Primary, secondary, and tertiary stress responses of juvenile seahorse Hippocampus reidi exposed to acute acid stress in brackish and seawater.

Seahorse Hippocampus reidi is a vulnerable species, inhabiting estuarine and coastal waters. The safety of acidic environments for fish has been considered in terms of ocean acidification in nature and decreasing pH in intensive aquaculture systems. This study aimed to investigate the effects of acute exposition (96 h) of juvenile seahorses to different pH (5, 6, 7, and 8) in brackish (BW - salinity 11) or seawater (SW - salinity 33). For that, we studied the responses of cortisol, oxidative stress, and survival, thus covering primary, secondary, and tertiary stress responses. In SW, cortisol levels were not altered for fish maintained at pH 5 and 8. However, in BW, cortisol was higher for fish kept at pH 5. Regarding secondary stress responses, only GST activity increased with acidification in SW. However, acidification in BW caused biochemical alterations at enzymatic level (SOD, GST, GPx) and glutathione metabolism, accompanied by reduction of antioxidant capacity (TEAC) and increased lipid peroxidation (TBARS). Survival was always above 90% and it did not differ significantly among pH levels. Our results suggest that H. reidi juveniles are more vulnerable to acidic exposure in BW than in SW.

Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus / Caracterização de enzimas digestivas do linguado brasileiro de cativeiro Paralichthys orbignyanus

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.

Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus.

Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Hemangioma esclerosante mandibular em Paralichthys orbignyanus: relato de caso / Sclerosing hemangioma mandibular in Paralichthys orbignyanus: case report

Neste trabalho, descreveu-se o primeiro caso de hemangioma esclerosante registrado em um exemplar adulto do linguado Paralichthys orbignyanus. Produzido a partir de reprodução artificial, o peixe em questão tinha aproximadamente 10 anos de idade e fazia parte de um plantel de reprodutores. Ao ser retirado do tanque, notou-se a presença de lesão mandibular com escoriações e focos hemorrágicos. Amostras do tumor foram coletadas da mandíbula para análise histopatológica. Microscopicamente foi observada uma proliferação de numerosos vasos sanguíneos rodeados por um estroma conectivo denso. A etiologia dessa neoplasia é desconhecida, mas o fato de o exemplar ter permanecido por muitos anos em cativeiro pode ter contribuído para o surgimento desse tipo de lesão, devido aos choques mecânicos contra a parede do tanque que acontecem esporadicamente.(AU)

In this study, we described the first case of sclerosing haemangioma in an adult Brazilian flounder Paralichthys orbignyanus. Produced by artificial reproduction, the fish was approximately 10 years old and was maintained at a breeding stock. When removed from the tank, mandibular lesion with excoriations and hemorrhagic foci were noted. Tumor samples were collected from the mandible for histopathological analysis. Proliferation of numerous blood vessels surrounded by dense connective stroma was observed microscopically. The etiology of this neoplasia is unknown, but the fact that the specimen remained in captivity for many years, may have contributed to the appearance of this type of lesion, due to sporadic mechanical shocks to the tank wall.(AU)

Elemental turnover rates and trophic discrimination in juvenile Lebranche mullet Mugil liza under experimental conditions.

The aim of this study was to determine the isotopic-turnover rate (RIT ) and trophic-discrimination factor (FTD ) in muscle tissues of Lebranche mullet Mugil liza fed an experimental diet (δ13 C = -27·1‰; δ15 N = 1·0‰). Juvenile M. liza exhibited a relatively fast RIT , with a half-life (t50 ) of only 16 and 14 days for δ13 C and δ15 N respectively and a nearly complete isotopic turnover (t95 ) of 68 and 60 days for δ13 C and δ15 N.

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

BACKGROUND: Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. OBJECTIVE: To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). MATERIALS AND METHODS: Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). RESULTS: The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. CONCLUSION: As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Crescimento e metabolismo do nitrogênio em juvenis de Trachinotus marginatus alimentados com diferentes níveis proteicos / Growth and nitrogenous metabolism of juveniles Trachinotus marginatus feeding with differently protein levels

O objetivo deste trabalho foi avaliar o crescimento e alterações no metabolismo do nitrogênio, além da composição do fígado e do músculo de juvenis de pampo Trachinotus marginatus alimentados com 43, 54 ou 64% de proteína bruta (PB43, PB54 e PB64, respectivamente). Grupos de 10 juvenis (1,47±0,14g) foram estocados em nove tanques de 50L, em sistema de recirculação de água marinha, durante 55 dias, alimentados quatro vezes ao dia com as rações pré-definidas. O ganho em peso, a taxa de crescimento específico e a conversão alimentar não apresentaram diferença significativa (P>0,05). Contudo, o acréscimo de proteína consumida aumentou o índice hepatossomático e a taxa de excreção de amônia pós-prandial, bem como diminuiu a retenção proteica. Os peixes alimentados com a dieta PB43 apresentaram menor concentração da transaminase glutâmico oxalacética hepática, além de menor concentração de triglicerídeos no músculo e fígado (P<0,05). A composição da carcaça, o teor de glicogênio e de proteínas totais para músculo e fígado não mostraram diferenças significativas (P>0,05), excetuando o maior teor de lipídios da carcaça no tratamento PB43. Concluímos que a melhor utilização da proteína por juvenis de pampo é observada quando eles são alimentados com uma dieta contendo 43% de proteína.

The experiment aimed to evaluate the growth, the nitrogen metabolism and liver and muscle composition of juvenile pompano Trachinotus marginatus fed with different protein levels (PB43, PB54, PB64). Groups of 10 juveniles (1.47±0.14g) were stocked in nine tanks with 50L in a marine recirculating aquaculture system during 55 days. Fish were fed four times a day. The weight gain, specific growth rate and feed conversion rate did not present significant differences (P>0.05). However, the increase in protein consumption augmented the hepatosomatic index, the pos prandial ammonia excretion rate and reduced the protein efficiency rate. Fish fed with PB43 presented lower hepatic glutamic oxaloacetic transaminase and lower concentration of triglycerides in muscle and liver (P<0.05). Carcass composition, glycogen and muscle and liver protein content did not present significant differences (P>0.05), with the exception of the lipid content in fish carcass fed with PB43. It is concluded that the better protein utilization in juvenile pompano is observed when they are fed with 43% protein.

Yersiniose em Trachinotus marginatus (pampo): diagnóstico histopatológico e imuno-histoquímico / Yersiniosis in Trachinotus marginatus (pampo): histopathological and immunohistochemical diagnostic

Descreveu-se um surto de yersiniose em uma criação de pampo Trachinotus marginatus estudado no Laboratório de Piscicultura Estuarina e Marinha da Universidade Federal do Rio Grande. A enfermidade manifestou-se por transtornos natatórios, exoftalmia com panoftalmite e pela clássica boca vermelha, nome pelo qual se denomina "enfermidade da boca vermelha" (EBV). Na necropsia, foram observados focos de hemorragia peritoneal, esplenomegalia e hepatomegalia. Microscopicamente, foi observada panoftalmite com infiltrados inflamatórios densos que afetavam quase todas as estruturas oculares. Esses infiltrados estavam constituídos por granulócitos, linfócitos, macrófagos e células granulares eosinofílicas. No exsudado, observaram-se estruturas pequenas, pouco coradas, de aspecto bacteriano Gram negativo. O estudo imuno-histoquímico, que se utilizou de um anticorpo monoclonal anti-Yersinia ruckeri, resultou positivo. Este é o primeiro surto conhecido de yersiniose em Trachinotus marginatus no Brasil.

In the "Laboratório de Piscicultura Estuarina e Marinha da Universidade Federal do Rio Grande" the rearing of Trachinotus marginatus (pompano) is studied, and in one of these rearing a yersioniosis outbreak occured. The disease was manifested by swimming disturbances, exophthalmiawith panophthalmitis and the classic red mouth, (red mouth disease (RMD). The necropsies revealed focus of peritoneal hemorrhage, esplenomegalyand hepatomegaly.Microscopically a panophthalmitis with dense inflammatory infiltrates was observed, which affected almost all ocular structures. These infiltrates were constituted by granulocytes, lymphocytes, macrophages and eosinophylicgranular cells (ECG), in the exudatessmall structures less colored with Gram negative bacteria aspect were observed. The immunohistochemical, which used a monoclonal antibody anti- Yersinia ruckeri waspositive. The RMD is caused by enterobacteriaGram negative, which affects both freshwater and saltwater fish, with predominance in salmonids. This is the first known yersiniosis outbreak in Trachinotus marginatus in Brazil and it is necessary to keep this disease in mind at the moment of rearing fish of this species.

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus / Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus.

Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.

Myxobolus sp. can cause in vivo myoliquefaction in the host Paralichthys orbignyanus (Osteichthyes, Paralichthydae).

We found intense myoliquefaction in vivo among specimens of wild and farmed South American flatfish Paralichthys orbignyanus Valenciennes, 1839. The soft flesh condition was attributed to Myxobolus sp. since the fish were not infected by myxosporeans which are usually associated with this condition (Kudoa spp.), and other causes (bacteria, fungi) were not found. The histopathology of the infection is described.

Immune adaptive response induced by Bicotylophora trachinoti (Monogenea: Diclidophoridae) infestation in pompano Trachinotus marginatus (Perciformes: Carangidae).

Fish have developed protective strategies against monogeneans through immunological responses. In this study, immune adaptive response to parasites was analysed in the pompano Trachinotus marginatus infested by Bicotylophora trachinoti. Hosts were pre-treated with formalin and after 10 days assigned to one of the following experimental treatments: (1) fish infested with remaining eggs of B. trachinoti; (2) fish infested with remaining eggs of B. trachinoti and experimentally re-infested by exposure to T. marginatus heavily infested with B. trachinoti. Samples were collected at 0, 15, and 30 days. Gills were dissected to check the presence of B. trachinoti. Blood was collected for haematological and biochemical assays. Spleen and head-kidney were dissected for phagocytosis assay. The spleen-somatic index was also calculated. Re-infested fish showed a faster and higher parasite infestation than infested ones. The parasite mean abundance at 15 days was 24.86+/-13.32 and 11.67+/-8.57 for re-infested and infested fish, respectively. In both groups, hosts showed an immune adaptive response to parasite infestation that was marked by an increased number of leukocytes. Also, phagocytosis (%) in spleen and head-kidney cells was stimulated after parasite infestation (92.50+/-3.73 and 66.00+/-9.54, respectively), becoming later depressed (77.39+/-6.69 and 53.23+/-9.14, respectively). These results support the hypothesis that monogenean infestation induces a biphasic response of the non-specific defence mechanisms in the pompano T. marginatus. This response is marked by an initial stimulation followed by a later depression of the non-specific defence mechanisms.