RESUMO

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

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RESUMO

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris­an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research­using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

A constante intensificação da aquicultura tem aumentado consideravelmente os níveis de estresse dos animais cultivados e, consequentemente, o número e a intensidade dos surtos de doenças. Logo, estudos sobre a resposta imune dos peixes, especialmente relacionados com a interação dos leucócitos de peixes com potenciais patógenos e xenobióticos, são de grande importância para o desenvolvimento de novas estratégias profiláticas e curativas. No presente trabalho, nós obtivemos sucesso ao isolar leucócitos oriundos do rim cranial de Astyanax lacustris ­ uma importante espécie de peixe Neotropical para a aquicultura e um modelo em potencial para pesquisas em aquicultura Neotropical ­ usando um protocolo de centrifugação com Percoll. Os leucócitos isolados foram incubados com lipossacarídeo (LPS) e, a expressão dos genes IL-1ß, IL-8, LysC, e LysG foi avaliada. Ainda, um novo protocolo para avaliação da atividade fagocítica dos leucócitos utilizando leveduras coradas com Vermelho Congo foi estabelecido. Os leucócitos isolados responderam à indução com LPS, exibindo up regulation dos genes IL-1ß e IL-8, duas das interleucinas pró-inflamatórias mais importantes para a resposta imune de vertebrados. Além do mais, a concentração ótima de leveduras para a avaliação da fagocitose foi de 106 células mL-1, resultando em uma capacidade fagocítica (PC) aceitável, mas sem excesso de leveduras durante o processo de contagem, garantindo maior precisão e eficácia do método. Até o presente momento, o presente estudo é o primeiro a investigar a expressão gênica e atividade fagocítica de leucócitos isolados de A. lacustris através da abordagem in vitro. Ainda, nossos resultados servirão de referência para futuros estudos em imunologia e toxicologia de peixes Neotropicais.

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Previous studies of contextual interference have shown that practicing several motor skills randomly (high contextual interference) facilitates retention and transfer in comparison to practicing the same tasks in a blocked order (low contextual interference). However, many studies have not supported this phenomenon in motor learning, and some researchers have questioned whether the effect can be tested by using only a few trials on the transfer test. The present study used a different methodological approach in which the number of test trials was increased to assess whether the contextual interference effect is sustained over an extended number of trials in the transfer phase. Undergraduate students (N=32) were randomly allocated to either a blocked or random group. The participants practiced 80 acquisition trials in the dart-throwing task from distances of 300 cm and 420 cm, using two different grips. The transfer test after a 10-min. interval consisted of 40 trials with a new grip at a distance of 360 cm. The results did not support the contextual interference effect since there were no significant differences between groups on transfer. These findings suggest that the contextual interference effect may not be a global learning phenomenon which can be generalized to all learning situations.

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The efficiencies of a natural Typha spp wetland (Wn) formed on a river bed and its effluent treatment in a constructed wetland (Wc, subsurface horizontal flow) were investigated in northeastern Brazil (Paraiba State). The Wc system (12 tanks with stone gravel, 4.13 m2, 0.22 m3, 20 Typha spp rhizomes, m(-2) each, with 38, 29, and 19 mm x d(-1) hydraulic loadings, and 5, 7, and 10 days HRT) was fed daily with effluent from a Wn. Wn removal presented the highest values after Typha spp were cut during the 5th week. Removal values were (1st and 2nd periods or before and after cutting): 75% and 81% BOD5; 10-53% total phosphorus; 13%-55% ammonia; 89%-91% FC; 90-96% coliphages and bacteriophages. Wc removals increased with time with best results on 10 d HRT. Removals were also higher in the 2nd period: 74%-78% BOD5; 58%-82% ammonia; 90% FC; 94-98% FS; and 92%-96% coliphages and bacteriophages. Despite the high remaining values of FC (1.4 x 10(4) CFU/100 ml) and FX (4 x 10(3) CFU/100 ml), the removals were satisfactory and HRT dependent, suggesting a gradual optimization of the system with time. The Wc exhibited good efficiency for improving water quality from polluted river.

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