Biomonitoring of Microcystin and Aflatoxin Co-Occurrence in Aquaculture Using Immunohistochemistry and Genotoxicity Assays

This work investigated the effects of co-occurring aflatoxin B-1 (AFB(1)) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB(1) by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB(1) was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB(1) showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Depletion study and estimation of the withdrawal period for oxytetracycline in tilapia cultured in Brazil

Defining pharmacokinetic parameters and depletion intervals for antimicrobials used in fish represents important guidelines for future regulation by Brazilian agencies of the use of these substances in fish farming. This article presents a depletion study for oxytetracycline (OTC) in tilapias (Orechromis niloticus) farmed under tropical conditions during the winter season. High performance liquid chromatography, with fluorescence detection for the quantitation of OTC in tilapia fillets and medicated feed, was developed and validated. The depletion study with fish was carried out under monitored environmental conditions. OTC was administered in the feed for five consecutive days at daily dosages of 80 mg/kg body weight. Groups of ten fish were slaughtered at 1, 2, 3, 4, 5, 8, 10, 15, 20, and 25 days after medication. After the 8th day posttreatment, OTC concentrations in the tilapia fillets were below the limit of quantitation (13 ng/g) of the method. Linear regression of the mathematical model of data analysis presented a coefficient of 0.9962. The elimination half- life for OTC in tilapia fillet and the withdrawal period were 1.65 and 6 days, respectively, considering a percentile of 99% with 95% of confidence and a maximum residue limit of 100 ng/g. Even though the study was carried out in the winter under practical conditions where water temperature varied, the results obtained are similar to others from studies conducted under controlled temperature.

The fatty acid profiles and energetic substrates of two Nile tilapia (Oreochromis niloticus, Linnaeus) strains, Red-Stirling and Chitralada, and their hybrid

Protein and lipid content as well as the fatty acid (FA) composition of storage tissues were analysed in two varieties of Oreochromis niloticus (Red-Stirling and Chitralada) and their hybrid. The animals were maintained in cages for 11 months. The samples were taken when the animals weighed 10, 50, 100, 250 and 500 g. The results showed that changes in the metabolic processes occur during an increase in body mass in both varieties of tilapia and also their hybrid, but that these differences are not found in animals collected at the commercial weight. The protein content of the fillet and liver decreased with growth and the same protein content associated with growth was found for fillet lipid content. The genetic variety did not influence the FA profile of the fillet, but different genotypes had different hepatic FA compositions. Even with the same lipid content, the hepatocytes of Chitralada accumulated higher levels of polyunsaturated fatty acids (PUFA) n6 in triglycerides and increased C22:6n3 in the hepatocyte membranes. The higher n6PUFA content was compensated by a lower fraction of saturated FA in the hepatocyte triglycerides. The skin of Chitralada also had higher n6PUFA and C22:6n3 contents, suggesting a higher ability to deposit PUFA in the skin due to alterations in the liver synthetic pathway.

Effects of trophic levels (chlorophyll and phosphorous content) in three different water bodies (urban lake, reservoir and aquaculture facility) on gill morphology of Nile tilapia (Oreochromis niloticus)

Histological and ultrastructural analyses of gills were observed in Nile tilapia collected from three different waterbodies in southeast Brazil: an urban lake in a park in the city of São Paulo, a reservoir in a rural city, and a commercial aquaculture facility. These aterbodies were analyzed and classified as hypereutrophic, eutrophic, and supereutrophic, respectively, with 310.00, 94.00, 28.00 of phosphate (PO2_ 4 lg L _1) and 65.49, 24.95, 12.83 of chlorophyll (lg L _1). A significant difference in the histological alterations index (HAI) was observed only in fish from the urban lake, with the presence of cell hypertrophy, hyperplasia, aneurism, and other alterations. When compared to the other groups, a large quantity of rodlet cells was also observed in the urban group. These results demonstrate the correlation of eutrophic states of water with gill morphology. Also discussed is the premise that large amounts of organic material dissolved in water can alter the morphology of the fish gills

Effect of the inclusion of fish residue oils in diets on the fatty acid profile of muscles of males and females lambari (Astyanax altiparanae)

This study evaluated the effects of two lipids sources of fish residue (tilapia and salmon) compared with a vegetable oil source (soybean oil) on the fatty acid profiles of male and female lambari. This experiment was developed in a completely randomized experimental design in a 3 x 2 factorial arrangement, totaling 6 treatments resulting from the combination of the three experimental diets for both sexes, with four replications for each treatment. This study involved 120 male (2.58 +/- 0.13 g) and 72 female lambari (4.00 +/- 0.09 g), fed the experimental diets twice a day until apparent satiation for a period of 60 days. Oleic, linoleic, palmitic and stearic fatty acids were found at higher concentrations in all experimental oils and diets, as well in the muscle of male and female lambari. The low amounts of arachidonic, eicosapentaenoic and docosahexaenoic acids in the experimental diets and subsequent greater concentrations in muscle tissue, suggested that lambari are able to desaturate and elongate the chain of fatty acids with 18 carbons. The fish of both sexes that received the diet with soybean oil showed high levels of n-6 fatty acids, especially of C18: 2n-6 and low levels of eicosapentaenoic and docosahexaenoic acids. The diet with salmon residue oil promoted higher levels of fatty acids of the n-3 series and resulted in the best n-3/n-6 ratio in the muscle of male and female lambari. The oils from fish residues can be a substitute for traditional fish oil and its use in the lambari diets does not impair its growth.

Molecular characterization by amplified fragment length polymorphism and aflatoxin production of Aspergillus flavus isolated from freshly harvested peanut in Brazil

Brazil contributes substantially to the global peanut production, and the state of Sao Paulo is the largest producer in the country. Peanut crops can be contaminated by Aspergillus flavus strains producing aflatoxins, which are highly toxic and carcinogenic. Thus, the production of high-quality peanuts is crucial both for the commercial peanut industry and as a matter of public health. In this study, we used amplified fragment length polymorphism analysis (AFLP) to investigate the genetic variability among A. flavus strains isolated from fresh peanuts harvested in four different regions in the state of Sao Paulo, and to determine whether the molecular genetic profiles correlated with aflatoxin production or sclerotia formation. AFLP analysis generated 78 fragments ranging from 27 to 365 base pairs in length. Thirteen percent were not polymorphic. Genotyping identified twelve groups of A. flavus. On the basis of the polymorphisms identified, similarity between the isolates ranged from 37% to 100%. Of all isolates collected, 91.7% produced aflatoxins and 83.9% produced small sclerotia. Statistical analysis failed to suggest any relationship between the presence of sclerotia and mean levels of aflatoxins B-1 and B-2. Furthermore, a dendrogram based on AFLP data revealed substantial genetic variability among the A. flavus strains, but showed no correlation between dendrogram groups separated by molecular genetic features and production of aflatoxins B-1 or B-2 or the formation of sclerotia.

Mycoflora and mycotoxins in field samples of Brazil nuts

Fungal and mycotoxin contamination was investigated in field samples of nuts, shells and pods of the Brazil nut collected during different periods in Itacoatiara, State of Amazonas, Brazil: day 0, samples still on the tree: days 5, 10 and 15, samples in contact with soil for 5, 10 and 15 days, respectively. The most prevalent fungi were Aspergillus flavus in fruit pods and nuts and Fusarium spp. in shells. Penicillium spp. and A. flavus were isolated from soil, and Fusarium spp. and Penicillium spp. from air. Aflatoxins and cyclopiazonic acid were not detected in any of the samples analyzed. The high frequency of isolation of aflatoxigenic A. flavus strains from soil and Brazil nuts increases the chance of aflatoxin production in these substrates. These findings suggest a possible contamination before drying and indicate soil as the main source of fungal contamination of Brazil nuts. (c) 2012 Elsevier Ltd. All rights reserved.

Protein diets promote the maturation of oocytes and spawning of Piaractus mesopotamicus kept in cages

The objective of this study was to evaluate the effects of balanced diets on the maturation of oocytes and the reproductive performance of P. mesopotamicus in cages. A completely random design with 224 fish in 16 cages measuring 5 m(3) was employed for this purpose. The treatments consisted of diets containing 18, 24, 30, and 36% crude protein (CP) provided ad libitum. The external and internal morphological characteristics of the specimens were examined, as well as: the position of the germinal vesicle, the distribution of oocyte diameters, the fertilization and hatching rates, the number of oocytes released, the total number of oocytes, the remaining weight and total weight of the ovaries, the gonadosomatic index, the condition factor (K), and the histology of the oocytes and ovaries post-spawning and during ovarian regression. The diameters of the oocytes collected before the first hormonal application displayed a unimodal distribution for the lowest protein content and a polymodal distribution for the other treatments. A similar situation was seen during spawning. The lowest fertilization and hatching rates were found as a consequence of the treatment with 30% CP (P < 0.05). The greatest hatching rate occurred in the females fed 18% CP. The greatest total oocyte weight was found in the specimens that received between 30 and 36% CP. The lowest K index was found in the females fed 36% CP. In conclusion, a diet containing 18% CP satisfies the reproductive requirements of females adapted to this system.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B-1, while 47.2% aflatoxins B-1 and B-2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson's correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB(1) and AFB(2), but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Aggressiveness Overcomes Body-Size Effects in Fights Staged between Invasive and Native Fish Species with Overlapping Niches

Approximately 50 years ago, Nile tilapia were accidentally introduced to Brazil, and the decline of pearl cichlid populations, which has been intensified by habitat degradation, in some locations has been associated with the presence of Nile tilapia. There is, however, little strong empirical evidence for the negative interaction of non-native fish populations with native fish populations; such evidence would indicate a potential behavioural mechanism that could cause the population of the native fish to decline. In this study, we show that in fights staged between pairs of Nile tilapia and pearl cichlids of differing body size, the Nile tilapia were more aggressive than the pearl cichlid. Because this effect prevailed over body-size effects, the pearl cichlids were at a disadvantage. The niche overlap between the Nile tilapia and the pearl cichlid in nature, and the competitive advantage shown by the Nile tilapia in this study potentially represent one of several possible results of the negative interactions imposed by an invasive species. These negative effects may reduce population viability of the native species and cause competitive exclusion.

Antioxidant defenses and biochemical changes in the neotropical fish pacu, Piaractus mesopotamicus: Responses to single and combined copper and hypercarbia exposure

This study investigated the potentially detrimental effects of copper and elevated aquatic CO2 (hypercarbia), alone or in combination, on pacu, Piaractus mesopotamicus. Fish were exposed for 48 h to control (no copper addition in normocarbia), to 400 mu g Cu2+L-1, to hypercarbic (1% CO2; PCO2=6.9 mm Hg) water and to 400 mu g Cu2+L-1+ hypercarbia. In liver the single factors caused an increase in lipid hydroperoxide concentration that was not observed when the factors were combined. Copper exposure elicited increased hepatic superoxide dismutase activity, irrespective of aquatic CO2 level. On the other hand, the effects of copper on hepatic glutathione peroxidase activity were dependent on water CO2 levels. The two stressors combined did not affect hepatic catalase activity. Hypercarbic water caused a decline in plasma glucose concentration, but this was not observed when hypercarbia was combined with copper exposure. Copper caused a decrease in branchial Na+/K+-ATPase activity that was independent of water CO2 level. Copper caused an increase in branchial metallothionein concentration that was independent of water CO2 level. Thus, branchial metallothionein and Na+/K+-ATPase were effective biomarkers of copper exposure that were not affected by water CO2 level. (C) 2012 Elsevier Inc. All rights reserved.

Behavioral characterization of the alarm reaction and anxiolytic-like effect of acute treatment with fluoxetine in piaucu fish

In Ostariophysan fish, the detection of the alarm substance liberated into the water as a consequence of an attack by a predator elicits an alarm reaction or anti-predatory behavior. In this study, experiments were performed to: (i) describe and quantitatively characterize the behavioral and ventilatory responses in piaucu fish (Leporinus macrocephalus), individually and as part of a school, to conspecific alarm substance (CAS) and; (ii) test the effect of acute fluoxetine treatment on alarm reaction. Histological analysis revealed the presence of club cells in the intermediate and superficial layers of the epidermis. The predominant behavioral response to CAS was freezing for fish held individually, characterized by the cessation of the swimming activity as the animal settles to a bottom corner of the aquarium. Fish exposed to CAS showed decrease in the mean ventilatory frequency (approximately 13%) relative to control. In schools, CAS elicited a biphasic response that was characterized by erratic movements followed by increased school cohesion and immobility, reflected as an increased school cohesion (65.5% vs. -5.8% for controls) and in the number of animals near the bottom of the aquarium (42.0% vs. 6.5% for controls). Animals treated with single i.p. injections of fluoxetine (10 mu g/g b.w.) did not exhibit alarm behavior following CAS stimulation. These results show that an alarm pheromone system is present in piaucu fish, evidenced by the presence of epidermal club cells and an alarm reaction induced by CAS and consequently of a chemosensory system to transmit the appropriate information to neural structures responsible for initiating anti-predator behavioral responses. In addition, fluoxetine treatment caused an anxiolytic-like effect following CAS exposure. Thus, the alarm reaction in piaucu can be a useful model for neuroethological and pharmacological studies of anxiety-related states. (C) 2011 Elsevier Inc. All rights reserved.

Mycobiota and mycotoxins in Brazil nut samples from different states of the Brazilian Amazon region

The objective of this study was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in Brazil nut samples collected in different states of the Brazilian Amazon region: Acre, Amazonas, Amapa, and Para. A total of 200 husk samples and 200 almond samples were inoculated onto Aspergillus flavus-parasiticus agar for the detection of fungi. Mycotoxins were analyzed by high-performance liquid chromatography. The mycobiota comprised the following fungi, in decreasing order of frequency: almonds - Phialemonium spp. (54%), Penicillium spp. (16%), Fusarium spp. (13%), Phaeoacremonium spp. (11%), and Aspergillus spp. (4%), husks - Phialemonium spp. (62%), Phaeoacremonium spp. (11%), Penicillium spp. (10%), Fusarium spp. (9%), and Aspergillus spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins were detected in 22 (11%) of the 200 almond samples, with 21 samples presenting aflatoxin B-1 levels above 8 mu g/kg, the limit established by the European Commission for Brazil nuts for further processing. Nineteen (9.5%) of the 200 husk samples contained aflatoxins, but at levels lower than those seen in almonds. Cyclopiazonic acid (CPA) was detected in 44 (22%) almond samples, with levels ranging from 98.65 to 1612 mu g/kg. Aspergillus nomius and A. flavus were the most frequent Aspergillus species. The presence of fungi does not necessarily imply mycotoxin contamination, but almonds of the Brazil nut seem to be a good substrate for fungal growth. (C) 2012 Elsevier B.V. All rights reserved.

Development and optimization of biodegradable films based on achira flour

The influence of glycerol concentration (C-g), process temperature (T-p), drying temperature (T-s), and relative humidity (RH) on the properties of achira flour films was initially assessed. The optimized process conditions were C-g of 17g glycerol/100g flour, T-p of 90 degrees C, T-s of 44.8 degrees C, and RH of 36.4%. The films produced under these conditions displayed high mechanical strength (7.0 MPa), low solubility (38.3%). and satisfactory elongation values (14.6%). This study showed that achira flour is a promising source for the development of biodegradable films with good mechanical properties, low water vapor permeability, and solubility compared to films based on other tubers. (c) 2011 Elsevier Ltd. All rights reserved.