Does exposure to moonlight affect day/night changes in melatonin and metabolic parameters in Amazonian fish?

Lunar cycle modulates the rhythmic activity patterns of many animals, including fish. The effect of the moonlight cycle on daily melatonin and metabolic parameters was evaluated in matrinxã (Brycon amazonicus) subjected to external natural lighting. Eighty juvenile were distributed in 4 tanks of 1m3 (20 fish/tank) and divided into two groups. One group was exposed to the full moon and the other group to the new moon for 30 days, which corresponds to the duration of the lunar period. At the end of the lunar phase, 6 fish from each group were anesthetized to collect blood, tissue and eye samples at midday and midnight. The comparison between the light and dark periods revealed a significant increase in plasma and ocular melatonin in the last period. However, there was no significant difference for plasma melatonin between moons. Ocular melatonin presented higher concentrations during the new moon. Glucose, total proteins, cortisol, liver glutathione and gill lipid peroxidation were higher in the full moon compared to in the new moon. Plasma triglyceride was higher during the night for the full moon, and the opposite was found for the new moon. Total cholesterol values were higher at night regardless the moon phase. Glutathione in the gills and lipid peroxidation in the liver showed no significant differences. These results highlight the importance of considering both the day and lunar cycles for melatonin and metabolic parameters in species of commercial interest and susceptible to stressful situations in rearing conditions.

Eugenol and Lippia alba essential oils as effective anesthetics for the Amazonian freshwater stingray Potamotrygon wallacei (Chondrichthyes, Potamotrygonidae).

This study assessed the potential of eugenol and the essential oil of Lippia alba (EOLA) in providing suitable anesthetic induction and recovery times, and their consequent effects on the blood and respiratory physiology, as well as the gill architecture of an Amazonian freshwater stingray, Potamotrygon wallacei, shortly after reaching the recovery and 48 h later. Juveniles of P. wallacei were exposed to increasing concentrations of eugenol (75, 100, 125, and 150 µL L-1) and EOLA (150, 175, 200, and 225 µL L-1) in an immersion bath. Anesthetic induction was found to be faster with the use of eugenol compared to EOLA. On the other hand, the stingrays anesthetized with eugenol displayed a longer recovery time than those exposed to EOLA. The highest concentrations of eugenol caused moderate to severe histological changes in the gills. No significant changes were found for hematocrit and plasma energy metabolites in the stingrays anesthetized with all concentrations of both eugenol and EOLA shortly after reaching the recovery from the ansthesia, when compared to those recovered after 48 h. Investigations regarding the potential use of these natural anesthetics are unprecedented for freshwater stingray species, and 200 µL L-1 EOLA is recommended as the most suitable anesthetic for use in juveniles of P. wallacei.

Morphological, cytochemical and ultrastructural aspects of blood cells in freshwater stingray species in the middle Rio Negro basin of Amazonian Brazil.

In the present work, we examined the morphology, dimensions, cytochemical staining reactions and ultrastructure of blood cells from three freshwater stingray species, Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba, living in the waters of the middle Rio Negro basin (Barcelos, Amazonas, Brazil). We identified erythrocytes, erythroblasts, thrombocytes and four types of leukocytes (basophils, heterophils, lymphocytes and monocytes) in the blood of these stingray species. In all the freshwater stingray species studied, the shapes and dimensions of these cells were similar to those of marine elasmobranchs. Positive PAS staining occurred in heterophils and thrombocytes, and weak staining occurred in lymphocytes and monocytes, while metachromasia only occurred in basophils. Positive Sudan Black B staining was observed in thrombocytes and lymphocytes, and weak staining occurred in heterophils. Basophils and heterophils were the only cells with positive bromophenol blue staining, while no peroxidase staining was observed in any of the four leukocyte types. This is the first study to establish the dimensions and cytochemical staining profiles of blood cells in Amazonian stingray species. Because these elasmobranch species are exported as ornamental fish to countries worldwide, this study can contribute to establishing standards for blood constituents that may be helpful in assessing the health and welfare of these fish in artificial systems.

Acute toxicity of a deltamethrin based pesticide (DBP) to the Neotropical electric fish Microsternarchus cf. bilineatus (Gymnotiformes)

A deltametrina é um inseticida piretróide amplamente utilizado no controle de pragas na agricultura brasileira. O uso intensivo e desordenado desse pesticida na Amazônia pode carreá-lo aos ecossistemas aquáticos de várias maneiras, mas principalmente por escoamento e lixiviação. O presente estudo foi concebido para determinar a toxicidade aguda (LC50) de um pesticida à base de deltametrina (PBD) e caracterizar seus efeitos sobre dois biomarcadores bioquímicos, a glutationa-S-transferase (GST) e a acetilcolinesterase (AChE), em tecidos do peixe elétrico Microsternarchus cf. bilineatus. Os peixes foram expostos a concentrações de 1, 2, 3, 4 e 5 µg L-1 de PBD por até 96 horas. Para cada tratamento, foi analisada a atividade absoluta das enzimas GST (músculo e fígado) e AChE (músculo e tecido nervoso). A CL50-96 h para Microsternarchus cf. bilineatus foi de 2,15 µg L-1, a menor concentração registrada para um peixe amazônico até o momento. Nenhuma das concentrações testadas deste inseticida afetou a atividade da AChE para o período de exposição testado. Um aumento significativo da atividade de GST no músculo foi detectado somente para as concentrações de 2 e 3 µg L-1. (AU)

Acute toxicity of a deltamethrin based pesticide (DBP) to the Neotropical electric fish Microsternarchus cf. bilineatus (Gymnotiformes) / Toxicidade aguda de um pesticida à base de deltametrina (PBD) no peixe elétrico neotropical Microsternarchus cf. bilineatus (Gymnotiformes)

Deltamethrin is a pyrethroid insecticide widely used in pest control in Brazilian agriculture. The intensive and disordered use of this pesticide in the Amazon region can drive it into aquatic ecosystems in several ways, but mainly by runoff and leaching. The present study was conceived to determine the acute toxicity (LC50) of a deltamethrin based pesticide (DBP) and to characterize its effects on two biochemical biomarkers, glutathione-S-transferase (GST) and acetylcholinesterase (AChE), in tissues of the electric fish Microsternarchus cf. bilineatus. Fishes were exposed to concentrations of 1, 2, 3, 4 and 5 g L-1 of DBP for up to 96 hours. For each treatment, the absolute activity of the enzymes GST (muscle and liver) and AChE (muscle and nervous tissue) were analyzed. The LC50-96 h for Microsternarchus cf. bilineatus was 2.15 g L-1, the lowest concentration registered for an Amazonian fish species so far. None of the concentrations tested of this insecticide affected AChE activity for the exposure period tested. A significant increase in muscle GST activity was detected only for concentrations of 2 and 3 g L-1.(AU)

A deltametrina é um inseticida piretróide amplamente utilizado no controle de pragas na agricultura brasileira. O uso intensivo e desordenado desse pesticida na Amazônia pode carreá-lo aos ecossistemas aquáticos de várias maneiras, mas principalmente por escoamento e lixiviação. O presente estudo foi concebido para determinar a toxicidade aguda (LC50) de um pesticida à base de deltametrina (PBD) e caracterizar seus efeitos sobre dois biomarcadores bioquímicos, a glutationa-S-transferase (GST) e a acetilcolinesterase (AChE), em tecidos do peixe elétrico Microsternarchus cf. bilineatus. Os peixes foram expostos a concentrações de 1, 2, 3, 4 e 5 g L-1 de PBD por até 96 horas. Para cada tratamento, foi analisada a atividade absoluta das enzimas GST (músculo e fígado) e AChE (músculo e tecido nervoso). A CL50-96 h para Microsternarchus cf. bilineatus foi de 2,15 g L-1, a menor concentração registrada para um peixe amazônico até o momento. Nenhuma das concentrações testadas deste inseticida afetou a atividade da AChE para o período de exposição testado. Um aumento significativo da atividade de GST no músculo foi detectado somente para as concentrações de 2 e 3 g L-1.(AU)

Sperm characterization of the Amazonian freshwater cururu stingray Potamotrygon wallacei (Potamotryogonidae): basic knowledge for reproduction and conservation plans.

This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 µm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.

The metabolic cost of nesting: body condition and blood parameters of Caiman crocodilus and Melanosuchus niger in Central Amazonia.

Although nesting ecology is well studied in several crocodilian species, it is not known how nest attendance influences physiology and body condition of nesting females. In this study, we describe body condition and serum biochemical values of nesting female, non-nesting female and male spectacled caiman (Caiman crocodilus) and black caiman (Melanosuchus niger) in two areas of Central Amazonia. We also evaluated the effect of nest age and nest distance to water on body condition and blood parameters of nesting females. Body condition and plasmatic concentrations of glucose, triglycerides, lactate and uric acid of nesting females were significantly different from those of non-nesting females and males in C. crocodilus, but not in M. niger. Our study also demonstrated that nest age and distance to water had a negative effect on female body condition in C. crocodilus, but not in M. niger. Female C. crocodilus attending older nests or nests built further away from permanent water bodies tended to have lower body condition. Our results demonstrate that the nesting strategy of C. crocodilus has a metabolic cost associated with nest attendance for nesting females, which appear to depend on accumulated energetic reserves during nest attendance. In contrast, nest attendance had little effect on the physiology of female M. niger.

Características bioquímicas e hematológicas do pirarucu Arapaima gigas Schinz, 1822 (Arapaimidae) de cultivo semi-intensivo na Amazônia / Biochemical and hematological characteristics of the pirarucu Arapaima gigas Schinz, 1822 (Arapaimidae) from semi-intensive culture in the Amazon

O presente estudo comparou os níveis plasmáticos de glicose, proteínas totais, uréia, triglicerídeos e colesterol, a contagem de eritrócitos, leucócitos e trombócitos entre alevinos e juvenis de Arapaima gigas (Osteoglociformes, Arapaimidae) de uma piscicultura semi-intensiva de Manaus, estado do Amazonas, Brasil. Os alevinos de A. gigas apresentaram níveis significativamente (p<0,05) menores de proteínas totais, triglicerídeos, uréia e Volume Corpuscular Médio (VCM) e maior concentração de glicose, colesterol e hemoglobina, contagem de eritrócitos, hematócrito e Concentração da Hemoglobina Corpuscular Média (CHCM) quando comparados aos juvenis. Porém, não houve diferença significativa (p>0,05) no número de trombócitos e leucócitos totais entre alevinos e juvenis. Os juvenis de A. gigas apresentaram menor quantidade de linfócitos e maior quantidade de monócitos, neutrófilos e eosinófilos, quando comparados aos alevinos. Em estudos futuros, espécimes de A. gigas com outras idades também deverão ser comparados para melhor entendimento dos efeitos da idade neste peixe amazônico, pois estas informações, além de possibilitarem uma melhor compreensão da fisiologia desta espécie, poderão auxiliar no estabelecimento de estratégias para seu manejo.

The present study compared the biochemical and hematological parameters of fingerlings and juveniles Arapaima gigas (Osteoglociformes, Arapaimidae) from a semi-intensive fish farming in Manaus, Amazon State, Brazil. Fingerlings A. gigas had lower levels of total protein, triglycerides, urea and mean cell volume (MCV), but higher (p<0.05) glucose, cholesterol and hemoglobin concentration, red blood cell counts, hematocrit and cell hemoglobin concentration (MCHC) when compared to juveniles. However, there was not significant difference (p>0.05) in the total thrombocytes and total white blood cell counts between fingerlings and juveniles. Juveniles A. gigas presented lower lymphocytes number and higher monocytes, neutrophils and eosinophils number when compared to the fingerlings. In further studies, A. gigas specimens with other ages should also be compared for a better understanding of the ontogenic development in this Amazon fish, because this information besides increase the knowledge of the physiology of this species, can aid in the establishment of strategies for its adequate handling.

Comparative study on hematological parameters of farmed matrinxã, Brycon amazonicus Spix and Agassiz, 1829 (Characidae: Bryconinae) with others Bryconinae species / Estudo comparativo sobre parâmetros hematológicos de matrinxã Brycon amazonicus Spix e Agassiz, 1892(Characidae: Bryconinae) criados em cativeiro, com outras espécies de Bryconinae

The aim of this study was not only to determine the red blood cells parameters, thrombocyte and leukocyte counts in farmed Brycon amazonicus (matrinxã), to compare these parameters among Bryconinae species from literature, and also to investigate the presence of special granulocytic cells in these fish. The results of the blood cells parameters here established for farmed B. amazonicus, a species of great economic importance in Brazilian aquaculture, could help a better understanding of the blood features in natural populations of this Amazon species. Blood parameters varied between Bryconinae species investigated, mainly the red blood cell counts, hemoglobin, hematocrit and mean corpuscular volume (MCV). The presence of the blood granulocytes, neutrophils and heterophils in matrinxã suggest that both leukocytes can be a characteristic for Bryconinae family. Furthermore, it indicates that the existence of special granulocytic cells in the blood of Bryconinae species from literature is an artifact, and this was herein discussed.

O objetivo deste estudo foi determinar os parâmetros eritrocíticos, as contagens de trombócitos e de leucócitos de espécimes de Brycon amazonicus (matrinxã), criados em cativeiro e compará-los com aqueles descritos na literatura para outras espécies de Bryconinae. Além disso, foi ainda investigada a presença de células granulocíticas especiais nestes peixes. Os resultados dos parâmetros sangüíneos apresentados para B. amazonicus podem ajudar a entender melhor as características sangüíneas em população natural desta espécie de grande importância para a aqüicultura brasileira. Os parâmetros sangüíneos das espécies de Bryconinae investigadas apresentaram variação interespecíficas principalmente a contagem de eritrócitos, hemoglobina, hematócrito e volume corpuscular médio (VCM). A presença dos granulócitos sangüíneos, neutrófilos e heterófilos em matrinxã sugere que esta pode ser uma característica das espécies pertencentes à subfamília Bryconinae. Além disso, indica que a existência de células granulocíticas especiais no sangue de espécies de Bryconinae da literatura é um artefato, e isto foi aqui discutido.

Tolerance to temperature, pH, ammonia and nitrite in cardinal tetra, Paracheirodon axelrodi, an amazonian ornamental fish / Tolerância a temperatura, pH, amônia e nitrito do cardinal tetra, Paracheirodon axelrodi, um peixe ornamental da Amazônia

Poor water quality condition has been pointed out as one of the major causes for the high mortality of ornamental fishes exported from the state of Amazonas, Brazil. The purpose of the current study was to define water quality standards for cardinal tetra (Paracheirodon axelrodi), by establishing the lower and higher for lethal temperature (LT50), lethal concentration (LC50) for total ammonia and nitrite and LC50 for acid and alkaline pH. According to the findings, cardinal tetra is rather tolerant to high temperature (33.3 ºC), to a wide pH range (acid pH=2.9 and alkaline pH=8.8) and to high total ammonia concentration (23.7 mg/L). However, temperatures below 19.6 ºC and nitrite concentrations above 1.1 mg/L NO2- may compromise fish survival especially during long shipment abroad.

A má qualidade da água tem sido apontada como uma das maiores causas da alta mortalidade dos peixes ornamentais exportados pelo Estado do Amazonas, Brasil. A proposta deste estudo foi definir padrões de qualidade da água para o cardinal tetra (Paracheirodon axelrodi), estabelecendo a menor e a maior temperatura letal (LT50), a concentração letal (LC50) para amônia total e nitrito e LC50 para pH ácido e alcalino. De acordo com os resultados, o cardinal tetra é mais tolerante a temperaturas elevadas (33,3 ºC), a amplos limites de pH (pH ácido = 2,9 e pH alcalino = 8,8) e a alta concentração de amônia (23,7 mg/L). Entretanto, temperaturas abaixo de 19,6 ºC e concentrações de nitrito acima de 1,1 mg/L NO2- podem comprometer a sobrevivência dos peixes, especialmente durante longos períodos de transporte para o exterior.

Methodological limitations of counting total leukocytes and thrombocytes in reptiles (Amazon turtle, Podocnemis expansa): an analysis and discussion / Limitações metodológicas de contagens de leucócitos e trombócitos totais em répteis (tartaruga da Amazônia, Podocnemis expansa): uma análise e discussão

The aim of this paper is to compare three different methods for counting white blood cells [WBC] (Natt and Herrick method, estimation with 1,000 and 2,000 erythrocytes) and three methods for counting total thrombocytes [TT] (Wojtaszek method, estimation with 1,000 and 2,000 erythrocytes) in a South American freshwater turtle species, Podocnemis expansa, Schweigger 1812 (Reptilia, Pelomedusidae). Direct WBC counts using the Natt and Herrick method showed limitations, which are discussed here. The WBC and TT counts using 1,000 erythrocytes from blood smears are not recommended for Amazon turtles nor other reptilian species, since wide variation in counts can be observed. Estimation methods for determining WBC and TT based on 2,000 erythrocytes of blood smears were most acceptable because they allow a differentiation between leukocytes and thrombocytes and also had a smaller variation. The methods investigated here for the Amazon turtle, which have been widely used in other reptile species, provided evidence that the most acceptable method is not that of using diluted stains and a hemocytometer.

O objetivo deste estudo foi comparar três diferentes métodos para contar leucócitos totais [LT] (método de Natt & Herrick, de estimação em 1000 e 2000 eritrócitos) e três métodos para contar trombócitos totais [TT] ( método de Wojtaszek, de estimação em 1000 e 2000 eritrócitos) em uma espécie de tartaruga de água doce da América do Sul, Podocnemis expansa, Schweigger 1812 (Reptilia, Pelomedusidae). As contagens diretas de LT usando o método de Natt & Herrick mostraram limitações que são aqui discutidas. As contagens de LT e TT usando estimativa em 1000 eritrócitos na extensão sanguínea não são recomendadas para tartaruga-da-Amazônia nem para outras espécies de répteis, pois houve ampla variação nestas contagens. Os métodos para determinar LT e TT baseados em 2000 eritrócitos nas extensões sanguíneas foram mais aceitáveis porque eles permitem uma diferenciação entre leucócitos e trombócitos, além disso, teve uma variação menor. Os métodos aqui investigados para tartaruga-da-Amazônia, os quais são amplamente usados em outras espécies de répteis, proveram evidências de que o método mais aceitável não é o que usa corantes diluentes e um hemocitômetro.

Effect of high levels of dietary vitamin C on the blood responses of matrinxã (Brycon amazonicus).

Juvenile matrinxã (Brycon amazonicus) were fed with different dietary vitamin C (ascorbic acid or AA) concentrations for 2 months in order to evaluate its effect on the physiological condition of this species. Five treatments were tested: Reference diet-RD (350 mg AA kg(-1) and 45% crude protein commercial feed); C400, C500, C600 and C800 with supplementation of 400, 500, 600 and 800 mg AA kg(-1). Fish fed 800 mg AA kg(-1) supplemented diets presented greater weight gain and survival. Hematocrit, hemoglobin concentrations and erythrocytes showed significant increase (p<0.05) in C600 and C800 relatively to the others. Total leukocyte values were significantly higher in fish fed vitamin C supplemented diets relatively to those fed RD and leukocytes count of C600 and C800 were the highest among the treatments. Monocytes were the only cells of fish defense mechanism that presented variation, and C600 and C800 presented the lowest values (P<0.05). Glucose level was significantly different relatively to RD only in C400 and the plasmatic total protein values for fish fed vitamin C supplemented diets were higher than for fish fed the RD. Fish plasmatic ions (Na(+) and Cl(-)) were significantly lower for C500, C600 and C800 relatively to C400 and RD. The physiological profile and the growth performance of matrinxã suggest that diet supplemented with 800 mg AA kg(-1) can improve its performance and increase the survival rate.

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters.

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg-1 of vitamin C (ascorbic acid or AA) and vitamin E (alpha-tocopherol or alpha-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p<0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg-1 and on the hemoglobin concentration on treatment with 500 mg alpha-T kg-1 relatively to control. Mean corpuscular volume presented a significant decrease (p<0.05) on treatment with 800 and 1200 mg AA kg-1 when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p<0.05) on treatment with 500 mg alpha-T kg-1. Only in vitamin C treatments, we noticed a significant increase (p<0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg-1, had high total protein values compared to control. Fish treated with 800 or 1200 mg alpha-T kg-1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg-1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.

The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture.

This study evaluated the efficacy of dietary vitamin C (ascorbic acid or AA), vitamin E (alpha-tocopherol or alpha-T), and C+E supplementation on the blood parameters of Arapaima gigas grown in net cages for 45 days. Four treatments were tested: control (commercial feed); C800; E500 and C+E (800+500) with supplementation of 800 mg AA kg(-1), 500 mg alpha-T kg(-1) and 800+500 mg AA+alpha-T kg(-1), respectively. Hematocrit (Ht), red blood cells (RBC), and hemoglobin concentration (Hb) (oxidative status indicators), thrombocytes and leukocytes (immunological indicators), plasma protein and glucose were evaluated. Fish fed vitamin C and C+E supplemented diets showed greater weight gain and survival. Dietary vitamin C and C+E diet supplementation resulted in increased Ht, Hb, RBC, MCHC, total leukocytes, total proteins, thrombocytes and eosinophils compared to the control and alpha-T. The alpha-tocopherol-supplemented diet reduced the number of total thrombocytes, lymphocytes and neutrophils and increased glucose and eosinophils relatively to the control. In general, leukocytes and thrombocytes were good indicators of the efficiency of vitamin on the defense mechanism of the A. gigas reared in cages. Results indicate that high alpha-T diet supplementation provides no benefit for the maintenance of the oxidative or the immunological status of A. gigas. However, it was demonstrated that high dietary AA improves A. gigas immunological status. Red blood cell indices and immune system indicators showed no synergistic effect between the vitamins after supplementing the A. gigas diet with alpha-T+AA.