Metabolic changes in Brycon cephalus (Teleostei,Characidae) during post-feeding and fasting

Metabolic changes during the transition from post-feeding to fasting were studied in Brycon cephalus, an omnivorous teleost from the Amazon Basin in Brazil. Body weight and somatic indices (liver and digestive tract), glycogen and glucose content in liver and muscle, as well as plasma glucose, free fatty acids (FFA), insulin and glucagon levels of B. cephalus, were measured at 0, 12, 24, 48, 72, 120, 168 and 336 h after the last feeding. At time 0 h (the moment of food administration, 09.00 h) plasma levels of insulin and glucagon were already high, and relatively high values were maintained until 24 h post-feeding. Glycemia was 6.42+/-0.82 mM immediately after food ingestion and 7.53+/-1.12 mM at 12 h. Simultaneously, a postprandial replenishment of liver and muscle glycogen reserves was observed. Subsequently, a sharp decrease of plasma insulin occurred, from 7.19+/-0.83 ng/ml at 24 h of fasting to 5.27+/-0.58 ng/ml at 48 h. This decrease coincided with the drop in liver glucose and liver glycogen, which reached the lowest value at 72 h of fasting (328.56+/-192.13 and 70.33+/-14.13 micromol/g, respectively). Liver glucose increased after 120 h and reached a peak 168 h post-feeding, which suggests that hepatic gluconeogenesis is occurring. Plasma FFA levels were low after 120 and 168 h and increased again at 336 h of fasting. During the transition from post-feeding to fast condition in B. cephalus, the balance between circulating insulin and glucagon quickly adjust its metabolism to the ingestion or deprivation of food.     

Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion

Methyl parathion (MP) is a widely used organophosphorus pesticide, which has been related to a broad spectrum of toxic effects on environmental organisms. The present study investigated the changes in the protein profile of enriched membrane fraction from zebrafish (Danio rerio) brain exposed to three concentrations (0.5, 1 and 2 mg/L) of MP. 2-DE revealed that the abundance of 21 protein spots was significantly changed by MP stress. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database search, 16 protein spots were identified as membrane proteins, among which 8 were down-regulated, while 8 were up-regulated. These proteins are mainly involved in oxidative stress response, signal transduction, metabolism, protein synthesis and degradation, neuroplasticity and regeneration as well as synaptic transmission. These results may aid our understanding of the mechanism of MP-induced neurotoxicity and provide the possibility of the establishment of candidate biomarkers of MP.     

Sub-lethal effects of the organophosphate folidol 600 (methyl parathion) on Callichthys callichthys (pisces:teleostei)

1. The sublethal effects of Folidol 600, an organophosphate containing methyl parathion as its active principle, were analysed in the fish Callichthys callichthys.2. After an intracoelomatic application of 500 mg/kg of Folidol 600, the behavior and the levels of plasmatic cholinesterase were analysed along the time, in acute experiments.3. Plasma cholinesterase activity was inhibited by 90% after 4 hr from the initial time, keeping this level of inhibition for 4 days.4. It recovered slowly in the next 8 to 10 days, reaching about 80–90% of the normal levels around the 35th day after the beginning of the experiment.5. Many behavioral changes were observed in the first 96 hr, such as: lowering of motility with loss of equilibrium and frequent unnatural body positions; uncoordinated movements leading to incapacity of food intake; and increase in the levels of respiratory frequency.     

Frugivory by the fish Brycon hilarii (Characidae) in western Brazil

Frugivory and seed dispersal have been poorly studied in Neotropical freshwater fishes. We studied frugivory and seed dispersal by the piraputanga fish (Brycon hilarii, Characidae) in the Formoso River, Bonito, western Brazil. We examined the stomach contents of 87 fish and found the diet of piraputanga consisted of 24% animal prey (arthropods, snails, and vertebrates), 31% seeds/fruits and 45% other plant material (algae/macrophytes/leaves/flowers). The piraputangas fed on 12 fruit species, and were considered as seed dispersers of eight species. Fruits with soft seeds larger than 10 mm were triturated, but all species with small seeds (e.g. Ficus, Psidium) and one species with large hard seed (Chrysophyllum gonocarpum) were dispersed. Piraputangas eat more fruits in the dry season just before the migration, but not during the spawning season. Fish length had a positive relation with the presence of fruits in their guts. The gallery forest of the Formoso River apparently does not have any plant species that depend exclusively on B. hilarii for seed dispersal because all fruit species are also dispersed by birds and mammals. Based on seed size and husk hardness of the riparian plant community of Formoso River, however, the piraputangas may potentially disperse at least 50% of the riparian fleshy fruit species and may be particularly important for long-distance dispersal. Therefore, overfishing or other anthropogenic disturbances to the populations of piraputanga may have negative consequences for the riparian forests in this region.     

Ultrastructure of the semicystic spermatogenesis in the South American freshwater characid Hemigrammus marginatus (Teleostei,Characiformes)

Semicystic, a rare type of spermatogenesis, was detected in the characid Hemigrammus marginatus and characterized by cysts hatching during the spermatid phase and maturation of the spermatozoa being completed at the lumen of the anastomosed seminiferous tubules. Primary spermatogonia, or type A, are distributed along the entire length of the seminiferous tubules, in an unrestricted spermatogonial pattern. H. marginatus spermiogenesis is included in type I, mainly characterized by presence of nucleus rotation. During this process, a vesicle resembling the acrosomal vesicle is visualized at the anterior region close to the nucleus of the early spermatids, however this structure did not remain in the spermatozoa. In H. marginatus, the spermatozoon is uniflagellated, primitive, type I aquasperm, with a rounded head, a short midpiece and a long flagellum with the axoneme in a 9 + 2 microtubules arrangement and no lateral fins. Residual spermatozoa are reabsorbed by Sertoli cells. Unusual biflagellate spermatozoa with three long cytoplasmatic projections originating in the midpiece are rarely observed and have not been registered in other characiforms. Ultrastructural characteristics of the spermatogenesis and spermatozoa observed in the present work provide important subsidies to systematic and phylogeny studies of Characidae fishes included in Incertae sedis groups, such as H. marginatus.     

Insectivore to frugivore: ontogenetic changes in gut morphology and digestive enzyme activity in the characid fish Brycon guatemalensis from Costa Rican rain forest streams

Brycon guatemalensis , a Neotropical characid fish, consumes an entirely terrestrial diet, shifting from eating insects as juveniles to fruits and leaves as adults. Juvenile and larger‐sized fish collected in the Rio Puerto Viejo at the La Selva Biological Station in Costa Rica were studied to test the hypotheses that, with ontogeny, (1) relative gut length increases, (2) pyloric caeca arrangement and number remain unchanged and (3) pepsin, trypsin and lipase activities decrease, while α‐amylase activity increases. These hypotheses were mainly supported in that larger fish had longer guts, unchanged pyloric caeca arrangement but fewer caeca, and, at both environmental and standard temperatures for the enzyme assays, lower pepsin and trypsin activities but higher α‐amylase activities than the juveniles. Only lipase, among the digestive enzymes, exhibited the unexpected outcome of either not differing significantly in activity (per g of tissue) between juveniles and larger fish or being significantly higher (per mg of protein) in larger fish. The overall results support the view that B. guatemalensis is specialized morphologically and biochemically to function first as a carnivore and then as a herbivore during its life history.     

Genetic monitoring of the Amazonian fish matrinchã (Brycon cephalus) using RAPD markers: insights into supportive breeding and conservation programmes

The importance of genetic evaluations in aquaculture programmes has been increased significantly not only to improve effectiveness of hatchery production but also to maintain genetic diversity. In the present study, wild and captive populations of a commercially important neotropical freshwater fish, Brycon cephalus (Amazonian matrinchã), were analyzed in order to evaluate the levels of genetic diversity in a breeding programme at a Brazilian research institute of tropical fish. Random Amplified Polymorphic DNA fingerprinting was used to access the genetic variability of a wild stock from the Amazon River and of three captive stocks that correspond to consecutive generations from the fishery culture. Although farmed stocks showed considerably lower genetic variation than the wild population, a significantly higher level of polymorphism was detected in the third hatchery generation. The results seem to reflect a common breeding practice on several hatchery fish programmes that use a small number of parents as broodstocks, obtaining reproductive success with few non‐identified mating couples. The obtained data were useful for discussing suitable strategies for the genetic management and biodiversity conservation of this species.     

Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes)

The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide = dimethyl sulfoxide (DMSO) and methyl glycol = methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60 °C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at −170 °C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60 °C for 8 s, of regardless straw size: 74 ± 7% motile sperm, 47 ± 4 s of motility duration, 69 ± 3% intact sperm, 64 ± 4% fertilization and 63 ± 3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.     

Developmental biology of medaka fish (Oryzias latipes) exposed to alkalinity stress

Alkalinity stress is common in cultured aquatic animals and considered to be one of the major stress factors for fishes when they are transferred to saline‐alkali waters. To evaluate potential effects of alkalinity on the developmental biology of Oryzias latipes, fertilized eggs, larvae and breeding fish were exposed to different carbonate alkalinity concentrations of 1.5–64.5 meq l^?1, for 9, 120, and 60 days, respectively. The mortality of embryos significantly increased when exposed to the high concentrations (16.5–64.5 meq l^?1). Although more than 50% of survived embryos hatched in 16.5 and 31.4 meq l^?1 concentrations of carbonate alkalinity, most were not able to swim up after hatching. Morphological abnormalities such as coagulated embryos, halted embryo development, and hatching failure were observed at stages 15, 29–33 and 38 in high concentrations (31.4, 64.5 meq l^?1). Almost all larvae in 16.5 and 31.4 meq l^?1 treatments died 70 d post‐hatch. Growth of juveniles exposed to carbonate alkalinity of 5.3 and 8.8 meq l^?1 was not significantly different at 70 d and 120 d post‐hatch. The number of eggs released by breeders, the fertilization rate and the hatching rate of eggs were significantly lower in the 31.4 meq l^?1 treatment than in other treatments. Although medaka are capable of surviving in high alkalinities (31.4, 64.5 meq l^?1) for an extended period of time, these conditions are stressful to the fish, especially at the embryonic and reproductive stages.     

Blood sugar as an indicator of stress in the freshwater fish, Labeo capensis (Smith)

Factors involved in the determination of the blood sugar level of L. capensis have been studied. The stress of capture and transportation produces a marked increase in the blood sugar concentration. The relationship between blood sugar concentration and oxygen saturation of water and between blood sugar concentration and ammonium ion concentration was also investigated. Increases in ammonium ion concentration and decreases in oxygen saturation of water produce increases in the blood sugar concentration. The results are discussed in relation to the phenomenon of 'asphyxiation hyper-glycemia'.     

Trichlorfon, an organophosphorus insecticide, depresses the immune responses and resistance to Lactococcus garvieae of the giant freshwater prawn Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.     

Evaluation of genotoxic and oxidative stress response to dimethoate in freshwater fish Channa punctatus (Bloch)

Abstract

Dimethoate is one of the organophosphate insecticides widely used in agriculture throughout the world and is an inhibitor of cholinesterase in animals. The objective of the present study was to detect oxidative stress and DNA damage induced by dimethoate in the freshwater fish Channa punctatus (C. punctatus). The LC₅₀-96 h value of technical grade dimethoate was estimated at 19.10 μg L^-1 in a semi-static system and, on the basis of the LC₅₀ value, three concentrations were determined. The fish were exposed to these concentrations of dimethoate for 96 h and samplings were done at 24 and 96 h for assessment of oxidative stress and DNA damage. After exposure to dimethoate, the level of superoxide dismutase declined while lipid peroxidation, glutathione, induction of micronucleus and DNA damage were increased in C. punctatus as the concentration and exposure time increased. Thus our results suggest that dimethoate induces genotoxic effects which invariably accompanied and correlated with oxidative stress.     

Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata)

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.     

Protective role of Spirulina feed in a freshwater fish (Poecilia reticulata Peters) exposed to an azo dye-methyl red

Acute toxicity of an azo dye-methyl red (5-40 ppm) was examined under starving conditions, on two groups of Poecilia reticulata--a freshwater fish, fed on different diets prior to their exposure to dye. Besides natural feed, fish of group-1 also received Spirulina feed for one month (feed population), whereas those of group-2 received only natural feed (non-feed population). The mortality data revealed non-feed population to be more tolerant to feed stress during acute toxicity study, whereas feed population exhibited better tolerance to the combined stress of both feed and methyl red; especially at higher concentrations of the latter. RBCs in methyl red treatments acquired different shapes (poikilocytosis) and an increase in their size (anisocytosis) was also noticed. Percentage of such abnormal RBCs was almost equal in both feed and non-feed populations, except at a lower concentration (5 ppm), at which percentage of poikilocytic RBCs was lesser in the feed population. RBC counts in the control non-feed fish (34.5 x 10(4)/mm3) were significantly lower than control feed population (50.0 x 10(4) /mm3). Their number decreased with an increase in methyl red concentrations in non-feed population (9-26%), but percent reduction in RBC counts was almost similar (20-26%) at various concentrations of methyl red (5-30 ppm) in the feed population. Despite reduction in RBC counts, feed population did not suffer from anemia in methyl red treatments, as evident by their RBC counts which were almost equal to control fish of non-feed population. The results suggest that Spirulina feed improves tolerance of test organism towards methyl red manifested by noticeable reduction in the cytotoxic effects on RBCs and a lower mortality rate at higher concentrations of dye.     

Dietary sodium butyrate ameliorated the blood stress biomarkers,heat shock proteins,and immune response of Nile tilapia (Oreochromis niloticus) exposed to heat stress

The present study investigated the effects of sodium butyrate (SB) on the growth performance, histomorphology, immune response, and stress related markers of Nile tilapia subjected to heat stress. SB was incorporated at 0, 0.5, 1, 1.5, and 2 g per kg diet and fed to fish for 8 weeks. The obtained results revealed significantly improved growth performance with a decreased feed conversion ratio in the fish fed SB (P < 0.05). In the anterior, middle, and distal parts of the intestine, villus length and width and internal villi distance as well as the number of goblet cells were increased in the fish fed SB (P < 0.05). The blood total protein, hemoglobin, and white and red blood cell counts showed a significant quadratic influence (P < 0.05). The survival rate for Nile tilapia exposed to heat stress for 48 h revealed that the SB fed groups had noticeably higher survival rates. Dietary SB significantly increased the phagocytic index and lysozyme and phagocytic activities both before and after heat stress (P < 0.05). After heat stress, blood glucose decreased significantly with SB feeding at 0.5, 1, or 1.5 g per kg diet, while cortisol was reduced in fish fed 1.5 or 2 g per kg diet (P < 0.05). Additionally, in fish fed SB, superoxide dismutase (SOD), catalase, and glutathione peroxidase activities were significantly increased both before and after heat stress, while malondialdehyde was decreased by SB feeding (P < 0.05). Liver heat shock protein 70 and SOD gene expression were significantly upregulated in fish fed on SB at 1 g per kg diet (P < 0.05). Thus, supplementation with SB at 1–2 g per kg diet can be used effectively in tilapia diets for improving growth, feed efficiency, and immune response as well as for tolerance to heat stress.     

Oxidative stress, DNA damage and osmolality in the Pacific white shrimp, Litopenaeus vannamei exposed to acute low temperature stress

To evaluate the genotoxic, physiological and immunological effects of short-term acute low temperature stress on the Pacific white shrimp, Litopenaeus vannamei, we rapidly transferred shrimp from tanks at 23±2 °C to aquaria at the same temperature (controls) or 12±2 °C for 12 h. Changes in the shrimp hemocyte respiratory burst activity and DNA damage were examined during and after exposure to the temperature stress using flow cytometry and the comet assay, respectively. We also monitored changes in the total hemocyte count, malondialdehyde levels, total protein concentration and osmolality in shrimp plasma. The results show that hemocyte respiratory burst activity, malondialdehydes levels and hemocyte DNA damage in the plasma all increased significantly after exposure to 12±2 °C for 3 h. In contrast, total hemocyte count, total protein concentration and osmolality in the plasma decreased compared to the controls. We conclude that acute low temperature can induce oxidative stress, DNA damage, lipid peroxidation and changes in osmolality in L. vannamei.