained growth rates of 0.16, 0.23 and 0.31 mm dayÀ1, respectively in wild juvenile queen conch fed on natural food Most studies on the digestive tract and digestive gland of microphagous prosobranch Gastropods have been performed on species living in intertidal environments in order to investigate the influence of tidal variations upon the digestive gland cycle (Nelson & Morton 1979) Queen conch natural feed is a complex mixture of macroalgae, microbenthos and biofilm involving ingestion of sediment (Stoner & Waite 1991; Shawl et al 2008; Serviere et al 2009) However, real nutrient requirements for queen conch, in terms of energy level, protein and micronutrients, are unknown (Amber et al 2011) Furthermore, shell growth and weight are not precise indices of optimal growth and assimilation (Lucas & Beninger 1985) The goals of this study were to compare the status of the digestive gland of juvenile queen conch fed with different formulated diets with increasing levels of proteins and lipids using histochemical techniques and to evaluate the growth and survival of conchs fed with these diets An experimental aquaculture facility was set up at Xcaret Marine Park, south of Cancun (Mexico), to raise juvenile queen conch received from Ocean Reef Aquarium society (ORA) in Fort Pierce, Florida Experimental cultures were set up in 45-L raceways with a density of 1.5 conchs LÀ1 Aquaria were filled with sand-filtered sea water (5 lm) which was continuously oxygenated using an air pump Sea water was renewed (4 times dayÀ1), and raceways were cleaned twice a week to avoid the development of microalgae and biofilm which could be used as a food source Juvenile queen conchs were fed 150 mg of diet per conch each day for months, and the uneaten food was removed daily Two experimental treatments and a control were set up The experimental treatments were as follows: (i) conchs were given formulated feed (diets 1, 3, 4, 5, and 9, see Table 1) for 84 days and (ii) conchs were given formulated feed (diets 2, and 8) for 84 days and then returned to a natural diet (a biofilm of 50% of red and green algae and 50% of seagrass and sand) for 21 days The main ingredients used in these formulated diets were fish meal, soy flour, wheat flour, spiruline, corn starch, fish oil, vegetable oil and soy lecithin Each of the nine diets was tested using three replicates of 30 conchs each, giving a total of 810 conchs on experimental diets The control comprised two replicates of 30 conchs each (n = 60 conchs), which were kept on a natural food diet (a biofilm of 50% of red and Table Biochemical composition of experimental diets used to feed juveniles of Strombus gigas Biochemical composition (g kgÀ1) Diets Protein Lipids Crude fibre Ash NFE 193 190 190 277 278 327 365 373 381 26 66 79 35 55 69 45 56 82 04 04 04 08 08 14 09 13 10 71 61 66 87 90 97 111 111 109 706 679 661 593 569 493 470 447 418 NFE, nitrogen-free extract green algae and 50% of seagrass and sand) for 84 days The nine formulated diets were tested (Table 1), containing levels of protein from 190 to 380 g kgÀ1 and levels of lipids from 26 to 82 g kgÀ1 The nutritional status of queen conchs was analysed using the following indices: siphonal length, flesh weight and histological features of the digestive gland Juveniles of S gigas were measured and weighed at the beginning of the experiment (T0) and at days 21, 42, 63 and 84 At the beginning of the experiment, juveniles from the wild, in the same size range as the conchs from ORA, were dissected and prepared for the histological analysis of the digestive gland Likewise, three juvenile queen conch from the Florida hatchery were also analysed before the start of the experiment, and another three juveniles of each replicate receiving one of nine diets (n = conchs) were examined at the end of the experiment Histological examination involved cutting the visceral mass of each individual into two sections: a distal part, containing only digestive gland and connective tissue, and a mid part also containing stomach Sampled sections were fixed in alcoholic Bouin fluid and processed using standard histological techniques (Gabe 1968; Luna 1969) After dehydration in ethanol series, and clearing with Clarene, the sections were embedded in Paraplast wax Sections of lm thick were stained with a trichrome stain following Gabe (1968) which included Alcian blue (Hycel de Mexico, SA de Cv Zapopan, Jalisco, Mexico) at pH 2.5 to differentiate proteoglycans Slides were also treated by the Periodic acid-Schiff (PAS) reaction for glucide detection (Gabe 1968) The slides were examined and pictures were taken with a Nikon DXm 1200F digital camera mounted on a Nikon microscope All the pictures were corrected for contrast and colour (Photoshop software, Adobe Photoshop CF version 9.0, San Jose, CA, USA) For every juvenile, two microscope slides were prepared with five sections per slide (distal part) The Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd incidence of blue granules (Gros et al 2009) was obtained by counting the total number observed in three fields of the five sections under 409 magnification and calculating the mean and standard deviation for each diet A feed index was established as the sum of blue granules counted in the large cells of adenomers These counts were transformed into granule area (lm2), using the circle area formula for the granules divided by the image area (lm2) observed on histological slides of digestive glands at 40 magnification (which was always 37 368 lm2), and multiplied by 100 The blue colour of the granules observed in the digestive cells after staining with Alcian blue indicated that they were proteoglycan components Such conspicuous secretory granules are good markers that demonstrate the digestive cell secretion being delivered to the stomach One microscope slide was prepared with the mid part (containing stomach) as a control of histochemical analysis The stomach epithelium that contains mucocytes stained blue A non-parametric Tukey test (Sokal & Rohlf 1995) was used to test for significant differences (P < 0.05) among diets in the feed index and growth rates in siphonal length and whole body weight In wild juvenile conchs, the digestive gland has an array of adenomers (Fig 1a) similar to those described in Figure (a) Digestive gland from a wild juvenile with primary ducts and adenomers; (b) primary duct with two types of epithelium; low and simple ( ) similar to the secondary ducts epithelium and plicate ciliated epithelium (Δ) typical of primary ducts; (c) stomach wall with mucocytes (◊) stained by Alcian blue that constitutes a positive control for various diets as mucocytes are stained blue even if the digestive gland granules are not; (d) adenomers of a digestive gland section containing with the two cell types Large digestive or secreting cells with large blue granules (star) and crypt cells with sporozoa-like microorganisms belonging to the Apicomplexa group (arrow) Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd adults All these secreting structures are connected to small ducts, which join larger ducts attached to the stomach The small secondary ducts are lined with a simple epithelium composed of a single cell type The larger primary ducts have two areas (Fig 1b), one similar to the small duct epithelium devoid of cilia and another composed of ciliated cells and mucocytes The connective tissue surrounding the digestive gland possesses two characteristic cell types, small round amoebocytes stained red by PAS and blue granules in the large cells stained blue by Alcian blue Both may play a role in the transfer of metabolites The functional glandular structure comprises two cell types (Figs 1d & 2a): tall (80 lm) and narrow ( 0.05) in survival, between group a and b, were evident during the experiment, while group c larvae showed a significantly (P < 0.05) lower survival relative to both groups a and b starting from day ph till the end of the experiment On day ph, a significant (P < 0.05) increase in TL was only observed in group B larvae relative to control (group A: 3.90 ± 0.20 mm; group B: 4.24 ± 0.10 mm; and group C: 4.16 ± 0.10 mm, respectively) Concerning total weight, both group B and C showed a significant (P < 0.05) increase in body weight relative to control (group A: 1.70 ± 0.10 mg, group B: 2.10 ± 0.11 mg and group C: 1.97 ± 0.12 mg, respectively) at this same sampling point On day 10 ph, a significant increase (P < 0.05) in both TL and body weight of group B larvae, relative to group A and C, was detected (group B: 6.83 ± 0.21 mm, 6.56 ± 0.21 mg; group A: 6.43 ± 0.20 mm; 6.10 ± 0.20 mg; and group C: 6.11 ± 0.20 mm and 5.8 ± 0.22 mg, respectively) Larval total lipid content at days ph resulted similar among the experimental groups (80 lg per larva) On the contrary, at 10 dph, larval total lipid content was higher in the control group (120 lg per larva) and resulted progressively reduced in the other experimental groups (80 lg per larva, group B and 40 lg per larva, group C, respectively) In Table are reported EPA, DHA, DHA/EPA and PUFA x3 levels in live preys (A) and in A polymnus larvae sampled at and 10 days ph (B) From Table 1A, it was evident that Acartia tonsa nauplii originated from months cold-stored eggs had lower content in EPA, DHA, DHA/ EPA and PUFA x3 respect to rotifers and nauplii obtained from a continuous A tonsa culture However, considering the second feeding period (days 7–10 ph), the lowest EPA, DHA and DHA/EPA levels were observed in Artemia nauplii, while the lowest PUFA x3 in copepodites/copepods originated from months cold-stored Acartia tonsa eggs Considering the lipid profiles obtained from the fish larvae fed different diets (Table 1B), at day ph, an higher DHA content and a higher DHA/EPA ratio was detected in larvae fed copepod nauplii obtained from the continuous culture (group B) relative to A and group C larvae At day 10 ph, A polymnus larvae fed copepods hatched from month cold-stored eggs showed the lowest EPA, DHA and PUFA x3 levels, while group A and B did not reveal major differences except for total x3 levels reflecting, at this sampling time those of the live preys used for the feeding trial Aquaculture Nutrition 18; 685–696 ª 2012 Blackwell Publishing Ltd Table A, B – Acartia tonsa nauplii originated from months cold-stored eggs had lower content in eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), DHA/EPA and PUFA x3 respect to rotifers and nauplii obtained from a continuous A tonsa culture However, considering the second feeding period (days 7–10 ph), the lowest EPA, DHA and DHA/EPA levels were observed in Artemia nauplii, while the lowest PUFA x3 in copepodites/copepods originated from months cold-stored Acartia tonsa eggs At day ph, a higher DHA content and a higher DHA/EPA ratio was detected in larvae fed copepod nauplii obtained from the continuous culture (group B) relative to A and group C larvae (B) At day 10 ph, A polymnus larvae fed copepods hatched from month cold-stored eggs showed the lowest EPA, DHA and PUFA x3 levels, while group A and B did not reveal major differences except for total x3 levels reflecting, at this sampling time those of the live preys used for the feeding trial (A) Feeding period 1–7 dph Fatty acid% Enriched rotifers EPA DHA DHA/EPA Iw3 5.2 22.9 4.4 33.1 ± ± ± ± 0.05 1.25 1.25 1.32 Feeding period 7–10 dph AT nauplii months A.T nauplii Enriched Artemis A.T copepodites /adults months A.T copepodites /adults 3.5 6.4 1.82 12.34 1.4 2.1 1.5 7.3 1.9 2.2 1.1 40.1 5.1 16.2 3.17 26.4 4.1 7.5 1.82 16.8 ± ± ± ± 0.02 0.03 0.03 ± ± ± ± 0.07 0.04 0.08 0.09 (B) dph ± ± ± ± 0.05 0.07 0.08 1.05 ± ± ± ± 0.08 0.06 0.1 1.2 ± ± ± ± 0.07 0.06 0.09 0.09 10 dph Fatty acid % Group A Group B Group C Group A Group B Group C EPA DHA DHA/EPA Iw3 1.32 3.87 2.94 10.63 0.98 4.39 4.46 8.13 1.23 2.87 2.33 7.12 2.21 8.64 3.91 21.13 1.57 8.97 5.70 13.42 0.57 2.36 4.16 3.75 ± ± ± ± 0.23 0.99 0.04 1.04 ± ± ± ± 0.40 0.92 1.00 0.80 On day ph, a significant higher level (P < 0.05), because of a positive effect of copepod feeding, of IGFI, TR-a and -b gene expression (compared to control, group A) was evidenced in group B larvae fed cultured copepods (Figs 2a & 3a,c), while no significant differences were observed for PPAR-aÀb relative to control (Fig 4a,c) About group C larvae (5 days ph), fed on copepods hatched from coldstored eggs, a significant higher level (P < 0.05) in gene expression, relative to control, was observed for all the genes analysed except for PPAR-a, PPAR-b and TR-a (P > 0.05) (Figs 2a,c, 3a,c & 4a,c) At day 10 ph, group B larvae only showed a significant higher level (P < 0.05) in TR-b and IGF I gene expression relative to control (group A), while group C larvae, fed copepods hatched from coldstored eggs, only showed a significant higher level (P < 0.05) of IGF I gene expression (Figs 2b, 3b,d & 4b,d) Finally, group B larvae showed a significant (P < 0.05) lower level in MSTN gene expression relative to control during both sampling times, while group C showed a significant decrease only at day 10 ph (Fig 2c,d) It is well established that copepods are a valuable source of food for marine aquaculture not only for their biochemical Aquaculture Nutrition 18; 685–696 ª 2012 Blackwell Publishing Ltd ± ± ± ± 0.70 0.82 1.07 0.58 ± ± ± ± 0.70 0.72 1.40 ± ± ± ± 0.40 0.67 0.78 1.07 ± ± ± ± 0.18 0.31 0.35 0.77 characteristics that match the nutritional requirements of the larvae but also for their small size and typical movement that may improve the predatory activity in fish larvae (Delbare et al 1996; Sargent et al 1997, 1999; Støttrup et al 1999; Payne & Rippingale 2001; Drillet et al 2011) In fact, several studies have shown that the inclusion of copepods in the larval fish diet may reduce mortality, deformities and in turn improve the quality and the number of the reared species (Støttrup 2000) For example, the inclusion of harpacticoid or calanoid copepods was able to improve growth and survival in both ornamental species such as clownfishes (Olivotto et al 2008a, 2010), the long snout seahorse (Olivotto et al 2008c), the baggai cardinal fish (Vagelli 2004), and the lemonpeel angelfish (Olivotto et al 2006a) and several finfish such as turbot (Støttrup & Norsker 1997), golden snapper (Schipp et al 1999) and flounder (Støttrup & Holmstrup 2004) At date, while methods for consistently produce small batches of copepods have been reported for several species, a reliable production of large numbers of copepods is still difficult (Murray & Marcus 2002; Drillet et al 2011; Olivotto et al 2011b) In fact, owing to a lack of knowledge about large scale cultivation of copepods, fish farms using copepods generally harvest them in situ and in large outdoor tanks under semi-controlled conditions (van der Meeren & Naas 1997; Sørensen et al 2007) However, this production may (a) (b) (c) Figure Insulin-like growth factors I (IGF I) (a, b) and myostatin (MSTN) (b, c) gene expression in and 10 days ph larvae fed standard rotifers and Artemia diet (group a), copepods of a continuous culture (group b) and copepods originated from months cold-stored eggs (group c) While group b larvae showed a significant increase in IGF I gene expression both at and 10 days ph sampling, group c larvae showed a significant increase in its gene expression only at days ph Myostatin (MSTN) showed, in group b larvae, a significant decrease in its gene expression during both samplings, while in group c, a significant lower gene expression was detected only at day 10 ph Values with different letters indicate statistical significance (P < 0.05) (d) (a) (c) (b) (d) result very instable and could limit the annual fish production as well as improve the possibility to introduce in the rearing systems parasites that use copepods as an intermediate host (Olivotto et al 2010) Figure TR-a (a, b) and TR-b (b, c) gene expression in and 10 days ph larvae fed standard rotifers and Artemia diet (group a), copepods of a continuous culture (group b) and copepods originated from months cold-stored eggs (group c) TR-a gene expression was significantly higher then control only in days ph group b larvae Considering TR-b gene expression, it was significantly higher than control during both and 10 ph samplings in group b larvae, while group c larvae only showed a significantly higher gene expression at days ph Values with different letters indicate statistical significance (P < 0.05) A valid alternative may thus be represented by the development of viable techniques for long-term storage of copepod eggs Both subitaneous and diapauses eggs have been investigated for storage and use in aquaculture (Støttrup Aquaculture Nutrition 18; 685–696 ª 2012 Blackwell Publishing Ltd (a) (c) et al 1999; Murray & Marcus 2002; Drillet et al 2006a,b; Peck & Holste 2006; Ohs et al 2009) As a successful induction of mass production of diapauses eggs has never been reported, particular attention has been given to the cold storage of subitaneous eggs These eggs are easily produced, occur during normal reproduction and not need to remain in a refractory phase before hatching (Peck & Holste 2006) In particular, cold storage of A tonsa eggs has been deeply investigated in the last years, and results obtained in different laboratories suggest that eggs remain viable for several months (Drillet et al 2006a,b) and that their viability decreases linearly by 4% every 20 days of storage (Peck & Holste 2006) This information suggests that subitaneous A tonsa eggs can be stored for periods of time, which could prove useful for feeding fish larvae It is well established that the quality of cold-stored eggs decreases during the storage period, especially considering their fatty acid content (Drillet et al 2006a,b, 2007) Currently, there is no reported research investigating the effects of fatty acids modification in copepods hatched from coldstored eggs on larval fish survival and development For this reason, the aim of the present study was to compare the effects of a standard rotifers/Artemia diet to one based on A tonsa produced through a continuous small scale system and a diet based on A tonsa hatched from months cold-stored eggs In the present study, concerning survival, no significant differences between the three experimental Aquaculture Nutrition 18; 685–696 ª 2012 Blackwell Publishing Ltd (d) * Figure Peroxisome proliferator-activated receptors (PPAR)-a (a, b) and PPAR-b (b, c) gene expression in and 10 days ph larvae fed standard rotifers and Artemia diet (group a), copepods of a continuous culture (group b) and copepods originated from months cold-stored eggs (group c) Only a significant decrease in PPAR-b gene expression was detected in group c larvae at 10 days ph respect to control Values with different letters indicate statistical significance (P < 0.05) (b) groups were observed till day ph This can be related to the fact that larvae fed copepods were fed one-third and half of the number rotifers and Artemia provided to control group However, after this time, group C larvae, fed copepods hatched from cold-stored eggs, showed a significant lower survival relative to control and group B larvae even if morphometric measurements (TL, WW) evidenced that at days ph, both group B and C larvae showed better growth performances relative to control However, at 10 days ph, the larvae fed copepods hatched from coldstored eggs (group C) resulted in the lowest TL and WW as well as in the lowest total lipid content As recent studies showed a positive role of HUFAs administration on larval fish growth and survival (Evjemo et al 2003; Vagelli 2004; Avella et al 2007; Olivotto et al 2008a,b, 2009, 2010, 2011a,b), the different survival and growth rates observed in the three experimental groups may be related to the different fatty acid composition of live preys used in this study This is in accord with the fact that the gut system of fish larvae has initially high assimilation capability of fatty acids and low protein digestibility, remarking the importance of fatty acids in this early part of larval history In fact, during the experiment, a relationship between larval survival, growth, metamorphosis and fatty acid composition of live preys and larvae can be observed Lower growth and survival can thus be related to fatty acid deficiencies, which are known to cause a general decrease of larval health, poor growth, low feed efficiency, anaemia and high mortality (Sargent et al 1999; Bell et al 2003; Olivotto et al 2003, 2005, 2006a,b; Faulk & Holt 2005) As growth and ontogeny and the addition of new/ improved physiological competence follow a well-defined and genetically programmed sequence in which gene transcription and hormone regulation play a crucial role, molecular data may be useful to monitor fish growth and development Consequently, gene expression can be used to generate useful insights linking biotic and abiotic conditions and individual’s performance The search for molecular markers can be approached looking for them among those genes whose expression could reasonably be modified by the different conditions, including nutrition (Carnevali et al 2006; Olivotto et al 2009; Avella et al 2010, 2011) It is well known that fish growth is controlled by the complex processes of myogenesis, which are regulated by several extrinsic regulators such as the growth factors IGF and MSTN Final growth is thus determined by the interplay of these positive and negative signals As an up-regulation of IGFs gene expression and a down-regulation of MSTN gene expression has recently been observed in clownfish larvae fed a diet rich in HUFAs and with high DHA/EPA ratio (Avella et al 2007; Olivotto et al 2011a,b), the differences in IGF I and MSTN gene expression observed among the different experimental groups may be related to the different fatty acid composition of the larvae and their energy status In fact, HUFAs are able to act directly on the genome, via specific nuclear receptors, the PPARs, involved in several biological processes such as skeletal development during ontogenesis, adipogenesis, lipid homoeostasis, lipid metabolism regulation, (Burdick et al 2006) and are thus considered good fatty acid sensors (Grimaldi 2007; Learver et al 2008) The role of PPARs in lipid metabolism regulation is evident also in the present study In days ph-larvae, the x-3 fatty acid percentage and the total lipid content did not show significant differences among the experimental groups and, as a consequence, no differences in PPAR-a, -b gene expression were detected On the contrary, on day 10 ph, the lowest PPAR-b expression was detected in group C fed copepods originated from cold-stored eggs that showed the lowest total lipid content, and the highest mortality Also, time to metamorphosis was affected by the diet in this study The requirement of nutritional reserves to undergo metamorphosis has been confirmed in a variety of marine organisms (Youson 1988; Pechenik & Cerulli 1991; Pfeileret 1999; Olivotto et al 2011a) with thyroid activity considered crucial at this regard As TRs mRNA levels are directly modulated by thyroid hormones concentrations (Olivotto et al 2011a) and as the highest TRs levels occur simultaneously with metamorphosis climax (Yamano & Miwa 1998; Galay-Burgos et al 2008), we can hypothesize that the higher TRs gene expression detected in 10 days ph group B larvae relative to group A and C caused the earlier metamorphosis in this group Subitaneous A tonsa eggs can switch to a quiescent stage when transferred to low temperatures undergoing important biochemical changes that can negatively affect their viability to hatch and their biochemistry This study demonstrated that after a months cold storage period, the quality of copepods obtained from those eggs was suboptimal for A polymnus larval rearing In fact, larvae fed those copepods showed lower growth and survival performances respect to larvae fed on a standard rotifer/Artemia diet or on A tonsa obtained from a continuous culture However, further research should both focus on the optimization of new storage techniques and on the search of new microalgae with better fatty acid profiles to improve the quality of copepod eggs and nauplii Special thanks to Beatrice Migliarini and Francesca Maradonna for their help at various stages of this study Funding for this study was provided by the ‘Fondi di Ateneo 2010’ to Ike Olivotto Ackman, R.G 1980 Fish lipids, Part In: Advances in Fish Science and Technology (Connell, J.J ed.), pp 86–103 Fishing News Books, Farnham, UK Avella, A.M., Olivotto, I., Gioacchini, G., Maradonna, F & Carnevali, O (2007) The role of fatty acids enrichments in the larviculture of false percula clownfish Amphiprion ocellaris Aquaculture, 273, 87–85 Avella, M.A., Olivotto, I., Silvi, S., Place, A.R & Carnevali, O (2010) Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish Am J Physiol Regul Integr Comp Physiol., 298, R359–371 Avella, M.A., Olivotto, I., Silvi, S., Ribecco, C., Cresci, A., Palermo, F., Polzonetti, A & Carnevali, O (2011) Use of Enterococcus faecium to improve common sole (Solea solea) larviculture Aquaculture, 315, 384–393 Aquaculture Nutrition 18; 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