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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.

Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut.

Amino acids (AA) regulate key metabolic pathways, including some immune responses. Therefore, this study aimed to assess whether an increased availability of dietary AA can mitigate the expected increase in plasma cortisol and metabolites levels due to high stocking density and its subsequent immunosuppression. Senegalese sole (Solea senegalensis) were maintained at low stocking density (LSD; 3.5 kg m−2) or high stocking density (HSD; 12 kg m−2) for 18 days. Additionally, both treatments were fed a control or a high protein (HP) diet (LSD, LSD HP, HSD and HSD HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. HSD was effective in inducing a chronic stress response after 18 days of treatment since fish held at HSD presented higher plasma cortisol, glucose and lactate levels. Moreover, this increase in stress indicators translated in a decrease in plasma lysozyme, alternative complement pathway (ACP) and peroxidase activities, suggesting some degree of immunosuppression. Interestingly, while plasma glucose and lactate levels in HSD HP specimens decreased to similar values than LSD fish, plasma lysozyme, ACP and peroxidase activities increased, with even higher values than LSD groups for ACP activity. It is suggested that the HP diet may be used as functional feed since it may represent a metabolic advantage during stressful events and may counteract immunosuppression in sole.

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.

The present study aimed to assess the effects of increased availability of dietary amino acids (AA) on brain monoamine neurotransmitters and the metabolic processes resulting from stressful situations in fish. Senegalese sole (Solea senegalensis) juveniles (24.2 ± 0.4 g wet mass) were weekly subjected to an acute handling stressor (HDLG) or remained undisturbed (CTL). Additionally, both treatments were fed a control or a high protein (HP) diet (CTL, CTL HP, HDLG and HDLG HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. Repeated handling induced a stress response after 14 and 28 days in fish held at both HDLG and HDLG HP treatments. While dietary treatment and handling stress activated the serotonergic system at 14 days, these effects were not observed after 28 days. In addition, the HP diet minimized the decrease in plasma indispensable AA due to repeated handling stress after 28 days. It was concluded that HP diet decreased post-stress plasma glucose and lactate levels in HDLG HP specimens only at 14 days of treatment. Moreover, dietary treatment was also effective in stimulating DA synthesis and release, thus dietary phenylalanine supplementation can increase DA biosynthesis in fish.

The use of proteomics in fish research is at a relatively early stage compared to terrestrial vertebrates. It has already been applied in the field of fish nutrition, enabling to point out metabolic changes occurring in response to dietary manipulations such as a variation in energy content, dietary nitrogen compostion, lipid composition or the incorporation of plant protein sources in aquafeeds. NR, M de V and TSS acknowledge financial support from Fundação para a Ciência e Tecnologia, Portugal, through grants SFRH/BDP/65578/2009, SFRH/BD/40698/2007 and SFRH/BD/41392/2007 respectively. The experiments presented here were partly funded by Fundação para a Ciência e Tecnologiam through projects PDTC/MAR/105152/2008 (SPECIAL_K), PPCDT/MAR/61623/2004 (SAARGO), PTDC/MAR/716851/2006 (HYDRAA), prize CERATONIA given by Caixa Geral de Depósitos and University of Algarve, and Research Council of Norway through project 165203/S40.

Temperature is a factor known to have a marked influence on metabolism and consequently on development, inducing ontogenetic plasticity in fish larvae. In this study, three different temperatures (15, 18 and 21°C) were assayed during Solea senegalensis embryonic development prior to hatching. After hatching all larvae were reared at the same temperature (21.0 ± 0.1°C) until 30 days. We observed that lowering the incubation temperature from 21 to 15°C reduced the incidence of skeletal deformities from 80 to 60%. Intermediate incubation temperature (18°C), resulted in larvae that displayed a rate of abnormalities close to that observed at 15°C (64%). A similar typology of deformities was observed in fish from all rearing temperatures, with caudal vertebra being the most affected structures and with a low incidence of cranial and appendicular deformities. The effect of temperature in developmental plasticity will lead to osteological abnormalities mainly affecting the caudal vertebrae at 18°C (30%) and particularly the preural vertebra at 15°C (55%). Our results, clearly demonstrate a significant effect of water temperature during egg incubation on the skeletal development of Senegalese sole, indicating that 18°C is the most appropriated temperature. These findings highlight the importance of tightly controlling temperature regimes during the early development of S. senegalensis, and the need for optimizing rearing protocols in order to avoid skeletal disorders or impaired growth. published

In order to investigate the effects of dietary protein hydrolysates (PH) on larval growth performance, skeleton quality and proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding with live feed and three microencapsulated diets differing in the molecular weight of their PH fraction (Control – inclusion of CPSP-90; H – inclusion of a high amount in 0.5–30 kDa hydrolysates; L – inclusion of a high amount in

Gilthead seabream is the main aquaculture species produced in Portugal, as well as in the Mediterranean. However, the winter disease syndrome, a series of metabolic and immune system disorders caused by low temperatures, affects seabream culture, leading to important industrial losses, either as mortalities or growth decreases during spring. These effects can be minimized through dietary optimisation, either improving diets for the end of summer season, or feeding seabream with a fortified diet during autumn/winter. However, the development of a fortified diet for autumn/winter is hindered by the lack of tools to evaluate fish metabolic and physiological condition. Nevertheless, the recent development of new generation molecular tools provides good perspectives to supply molecular indicators of condition that would support this task.

The physiological responses of Senegalese sole to a sudden salinity change were investigated. The fish were first acclimated to an initial salinity of 37.5 ppt for 4 h. Then, one group was subjected to increased salinity (55 ppt) while another group was subjected to decreased salinity (5 ppt). The third group (control group) remained at 37.5 ppt. We measured the oxygen consumption rate, osmoregulatory (plasma osmolality, gill and kidney Na+,K+-ATPase activities) and stress (plasma cortisol and metabolites) parameters 0.5 and 3 h after transfer. Oxygen consumption at both salinities was higher than for the control at both sampling times. Gill Na+,K+-ATPase activity was significantly higher for the 55 ppt salinity at 0.5 h. Plasma osmolality decreased in the fish exposed to 5 ppt at the two sampling times but no changes were detected for high salinities. Plasma cortisol levels significantly increased at both salinities, although these values declined in the low-salinity group 3 h after transfer. Plasma glucose at 5 ppt salinity did not vary significantly at 0.5 h but decreased at 3 h, while lactate increased for both treatments at the first sampling time and returned to the control levels at 3 h. Overall, the physiological response of S. senegalensis was immediate and involved a rise in oxygen consumption and plasma cortisol values as well as greater metabolite mobilization at both salinities.

The Senegalese sole (Solea senegalensis) is characteristic from southern Europe and Mediterranean. It has recently been adapted for aquaculture production since it is well accepted by consumers and reaches high commercial values. After the initial description of the ontogenic events of skeletogenesis, systematic evaluation of the incidence of skeletal malformations by several groups revealed high levels of skeletal deformities reaching up to 90% of the population.

Skeletogenesis is a continuous process occurring during fish larval development that is influenced by vitamin K VK, an essential factor for the correct formation of coagulation factors and skeletal matrix proteins. High levels of skeletal deformities are recorded in most commonly cultured species like the sparids, sea bass and flatfish like the Senegalese sole. The appearance of malformations takes place during the larval stages, when skeletal structures are forming. This work was funded by project PDTC-MAR-105152-2008 (SPECIAL K) from the Foundation of Science and Technology.

Physiological responses after an acute handling stress and their subsequent effects on innate immune parameters, plasma free amino acids (AA) and liver energy substrates were assessed in Senegalese sole (Solea senegalensis). Eight groups of six specimens (136.1± 58.4 g wet weight) were maintained undisturbed, while other eight groups of six specimens were used for acute stress challenge (air exposed during 3 min). A group of six specimens was sampled for blood and head-kidney collection immediately after air exposure (time 0), while the remaining groups were sampled at 5 and 30 min, 1, 2, 4, 6 and 24 h. Undisturbed fish were sampled at the same times and used as control. Fish were fasted for 24 h prior to air exposure and sampling. Plasma cortisol, glucose, lactate and osmolality levels increased immediately after stress peaking at 1 h in air exposed fish. Changes in plasma free AA were also observed at 1 and 24 h after stress. In liver, glycogen levels significantly decreased at 30 min and 1 h, while triglycerides values significantly increased at 1, 2 and 4 h in air exposedfish. In addition, total AA levels in liver augmented significantly at 2 h holding high until 24 h in air exposed specimens. The respiratory burst of head-kidney leucocytes from air exposed fish was significantly higher than that from control groups at 2 and 6 h after air exposure. On the other hand, plasma lysozyme activity significantly decreased at 4 h after acute stress in air exposed fish, while plasma alternative complement pathway followed an inverse linear relationship with respect to cortisol showing the lowest value at 1 h after air exposure. The present study suggests that Senegalese sole presents a stress response comparable to that observed in other teleosts. While some indispensable AA may be used for the synthesis of compounds related to the stress response or fatty acid transport, dispensable AA were probably mainly employed either as energy sources or in gluconeogenesis. Moreover, results from non-specific immune parameters assessed suggest that cortisol may act as regulator of the innate immune system.

O cultivo de larvas de peixes marinhos é caracterizado por altas mortalidades e por problemas de qualidade, nomeadamente malformações esqueléticas. A solução de parte destes problemas passa por uma melhor compreensão dos requisitos nutricionais. Este trabalho pretende fazer uma revisão de trabalhos recentes que demonstram como os aminoácidos, os ácidos gordos e a vitamina K, mas também o regime alimentar, podem influenciar a qualidade e a performance das larvas de peixes marinhos.

The Senegalese sole (Solea senegalensis) is a highly valued species, with particular interest for the southern European aquaculture industry. For the last two decades this species has been transposed for industrial production, but it still presents some constraints as the high frequency of vertebral deformities observed under intensive rearing. The patterns of osteological deformities have previously been described and found to affect mostly the haemal and caudal vertebrae, but no studies have been conducted in order to identify causes for such deformities.

Temperature-induced responses at the level of muscle growth potential have been observed in teleost fish species, concomitantly with gene expression changes (Johnston and Hall 2004). Incubation temperature has been demonstrated to influence the muscle cellularity at hatch and during larval development, and these effects are highly species-specific. Changes in the relative timing and expression level of myogenic genes were found to be related with thermal plasticity of muscle phenotype (Fernandes, Mackenzie et al. 2006). The eggs of the flatfish Senegalese sole (Solea senegalensis) in aquaculture conditions are normally incubated within the 16-20ºC range, but influence of temperature on muscle cellularity and expression of growth-related genes is unknown. Understanding these effects may contribute to improve growth conditions and juvenile production.

Senegalese sole is distributed along the Mediterranean and southwestern Europe, reaching a market price of 20-25 €/kg - Promising species for aquaculture diversification - Control and intensification of culture is reasonably achieved - The high incidence of skeletal anomalies in intensively reared fish represents a constraint to commercial production - Samples from Portugal and Greece were analyzed towards identifying skeletal differences due to rearing conditions

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.

In order to investigate the effects of dietary protein hydrolysate on larval skeleton quality and larvae proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding onwards with live feed and three microencapsulated diets ir differing only on the molecular weight of their protein hydrolysate fraction.

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.

Submitted by Paulo Gavaia (pgavaia@ualg.pt) on 2014-05-30T10:05:46Z No. of bitstreams: 1 Avanços recentes em nutrição de larvas de peixes.pdf: 75116 bytes, checksum: 728a03d131b35c9646da86008536f71d (MD5) Approved for entry into archive by Pedro Sousa (a19661@ualg.pt) on 2014-06-25T09:13:19Z (GMT) No. of bitstreams: 2 Avanços recentes em nutrição de larvas de peixes.pdf: 75116 bytes, checksum: 728a03d131b35c9646da86008536f71d (MD5) 8753041680192686.zip: 69092 bytes, checksum: 482c5f4899029bb21c5d8512b89342fe (MD5) Made available in DSpace on 2014-06-25T09:13:19Z (GMT). No. of bitstreams: 2 Avanços recentes em nutrição de larvas de peixes.pdf: 75116 bytes, checksum: 728a03d131b35c9646da86008536f71d (MD5) 8753041680192686.zip: 69092 bytes, checksum: 482c5f4899029bb21c5d8512b89342fe (MD5) Previous issue date: 2009

The appearance of skeletal deformities is a major problem associated to intensive, large-scale culture of fish species, affecting growth, development and ultimately, survival of produced fish. Although the mechanisms working behind it are still unclear, it is known that environmental, genetic and nutritional factors play fundamental roles. Given its importance as a vertebrate model to study skeleton-related diseases, the selection of an appropriate dietary protocol is a critical step in order to diminish the nutritional effect on bone and cartilage development. To better understand the effect of diet on skeletal development and deformities, as well as on larvae and juvenile global performance, we have tested four distinct dietary regimes, composed of rotifers Brachionus sp., Artemia nauplii and dry, formulated food.

In aquaculture, skeleton deformities are a serious problem as they affect growth, morphology, survival of fish and also increase productions costs and decrease market value of the final products. Since vitamin K is known for playing and important role in bone metabolism, the aim of this study was to investigate the effects of dietary vitamin K1 supplementation on skeleton deformities and on the expression of proteins involved involved in skeleton development of Senegalese sole.

Submitted by Cláudia Aragão (caragao@ualg.pt) on 2014-07-03T09:38:26Z No. of bitstreams: 1 High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles.pdf: 206399 bytes, checksum: d3f961b9099d365012408f6d70d54770 (MD5) Approved for entry into archive by Filipa Custódio (fccustodio@ualg.pt) on 2014-07-03T15:48:51Z (GMT) No. of bitstreams: 2 0467513760195905.zip: 187432 bytes, checksum: 5e49ca88a3def472a5b2901f4ad0ead2 (MD5) High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles.pdf: 206399 bytes, checksum: d3f961b9099d365012408f6d70d54770 (MD5) Made available in DSpace on 2014-07-03T15:48:51Z (GMT). No. of bitstreams: 2 0467513760195905.zip: 187432 bytes, checksum: 5e49ca88a3def472a5b2901f4ad0ead2 (MD5) High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles.pdf: 206399 bytes, checksum: d3f961b9099d365012408f6d70d54770 (MD5) Previous issue date: 2008

Reliable juvenile production or recruitment requires high numbers of healthy fish larvae. Despite considerable progress in marine fish farming in the past 20 years, juvenile fish production is still fraught with problems which arise during the larval phase. In fisheries, juvenile recruitment in some populations has not recovered despite long-term moratoria on captures and protection of the broodstock. These issues highlight the growing importance of multidisciplinary fish larval research.

Submitted by Paulo Gavaia (pgavaia@ualg.pt) on 2014-05-30T10:27:14Z No. of bitstreams: 1 Effect of pre-weaning feeding regime on weaning performance of Senegalese sole, Solea senegalensis (Kaup, 1858).pdf: 328434 bytes, checksum: aee6e779dae7e86c9ed402c159644123 (MD5) Approved for entry into archive by Pedro Sousa (a19661@ualg.pt) on 2014-06-25T10:35:29Z (GMT) No. of bitstreams: 2 Effect of pre-weaning feeding regime on weaning performance of Senegalese sole, Solea senegalensis (Kaup, 1858).pdf: 328434 bytes, checksum: aee6e779dae7e86c9ed402c159644123 (MD5) 8753041680192686.zip: 295206 bytes, checksum: 7a92aa469c468cff66f427c2e20c9800 (MD5) Made available in DSpace on 2014-06-25T10:35:29Z (GMT). No. of bitstreams: 2 Effect of pre-weaning feeding regime on weaning performance of Senegalese sole, Solea senegalensis (Kaup, 1858).pdf: 328434 bytes, checksum: aee6e779dae7e86c9ed402c159644123 (MD5) 8753041680192686.zip: 295206 bytes, checksum: 7a92aa469c468cff66f427c2e20c9800 (MD5) Previous issue date: 2005

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 Os ácidos gordos (AG) essenciais presentes nas dietas são tidos como factores críticos no sucesso do cultivo larvar. Tem sido dado um maior destaque aos ácidos docosahexaenóico (DHA) e eicosapentaenóico (EPA) devido às suas funções na integridade celular membranar e predominância nos tecidos. A importância do ácido araquidónico (ARA) no crescimento, sobrevivência e resistência ao stress é também cada vez mais reconhecida. Realizou-se um estudo para determinar os efeitos que diferentes concentrações de ARA em microdietas causam no crescimento, sobrevivência e perfil de ácidos gordos das larvas de dourada. As larvas foram alimentadas durante 25 dias com microdietas, incluindo um período inicial de coalimentação com rotíferos enriquecidos. Foram testadas, em triplicado, quatro microdietas experimentais que diferiam nos níveis de ARA, variando entre 0,4% e 3,0% da matéria seca (ARA0,4/ ARA0,8/ ARA1,5/ ARA3,0). As microdietas apresentavam níveis de HUFA n-3 de 4,6% e um rácio DHA/EPA de 1,4. Mediu-se o crescimento com base no peso seco e taxa de crescimento relativo, e obteve-se a sobrevivência por contagem directa. O perfil em AG dos lípidos totais das larvas foi determinado por cromatografia em fase gasosa. Após 34 dias, não se registaram diferenças significativas no crescimento e sobrevivência, apesar de existir uma tendência nos tratamentos intermédios para um menor crescimento. A composição lipídica dos tecidos reflectiu o perfil de AG das dietas, particularmente quanto ao ARA. O DHA manteve-se constante, enquanto o EPA diminuiu com o aumento de ARA, mesmo com níveis semelhantes de EPA nas dietas, sugerindo uma competição entre estes AG para a incorporação nos tecidos. Até 1,5% nas dietas, o ARA mostrou ser preferencialmente depositado nos tecidos, sendo metabolizado em concentrações superiores (3,0%). O DHA e alguns AG saturados foram preferencialmente retidos enquanto que o EPA e os ácidos linoleico (LA), α- linolénico (ALA) e oleico (OA) foram metabolizados, provavelmente para fins energéticos. Nas condições testadas, as larvas de dourada mostraram uma boa tolerância à variação de ARA nas dietas, de 0,4% a 3,0%. No entanto, o crescimento não foi afectado nem se verificou uma melhoria da sobrevivência.

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.

Tese dout., Aquacultura, Universidade do Algarve, 2008 Diplodus sargus is a potential species for aquaculture. Constraints to its production are a high incidence of skeletal deformities and stagnation of the growth rate in juvenile stage, possibly caused by dietary imbalances. This thesis is focused on the amino acid (AA) requirements of Diplodus sargus larvae, on the formulation of diets with AA supplementation and also identifies larvae skeletal deformities patterns (Chapter 2). D. sargus AA requirements were estimated using the AA profiles from larval carcass. It was observed that the AA profiles were relatively constant during larval ontogeny, and dietary imbalances were identified in rotifers and Artemia (Chapter 3). Tyrosine, lysine, tryptophan and arginine supplementation was tested by tube feeding and lower gut absorptions efficiencies were observed for these AA. Also, these AA do not seem to be limiting D. sargus growth (Chapter 4 and 5). Based on the results from Chapter 3, a microencapsulated casein diet with a balanced AA profile was formulated and was compared to an unbalanced AA diet and to a live feed diet (control) (Chapter 6). Results showed lower ammonia excretion and less skeletal deformities in larvae fed a balanced diet. Tryptophan supplementation in the diet was tested because of its involvement in skeleton ossification (Chapter 7). However, a decrease on larval growth and no effect on skeletal deformities were observed. The effect of tyrosine and phenylalanine supplementation on D. sargus larvae was tested and a higher survival to a temperature stress test was observed in larvae fed a tyrosine supplementation (Chapter 8). Larvae given a phenylalanine supplementation had a decreased incidence on vertebral compressions. In conclusion, an AA balanced diet reduces skeletal deformities and improves nitrogen utilization. Skeletal deformities do not seem related to tryptophan deficiency. Supplementation of phenylalanine and tyrosine lead to a decrease on skeletal deformities and better resistance to a stress test, respectively. From the AA studied; tryptophan, lysine, phenylalanine, arginine and tyrosine; none seems to be limiting D. sargus larval growth Faculdade de Ciências e Tecnologia (FCT) Instituto de Investigação das Pescas e do Mar (IPIMAR)