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The role of the carapace in the uptake and storage of newly accumulated metals was investigated in the green crab exposed to environmentally relevant concentrations of calcium ([Ca] = 389 mg L-1 or 9.7 mmol L-1), zinc ([Zn] = 82 µg L-1 or 1.25 µmol L-1), and nickel ([Ni] = 8.2 µg L-1 or 0.14 µmol L-1) in 12 °C seawater, using radio-tracers (45Ca, 65Zn, 63Ni). After 24-h exposure, carapace exhibited the highest concentration of newly accumulated Ca, whereas carapace and gills exhibited the highest concentrations of both newly accumulated Zn and Ni relative to other tissues. For all three metals, the carapace accounted for >85 % of the total body burden. Acute temperature changes (to 2 °C and 22 °C) revealed the highest overall temperature coefficient Q10 (2.15) for Ca uptake into the carapace, intermediate Q10 for Ni (1.87) and lowest Q10 (1.45) for Zn. New Ca uptake into the carapace continued linearly with time for 24 h, new Zn uptake gradually deviated from linearity, whereas Ni uptake reached a plateau by 6 h. Attachment of a rubber membrane to the dorsal carapace, thereby shielding about 20 % of the total crab surface area from the external water, eliminated both new Zn and Ni incorporation into the shielded carapace, whereas 36 % of new Ca incorporation persisted. When recently euthanized crabs were exposed, new Zn uptake into the carapace remained unchanged, whereas Ca and Ni uptake were reduced by 89 % and 71 %, respectively. We conclude that the carapace is a very important uptake and storage site for all three metals. All of the uptake of new Zn and new Ni, and most of the uptake of new Ca into this tissue comes directly from the external water. For Zn, the mechanism involves only physicochemical processes, whereas for Ca and Ni, life-dependent processes make the major contribution.

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The present study was aimed at evaluating the in vivo effects of microplastics (MP), in terms of oxidative stress and histopathological effects, in two crustacean species: Procambarus clarkii and Leptuca pugilator. In addition, MP accumulation in the hepatopancreas (HP) of both species was also determined. Adults of both crayfish and crabs were exposed for one month to fluorescent polystyrene beads (size: 1 µm) at nominal concentrations of 1000 or 5000 particles/mL. During the exposure, animals were maintained under controlled feeding, aeration, temperature, and photoperiod conditions. At the end of the exposure, HP and hemolymph (HL) samples were harvested for analysis of oxidative damage and total antioxidant levels. Additionally, the presence of MPs in both tissues was confirmed. Significant differences with the control groups were observed in lipid peroxidation levels in HP in animals exposed to the lowest concentration in P. clarkii and to the highest concentration in L. pugilator. A marked increase in antioxidant levels was also observed in the HL at both concentrations in P. clarkii, and at the highest MPs concentration in L. pugilator. Moreover, several histopathological changes were detected in both gills and HP, including hypertrophied lamellae, lifting or collapse of gill epithelia, loss of normal shape of hepatopancreatic tubules, and epithelial atrophy in the HP tissue. We conclude that exposure to MP beads at selected concentrations results in oxidative damage, induces histopathological changes in gills and HP, and triggers an antioxidant response in two crustacean species.

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The production chain of Penaeus vannamei require detailed quality evaluation from farm to processing plant, to maintain the quality standards. We evaluated the impact of production chain steps in main defects of P. vannamei as red hepatopancreas, detached cephalothorax and post-mult. Red hepatopancreas and detached cephalothorax exhibited the highest defect incidence, which are related to capture method and post-harvest handling procedures. Red hepatopancreas showed a positive correlation (r = 0.56; P = 0.008) with post-molts. The Principal Component Analysis (PCA) explained 53.91% of total data variance, where the second principal component (PC2) contributed with 19.39% separating shrimp defects and management steps based on square cosines of red hepatopancreas and transport time. These results suggested that steps of production chain as well as post mortem process are directly related to shrimp defects, and the knowledge of these factors can contribute to improve the product quality.

A cadeia produtiva do Penaeus vannamei exige avaliações detalhadas quanto à qualidade do produto desde o campo até a planta de processamento, considerando a importância da manutenção dos padrões de qualidade. Foi avaliado o impacto das etapas da cadeia produtiva nos principais defeitos do P. vannamei como hepatopâncreas vermelho, cefalotórax desprendido e pós-mudas. Hepatopâncreas e o cefalotórax desprendido foram os defeitos de maior incidência, e estão relacionados ao método de despesca e manuseio pós-abate. O hepatopâncreas vermelho apresentou correlação positiva (r = 0,56; P = 0.008) com pós-muda. A Análise de Componentes Principais (ACP) explicou 53,91% da variância total dos dados, onde o segundo componente principal (PC2) contribuiu com 19,39% separando os defeitos do camarão e as etapas de manejo com base nos cossenos quadrados do hepatopâncreas vermelho e tempo de transporte. Esses resultados sugerem que as etapas da cadeia produtiva bem como o processo post mortem estão diretamente relacionados aos defeitos dos camarões, e o conhecimento desses fatores pode contribuir para a melhoria da qualidade do produto.

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The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.(AU)

A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.(AU)

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The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.

A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.

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Abstract The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.

Resumo A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.

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Environmental cues synchronize endogenous rhythms of many physiological processes such as hormone synthesis and secretion. Little is known about the diurnal pattern of hormones and gene expression of the Callinectes sapidus molt cycle. We aimed to investigate in the eyestalk and hepatopancreas of premolt and intermolt C. sapidus the following parameters: 1) the diurnal expression of the ecdysteroid receptor CasEcR isoforms, and the molt inhibiting hormone CasMIH; 2) the diurnal hemolymph ecdysteroid and melatonin levels; and 3) melatonin effects on the transcripts of the above-mentioned genes in intermolt C. sapidus. Ecdysteroid levels were higher in the premolt than the intermolt animals at all time points evaluated (ZTs). Premolt crabs displayed a variation of ecdysteroid concentration between time points, with a reduction at ZT17. No difference in the melatonin level was seen in either molt stage or between stages. In the eyestalk of intermolt animals, CasEcR expression oscillated, with a peak at ZT9, and premolt crabs have a reduction at ZT9; CasMIH transcripts did not vary along 24 h in either molt stage. Moreover, the evaluated eyestalk genes were more expressed at ZT9 in the intermolt than the premolt crabs. In the hepatopancreas, CasEcR expression showed a peak at ZT9 in premolt crabs. Exogenous melatonin (10-7 mol/animal) reduced the expression of both genes in the eyestalk at ZT17. In the hepatopancreas, melatonin markedly increased the expression of the CasEcR gene at ZT9. Taken altogether, our results are pioneer in demonstrating the daily oscillation of gene expression associated to molt cycle stages, as well as the daily ecdysteroid and melatonin levels and the remarkable influence of melatonin on the molt cycle of C. sapidus.

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This work aimed to assess the effect of the probiotic strain, Lactobacillus plantarum, on the levels of leptin, IGF-1 and their receptors on the hepatopancreatic tissues of Nile tilapia (Oreochromis niloticus) and then correlate fish growth performance and gut microbiological parameters. Fish juveniles (±23g) were reared in a recirculation system with constant aeration and temperature (25°C). They were distributed into six polyethylene tanks (45L) and fed twice a day at 5% of the tank biomass with the respective diets: control (commercial diet without probiotic) and supplemented with L. plantarum inoculum (1 x 108 CFU mL-1), both in triplicate. After 30 days of feeding, L. plantarum-fed fishes showed greater weekly growth rate, final weight, and feed conversion rate, in addition to higher count of lactic-acid bacteria and lower count of pathogenic bacteria in the intestinal tract, when compared to the control group. The immunostaining intensity for IGF-1 and leptin hormones was lower after L. plantarum supplementation than in the control group, with no change in the level for receptors. This reduction could implicate important changes in fish metabolism and homeostasis.(AU)

O presente trabalho teve como objetivo avaliar o efeito da cepa probiótica Lactobacillus plantarum sobre os níveis de leptina, IGF-1 e seus receptores no tecido hepatopancreático de tilápia-do-nilo (Oreochromis niloticus) e correlacionar com o desempenho zootécnico e os parâmetros microbiológicos intestinais dos peixes. Juvenis de tilápia-do-nilo (±23g) foram distribuídos em seis tanques de polietileno (45L) conectados a um sistema de recirculação, com aeração e temperatura constantes (25°C). Os peixes foram alimentados duas vezes ao dia, a 5% da biomassa do tanque, com as respectivas dietas: controle (dieta comercial sem probiótico) e suplementada com L. plantarum (1 x 108 UFC mL-1), ambas em triplicata. Após 30 dias de cultivo, os peixes alimentados com L. plantarum apresentaram maiores ganho de peso semanal, peso final e conversão alimentar, bem como maior contagem de bactérias ácido-láticas e menor contagem de bactérias patogênicas no trato intestinal das tilápias alimentadas com dieta probiótica, em comparação ao grupo controle. A intensidade da imunomarcação para os hormônios IGF-1 e leptina foi menor com a suplementação de L. plantarum do que no grupo controle, sem alterar os níveis de seus receptores. Essa redução pode implicar mudanças importantes no metabolismo e na homeostase dos peixes.(AU)

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The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This crustacean shifts its metabolism to survive under extreme environmental conditions such as hypoxia, although for a limited time. Glucose-6-phosphatase (G6Pase) is a key enzyme contributing to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. To our knowledge, there are no current detailed studies about cDNA or gene sequences of G6Pase from any crustacean reported. Herein we report the shrimp P. (L.) vannamei cDNA and gene sequences. The gene contains seven exons interrupted by six introns. The deduced amino acid sequence has 35% identity to other homolog proteins, with the catalytic amino acids conserved and phylogenetically close to the corresponding invertebrate homologs. Protein molecular modeling predicted eight transmembrane helices with the catalytic site oriented towards the lumen of the endoplasmic reticulum. G6Pase expression under normoxic conditions was evaluated in hepatopancreas, gills, and muscle and the highest transcript abundance was detected in hepatopancreas. In response to different times of hypoxia, G6Pase mRNA expression did not change in hepatopancreas and became undetectable in muscle; however, in gills, its expression increased after 3 h and 24 h of oxygen limitation, indicating its essential role to maintain glycemic control in these conditions.

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In this study, we analyzed biomarkers and heavy metals in Ucides cordatus specimens caught in mangrove areas [A1 = reference (low impact area); A2, A3 and A4 = port complex (potentially impacted area)] of the Amazonian northern coast of Brazil. Sediments and crab muscles sampled from the potentially impacted mangroves showed high levels of metals. Additionally, catalase and glutathione S-transferase enzymes in the gills and hepatopancreas of the collected crabs were elevated in crabs from A2, A3 and A4 relative to those from A1. The histological characteristics of the gills and hepatopancreas differed in crabs collected from A2, A3 and A4 compared with those collected from A1. The crabs were larger and heavier in the rainy season in A1 and A3 and heavier in the dry season in A4. Heavy metal analyses of sediments, crab muscles and biological responses indicated that the animals are subjected to stress levels differentiated in the mangroves due to a gradient (A4>A3>A2>A1) of anthropogenic pressure in São Marcos Bay. In conclusion, the analyses of metals in the sediment and in the muscles of crabs, as well as the biochemical biomarker and histological analyses, suggest that crabs from mangroves in the port complex region are subject to pollutants that compromise their health.

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Abstract Study aimed to evaluate the effect of replacing fish meal with different levels of full fat soybean meal (FFSBM) on growth and digestive enzyme activities in the stomach, Liver and intestine for Clarias gariepinus. Four diets (D1, D2, D3 and D4) were formulated with 0, 15, 20 and 20 g 100-1 protein + DL-methionine by alternating FFSBM with fish meal. The growth of C. gariepinus was found to be significantly decreased when FFSBM replacement increased. Final body weight was 89.69, 79.70, 70.82 and 68.29 g for fish fed on D1, D2, D3 and D4, respectively, with significant differences between treatments. Specific growth rate (SGR) ranged between 3.11 to 2.78%. Proteolytic activity was higher only with alkaline pHs, whereas only very low activity was shown with acidic. Results of liver showed approximately similar results at acid and alkaline. In contrast, higher proteolytic activity in the stomach was observed at acid pHs 3.0 and 4.0 μg tyrosine-1 minute-1 mg-1 protein) whereas lower values were observed at neutral pH 7.0 g tyrosine-1 minute-1 mg-1 protein for catfish fed on the experimental diets. Moreover, trypsin activity was higher for the stomach, followed by the intestine and liver. However, higher amount of amylase observed in the liver than intestine and stomach.

Resumo Objetivou-se avaliar o efeito da substituição da farinha de peixe por diferentes níveis de farelo de soja integral (FSI) sobre o crescimento e atividades de enzimas digestivas no estômago, fígado e intestino de Clarias gariepinus. Quatro dietas (D1, D2, D3 e D4) foram formuladas com 0, 15, 20 e 20 g 100-1 proteína + DL-metionina, alternando FSI com farinha de peixe. O crescimento de C. gariepinus foi significativamente reduzido quando aumentou a substituição de farinha de peixe por FSI . O peso corporal final foi de 89,69, 79,70, 70,82 e 68,29 g para peixes alimentados com D1, D2, D3 e D4, respectivamente, com diferenças significativas entre os tratamentos. Taxa de crescimento específico (TCE) variou entre 3,11 a 2,78%. A atividade proteolítica foi maior apenas com pHs alcalinos, enquanto que atividades muito baixas foram somente mostradas com ácido. Os resultados no fígado mostraram valores aproximadamente semelhantes tanto em meio ácido quanto alcalino. Em contraste, observou-se maior atividade proteolítica no estômago em pH ácido (3,0 e 4,0 g de tirosina-1 minuto-1 mg-1 de proteína), ao passo que valores mais baixos foram observados em pH neutro (7,0 g de tirosina-1 minuto-1 mg-1 de proteína) para os bagres que se alimentaram com as dietas experimentais. Além disso, a atividade da tripsina foi maior para o estômago, seguida pelo intestino e pelo fígado. No entanto, observou-se maior quantidade de amilase no fígado,intestino e estômago.

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Vibrio parahaemolyticus strains have been identified as the causative agent of acute hepatopancreas necrosis disease (Vp AHPND+) in shrimp. In the present work, a highly virulent (M0904) and less virulent (M0607) strain of Vp AHPND+ were used in experimental infections of Penaeus vannamei. Throughout the experiment, histopathological analyses were conducted on shrimp tissues, and samples from the bottom (B) and water column (WC) of each aquarium were taken to estimate bacterial growth. The first deaths of shrimp challenged with Vp M0904 occurred 4 h post-infection (hpi), reached 50% mortality at 17 hpi, and showed more acute pathological progression associated with AHPND despite both strains having similar bacterial densities from 0-4 hpi. The first deaths of shrimp inoculated with Vp M0607 occurred at 13 hpi, a difference of 9 h. These shrimps displayed reduction of reserve vacuoles in R and B cells of the hepatopancreas, and reached 50% mortality at 27 hpi. As bacterial growth in the WC and B in the first hours was similar for both strains, bacterial density was not a significant factor in Vp virulence. Since pirAB toxin is the causative agent of AHPND, it is possible that there is a threshold level at which the toxin damages hepatopancreatic tissues, so production or secretion of the toxin can be dependent of the Vp strain. We propose new AHPND stage: remission with 3 main routes for the disease outcome after the remission stage (re-infection, chronic effect and recuperation). Hepatopancreatic embryonic cells can be used as a biological indicator of this stage.

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The Caribbean spiny lobster Panulirus argus (Latreille, 1084) sustains economically valuable fisheries throughout the wider Caribbean region. This species is currently affected by the pathogenic virus Panulirus argus Virus 1 (PaV1) that causes a systemic and chronic-degenerative infection in juvenile spiny lobsters P. argus. To date, there is no available information regarding the host alterations induced by this pathogen at the molecular level. In the present study, comparative proteomic analyses of the changes in the hepatopancreas between infected and non-infected juvenile lobsters were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) coupled to synchronous precursor selection (SPS)-based MS3. We identified a total of 636 proteins, being 68 down-regulated and 71 up-regulated proteins. Among the down-regulated proteins, we identified several enzymes involved in the metabolism of hormones and lipids, digestive proteases and glycosidases, while proteins associated with the histone core, protein synthesis, immune response and RNA regulation were up-regulated. Several misregulated enzymes involved in the regulation of neuromodulators were also identified. RT-qPCR assays were used to validate the expression of transcripts encoding for selected differential proteins that were in concordance to proteomic data, as well as the tendency observed in the enzymatic activities of trypsin, chymotrypsin, and glycosidase. In a similar way, we observed glycogen reduction in muscle, and an increase in plasma acylglycerides and glucose, which may be explained by proteomic data. This study provides the first insight into the molecular changes in the hepatopancreas of Caribbean spiny lobsters associated to PaV1 infection. Data provided herein would help to clarify the origin of the molecular misregulations observed at macroscopic level in this host-pathogen interaction.

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Gambusia holbrooki es el pez de agua dulce con mayor distribución en Chile y el mundo, pero los estudios que abordan la morfología e histología hepática del pez son escasos. El hígado es utilizado para evidenciar efectos subletales de contaminantes ambientales y es preciso contar con una descripción histomorfológica del hígado para futuros estudios comparativos. El presente estudio tiene como objetivo describir patrones histomorfológicos del hígado de Gambusia holbrooki, para ello se colectaron 97 individuos adultos de los sistemas límnicos de los valles de Lluta y Azapa (Extremo norte de Chile), para observar patrones morfológicos comunes en ambas poblaciones, se utilizó técnicas histológicas de rutina e histoquímica. Las evidencias demostraron que el hígado contiene tejido pancreático y su arquitectura tisular es trabecular con mayor presencia de capilares sinusoides.

Gambusia holbrooki is the freshwater fish with the greatest distribution in Chile and the world. However, studies dealing with morphology and liver histology of fish are scarce. The liver commonly shows the sublethal effects of environmental pollutants and there should be a histomorphological description of the liver for further comparative studies. The present study aims to describe histomorphological patterns of the liver of Gambusia holbrooki. A total of 97 adult individual specimens were collected from the ecosystems in the valleys of Lluta and Azapa (Region of Arica and Parinacota), to observe patterns morphologically common in both populations. Routine histological and histochemical techniques were used for analysis. The evidence showed that the liver contains pancreatic tissue, and that tissue architecture is trabecular with greater presence of capillary sinusoids.

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The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

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Glyphosate-based herbicides (GBH), including Roundup®, are the most used herbicides in agricultural and non-agricultural areas, which can reach aquatic environments through drift during application or surface runoff. Some studies, mostly in fish, demonstrated that GBH caused oxidative stress in non-target animals. However, only few information is available on the GBH effects in the antioxidant and stress proteins of many other organisms, such as freshwater crustaceans. Thus, we aimed to investigate the effects of environmentally relevant GBH concentrations on the relative transcript expression (RTE) of the superoxide dismutase (sod1), catalase (cat), selenium-dependent glutathione peroxidase (gpx), glutathione-S-transferase (gst), thioredoxin (txn), heat shock protein (hsp70 and hsp90) in the hepatopancreas of the ecologically important freshwater prawn Macrobrachium potiuna. Moreover, this study aimed to assess the gender-differences responses to GBH exposure. Male and female prawns were exposed to three Roundup WG® concentrations (0.0065, 0.065 and 0.28 mg of glyphosate/L) and a control group (0.0 mg/L) for 7 and 14 days. In general, males had an under-expression of the studied genes, indicating an oxidative stress and possible accumulation of ROS in the hepatopancreas. In the opposite, females had an overexpression of the same genes, indicating a more robust antioxidant system, in order to cope with the possible ROS increase after Roundup WG® exposure. Therefore, results confirmed that gender could be a confounding factor in ecotoxicological assessment of GBH effects. Additionally, this work highlights that sod1, cat, gpx, gst, txn, hsp70 and hsp90 gene expressions seem to be useful biomarkers to investigate the oxidative stress caused by Roundup WG® in Macrobrachium sp.

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This research was to evaluate the load and mobility of Cu in juvenile Litopenaeus vannamei after exposure (48 h) to sublethal concentration of nitrite (5.3 mg/L NO2--N) at a salinity of 3.0 g/L. The hypothesis is that such exposure causes a Cu mobility in the tissues of shrimp. The Cu concentration in exoskeleton, hepatopancreas, muscle and hemolymph in the control group were 38.9 ± 3.0, 2478 ± 256, 11.9 ± 0.2 µg/g (dw) and 95.4 ± 19.1 µg/mL, while in the nitrite exposure, were 46.0 ± 0.1, 1546 ± 173, 11.3 ± 0.3 µg/g (dw) and 118.2 ± 10.5 µg/mL, respectively. Only hepatopancreas exhibited a significant (p < 0.05) reduction (37.6%) between the control and the nitrite exposure. This is evidence that nitrite has a significant effect on Cu accumulation in hepatopancreas when shrimp are exposed to sublethal levels in a salinity of 3 g/L. Results confirm the hypothesis that Cu mobility was only significant in hepatopancreas.

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Study aimed to evaluate the effect of replacing fish meal with different levels of full fat soybean meal (FFSBM) on growth and digestive enzyme activities in the stomach, Liver and intestine for Clarias gariepinus. Four diets (D1, D2, D3 and D4) were formulated with 0, 15, 20 and 20 g 100-1 protein + DL-methionine by alternating FFSBM with fish meal. The growth of C. gariepinus was found to be significantly decreased when FFSBM replacement increased. Final body weight was 89.69, 79.70, 70.82 and 68.29 g for fish fed on D1, D2, D3 and D4, respectively, with significant differences between treatments. Specific growth rate (SGR) ranged between 3.11 to 2.78%. Proteolytic activity was higher only with alkaline pHs, whereas only very low activity was shown with acidic. Results of liver showed approximately similar results at acid and alkaline. In contrast, higher proteolytic activity in the stomach was observed at acid pHs 3.0 and 4.0 g tyrosine-1 minute-1 mg-1 protein) whereas lower values were observed at neutral pH 7.0 g tyrosine-1 minute-1 mg-1 protein for catfish fed on the experimental diets. Moreover, trypsin activity was higher for the stomach, followed by the intestine and liver. However, higher amount of amylase observed in the liver than intestine and stomach.(AU)

Objetivou-se avaliar o efeito da substituição da farinha de peixe por diferentes níveis de farelo de soja integral (FSI) sobre o crescimento e atividades de enzimas digestivas no estômago, fígado e intestino de Clarias gariepinus. Quatro dietas (D1, D2, D3 e D4) foram formuladas com 0, 15, 20 e 20 g 100-1 proteína + DL-metionina, alternando FSI com farinha de peixe. O crescimento de C. gariepinus foi significativamente reduzido quando aumentou a substituição de farinha de peixe por FSI . O peso corporal final foi de 89,69, 79,70, 70,82 e 68,29 g para peixes alimentados com D1, D2, D3 e D4, respectivamente, com diferenças significativas entre os tratamentos. Taxa de crescimento específico (TCE) variou entre 3,11 a 2,78%. A atividade proteolítica foi maior apenas com pHs alcalinos, enquanto que atividades muito baixas foram somente mostradas com ácido. Os resultados no fígado mostraram valores aproximadamente semelhantes tanto em meio ácido quanto alcalino. Em contraste, observou-se maior atividade proteolítica no estômago em pH ácido (3,0 e 4,0 g de tirosina-1 minuto-1 mg-1 de proteína), ao passo que valores mais baixos foram observados em pH neutro (7,0 g de tirosina-1 minuto-1 mg-1 de proteína) para os bagres que se alimentaram com as dietas experimentais. Além disso, a atividade da tripsina foi maior para o estômago, seguida pelo intestino e pelo fígado. No entanto, observou-se maior quantidade de amilase no fígado,intestino e estômago.(AU)

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Global trends in pesticide use can increase aquatic pollution and affect resident fisheries. Crabs exposed to organophosphate pesticides, such as chlorpyrifos, may increase production of reactive oxygen species (ROS), affecting the pro-oxidant/antioxidant balance. Zichiopsis collastinensis crabs were exposed to environmentally relevant concentrations of chlorpyrifos (0.1 and 0.5 µg L-1). Effects on the oxidative stress enzymes catalase, superoxide dismutase, glutathione S-transferases, glutathione reductase, and on thiobarbituric acid reactive substances and hydrogen peroxide concentrations were evaluated at four intervals during 96 h exposures. Exposures caused decreased GST activity and increased H2O2 levels in gills. There were modifications of GST, CAT and SOD activities in the hepatopancreas after 12 h of exposure, and an increase of H2O2 levels at every exposure interval observed. The present study proved that chlorpyrifos lead to oxidative stress in Z. collastinensis. However other enzymatic/non-enzymatic responses should be further investigated in order to be included as part of a battery of biomarkers, together with H2O2 levels, which is a parameter highly recommended to be taken into account.

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BACKGROUND: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. OBJECTIVE: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. METHODS: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. RESULTS: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases ß sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. CONCLUSION: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

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