Luís E.C. Conceição

A review of the culture potential of Solea solea and S. senegalensis

A number of scientific studies have investigated aspects of soles(Solea soleaandS. senegalensis) ecology, population genetics and biology in their natural environment, and the species have been extensively studied in captivity during the last decade. Studies on the genetic population structure of sole indicate that several distinct breeding populations exist within its distributional range in European waters. Recent studies suggest a phylogenetic relatedness ofS. soleaand S. senegalensis, being found as closest sister lineages in most reconstructions. However, studies on molecular genetics and morphological traits give diagnostic differences that consistently lead to their taxonomic separation at the specific rank. Studies show that sole spawn readily in captivity, and the buoyant, fertilized eggs are easily collected. Stocking density during maturation should be 1–1.5kg/m2, and temperature should be kept above 16°C (S. senegalensis) or between 8 and 12°C (S. solea). In nature, the onset of spawning is related to a rise in temperature occurring during spring (March–June). Salinity should be kept constant around 33–35‰ and the fish reared under simulated natural photoperiod (LDN). In other cultured flatfish species, a change in the photoperiod is the key environmental signal used to manipulate and control maturation, but at present time there are no published work that verifies or contradicts this for either S. senegalensisor S. solea. Studies indicate that a mixture of inert and live food may increase the weaning success of sole fry, and this can be further enhanced by using attractants in the dry feed. Future experiments are needed to determine the ideal time to commence weaning and determine the minimum duration of this period. Studies on alternative feeding strategies are also required. The effect of temperature and photoperiod on juvenile growth has not been studied systematically in neither of the two species and the relative importance of a direct photoperiod effect on growth in sole therefore remains to be defined.

The supply of amino acids during early feeding stages of marine fish larvae: a review of recent findings

In marine fish larvae, the sum of protein deposition, turnover and catabolism necessary for their rapid growth dictates a high amino acid (AA) requirement. Once the yolk is exhausted, the digestive tract becomes the vital organ that ensures a steady supply of dietary AA to the growing larval tissues. In this paper, we discuss the demand and availability of AA (free and polymerised pools) in relation to larval digestive capacity. The sources of AA from compound and live diets are described, and the early regulatory roles of cholecystokinin (CCK) and a retrograde peristaltic activity are highlighted.

Amino acid requirements of fish larvae and post-larvae: new tools and recent findings

This paper reviews methodologies and recent findings in the study of the amino acid (AA) metabolism of fish larvae and post-larvae, in order to better understand the AA requirements. The larval indispensable AA (IAA) profile can be used as index of the IAA requirements. When turbot larvae and live food IAA profiles are compared, the profile of the latter seems to be deficient in some IAA. However, the larval IAA profile is only a rough indicator of AA requirements. A more precise estimate of the ideal dietary IAA profile implies the knowledge of the relative bioavailabilities of the individual AA, in particular, eventual differential rates of absorption and catabolism. Metabolic budgets (including unabsorbed AA, AA oxidation and AA retention) can be estimated using an in vivo method based on controlled tube-feeding of AA mixes containing a 14C-labelled AA. Results with fasted post-larval Senegal sole (Solea senegalensis) and fasted herring (Clupea harengus) larvae show a high retention of labelled doses of IAA (>60%) in the body, compared to catabolism as measured by liberated 14CO2 ( 40%) and a lower retention (<57%). So, from the onset of exogenous feeding, fish larvae have high catabolic losses of AA, but use DAA preferentially to IAA as energy substrates. A new method combining the use of 13C-labelled live food and 13C-NMR spectroscopy can be used to study simultaneously the relative bioavailability of several individual AA in fish larvae. In larval gilthead seabream (Sparus aurata) fed on rotifers, relative bioavailabilities (a combined measure of absorption efficiency and rate of catabolism) vary between AA being high for aspartate, glutamate and lysine and low for threonine. These estimates of relative biovailability of individual AA together with the IAA profiles of the larval seabream indicate that rotifers are deficient in threonine and leucine for larval seabream, threonine being the first limiting AA for protein synthesis. In order to define ideal IAA profiles for larval fish, further studies are needed on the factors affecting the relative bioavailability of IAA, such as species, age, developmental stage, temperature and the dietary nitrogen molecular form(s). Estimates of relative bioavailability of individual AA together with the IAA profile of the larval protein allow to determine the ideal dietary IAA profile for a given species.

In vivo studies of digestion and nutrient assimilation in marine fish larvae

Abstract This paper describes a method for quantifying the functionality of the digestive system in fish larvae. The system described can provide data for the gut absorption, oxidation and retention (assimilation) of nutrients. A tube-feeding setup (originally described in Aquaculture 116 (1993) (341–352) using 14 C-labelled dietary nutrients formed the basis of a new incubation system. This permitted unabsorbed nutrients evacuated from the gut to be distinguished from molecules originating from catabolism of the absorbed nutrient, both of which are present in the incubation water. The system is based on the release, transfer and entrapment of metabolically produced 14 C–CO 2 through manipulation of the water pH. The efficiency of the trap has been validated and tested, and provides 100.0±1.3% (S.D.) recovery. The usefulness of the method is demonstrated in a study in which Atlantic halibut post-larvae (46 days post first feeding) were fed a 14 C-labelled protein diet. These data show that this protein has a digestibility of 42% for halibut post-larvae. If oxidation had not been measured through the use of the CO 2 trap, digestibility would have been greatly underestimated (at about 25%).

Dietary lipid level affects growth performance and nutrient utilisation of Senegalese sole (Solea senegalensis) juveniles

Over the last few years, several aspects of Senegalese sole (Solea senegalensis) culture have been developed and optimised but the dietary lipid level for optimal growth has never been determined. Hence, five isonitrogenous diets (56 % dietary protein) with increasing dietary lipid levels (4, 8, 12, 16 and 20 % DM) were fed to satiation to triplicate groups of twenty fish (mean initial weight 10 g). Fifteen tanks were randomly assigned one of the five diets. Feed was distributed using automatic feeders, and fish were fed over a 16-week period. At the end of the experiment the fish fed on diets containing the two lowest dietary lipid levels (4 and 8 %) showed a 3-fold body-weight increase with a significantly higher daily growth index than fish fed higher lipid levels (1.2 v. 0.8). Moreover, these fish displayed a significantly lower dry feed intake (12 g/kg per d) and feed conversion ratio (1.0) compared with fish fed higher lipids levels (16-19 g/kg per d; feed conversion ratio 2.0). Low dietary lipid levels ( < 12 %) significantly improved nutrient retention and gain and hence growth, without major effects on whole-body composition. Despite the slight alteration in n-3 PUFA muscle content in the fish fed low-fat-diets, this fish fed low dietary lipid still remains a rich n-3 PUFA product and generally maintained its nutritional value. These results evidenced a low lipid tolerance of Senegalese sole juveniles and suggest a maximal dietary inclusion level of 8 % lipids for both optimal growth and nutrient utilisation without compromising flesh quality.

Long chain polyunsaturated fatty acid synthesis in a marine vertebrate: ontogenetic and nutritional regulation of a fatty acyl desaturase with Δ4 activity.

Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally characterized fatty acyl desaturase and elongase cDNAs corresponding to Δ4fad (with some Δ5 activity for the n-3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to higher or lower dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Δ4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Δ4fad was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway.

Dietary protein:lipid ratio and lipid nature affects fatty acid absorption and metabolism in a teleost larva

Studies with teleost larvae have reported poor performance associated with quantitative lipid imbalances in the diet. The present study examined the effect of dietary protein:neutral lipid ratio on fatty acid (FA) absorption efficiency and metabolism in larval Senegalese sole. In addition, the effect of lipid class (triolein (TRI) and l-3-phosphatidylcholine-1,2-di-oleoyl (PC)), carbon number and degree of saturation of the labelled NEFA, stearic acid (SA), oleic acid (OA) and DHA) was tested. FA absorption was determined by tube feeding [1-14C]-labelled lipids and NEFA after a single meal of either non-enriched Artemia (NEA) or Artemia enriched on a soyabean oil emulsion (EA), or after feeding these diets over an extended period of time (18 d). The tested dietary protein:lipid ratios had no short-term influence but long-term feeding of a diet higher in neutral lipid (EA) increased lipid accumulation within the gut epithelium and resulted in lower FA absorption (higher label evacuation and lower retention of dietary FA), which may partially explain the trend for lower growth observed with this diet. The lipids and NEFA, showed different digestive and metabolic properties, independent of feeding regime. FA absorption increased with unsaturation, being lowest for SA, followed by OA, and highest for DHA. In addition, sole larvae had a lower capacity to digest and absorb FA esterified to TRI, compared with PC, with the order of decreasing absorption being NEFA>PC>>TRI. Moreover, larvae appeared to discriminate between the source of OA, as this FA in the free form or esterified to PC was catabolised less than TRI.

Can We Predict Personality in Fish? Searching for Consistency over Time and across Contexts

The interest in animal personality, broadly defined as consistency of individual behavioural traits over time and across contexts, has increased dramatically over the last years. Individual differences in behaviour are no longer recognised as noise around a mean but rather as adaptive variation and thus, essentially, raw material for evolution. Animal personality has been considered evolutionary conserved and has been shown to be present in all vertebrates including fish. Despite the importance of evolutionary and comparative aspects in this field, few studies have actually documented consistency across situations in fish. In addition, most studies are done with individually housed fish which may pose additional challenges when interpreting data from social species. Here, we investigate, for the first time in fish, whether individual differences in behavioural responses to a variety of challenges are consistent over time and across contexts using both individual and grouped-based tests. Twenty-four juveniles of Gilthead seabream Sparus aurata were subjected to three individual-based tests: feed intake recovery in a novel environment, novel object and restraining and to two group-based tests: risk-taking and hypoxia. Each test was repeated twice to assess consistency of behavioural responses over time. Risk taking and escape behaviours during restraining were shown to be significantly consistent over time. In addition, consistency across contexts was also observed: individuals that took longer to recover feed intake after transfer into a novel environment exhibited higher escape attempts during a restraining test and escaped faster from hypoxia conditions. These results highlight the possibility to predict behaviour in groups from individual personality traits.

Dietary TAG source and level affect performance and lipase expression in larval sea bass (Dicentrarchus labrax)

The influence of dietary TAG source (fish oil, triolein, and coconut oil) and level (7.5 and 15% of the diet) on growth, lipase activity, and mRNA level was studied in sea bass larvae, from mouth opening until day 24 and from day 37 to 52. Fish oil and triolein induced better growth in both experiments, this being significant at a higher dietary level. Coconut oil significantly decreased growth at the higher level, possibly as the result of an excessive supply of medium-chain TAG. Growth was not related to lipase specific activity, suggesting a production in excess to dietary needs. Body lipid content was positively related to dietary lipid level and was affected by lipid quality. In addition, larval FA composition generally reflected that of the diet. The source of dietary lipid, but not the quantity, was shown to affect lipase activity significantly. Coconut oil diets induced the highest lipase activity, whereas the effect of fish oil was age dependent-it was similar to coconut oil at day 24 but induced the lowest lipase activity in 52-d-old larvae. The differential lipase response was probably caused by differences in the FA composition of the diet, related to the specificity of lipase toward FA differing in chain length and degree of saturation. No significant differences were found in lipase/glyceraldehyde-3-phosphate dehydrogenase mRNA, which suggests the existence of a posttranscriptional regulation mechanism.

Metabolic budgets for lysine and glutamate in unfed herring (Clupea harengus) larvae

Abstract Fish larvae are thought to have little control of their amino acid (AA) metabolism, leading to higher AA requirements than adult fish and mammals. Therefore, it is important to know to what extent fish larvae have the capacity to spare indispensable amino acids (IAA) at the expense of dispensable amino acids (DAA). This study intended to estimate a metabolic budget both for an IAA and a DAA in fasting herring larvae. A mix of crystalline amino acids solubilised in 1/3 seawater and added as either l - 14 C-glutamate or l - 14 C-lysine, was tube-fed to first feeding and 47-day-old (pre-metamorphosis) herring larvae. Both when glutamate and lysine were given in high and low doses, the following components of the metabolic budget were estimated: total AA assimilation, retention of AA in the free AA and in the protein pools, AA conversion into lipid and AA oxidation. First feeding herring larvae absorbed around 93% of both lysine and glutamate. However, 23% of the tube-fed lysine was oxidised and 70% was retained in the body, while for glutamate oxidation was as high as 76% and retention is only 17%. A similar picture was observed for 47-day-old larvae. Here, oxidation was 22% and 62% and retention 63% and 32%, for lysine and glutamate, respectively. Therefore, herring larvae use glutamate preferentially to lysine as an energy substrate from first feeding onwards. These results suggest that fish larvae have better capacity of regulating AA catabolism than previously believed.