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- Novel methodologies in marine fish larval nutritionPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Major gaps in knowledge on fish larval nutritional requirements still remain. Small larval size, and difficulties in acceptance of inert microdiets, makes progress slow and cumbersome. This lack of knowledge in fish larval nutritional requirements is one of the causes of high mortalities and quality problems commonly observed in marine larviculture. In recent years, several novel methodologies have contributed to significant progress in fish larval nutrition. Others are emerging and are likely to bring further insight into larval nutritional physiology and requirements. This paper reviews a range of new tools and some examples of their present use, as well as potential future applications in the study of fish larvae nutrition. Tube-feeding and incorporation into Artemia of 14C-amino acids and lipids allowed studying Artemia intake, digestion and absorption and utilisation of these nutrients. Diet selection by fish larvae has been studied with diets containing different natural stable isotope signatures or diets where different rare metal oxides were added. Mechanistic modelling has been used as a tool to integrate existing knowledge and reveal gaps, and also to better understand results obtained in tracer studies. Population genomics may assist in assessing genotype effects on nutritional requirements, by using progeny testing in fish reared in the same tanks, and also in identifying QTLs for larval stages. Functional genomics and proteomics enable the study of gene and protein expression under various dietary conditions, and thereby identify the metabolic pathways which are affected by a given nutrient. Promising results were obtained using the metabolic programming concept in early life to facilitate utilisation of certain nutrients at later stages. All together, these methodologies have made decisive contributions, and are expected to do even more in the near future, to build a knowledge basis for development of optimised diets and feeding regimes for different species of larval fish.
- Recent advances in nutrition of fish larval | Avanços recentes em nutrição de larvas de peixesPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.
- Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatusPublication . F Riesco, Marta; Raposo de Magalhães, Cláudia; Engrola, Sofia; Martinez-Paramo, Sonia; Mira, Sara; Cunha, Maria Emilia; Cabrita, ElsaThe dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and poor sperm quality is a limiting factor in reproduction success. The present study aimed at evaluating two main aspects in the design of a cryopreservation protocol-extender additives (taurine, glucose, cholesterol, BSA) and sperm containers (0.5 mL straws, 2 mL cryovials and 5 mL macrotubes). Sperm quality was assessed through the evaluation of the percentage of motile cells, viable cells, DNA fragmentation, lipid peroxidation and apoptosis. Some specific techniques, such as Caspase 3/7 detection, which provides information on membrane integrity and cell death, detecting early and late apoptotic and necrotic events, were required to establish an optimized cryopreservation protocol for this species. Taurine was the most suitable cryopreservation additive in terms of viable cells and cholesterol presented the highest percentage of necrotic cells in this study. Caspase 3/7 assay enabled us to detect necrotic damage induced by cryopreservation. Statement of relevance: The development of reproductive tools in dusky grouper, a potential species for aquaculture production, emerges as important tool to decrease the number of wild males maintained in captivity. A cryopreservation protocol was previously described for this species although several constraints in terms of sperm quality were detected. Our work provided new evidences that cryopreservation protocols can be improved through the addition of certain additives and use of appropriate sperm containers. Specific sperm analysis was crucial to identify and establish the most suitable conditions for breeders management and species conservation purposes. (C) 2016 Elsevier B.V. All rights reserved.
- Dietary protein complexity modulates growth, protein utilisation and the expression of protein digestion-related genes in Senegalese sole larvaePublication . Canada, Paula; Conceicao, Luis E. C.; Mira, Sara; Teodósio, Rita; Fernandes, Jorge M. O.; Barrios, Carmen; Millan, Francisco; Pedroche, Justo; Valente, Luisa M. P.; Engrola, SofiaGiven its complex metamorphosis and digestive system ontogeny, Senegalese sole larvae capacity to digest and utilize dietary protein is likely to change throughout development. In the present study, we hypothesized that the manipulation of dietary protein complexity may affect Senegalese sole larvae capacity to digest, absorb and retain protein during metamorphosis, as well as the mRNA expression of genes encoding for the precursors of proteolytic enzymes of the digestive tract and the enterocyte peptide transporter PepT1, which may have further impact on somatic growth. Three diets were formulated using approximately the same practical ingredients, except for the main protein source. The Intact diet protein content was mostly based on intact plant protein where the target peptide molecular weight (MW) would be > 70 kDa. The PartH diet protein fraction was mostly based on a protein hydrolysate with a high content of 5-70 kDa peptides. The HighH diet protein fraction was mostly based on a protein hydrolysate with a high content of 5 kDa peptides. A growth trial was performed with larvae reared at 19 degrees C under a co-feeding regime from mouth opening. The transcription of pga, tryp1c, ialp, ampn and pepT1 (encoding respectively for PepsinogenA, Trypsinogen1C, Intestinal alkaline phosphatase, Aminopeptidase N and for the enterocyte peptide transporter 1) was quantified by qPCR, during the metamorphosis climax (16 DAH) and after the metamorphosis was completed (28 DAH). An in vivo method of controlled tube-feeding was used to assess the effect on the larvae capacity to utilize polypeptides with different MW (1.0 and 7.2 kDa) representing a typical peptide MW of each of the hydrolysates included in the diets. The PartH diet stimulated growth in metamorphosing larvae (16 DAH), whereas the Intact diet stimulated growth after 36 DAH. The Intact diet stimulated the larvae absorption capacity for 1.0 kDa peptides at 16 DAH, which may have contributed for enhanced growth in later stages. The PartH diet stimulated the transcription of tryp1c and pept1 at 28 DAH, which seemed to reflect on increased post-larvae capacity to retain dietary 7.2 kDa polypeptides. That may indicate a possible strategy to optimize the digestion and utilisation of the PartH dietary protein, though it did not reflect into increased growth. The Intact diet promoted the transcription of pepsinogenA, which may reflect a reduced gastrointestinal transit time, which could have enhanced the dietary nutrients assimilation, ultimately improving growth. The present results suggest that, whereas pre-metamorphic sole larvae utilize better dietary protein with a moderate degree of hydrolysis, post-metamorphic sole make a greater use of intact protein.
- The evolution of Cicada songs contrasted with the relationships inferred from mitochondrial DNA (Insecta, hemiptera)Publication . Fonseca, P. J.; Serrão, Ester; Pina-Martins, F.; Silva, P.; Mira, Sara; Quartau, J. A.; Paulo, O. S.; Cancela, LeonorThe molecular phylogeny of nine Palaearctic species of cicadas (Hemiptera, Cicadoidea) was inferred using two mitochondrial DNA genes, Cytochrome Oxidase I and II. The two main groups detected, namely species within Tettigetta and Tympanistalna, as well as the two species investigated in the genus Cicada, are robustly supported across the analytical methods. The structure of the song syllables, generated during single tymbal cycles of males of the analysed group of species is remarkably consistent in these two phyletic lines. This reflects the morphology and the mechanics of the tymbal. However the higher level song patterns, which depend on the activity of the central nervous system and have evolved to advertise receptive mates, do not seem to be consistent with either the inferred molecular topology or the basic tymbal cycle. The observed similarities between the molecular phylogeny and the basic tymbal cycles seem to reflect the basic conservative nature of the tymbal structure, while the discrepancy between the former and the calling song pattern is probably related to the high plasticity of the pattern generator in the central nervous system and dependent on species-specific selection.
- The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvaePublication . Canada, Paula; Engrola, S.; Mira, Sara; Teodósio, Rita; Fernandes, Jorge M. O.; Sousa, Vera; Barriga-Negra, Lúcia; Conceicao, Luis; Valente, Luisa M. P.The full expression of growth potential in fish larvae largely depends on an efficient protein utilization, which requires that all the indispensable amino acids (IAAs) are provided at an optimum ratio. The effect of supplementing a practical microdiet with encapsulated crystalline-AA to correct possible IAA deficiencies was evaluated in Senegalese sole larvae. Two isonitrogenous and isoenergetic microdiets were formulated and processed to have approximately the same ingredients and proximate composition. The control diet (CTRL) was based on protein sources commonly used in the aquafeed industry. In the supplemented diet (SUP) 8% of an encapsulated fish protein hydrolysate was replaced by crystalline-AA in order to increase the dietary IAA levels. The microdiets were delivered from mouth-opening upon a co-feeding regime until 51 days after hatching (DAH). The larvae capacity to utilize protein was evaluated using an in vivo method of controlled tube-feeding during relevant stages throughout development: pre-metamorphosis (13 DAH); metamorphosis climax (19 DAH) and metamorphosis completion (25 DAH). Somatic growth was monitored during the whole trial. A possible effect on the regulation of muscle growth was evaluated through muscle cellularity and the expression of related genes (myf5, myod2, myogenin, mrf4, myhc and mstn1) at metamorphosis climax (19 DAH) and at a juvenile stage (51 DAH). The SUP diet had a negative impact on larvae somatic growth after the metamorphosis, even though it had no effect on the development of Senegalese sole larvae capacity to retain protein. Instead, changes in somatic growth may reflect alterations on muscle growth regulation, since muscle cellularity suggested delayed muscle development in the SUP group at 51 DAH. Transcript levels of key genes regulating myogenesis changed between groups, during the metamorphosis climax and at the 51 DAH. The group fed the SUP diet had lower dnmt3b mRNA levels compared to the CTRL group. Further studies are needed to ascertain whether this would possibly lead to an overall DNA hypomethylation in skeletal muscle. (C) 2016 Elsevier B.V. All rights reserved.
- Avanços recentes em nutrição de larvas de peixesPublication . Conceicao, Luis; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo; Mira, Sara; Dias, J.Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.
- Marine green macroalgae: a source of natural compounds with mineralogenic and antioxidant activitiesPublication . Surget, Gwladys; Roberto, Vania Palma; Le Lann, Klervi; Mira, Sara; Guerard, Fabienne; Laizé, Vincent; Poupart, Nathalie; Leonor Cancela, M.; Stiger-Pouvreau, ValerieMarine macroalgae represent a valuable natural resource for bioactive phytochemicals with promising applications in therapeutics, although they remain largely under-exploited. In this work, the potential of two marine green macroalgae (Cladophora rupestris and Codium fragile) as a source of bioactive phenolic compounds was explored, and antioxidant, mineralogenic, and osteogenic activities were evaluated. For each species, a crude hydroalcoholic extract (CE) was prepared by solid/liquid extraction and fractionated by liquid/liquid purification into an ethyl acetate fraction (EAF) enriched in phenolic compounds and an aqueous fraction (AF). Antioxidant activity, assessed through radical scavenging activity and reducing power assay, was increased in EAF fraction of both species and closely related to the phenolic content in each fraction. Mineralogenic activity, assessed through extracellular matrix mineralization of a fish bone-derived cell line, was induced by EAF fractions (up to 600 % for C. rupestris EAF). Quantitative analysis of operculum formation in zebrafish larvae stained with alizarin red S further confirmed the osteogenic potential of EAF fractions in vivo, with an increase of more than 1.5-fold for both C. fragile and C. rupestris fractions, similar to vitamin D (control). Our results demonstrated a positive correlation between phenolic fractions and biological activity, suggesting that phenolic compounds extracted from marine green macroalgae may represent promising molecules toward therapeutic applications in the field of bone biology.