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- 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.
- Potential use of macroalgae Gracilaria gracilis in diets for European seabass (Dicentrarchus labrax): Health benefits from a sustainable sourcePublication . Passos, Ricardo; Correia, Ana Patrícia; Pires, Damiana; Pires, Pedro; Ferreira, Inês; Simões, Marco; Carmo, Beatriz do; Santos, Paulo; Pombo, Ana; Afonso, Clélia; Baptista, TeresaSeaweeds still possess a large undisclosed potential, mainly due to their constituent’s richness, which may have several uses for society. In aquaculture, they may play a role as an ecological sustainable aquafeed supplement to increase overall health and fight pathogenic outbreaks. This study aimed to evaluate the general health modulation that the inclusion of Gracilaria gracilis could accomplish in the diet of Dicentrarchus labrax. Dried algae at 2.5% and 5% and algal extract at 0.35% inclusion levels were supplemented to seabass diet to evaluate possible growth, haematological, immunological, antioxidant, metabolic, and intestinal morphological modulations. The supplementations did not impact growth or feed utilization, and barely affected the haematological profile and some metabolic parameters. Nevertheless, it caused a marked outcome on lysozyme, some oxidative stress biomarkers, and intestine morphology, suggesting beneficial consequences from the algal inclusion. Dried algae powder, with a 2.5% inclusion, boosted immune response, with higher plasmatic lysozyme and intestinal acid goblet cells and protected against oxidative damages by improved enzymatic and non-enzymatic responses. Thus, we provide evidence that dietary seaweed application may be a path towards a more sustainable aquaculture industry.
- Lidocaine supplementation in clove-oil and 2-phenoxyethanol anesthesia for gilthead seabream (Sparus aurata)Publication . Tchobanov, Carolina F.; Vaz, Mariana; Pires, Damiana; Passos, Ricardo; Antunes, Luís M.; Baptista, TeresaAnimal welfare and reducing stress during procedures are key objectives for success in animal production. Anesthesia has been used for procedures to reduce animal stress and its negative impact on welfare. This study aimed first to refine the concentrations of the anesthetic clove-oil (CO) and lidocaine (L) in gilthead seabream (Sparus aurata) juveniles (56.0 ± 15.09 g) and then combine clove-oil and 2-phenoxyethanol (2PHE) with the refined concentration of lidocaine. The concentrations of clove-oil (30, 45, and 60 mg L− 1), and the concentrations of lidocaine (2.5, 5, and 7.5 mg L− 1), were evaluated in the refinement trial. Based on these results, a second trial was performed with 45 mg L− 1 CO or 0.4 mL L− 1 2PHE as anesthetics alone or combined with 2.5 mg L− 1 of lidocaine. Results from this work showed an improvement in induction times for 2-phenoxyethanol when lidocaine was added (2PHE 179.53 ± 63.21 s; 2PHE + L 130.65 ± 40.16 s). Recovery time also showed a reduction for clove-oil when lidocaine was used (CO 349.90 ± 123.69 s; CO + L 250.11 ± 51.99 s). The use of lidocaine showed better results, reducing lactate and histological progressive alterations. Lidocaine showed stress-induced oxidative alterations when it was combined with 2-phenoxyethanol. Lidocaine exposure increased ALT, AST, histological regressive alterations for both anesthetics, and gene expression of hsp70 in the gills when clove-oil was used. Further studies are necessary to comprehend the synergistic effects of lidocaine when combined with synthetic and natural anesthetics and to discern potential acute or chronic toxic responses in fish. These insights will be crucial for refining anesthesia protocols and ensuring the well-being of aquatic species in aquaculture practices and research settings.
- Early immune modulation in European Seabass (Dicentrarchus labra) juveniles in response to Betanodavirus infectionPublication . Vaz, Mariana; Pires, Damiana; Pires, Pedro; Simões, Marco; Pombo, Ana; Santos, Paulo; Carmo, Beatriz do; Passos, Ricardo; Costa, Janina Z.; Thompson, Kim D.; Baptista, TeresaThe early host–pathogen interaction between European seabass (Dicentrarchus labrax) and Betanodavirus was examined by using juvenile fish infected intramuscularly with RGNNV (red-spotted grouper nervous necrosis virus). The time course selected for sampling (0–144 h post-infection (hpi)) covered the early stages of infection, with hematological, antioxidant and immunological responses examined. Early activation of the host’s immune system was seen in the first few hours post-infection (6 to 9 hpi), as evidenced by an increase in tnfa, cd28 and c3 expression in the head kidney of infected fish. Most hematological parameters that were examined showed significant differences between sampling times, including differences in the number of thrombocytes and various leukocyte populations. The plasma lysozyme concentration decreased significantly over the course of the trial, and most antioxidant parameters examined in the liver showed significant differences over the infection period. At 144 hpi, peak expression of tnfa and il-1b coincided with the appearance of disease symptoms, peak levels of virus in the brain and high levels of fish mortality. The results of the study show the importance of analyzing the early interactions between European seabass and Betanodavirus to establish early indicators of infection to prevent more severe outcomes of the infection from occurring.
- Effect of Levabon® Aquagrow E on the growth performance and immune response of European seabass (Dicentrarchus labrax) under stress conditionsPublication . Passos, Ricardo Miguel Henriques; Baptista, Teresa Maria Coelho; Lemos, Marco Filipe Loureiro; Novais, Sara CalçadaA intensificação dos métodos de cultivo em aquacultura está a levar a um abuso dos tratamentos com antibióticos. Isto é motivo para preocupação porque: 1) ocorre o desenvolvimento de bactérias resistentes aos antibióticos; 2) potencia a presença de resíduos de antibióticos na alimentação humana; 3) destrói as populações microbianas nos sistemas de aquacultura; 4) causa imunossupressão nos organismos. Esforços para criar alternativas aos antibióticos e que não sejam danosos para o ambiente resultaram na aplicação de pre- e probióticos. Neste trabalho vão ser avaliados os efeitos do prebiótico comercialmente disponível, Levabon® Aquagrow E (Biomin®, Áustria), no crescimento de robalos juvenis sob condições de stress, bem como a resposta dos peixes a diferentes agentes causadores de stress. Para este trabalho, os peixes foram expostos a stress por aumento de densidade, stress por hipoxia ou mantidos em condições controlo (não expostos a stress). Os peixes de cada condição foram submetidos a duas dietas distintas: ração comercial para aquacultura sem suplementação ou a mesma ração, mas com o suplemento de Levabon® a 6 g Kg-1. Para se atingirem os objetivos propostos foram feitas três amostragens, nas quais os peixes foram medidos e pesados e amostras de sangue foram obtidas, para avaliação do hematócrito, níveis de cortisol, glicose, fosfatase alcalina (ALP) e atividade bactericida, da lisozima e da peroxidase. Na última amostragem, o fígado e tecido muscular foram extraídos para a avaliação do stress oxidativo. Peixes alimentados com o prebiótico atingiram melhor crescimento, maiores níveis de glicose e lidaram melhor com os danos do stress oxidativo. O efeito estimulante dos mananoligossacarídeos (MOS) nas bactérias benéficas Bifidobacteria e Lactobacilli poderá ter resultado numa melhor digestão e assimilação dos nutrientes, potenciando o melhor crescimento. O nível de glicose, um bom indicador de stress crónico, apresentou uma tendência para aumentar em peixes alimentados com o suplemento ao longo da experiência, permitindo que possuíssem mais energia para responder às alterações metabólicas causadas pelo stress. O prebiótico também reduziu os danos oxidativos nos lípidos do fígado, causados pelo stress de densidade, bem como os danos no ADN no músculo causados pelo stress de hipoxia. Sendo que este produto atuou positivamente nos pontos-chave de um cultivo aquícola bem-sucedido (melhores taxas de crescimento, melhoria do sistema imune e menos problemas relacionados com o stress), pode-se afirmar que o Levabon® Aquagrow E tem potencial para ser lucrativo quando adicionado consistentemente à ração.
- Short-term immune responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicidaPublication . Santos, Paulo; Peixoto, Diogo; Ferreira, Inês; Passos, Ricardo; Pires, Pedro; Simões, Marco; Pousão-Ferreira, Pedro; Baptista, Teresa; Costas, BenjaminPhotobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 uL of PBS (placebo) or 100 uL of exponentially growing Phdp (1 x 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.
- Principais Patógenos que Afetam Peixes Cultivados em PortugalPublication . Pires, Damiana; Vaz, Mariana; Pires, Pedro; Passos, Ricardo; Carmo, Beatriz; Santos, Paulo; Tomás, Mafalda; Ferreira, Susana M. F.; Baptista, TeresaA mortalidade de peixes que ocorre nas explorações aquícolas, devida à ação de agentes patogénicos, constitui ainda um dos maiores entraves ao desenvolvimento económico do sector. Tendo em conta a sua origem, as doenças podem subdividir-se em não infeciosas e infeciosas, sendo estas últimas as de maior importância e a principal causa de perdas económicas elevadas. As doenças infeciosas agrupam-se em três categorias, de acordo com a sua etiologia viral, bacteriana e parasítica. Estas doenças são muitas vezes indicadoras de condições fisiológicas e patológicas, resultantes de alimentação inadequada, infeções subclínicas, ou condições ambientais adversas. Quando simultaneamente existem condições favoráveis para a multiplicação do patógeno e condições adversas para os peixes, observa-se o desenvolvimento da doença. Entre as condições adversas que podem diminuir a resposta imune dos peixes cultivados, provocando uma maior predisposição aos patógenos, enumeram-se: densidade elevada, baixos níveis de oxigénio, stress alimentar, stress provocado pelo transporte, acumulação de compostos azotados, alterações de pH, entre outros. Estas situações, tendo em conta as consequências, merecem uma atenção redobrada por parte dos aquicultores.
- Proximate composition and fatty acid profile of Gilthead seabream (Sparus aurata) fed with Pelvetia canaliculata supplemented diets: An insight towards the valorization of seaweed biomassPublication . Antunes, Madalena; Neves, Marta; Pires, Damiana; Passos, Ricardo; Carmo, Beatriz do; Tchobanov, Carolina F.; Forte, Sara; Vaz, Mariana; Baptista, Teresa; Tecelão, CarlaSeaweeds are a sustainable source of protein and lipids that may be used to replace fish by-products in aquaculture feed. This study aimed at using the macroalgae Pelvetia canaliculata as an ingredient in gilthead seabream (Sparus aurata) feed, either as freeze-dried powder or as algae residue (waste) that was obtained after the supplementation of sunflower oil. The formulated diets and the fish muscle were analyzed concerning the proximate composition and the fatty acid profile. The health lipid indices hypocholesterolemic/ hypercholesterolemic (h/H), atherogenic (AI), thrombogenic (TI), as well as n-3/n-6 and polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratios were calculated. Additionally, the peroxidizability index (PI) was determined. No differences were observed in the proximate composition of fish muscle regardless of the diet used. Fish fed a diet supplemented with 10% of algae waste (W10) stand out for the highest content in oleic acid (C18:1 n-9), and the lowest in both linoleic (C18:2 n-6) and palmitic (C16:0) fatty acids. All fish samples showed values of health lipid indices within the limits recommend for a nutritional balanced diet. These results highlight that fish fed diets supplemented with P. canaliculata are sources of healthy lipids that might be consumed on a regular basis to prevent cardiovascular diseases.