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- Effect of three diets on the growth and fatty acid profile of the common ragworm Hediste diversicolor (O.F. Müller, 1776)Publication . Santos, António; Granada, Luana; Baptista, Teresa; Anjos, Catarina; Simões, Tiago; Tecelão, Carla; Fidalgo e Costa, Pedro; Costa, José Lino; Pombo, AnaThe polychaete Hediste diversicolor has a high physiological tolerance to extreme environmental factors, being easily farmed and reproduced in different types of conditions. Both in the field and under laboratory conditions, this worm can feed on different types of food. In order to highlight the potential of H. diversicolor for aquaculture, specific growth rate (SGR), daily growth rate (DGR), survival rate and fatty acid profile of juvenile worms, fed with three different diets, were assessed. The experiments were conducted using juvenile polychaete from a controlled reproduction with wild adults. H. diversicolor individuals were fed with two commercial diets, seabream dry feed (Aquagold) and semi-wet pellets for reared sole (Moist Sole), and with a non-processed diet consisting on mackerel's fillets (Trachurus trachurus). Juveniles fed with Aquagold had the highest final individual weight (0.89 ± 0.10 g). The SGR was higher in H. diversicolor fed with Aquagold and Moist Sole, (6.49 ± 0.30% d−1 and 6.54 ± 0.06% d−1 , respectively. The highest DGR was observed for juveniles fed with Aquagold (0.146 ± 0.02 g d−1 ). The survival rate of ragworms under different treatments ranged from 96 to 100%. Regarding the protein content, the Moist Sole diet provided the highest percentage of protein in the reared worms (8.87%). Results showed that the total fat content of the diets was reflected in the fat content of the reared worms. The Moist Sole diet treatment had the highest fat content (2.25%) and individuals fed with seabream dry feed showed similar results (2.18%), while the lowest percentage was observed for the mackerel diet (0.85%). According to the fatty acid profile, the major fatty acids found in the juveniles fed with the three different diets were palmitic (C 16:0), with a higher value in the individuals fed with mackerel's fillets. Oleic (C 18:1 n9), eicosapentaenoic (C 20:5 n3), docosahexaenoic (C 22:6 n3) and stearic (C 18:0) acids presented high values in H. diversicolor fed with all the experimental diets. Statement of Relevance: The common ragworm Hediste diversicolor is a potential high quality fatty acids source for reared fish and shrimp. Previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of this polychaete (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms juveniles (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate diet to be used in commercial aquaculture.
- Detection of Betanodavirus in experimentally infected European seabass (Dicentrarchus labrax, Linnaeus 1758) using non‐lethal sampling methodsPublication . Ferreira, Inês Almeida; Costa, Janina Z.; Macchia, Valeria; Thompson, Kimberly Dawn; Baptista, TeresaOne of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non‐lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50/ml and 106 TCID50/ml, respectively), and samples collected 7, 15 and 30 days post‐infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non‐lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT‐qPCR). The presence of virus in non‐lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular‐infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme‐linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus‐specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.
- Optimization of phycobiliprotein pigments extraction from red algae Gracilaria gracilis for substitution of synthetic food colorantsPublication . Pereira, Tatiana; Barroso, Sónia; Mendes, Susana; Amaral, Renata A.; Dias, Juliana R.; Baptista, Teresa; Saraiva, Jorge A.; Alves, Nuno; Gil, Maria M.The extraction of phycobiliprotein (PBP) pigments from red algae Gracilaria gracilis was optimized using maceration, ultrasound-assisted extraction (ultrasonic water bath and ultrasonic probe), high pressure-assisted extraction, and freeze-thaw. The experimental conditions, namely homogenization time (t1), buffer concentration (C), treatment time (t2), biomass: buffer ratio (R), and pressure (P), were optimized using Response Surface Methodology (RSM). The yield of phycoerythrin (PE) extracted, determined spectroscopically, was used as the response variable. Maceration was the most efficient extraction method yielding 3.6 mg PE/g biomass under the optimal conditions (t1 = t2 = 10 min; C = 0.1 M; R = 1:50). Scanning Electron Microscopy (SEM) analysis of the biomass before and after the cell disruption treatments revealed a more efficient cell wall rupture with maceration.
- First report of Calceostoma glandulosum (Monogenea) in Argyromus regius: morphogical and molecular characterization and temperature effects on lifecyclePublication . Ribeiro, Maria Carolina; Lourenço-Marques, Cátia; Pousão-Ferreira, Pedro; Soares, FlorbelaThe increase in fish production in aquaculture reflects the growth of the world population. However, this expansion is accompa nied by challenges, such as the intensification of production systems through increased stocking density, which induces stress in fish, weakening their immune system and making them more susceptible to disease outbreaks, particularly those caused by parasites. Monogeneans are ectoparasites that attach to the external surfaces of fish and are characterised by their host-specificity. One of the emerging species in aquaculture, much appreciated in the Mediterranean region, is the meagre (Argyrosomus regius), whose production has been increasing due to its high-quality meat and excellent growth rates. However, the information available on the parasites that affect this species is limited, which makes it difficult to prevent and control disease outbreaks in aquaculture systems effectively. This study presents the first morphological and molecular characterisation of Calceostoma glandulosum, an ectoparasite of the Monopisthocotylea subclass, characterised by its lapel (L) in the cephalic area and an anchor-shaped haptor (Hp). The parasite’s life cycle, including eggs, larvae (oncomiracidium), and adults, has also been described. It was demonstrated that water temperature could influence the development of the eggs; at more favourable temperatures for the parasite, there was a higher percentage of developed and hatched eggs. The occurrence of the parasite in the farming tanks was higher when the temperature reached 20°C, and in vitro experiments have shown a hatching rate of 100% at 23°C. C. glandulosum reaches sexual maturity between 11 and 14 days and has a fertility period of 10 days. To assess the host-specificity of C. glandulosum for A. regius, eggs were investigated in other species (Sparus aurata, Diplodus sargus, and Seriola rivoliana). This work provides valuable insights into the behavior of monogeneans concerning environmental conditions and host interactions, offering critical information for implementing preventive measures in aquaculture.
- The influence of light and culture media on the growth of the red seaweed Gracilaria gracilis (Rhodophyta, Gracilariales) under laboratory conditionsPublication . Freitas, M.V.; Correia, A.; Ribeiro, J.; Pereira, C.; Afonso, C.; Gil, M.M.; Mendes, S.; Baptista, T.; Mouga, T.
- Functional and molecular mmune response of rainbow trout (Oncorhynchus mykiss) following challenge with Yersinia ruckeriPublication . Fajardo, Carlos; Santos, Paulo; Passos, Ricardo; Vaz, Mariana; Azeredo, Rita; Machado, Marina; Fernández-Boo, Sergio; Baptista, Teresa; Costas, BenjaminCurrently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30–70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout’s short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immunerelated genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1b, il-8, il-10, tnf-a1, tnf-a2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.
- Effect on health status and pathogen resistance of gilthead seabream (Sparus aurata) fed with diets supplemented with Gracilaria gracilisPublication . Passos, Ricardo; Correia, Ana Patrícia; Ferreira, Inês; Pires, Pedro; Pires, Damiana; Gomes, Emma; Carmo, Beatriz do; Santos, Paulo; Simões, Marco; Afonso, Clélia; Baptista, TeresaAquaculture intensification turns the reared populations prone to disease outbreaks, leading the industry to use antibiotics and chemotherapeutics. As the industry is searching for solutions, the development of sustainable aquafeeds with immunostimulant properties is emerging. This work aimed to assess the health benefits of Gracilaria gracilis as a supplement in Sparus aurata feed as well as its protective effect in a bacterial challenge. G. gracilis supplementation improved growth performance and influenced the haematological profile, namely in the constitution of the leukocytic population and erythrocytes size. Algae powder supplementation did not compromise the hepatic antioxidant systems and intestinal health and morphology were not affected by the algal supplementations. Algal extract improved lysozyme plasmatic concentration and a 5% algae powder inclusion was able to successfully protect the fish against a Photobacterium damselae subsp. piscicida infection, granting a 95% survival. In general, a 5% G. gracilis powder diet supplementation was able to improve growth, health, and bacterial resistance in gilthead seabream.
- Modulatory effect of Gracilaria gracilis on European seabass gut microbiota community and its functionalityPublication . Gonçalves, Ana Teresa; Simões, Marco; Costa, Cátia; Passos, Ricardo; Baptista, TeresaSeaweeds are an important source of nutrients and bioactive compounds and have a high potential as health boosters in aquaculture. This study evaluated the effect of dietary inclusion of Gracilaria gracilis biomass or its extract on the European seabass (Dicentrarchus labrax) gut microbial community. Juvenile fish were fed a commercial-like diet with 2.5% or 5% seaweed biomass or 0.35% seaweed extract for 47 days. The gut microbiome was assessed by 16S rRNA amplicon sequencing, and its diversity was not altered by the seaweed supplementation. However, a reduction in Proteobacteria abundance was observed. Random forest analysis highlighted the genera Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae. SparCC correlation network analysis suggested several mutualistic and other antagonistic relationships that could be related to the predicted altered functions. These pathways were mainly related to the metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae. This study shows the beneficial potential of Gracilaria as a functional ingredient through the modulation of the complex microbial network towards fish health improvement.
- Environmental limits of low salinity to Gracilaria gracilis development. Where do we draw the line?Publication . França, Marcela; Afonso, Clélia; Baptista, Teresa; Pombo, Ana; Mouga, Teresa
- In vitro evaluation of the antibacterial and antioxidant activities of extracts of Gracilaria gracilis with a view into its potential use as an additive in fish feedPublication . Afonso, Clélia; Correia, Ana; Freitas, Marta; Mouga, Teresa; Baptista, TeresaFish in aquaculture systems are subject to several stressors that inhibit the immune response and potentiate the development of disease and increased mortality. The inclusion of additives in the fish diet, namely seaweeds or their extracts, that are natural sources of bioactive compounds can be an important tool for promoting the health and well-being of these animals. The present study aims at the development of sustainable and effective methodologies for the extraction of bioactive compounds of the red seaweed Gracilaria gracilis, exploring its antibacterial and antioxidant potential and considering its potential use as an additive for functional fish feeds. The yield of the extraction methods was evaluated upon the use of sequential solid–liquid extraction techniques with ethanol and water as solvents, different extraction temperatures (room temperature: 40ºC and 70ºC), and extraction time. The results demonstrated that the adoption extraction times of 30 min. at 40ºC provided higher yields. We also evaluated the antioxidant capacity and the antibacterial properties of the obtained extracts against different strains that cause fish diseases by disk diffusion and broth microdilution methods. The antioxidant activity was determined by the DPPH reducing capacity method and quantification of total polyphenols content (TPC).With these results, we can establish extraction procedures that allow the future use of G. gracilis extracts, with antibacterial and antioxidant effects in a safe and effective way.