Implications of Salinity and Acidic Environments on Fitness and Oxidative Stress Parameters in Early Developing Seahorses Hippocampus reidi.

Water acidification affects aquatic species, both in natural environmental conditions and in ex situ rearing production systems. The chronic effects of acidic conditions (pH 6.5 vs. pH 8.0) in seahorses (Hippocampus spp.) are not well known, especially when coupled with salinity interaction. This study investigated the implications of pH on the growth and oxidative stress in the seahorse Hippocampus reidi (Ginsburg, 1933), one of the most important seahorse species in the ornamental trade. Two trials were carried out in juveniles (0-21 and 21-50 DAR-days after the male's pouch release) reared under acid (6.5) and control (8.0) pH, both in brackish water (BW-salinity 11) and seawater (SW-salinity 33). In the first trial (0-21 DAR), there was no effect of pH on the growth of seahorses reared in SW, but the survival rate was higher for juveniles raised in SW at pH 6.5. However, the growth and survival of juveniles reared in BW were impaired at pH 6.5. Compared to SW conditions, the levels of superoxide dismutase and DT-diaphorase, as well as the oxidative stress index, increased for juveniles reared in BW. In the second trial, seahorse juveniles were reared in SW at pH 8.0, and subsequently kept for four weeks (from 21 to 50 DAR) at pH 6.5 and 8.0. The final survival rates and condition index were similar in both treatments. However, the growth under acidic conditions was higher than at pH 8.0. In conclusion, this study highlights that survival, growth, and oxidative status condition was enhanced in seahorse juveniles reared in SW under acidic conditions (pH = 6.5). The concurrent conditions of acidic pH (6.5) and BW should be avoided due to harmful effects on the fitness and development of seahorse juveniles.

Exposure to salinity induces oxidative damage and changes in the expression of genes related to appetite regulation in Nile tilapia (Oreochromis niloticus).

Variations in water salinity and other extrinsic factors have been shown to induce changes in feeding rhythms and growth in fish. However, it is unknown whether appetite-related hormones mediate these changes in Nile tilapia (Oreochromis niloticus), an important species for aquaculture in several countries. This study aimed to evaluate the expression of genes responsible for appetite regulation and genes related to metabolic and physiological changes in tilapia exposed to different salinities. Moreover, the study proposed to sequence and to characterize the cart, cck, and pyy genes, and to quantify their expression in the brain and intestine of the fish by quantitative polymerase chain reaction (qPCR). The animals were exposed to three salinities: 0, 6, and 12 parts per thousand (ppt) of salt for 21 days. Furthermore, lipid peroxidation, reactive oxygen species, DNA damage, and membrane fluidity in blood cells were quantified by flow cytometry. The results indicated an increased expression of cart, pyy, and cck and a decreased expression of npy in the brain, and the same with cck and npy in the intestine of fish treated with 12 ppt. This modulation and other adaptive responses may have contributed to the decrease in weight gain, specific growth rate, and final weight. In addition, we showed oxidative damage in blood cells resulting from increasing salinity. These results provide essential data on O. niloticus when exposed to high salinities that have never been described before and generate knowledge necessary for developing biotechnologies that may help improve the production of economically important farmed fish.

Dietary Euterpe oleracea Mart. attenuates seizures and damage to lipids in the brain of Colossoma macropomum.

The Amazonian açai fruit (Euterpe oleracea) has shown promising anticonvulsant properties, comparable to those of diazepam (BDZ) in in vivo models submitted to pentylenetetrazole (PTZ). PTZ is a classic convulsant agent used in studies for the purpose of screening anticonvulsants and investigating the mechanisms of epilepsy. Herein, we aimed to determine, for the first time, the effect of dietary administration of lyophilized E. oleracea (LEO) on PTZ-induced seizures, using juvenile Colossoma macropomum fish (9.1 ± 1.5 g) as a model. A control diet (0.00% LEO) and two levels of LEO inclusion were established: 5.00% and 10.0% LEO (w/w). Fish were divided into five groups (n = 5): control (0.9% physiological solution; i.p.), PTZ (PTZ 150 mg kg-1; i.p.), PTZ LEO 5.00%, PTZ LEO 10.0%, and BDZ-PTZ (BDZ: diazepam 10 mg kg-1; i.p.). In addition to the electroencephalography (EEG), the lipid peroxidation (TBARS) was quantified in the brain, along with the characterization of behavioral responses. Fish receiving PTZ showed intense action potential bursts (APB), which overlapped with a hyperactive behavior. In PTZ LEO 5.00% and 10.0% groups, convulsive behavior was significantly reduced compared to the PTZ group. Fish fed 5.00% or 10.0% LEO and exposed to PTZ showed less excitability and lower mean amplitude in tracings. The inclusion of 10.0% LEO in the diet prevented the increase in mean amplitude of the EEG waves by 80%, without significant differences to the quantified mean amplitude of the BDZ-PTZ group. TBARS concentration was reduced by 60% in the brain of fish fed 10.0% LEO-enriched diets relative to the PTZ-administered group. The results of this study demonstrated the anticonvulsant and protective roles of LEO to the brain, and the dietary inclusion of LEO seems to be promising for the formulation of functional diets. Results of this study may boost the interest on the anti-seizurogenic properties of Euterpe oleracea, including the development of new approaches for the prevention of seizures in humans and animals with low epileptic threshold.

Temperature and hypoxia on oxidative stress responses in pacu Piaractus mesopotamicus.

The aim of this study was to verify the effects of the interaction between different temperatures and levels of dissolved oxygen in the oxidative stress parameters of pacu juveniles. A total of 81 pacu juveniles (61.7 ± 9.1 g) were exposed to three temperatures (18, 23, and 28 °C), acclimated for a period of 30 days, and then submitted to three levels of dissolved oxygen: control or normoxia (7 mg L-1); moderate hypoxia (4 mg L-1); and severe hypoxia (2 mg L-1) for 12 h. Glutathione-S-transferase (GST) activity, total antioxidant capacity against peroxyl radicals (ACAP), and protein thiol content (PSH) and LPO (lipid peroxidation) [measured by the TBARS] were measured in gill, liver, muscle and brain. The results indicated that the interaction between different temperatures and dissolved oxygen levels caused alterations in the antioxidant system and induced lipid and protein damage in pacu juveniles. In addition, the effects were organ specific. In conclusion, exposure to moderate and severe hypoxia affect oxidative stress parameters and have been shown to be organ-specific in pacu juveniles. The interaction between 23 °C and hypoxia caused greater disturbances in oxidative stress markers, such as PSH in the gills and liver and LPO in the muscle.

Oxidative stress responses of juvenile tambaqui Colossoma macropomum after short-term anesthesia with benzocaine and MS-222.

The present study aimed to evaluate the effects of benzocaine and tricaine methanesulfonate on oxidative stress parameters of juvenile tambaqui tissues. Fish (n=80) were anesthetized with benzocaine (100 mg L-1) or tricaine (240 mg L-1) and two control groups were used (non-anesthetized fish and fish exposed to ethanol-only). After anesthetic induction 10 fish/anesthetic were euthanized after 3, 12 and 24 hours post-anesthesia and tissue samplings (gills, liver and brain) were performed. Samples were submitted to analyses of enzyme activity glutathione-S-transferase (GST), cellular lipid peroxidation (TBARS) and total antioxidant capacity (ACAP). ACAP increased in gills of benzocaine treatment after 12 hours. The liver showed a reduction in ACAP of tricaine treatment after 12 hours. Both anesthetic treatments showed an increase of ACAP at 24 hours compared to control group. The activity of the GST enzyme increased in the gills for treatments benzocaine and tricaine after 3 and 12 hours. Liver showed increased GST activity (benzocaine after 24 hours and tricaine after 3 and 24 hours). Lipid damage decreased in gills (both anesthetics) and brain (tricaine) after 24 hours. The results demonstrate that benzocaine and tricaine did not cause oxidative damage in juvenile tambaqui under the experimental conditions herein established.

Oxidative stress responses of juvenile tambaqui Colossoma macropomum after short-term anesthesia with benzocaine and MS-222

ABSTRACT The present study aimed to evaluate the effects of benzocaine and tricaine methanesulfonate on oxidative stress parameters of juvenile tambaqui tissues. Fish (n=80) were anesthetized with benzocaine (100 mg L-1) or tricaine (240 mg L-1) and two control groups were used (non-anesthetized fish and fish exposed to ethanol-only). After anesthetic induction 10 fish/anesthetic were euthanized after 3, 12 and 24 hours post-anesthesia and tissue samplings (gills, liver and brain) were performed. Samples were submitted to analyses of enzyme activity glutathione-S-transferase (GST), cellular lipid peroxidation (TBARS) and total antioxidant capacity (ACAP). ACAP increased in gills of benzocaine treatment after 12 hours. The liver showed a reduction in ACAP of tricaine treatment after 12 hours. Both anesthetic treatments showed an increase of ACAP at 24 hours compared to control group. The activity of the GST enzyme increased in the gills for treatments benzocaine and tricaine after 3 and 12 hours. Liver showed increased GST activity (benzocaine after 24 hours and tricaine after 3 and 24 hours). Lipid damage decreased in gills (both anesthetics) and brain (tricaine) after 24 hours. The results demonstrate that benzocaine and tricaine did not cause oxidative damage in juvenile tambaqui under the experimental conditions herein established.

Ontogenic development of kidney, thymus and spleen and phenotypic expression of CD3 and CD4 receptors on the lymphocytes of cobia (Rachycentroncanadum).

In the present study was evaluated the ontogenic of immunocompetent organs of cobia up to 53 days after hatching (dah) through histology and immunohistochemistry techniques. The kidney was the first lymphohematopoietic organ to appear, at 1 dah, followed by the spleen at 5 dah and the thymus at 7 dah. The first CD3 receptors on the lymphocytes were observed in 27% of the thymic tissue at 7 dah and in 99% at 53 dah. The phenotypic expression of CD3 receptors was registered in 10% of the kidney at 8 dah and in 32% at 53 dah. CD4 receptors were observed in 5% and 63% of the thymic area at 7 and 53 dah, respectively. In the kidney, T4 lymphocytes were first observed at 13 dah in 9% of the organ and in 28% at 53 dah, defining the functional development of the specific system associated with immunological memory capacity.

Acclimation of juvenile Mugil liza Valenciennes, 1836 (Mugiliformes: Mugilidae) to different environmental salinities

Survival and physiological parameters associated with metabolism and osmoregulation were evaluated in juveniles of the Lebranche mullet Mugil liza acclimated to different water salinities (5, 10, 20, 30, and 40‰) for 15 days. Room temperature (25ºC) and photoperiod (12L:12D) were fixed. Fish were fed twice-a-day with commercial diet (28% crude protein) until satiation. After acclimation, whole body oxygen consumption was measured and fish were euthanized and sampled for blood, gills, and liver. Whole body oxygen consumption and plasma osmolality did not change in the range of salinities tested. The isosmotic point was estimated as 412.7 mOsmol kg-1 (13.5‰). Gill Na+,K+-ATPase activity tended to be lower at 20 and 30‰, while liver glycogen content was significantly higher at 20‰ than at 5 and 40‰. These results indicate that juvenile M. lizais able to acclimate for a short-period of time (15 days) to a wide range of salinities (5-40‰). This condition is achieved through adjustments in gill Na+,K+-ATPase activity and carbohydrate metabolism to regulate plasma osmolality and aerobic/energy metabolism. Therefore, our findings support the idea of catching juveniles M. lizain sea water and rear them in estuarine and marine waters.

A sobrevivência e parâmetros fisiológicos associados ao metabolismo e a osmorregulação foram avaliados em juvenis da tainha Mugil lizaaclimatada à diferentes salinidades (5, 10, 20, 30 e 40‰) por 15 dias. Foram fixadas a temperatura (25ºC) e o fotoperíodo (12L:12D) da sala onde os experimentos foram realizados. Os peixes foram alimentados duas vezes ao dia com ração comercial (28% de proteína bruta) até a saciedade. Após aclimatação, foi medido o consumo corporal de oxigênio e os peixes foram eutanasiados e foram coletadas amostras de sangue, brânquias e fígado. O consumo corporal de oxigênio e a osmolalidade plasmática não variaram na faixa de salinidade testada. O ponto isosmótico foi estimado em 412,7 mOsmol kg-1 (13,5‰). A atividade da Na+,K+-ATPase branquial tendeu a ser menor em 20 e 30‰, enquanto o conteúdo de glicogênio hepático foi significativamente maior em 20‰ do que em 5 e 40‰. Estes resultados indicam que o juvenil de M. liza é capaz de se aclimatar a uma ampla faixa de salinidade (5-40‰) por um curto período de tempo (15 dias). Esta condição é atingida através de ajustes na atividade da Na+,K+-ATPase branquial e no metabolismo de carboidratos para regular a osmolalidade plasmática e o metabolismo aeróbico/energético. Portanto, nossos achados suportam a ideia de que é possível capturar juvenis da tainha M. liza em água do mar e cultivá-los em águas estuarinas e marinhas.

Whole-body and muscle amino acid composition of Plata pompano (Trachinotus marginatus) and prediction of dietary essential amino acid requirements / Composición de aminoácidos de la carcasa y del músculo del Pámpano (Trachinotus marginatus) y estimación de las necesidades por aminoácidos esenciales / Composição de aminoácidos da carcaça e do músculo do Pampo prateado (Trachinotus marginatus) e estimativa das necessidades por aminoácidos essenciais

Background: knowing the essential amino acid (EAA) requirement values is fundamental to formulate good quality and cost-effective fish feeds. However, such requirements have been established for few fish species. The estimation of amino acid requirements based on amino acid composition of fish is a fast and reliable alternative. Objective: to determine whole-body and muscle amino acid composition of Plata pompano (Trachinotus marginatus) and estimate its EAA requirements. Methods: EAA requirements were estimated using A/E ratios [(Individual EAA / Total EAA) * 1000]. Results: hystidine, leucine, lysine and phenylalanine were present in higher concentrations in muscle tissue in comparison with the whole-body. On the other hand, arginine, isoleucine, methionine, tryptophan and valine concentration were not different between whole-body and muscle. A/E ratios for Plata pompano determined in the present study are similar to those reported in other fish species, although valine A/E ratios were slightly smaller. Conclusion: until dose-response experiments are conducted to precisely determine EAA requirements, the estimated EAA values using whole-body EAA-as proposed in this study-could be used to formulate diets for Plata pompano.

Antecedentes: conocer los requerimientos de aminoácidos esenciales (EAA) es fundamental para la formulación de raciones rentables y de buena calidad para peces. Sin embargo, dichos requerimientos se han establecido solo para unas pocas especies de peces. La determinación de las necesidades de aminoácidos basada en la composición de aminoácidos de los peces es un método alternativo rápido y viable. Objetivo: determinar la composición de aminoácidos del cuerpo completo y del músculo del Pámpano (Trachinotus marginatus) y estimar sus necesidades de EAA. Métodos: el requerimiento de aminoácidos esenciales se calculó utilizando el índice A/E [(Individual EAA/Total EAA) * 1000]. Resultados: la histidina, leucina, fenilalanina y lisina estaban en una mayor concentración en el músculo que en el cuerpo. Por otro lado, arginina, isoleucina, metionina, triptófano y valina no mostraron ninguna diferencia significativa entre la composición del músculo y el cuerpo. Los valores de A/E para Pámpano determinados en este estudio son similares a los reportados para otras especies de peces, pero no los de valina, que fue levemente menor. Conclusiones: hasta que no sean realizados experimentos de dosis-respuesta para determinar con precisión los requerimientos de EAA, el cálculo de los requerimientos de aminoácidos esenciales a partir de la concentración de aminoácidos del cuerpo -como se propone en este estudio- puede ser utilizado en la formulación de dietas para pámpano.

Antecedentes: estimar as necessidades por aminoácidos essenciais (AAE) é importante para formular dietas de boa qualidade e com bom custo-benefício. No entanto, poucas espécies de peixes possuem suas necessidades por (AAE) estabelecidas. A determinação das necessidades por (AAE) baseada na composição de aminoácidos do peixe é uma alternativa rápida e viável. Objetivo: determinar a composição de aminoácidos da carcaça e do músculo do Pampo prateado (Trachinotus marginatus) e estimar suas necessidades por (AAE). Métodos: as necessidades por AAE foi estimada usando o índice A/E [(AAE individual/AAE total) * 1000]. Resultados: histidina, leucina, lisina e fenilalanina estavam em maior concentração no músculo do que na carcaça. Por outro lado, arginina, isoleucina, metionina, triptofano e valina não apresentaram diferenças significativas entre a composição da carcaça e do músculo. O índice A/E determinado no presente estudo para o Pampo prateado é similar ao reportado para outras espécies de peixe, entretanto o valor do índice A/E para a valina foi ligeiramente menor. Conclusão: até que experimentos dose resposta sejam realizados para determinar com exatidão as necessidades por aminoácidos essenciais, os valores estimados para as necessidades por aminoácidos essenciais usando a composição da carcaça como proposto neste estudo pode ser usada na formulação de dietas para o Pampo prateado.

Thyroid gland development in Rachycentron canadum during early life stages.

The aim of this study was to describe the ontogeny of thyroid follicles in cobia Rachycentron canadum. Larvae were sampled daily (n=15 - 20) from hatching until 15 dah (days after hatching). Following, larvae were sampled every two days by 28 dah; a new sample was taken at 53 dah. The samples were dehydrated, embedded in Paraplast, and sections of 3 µm were dewaxed, rehydrated and stained with HE and PAS. A single follicle was already present 1 dah and three follicles were found 8 dah. The number of follicles increased up to 19 on 53 dah. The diameter of follicles and follicular cell height were lower 1 dah (6.83 ± 1.00 and 4.6 ± 0.01 µm), but increased from 8 dah (24.03 ± 0.46 µm e 6.43 ± 0.46 µm). From 8 dah, the presence of reabsorption vesicles was observed in the colloid and from the 19 dah some follicles did not present colloid. The early thyroid follicle appearance in cobia larvae as well as the high quantity of follicles without colloid and/or with vesicles even after the metamorphosis, might be the explanation of the fast growth of the cobia.

Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (-2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P less than 0.05) and significantly reduced 2 h later (P less than 0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P less than 0.05) at -2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

Micobacteriose por Mycobacterium marinum em "linguado" Paralichthys orbignyanus e em "barber goby" Elacatinus figaro: diagnóstico histopatológico e imuno-histoquímico / Micobacteriosis by Mycobacterium marinum in Brazilian flounder Paralichthys orbignyanus and barber goby Elacatinus figaro: histopathological and imunohistochemical diagnosis

As micobacterioses afetam várias espécies de peixes, tanto comerciais como ornamentais. É uma enfermidade sistêmica com formação de granulomas, conhecida como micobacteriose de peixes. O desenvolvimento da aquicultura tem aumentado o número de casos com essa enfermidade que muitas vezes é de difícil diagnóstico. Neste trabalho apresentamos dois casos de micobacterioses em Paralichthys orbignyanus e Elacatinus figaro provenientes do Laboratório de Piscicultura Estuarina e Marinha da Universidade Federal do Rio Grande (FURG). As micobactérias de peixes possuem menos ácidos graxos que as de mamíferos, por isso foi utilizado o protocolo de coloração de Fite-Faraco, uma vez que o protocolo de coloração clássico de Ziehl Neelsen pode resultar em falsos negativos. Para determinar o tipo de micobactéria foi utilizada a imuno-histoquímica com a qual se pode diagnosticar a presença de Mycobacterium marinum. A enfermidade pode ser transmitida para o homem e com esse trabalho ressalta-se a necessidade de fazer o diagnóstico correto e implantar medidas de proteção para as pessoas que estão em contato com os peixes infectados.

Mycobacteriosis affects various species of fish, including those cultured with commercial purposes. It is a chronic systemic with granuloma forming disease known as fish mycobacteriosis. In the last few years the interest of fish culture has been increasing due to the reduction of the natural fish stocks. However the intensification of fish culture has collaborated to the development of diseases such as mycobacteriosis, causing losses due to the chronic characteristics and difficult diagnosis. This study is to report two cases of mycobacteriosis observed at "Laboratório de Piscicultura Estuarina e Marinha of the Universidade Federal do Rio Grande (FURG)", one for Brazilian flounder Paralichthys orbignyanus and another for barber goby Elacatinus figaro. The Fite-Faraco staining protocol was used. Membranes of mycobacterial cells contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species. The fish mycobacterium is much less acid and alcohol fast than the tubercle bacillus and the classic Ziehl-Neelsen Staining Protocol may give false results. The presence of mycobacterial antigen lesions was studied by immunohistological methods using monoclonal antibodies to Mycobacterium marinum. Humans may be infected by these bacteria and develop cutaneous granuloma. In this paper the necessity is stressed for a correct diagnosis of the disease, contributing to the prevention of its establishment in aquatic facilities, and thus, preserving cultured fish as well as human beings from infection.

Identification, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 +or- 3 degrees C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.

Increased growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor I (IGF-I) gene transcription after hyperosmotic stress in the Brazilian flounder Paralichthys orbignyanus.

Growth hormone (GH) action is the result of an intracellular cascade initiated just after its interaction with the growth hormone receptor (GHR) located on the surface of target cells. This cascade culminates with the transcription of target genes, such as the insulin-like growth factors (IGFs), which are responsible for most GH biological effects. In addition to its central role in growth, fish GH is also involved with osmoregulatory control. Within this context, the objective of the present work was to isolate GH, GHR, and IGF-I cDNAs from the Brazilian flounder Paralichthys orbignyanus and evaluate whether these genes are induced by hyperosmotic stress. The obtained results indicated that GH mRNA had a significant peak only 24 h after hyperosmotic stress. In gills, GHR mRNA was significantly increased after 7 days. In liver, GHR and IGF-I mRNAs were significantly increased in 72 h and both reached even higher levels after 7 days. These results indicate that hyperosmotic stress can increase GH sensitivity in the gills and liver of P. orbignyanus and, consequently, improve IGF-I production. The management of this parameter could be useful in achieving better growth performance for this and other commercially important species in which GH has a direct correlation with osmoregulatory mechanisms.