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Background: Several miRNAs are known to control myogenesis in vertebrates. Some of them are specifically expressed in muscle while others have a broader tissue expression but are still involved in establishing the muscle phenotype. In teleosts, water temperature markedly affects embryonic development and larval growth. It has been previously shown that higher embryonic temperatures promoted faster development and increased size of Senegalese sole (Solea senegalensis) larvae relatively to a lower temperature. The role of miRNAs in thermal-plasticity of growth is hitherto unknown. Hence, we have used high-throughput SOLiD sequencing to determine potential changes in the miRNA transcriptome in Senegalese sole embryos that were incubated at 15 ºC or 21 ºC until hatching and then reared at a common temperature of 21 ºC. Results: We have identified 320 conserved miRNAs in Senegalese sole, of which 48 had not been previously described in teleosts. mir-17a-5p, mir-26a, mir-130c, mir-206-3p, mir-181a-5p, mir-181a-3p and mir-199a-5p expression levels were further validated by RT- qPCR. The majority of miRNAs were dynamically expressed during early development, with peaks of expression at pre-metamorphosis or metamorphosis. Also, a higher incubation temperature (21 ºC) was associated with expression of some miRNAs positively related with growth (e.g., miR-17a, miR-181-5p and miR-206) during segmentation and at hatching. Target prediction revealed that these miRNAs may regulate myogenesis through MAPK and mTOR pathways. Expression of miRNAs involved in lipid metabolism and energy production (e.g., miR-122) also differed between temperatures. A miRNA that can potentially target calpain (miR-181-3p), and therefore negatively regulate myogenesis, was preferentially expressed during segmentation at 15 ºC compared to 21 ºC. Conclusions: Temperature has a strong influence on expression of miRNAs during embryonic and larval development in fish. Higher expression levels of miR-17a, miR-181-5p and miR-206-3p and down-regulation of miR-181a-3p at 21 ºC may promote myogenesis and are in agreement with previous studies in Senegalese sole, which reported enhanced growth at higher embryonic temperatures compared to 15 ºC. Moreover, miRNAs involved in lipid metabolism and energy production may also contribute to increased larval growth at 21 ºC compared to 15 ºC. Taken together, our data indicate that miRNAs may play a role in temperature-induced phenotypic plasticity of growth in teleosts.

The present work examined the short- and long-term effects of three rearing temperatures on protein metabolism and growth trajectories of Senegalese sole larvae using 14C-labelled Artemia protein as feed. A first feeding trial was performed on larvae reared at 15, 18 and 21 °C (at 26, 17 and 14 days post-hatching (dph), respectively) and a second trial conducted on post-larvae after transfer to the same rearing temperature (~20 °C) (49, 35 and 27 dph, in larvae initially reared at 15, 18 and 21 °C, respectively). Temperature greatly influenced larvae relative growth rate (RGR) and survival, since growth at 15 °C was severely depressed. Protein digestibility and retention was highest at 18 °C during the first trial (85.35 ± 1.16 and 86.34 ± 2.33 %, respectively). However, during the second trial, post-larvae from 15 °C had the highest feed intake and protein digestibility (3.58 ± 1.54 and 75.50 ± 1.35 %, respectively), although retention was similar between treatments. Furthermore, after transfer to 20 °C larvae from 15 °C experienced compensatory growth, which was observed until 121 dph, and confirmed by RGR values, which were significantly higher at 15 ºC than at 21 ºC or 18 ºC. Results from the present study show that Solea senegalensis larval development, survival and protein digestion and retention are highly affected by thermal history.

Fertilised eggs of Senegalese sole were incubated at 15, 18 or 21 °C, and after hatching all larvae were reared at 21 °C until 30 days post-hatch. By this point larvae from the 18 or 21 °C temperature groups had 11 and 9% more muscle fibres than those from 15 °C, respectively. Hyperplastic growth during metamorphosis was higher in larvae from 18 °C. Embryonic temperature induced gene expression changes, albeit with a variable pattern throughout development. Myf5, myod2, myHC and fst mRNA levels were significantly higher at several stages prior to hatching in embryos incubated at 21 °C, whereas hsp90AB and hsp70 transcripts were present at higher levels in the 15 °C group. Myf5, myod1, myod2, pax7, myog, fst, igf-II, igf1r, hsp90AA and hsp90AB were expressed at higher levels during early development, particularly during somitogenesis. In contrast, mrf4, myHC, mylc2, igf-I, mstn1 and hsp70 were up-regulated at later stages of larval development, namely during and after metamorphosis. This study is the first example of thermal plasticity of myogenesis with prolonged effect in a flatfish.

In the present study, Senegalese sole eggs incubated at 20 °C were reared at three different temperatures (15 °C, 18 °C or 21 °C) during the pelagic phase, and then transferred to a common temperature (20 °C) from benthic stage until 100 days post-hatch (dph). Somatic growth, fast muscle cellularity and expression of 15 growth-related genes were compared at the same developmental stage for each temperature (pre-metamorphic larvae, metamorphic larvae, post-metamorphic larvae) and at 83 dph and 100 dph early juveniles. During pre-metamorphosis and metamorphosis, larvae from 21 °C weighed significantly more than those reared at 18 °C or 15 °C (P < 0.001). Relative growth rate (RGR) of pelagic larvae and survival of newly-settled larvae were also higher at 21 °C (P < 0.05). Furthermore, an increase in muscle growth towards the highest temperatures was observed concomitantly with an increase in gene expression, namely myogenic regulatory factors (MRFs), myosins, igf-I and fgf6. After transfer to a common temperature (20 °C), the 15 °C group initiated a process of compensatory growth, inverting relative growth rate values that became the highest, particularly between 83 dph and 100 dph (P < 0.05). This increased growth effort in the 15 °C group was accompanied by an up-regulation of gene expression in fast muscle, particularly in the 83 dph juveniles. Mrf4 and myHC were up-regulated at 15 °C and positive correlations with growth were also found for myog or pax7 (P < 0.05). In contrast, mstn1 was down-regulated at 15 °C (P < 0.05), suggesting a boost in muscle growth. By 100 dph, the 15 °C group had already reached the same weight as the 18 °C and the same length as the 21 °C ones. At this age, the 18 °C juveniles had the largest mean muscle fibre diameter (P < 0.001), and thus fibre hypertrophy seems to be a major growth mechanism relatively to the other groups. Our work shows that temperature during pelagic phase greatly influences the growth trajectory of Senegalese sole juveniles.

Texto integral disponível através do editor Myogenin (myog) encodes a highly conserved myogenic regulatory factor that is involved in terminal muscle differentiation. It has been shown in mammals that methylation of cytosines within the myog promoter plays a major role in regulating its transcription. In the present study, the Senegalese sole (Solea senegalensis) myog putative proximal promoter was identified and found to be highly conserved among teleosts. Therefore, it is plausible that it plays a similar role in controlling myog expression. Cytosine methylation of the myog promoter in skeletal muscle of Senegalese sole larvae undergoing metamorphosis was influenced by rearing temperature. A lower temperature (15°C) significantly increased myog promoter methylation in skeletal muscle, particularly at specific CpG sites, relatively to higher rearing temperatures (18 and 21°C). Myog transcription was downregulated at 15°C, whereas expression of dnmt1 and dnmt3b was upregulated, consistently with the higher myog methylation observed at this temperature. Rearing temperature also affected growth and fast muscle cellularity, producing larger fibers at 21°C. Taken together, our data provide the first evidence of an epigenetic mechanism that may be underlying the temperature-induced phenotypic plasticity of muscle growth in teleosts.

A large effort has been dedicated in the past years to the development of nutritional balanced inert diets for marine fish larvae in order to suppress the nutritional deficiencies of live feed. In this study growth performance, Artemia intake, protein digestibility and protein retention were measured for Senegalese sole (Solea senegalensis Kaup), in order to provide insight into how protein utilization affects growth performance. Three feeding regimes were tested: ST – standard live feed; ArtRL – live feed and 20%Artemia replacement with inert diet (dry matter basis) from mouth opening; ArtRH – live feed and 58%Artemia replacement with inert diet from mouth opening. Artemia intake and protein metabolism were determined at 6, 15 and 21 days after hatching using 14C-labelled Artemia protein and subsequent incubation in metabolic chambers. At the end of the experiment, sole fed exclusively with live feed were significantly larger than sole from Artemia replacement treatments. Protein digestibility decreased during sole ontogeny, and more sharply in ArtRH sole. Concomitantly retention efficiency increased during ontogeny but with a slight delay in ArtRH sole. Senegalese sole larvae growth and protein utilization is depressed when co-fed high levels of inert diet and Artemia, mostly during metamorphosis climax.

The present study intended to evaluate the effects of early introduction of inert diet in lipid digestibility and metabolism of sole, while larval feed intake, growth and survival were also monitored. Solea senegalensis larvae were reared on a standard live feed regime (ST) and co-feeding regime with inert diet (Art R). Trials using sole larvae fed with Artemia enriched with two different lipid emulsions, containing glycerol tri [1-14C] oleate (TAG) and L-3-phosphatidylcholine-1,2-di-[1-14C] oleoyl (PL), were performed at 9 and 17 days after hatching (DAH) to study lipid utilization. Co-feeding did not affect sole survival rates (ST 59.1 ± 15.9%; Art R 69.56 ± 9.3%), but was reflected in significantly smaller final weight at 16 DAH (ST 0.71 ± 0.20; Art R 0.48 ± 0.14 mg). Higher feed intake was observed in sole larvae fed on Artemia enriched with labeled PL at 9 DAH but not at 17 DAH. At 17 DAH, the smaller larvae (Art R treatment) ingested proportionally more Artemia in weight percentage, independently of enrichment. At 9 DAH lipid digestibility was equal among treatments and higher than 90%, while at 17 DAH it was higher in ST treatment (around 73%) compared to the Art R group (around 66%). Lipid retention efficiency at 9 DAH was higher in the Art R treatment, reaching values of 50%, while these values almost duplicated at 17 DAH, ranging up to 80% in both treatments without significant differences. These results show that co-feeding of live feed and inert diet from first-feeding in Senegalese sole has a toll in terms of growth and lipid digestibility but does not seem to compromise lipid metabolic utilization.

The objective of this workwas to evaluate the effect of a dietary amino acid imbalance, originating from the use of a soy protein concentrate (SPC) as the major protein source, on the growth performance and amino acid metabolism of Senegalese sole (Solea senegalensis) postlarvae. Senegalese sole (85.6724.6mg wet weight) were fed one of two experimental diets: one based on fish meal (FM) and another based on SPC. Diet were isonitrogenous (around 56% crude protein) and isoenergetic. Diet acceptability was very good and the growth rate was 6.9%day 1 for sole eating the FM diet and 6.0%day 1 for sole eating the SPC diet. Mass-speci¢c ammonia excretion and the activities of selected amino acid metabolic enzymes (ALAT, ASATand GDH) did not present significant differences between treatments, although this may have been due to the high variability found for these parameters in the SPC treatment. This variability may suggest different capacities of individual fish to adapt to the possible methionine dietary deficiency. The utilization of amino acids as a substrate for lipogenesis does not seem to be affected by the dietary protein source, since NAPDH-generating enzymes (G6PD and ME) had similar activities in both treatments. Amino acid metabolism in Senegalese sole postlarvae seems to be slightly affected by the dietary protein source. Nevertheless, the changes induced by the SPC diet do not seem to impair growth, at least at the high dietary protein level used in this experiment.

Dissertação de mest., Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009 Docosahexaenoic acid (DHA) is an essential fatty acid for marine fish larvae, with effects on growth and larval quality. DHA requirements for Senegalese sole (Solea senegalensis), a species of high commercial interest, are poorly understood. On this thesis a dose–response design in conjunction with a methodology based on tube feeding of 14C-[DHA], was employed in order to determine the dietary DHA requirements for Senegalese sole post-larvae. The effect of dietary DHA was examined on growth, survival, pigmentation and Artemia intake (at low light conditions). From 22 to 35 days after hatching (DAH) - pre-weaning period - post-larvae were fed frozen Artemia that had been enriched using one of four experimental emulsions containing four graduated concentrations of DHA. Final levels of DHA in the enriched Artemia metanauplii were 0.0 mg g-1DW (DHA-0), 5.1 mg g-1DW (DHA-5), 14.1 mg g-1DW (DHA-14) and 30.2 mg g-1DW (DHA-30). Afterwards and during the weaning period (41 to 61 DAH) all post-larvae were fed with a same moist feed. Significant differences in dry weight (DW) were found at the end of the pre-weaning period. Treatment DHA-0, induced a higher larval growth (2.84±1.04 mgDW) than treatment DHA-5 (2.38±0.74 mgDW). Light intensity influenced both final DW and RGR and there was a positive correlation for a higher growth (DW and RGR) as the light intensity increased. On weaning period treatments DHA-0 and DHA-30 showed significant higher DW and total length values than those presented in DHA-5 and DHA-14. On the radiolabelled Artemia intake test, and after 30 min in presence of Artemia (T30), larvae ingested significantly higher amounts than observed at 10 min, and a positive correlation between dietary DHA levels and Artemia fed within the T10 groups was also found. Tube feeding of [1- 14C]DHA resulted in no significant differences for absorption, retention and catabolism between larvae submitted to different experimental treatments. However, a slight tendency for an increase in the percentage of catabolised DHA with increased DHA dietary supply was also noted, with an apparent increase in catabolism between treatment DHA-5 and DHA-14. No major effects of dietary DHA levels ranging from 0.0 to 30.2 mg.g-1Artemia DW between 22 and 35DAH could be observed on larval performance. However, light intensity probably had an impact on post-larvae performance, mainly at high intensities. Furthermore, the tube feeding trial suggested a possible DHA requirment between 5.1 and 14.6 mg g-1 of Artemia dry weight.

Dissertação mest., Aquacultura e Pescas, Universidade do Algarve, 2009 The general aim of this study was to understand the underlying factors responsible for the individual variation in growth of juvenile Senegalese sole (Solea senegalensis). Senegalese sole is a species of high commercial value and high interest for aquaculture in Europe, therefore it would be important to understand these individual variations. Understanding the causes for individual variation in growth in relation with the feed intake, metabolic rate and response to stress contributes to maximize the production efficiency by reducing food waste and improving water quality. In order to identify the possible factors responsible for the individual variation in sole growth, its relationship with metabolic rate, stress response and feed intake was determined. Twelve fish with 18.49 ± 2.94g of initial weight were kept in standard conditions and feed once a day, during a period of six months. Individual oxygen consumption measurements in Sole juveniles were determined by flow-through respirometry at standard conditions, after the fish were fed a single meal and after a stressful condition. Blood samples for plasma cortisol, were taken from all fish at the start of the experiment (control, indicative of basal levels) and after stress exposure. Quantification of individual feed intake was performed by incorporation with radio-opaque ballotinis glass beads in a dry feed, followed by radiography to quantify the amount of ingested pellets. Individual fish exhibited pronounced variation in growth (final weight ranging from 30.45 to 67.53g), oxygen consumption of fasted fish (108.0 to 447.4 μmol/g/h), oxygen consumption of fed fish (174.7 μmol/g/h to 813.5 μmol/g/h), oxygen consumption after emersion stress (186.8 μmol/g/h to 376.7 μmol/g/h), and plasma cortisol after emersion stress (4.81 ng/ml to 46.45 ng/ml). Individual differences in metabolic rate do not contribute to explain individual differences in growth. However, after emersion stress individual differences in stress response seem to play a role in explaining individual differences in growth, what may result from different copying styles. Sole feed intake as measured by X-ray does not seem to be a good indicator of growth rate. The oxygen consumption increases both when fish are fed and after an emersion stress, as result of additive costs of growth and stress to the cost of maintenance. All together, this study suggests that individual variation of growth in Solea senegalensis is essentially due to genetic variation, and its reduction may accomplished when genetic selection programs are adopted. Furthermore, a study with a larger number of fish and over a larger time period would be important to check some of the trends found in this thesis between growth potential, metabolic rate and coping styles of Senegalese sole.