Linalool induces relaxation of the mantle of golden apple snail (Pomacea canaliculata).

The objective of this study was to evaluate the possible relaxing effect of essential oils (EOs) (Aloysia triphylla and Lippia alba) and phytochemicals (citral and linalool) in the gastropod Pomacea canaliculata. Animals were exposed to compounds at the concentrations range of 25-750 µL L-1. Magnesium chloride (MgCl2, 10-50 g L-1) and control group (ethanol 6.75 mL L-1, highest concentration used for treatment dilution) were also tested. The EOs, citral and MgCl2 had no relaxing effect at the concentrations range tested, and citral caused aversive behavior (closure of the operculum) from 90 µL L-1. Exposure to linalool at 25, 50, 100, 200 and 400 µL L-1 relaxed 28, 76, 88, 96 and 100% of the animals, respectively. The concentrations of 25, 50 and 400 µL L-1 differed statistically from each other, while 100 and 200 µL L-1 were equal to 50 and 400 µL L-1. All animals recovered up to 40 min, except at of 400 µL L-1. Linalool is effective for relaxing P. canaliculata and can be useful in management techniques that require relaxation. However, further studies are needed to certify whether linalool is appropriate for maintaining animal welfare in invasive procedures that require total insensitivity.

Essential oil of Ocimum gratissimum (Linnaeus, 1753): efficacy for anesthesia and transport of Oreochromis niloticus.

This study aimed to evaluate the essential oil of Ocimum gratissimum L. (EOOG) for anesthesia and in the transport of Oreochromis niloticus. Experiment I determined the time of anesthesia induction and recovery during anesthesia of O. niloticus exposed to different concentrations of EOOG (0, 30, 90, 150, and 300 mg L-1). Based on data from Experiment I, Experiment II evaluated the effect of 0, 30, and 90 mg L-1 EOOG on blood parameters and oxidative stress immediately after anesthesia induction and 1 h after recovery. Experiment III evaluated the effect of 0, 5, and 10 mg L-1 EOOG on blood variables immediately after 4.5 h of transport of juveniles. Concentrations between 90 and 150 mg L-1 EOOG were efficient for anesthesia and recovery. The use of 90 mg L-1 of EOOG prevented an increase in plasma glucose. Other changes in blood parameters and oxidative stress are discussed. The use of 10 mg L-1 EOOG in transport increased plasma glucose and decreased hematocrit values immediately after transport. It is concluded that the use of 90 and 150 mg L-1 EOOG causes anesthesia and recovery in O. niloticus within the time intervals considered ideal. The use of 90 mg L-1 EOOG favored stable plasma glucose soon after anesthesia induction and 1 h after recovery, but caused changes in the antioxidant defense system by increasing hepatic and kidney ROS. The transport of 12 g O. niloticus for 4.5 h can be performed with concentration of 5 mg L-1 of EOOG.

GABAa receptor subunits expression in silver catfish (Rhamdia quelen) brain and its modulation by Nectandra grandiflora Nees essential oil and isolated compounds.

Studies using silver catfish (Rhamdia quelen) as experimental models are often applied to screen essential oils (EO) with GABAergic-mediated effects. However, the expression of GABAa receptors in the silver catfish brain remains unknown. Thus, we assessed whether silver catfish express GABAa receptor subunits associated with sedation/anesthetic process and/or neurological diseases. Additionally, we evaluated the brain expression of GABAa receptor subunits in fish sedated with Nectandra grandiflora EO and its isolated compounds, the fish anesthetic (+)-dehydrofukinone (DHF), and dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL), which have GABAa-mediated anxiolytic-like effects in mice. The expression of the subunits gabra1, gabra2, gabra3, gabrb1, gabrd and gabrg2 in the silver catfish brain were assessed after a 24h-sedation bath by real time PCR. Since qPCR data rarely describes mechanisms of action, which are usually found through interactions with receptors, we also performed an antagonist-driven experiment using flumazenil (FMZ). Real-time PCR detected the mRNA expression of all targeted genes in R. quelen brain. The expression of gabra1 was decreased in fish sedated with ERM; EO increased gabra2, gabra3, gabrb1 and gabrg2 expression; SEL increased gabrb1, gabrd and gabrg2 expression. EO and compounds DFX, SEL and ERM induced sustained sedation in fish and FMZ-bath prompted the recovery from ERM- and DFX-induced sedation. Our results suggest that the EO, SEL, ERM and DFX sedative effects involve interaction with the GABAergic system. Our findings support the use of the silver catfish as robust and reliable experimental model to evaluate the efficacy of drugs with putative GABAergic-mediated effects.

Aeromonas hydrophila infection in silver catfish causes hyperlocomotion related to stress.

Aeromonosis is a fish disease that leads to haemorrhagic septicaemia and high mortality. The detection of early behavioural changes associated to this disease could be helpful in anticipating the initiation of treatment, increasing the probability of success. The influence of this disease on the hypothalamic-pituitary-interrenal (HPI) axis and on the brain expression of heat shock proteins (HSP) is little known. Therefore, the aim of this study was to evaluate the effect of Aeromonas hydrophila infection on individual behaviour and brain expression of genes related to stress (slc6a2, hsp90, hspa12a, hsd20b, hsd11b2, crh) in silver catfish (Rhamdia quelen). Thirty fish were divided into healthy and infected groups. The fish of the infected group were inoculated intramuscularly with 50 µL of bacterial suspension (6.4 × 108 CFU/mL), while control animals received 50 µL of saline. On day five post-infection, animals were submitted to the novel tank test, euthanized, and the brain was collected for molecular analysis. Infected fish swam more in the unknown aquarium and presented an increase in brain expression of genes related to HSP (hspa12a) and the route of cortisol synthesis (crh) when compared to uninfected fish. Therefore, this disease causes hyperlocomotion related to stress.

Diphenyl diselenide dietary supplementation protects against methylmercury-chloride-induced immunotoxicity in the head kidney and spleen of grass carp (Ctenopharyngodon idella) via regulation of purinergic signaling and the NLRP3 inflammasome.

This study aimed to evaluate whether dietary supplementation with diphenyl diselenide (Ph2Se2) would prevent the impaired immune and inflammatory responses elicited by methylmercury chloride (CH3HgCl) via protective effects on purinergic signaling in fish immune organs. Tissue and lymphocytic nucleoside triphosphate diphosphohydrolase (NTPDase) activity for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was downregulated in the head kidney and spleen of grass carp (Ctenopharyngodon idella) exposed to CH3HgCl. Concomitantly, adenosine deaminase (ADA) activity was upregulated. Further, nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome gene expression was upregulated in the spleen and head kidney of CH3HgCl-exposed grass carp. Dietary supplementation with Ph2Se2 ameliorated these CH3HgCl-mediated alterations on purinergic enzymes, and their activities returned to baseline levels (except NTPDase activity for ADP). Based on these results, purinergic signaling in immune organs and lymphocytes can be considered a pathway linked to pro-inflammatory effects during exposure to environmental CH3HgCl concentrations, which may contribute to mortality of the affected fish. Since dietary supplementation with 3 mg Ph2Se2/kg in the feed prevented the CH3HgCl-induced alterations, it can be considered a potential suitable treatment to prevent impaired immune and inflammatory responses caused by Hg.

Melaleuca alternifolia essential oil abrogates hepatic oxidative damage in silver catfish (Rhamdia quelen) fed with an aflatoxin-contaminated diet.

Mycotoxins are secondary metabolites produced by varieties of fungi that contaminate food and feed resources and are capable of inducing a wide range of toxicity. This problem is extensively aggravated due to the increasing replacement of fish meal by plant-derived proteins. Among the mycotoxins, aflatoxins have received a great deal of attention owing to their great prevalence in plant feedstuffs and to the detrimental effects on animals. The objective of this study was to evaluate whether dietary supplementation with tea tree (Melaleuca alternifolia) oil (TTO) would avoid or minimize the negative impacts on silver catfish (Rhamdia quelen) fed with aflatoxins-contaminated diets. Four treatments were tested: control (fish fed with a control diet); AFB (fish fed with a mycotoxin-contaminated diet - 1893 µg kg-1 of AFB1 and 52.2 µg kg-1 AFB2); TTO (fish fed with a control diet + 1 mL kg-1 of TTO), and TTO + AFB (fish fed with a mycotoxin contaminated diet - 2324 µg kg-1 of AFB1 and 43.5 µg kg-1 AFB2 + 1 mL kg-1 of TTO). Diets were tested in three replications and analyzed at days 5 and 10 of dietary intake. Significantly reduced antioxidant enzymes (SOD, GPx, and GST) and increased lipid peroxidation (LOOH) and protein carbonyl (PC) content in plasma and liver, with 16.6% mortality occurrence, were observed in the group fed aflatoxin-contaminated diet. Furthermore, aflatoxins also significantly increased plasmatic and hepatic ROS levels and decreased hepatic antioxidant capacity against peroxyl radical (ACAP) levels. Plasma cortisol levels were not altered by aflatoxicosis, but the intoxication induced hepatose. Notwithstanding, addition of TTO to the groups receiving aflatoxins showed a protective effect, avoiding the increase of ROS, LOOH, and PC levels in plasma and liver. Moreover, TTO treatment ameliorated the aflatoxin-associated liver damage. Thus, TTO supplementation at concentration of 1 mL kg-1 in feed may be used in fish to increase antioxidant status and reduce the negative effects caused by aflatoxins toxicity.

A caffeine-supplemented diet modulates oxidative stress markers and prevents oxidative damage in the livers of Nile tilapia (Oreochromis niloticus) exposed to hypoxia.

Recent evidence has revealed the involvement of oxidative stress and oxidative damage with health impairment and mortality in fish exposed to hypoxia. Thus, natural compounds with antioxidant and free-radical-scavenging properties, such as caffeine, might help to prevent or reduce hepatic damage elicited by hypoxia. Thus, the aim of this study was to evaluate whether dietary supplementation with caffeine could prevent or reduce oxidative damage in the livers of Nile tilapia (Oreochromis niloticus) exposed to hypoxia. Hepatic reactive oxygen species, lipid peroxidation levels, and xanthine oxidase (XO) activity were higher in fish exposed to hypoxia compared with normoxia. Hepatic catalase, glutathione peroxidase, and glutathione S-transferase activities, as well as the antioxidant capacity against peroxyl radical levels, were lower in fish exposed to hypoxia compared with normoxia. No significant difference between groups was observed regarding hepatic superoxide dismutase activity. Dietary supplementation with 8% caffeine prevented all alterations elicited by hypoxia. Based on this evidence, the use of dietary supplementation with 8% caffeine can be an interesting approach to preventing hepatic lipid damage and impairment of the antioxidant defense system elicited by hypoxia, and this effect can be mediated by protective effects on XO activity.

Grape pomace flour alleviates Pseudomonas aeruginosa-induced hepatic oxidative stress in grass carp by improving antioxidant defense.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen in aquaculture systems being associated to extensive liver damage caused by oxidative stress in both marine and freshwater fish. Dietary supplementation with natural antioxidants is considered a rational strategy to prevent hepatic diseases involved with oxidative stress. Bio-residues resulting from the wine industry, such as grape pomace, are potential sources of bioactive phenolic compounds that can be applied as supplement for animal production. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) was able to prevent or reduce the hepatic oxidative damage of grass carp, Ctenopharyngodon idella, experimentally infected by P. aeruginosa. Hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), thiobarbituric acid reactive substances, and protein carbonylation levels were higher in fish experimentally infected by P. aeruginosa compared to the control group. Hepatic superoxide dismutase and catalase activities and antioxidant capacity against peroxyl radical levels were also higher in fish experimentally infected by P. aeruginosa compared to the control group. Dietary supplementation with 300 mg/kg GPF prevented all alterations elicited by P. aeruginosa, with the exception of protein carbonylation levels. The dietary supplementation with 150 mg/kg GPF was not able to avoid alteration of the analyzed variables, being results similar to those infected (positive control). Based on these results, dietary supplementation with 300 mg/kg GPF prevented P. aeruginosa-induced liver damage in grass carp, and this protective effect occurred through prevention on excessive ROS and NOx production, as well as via prevention of lipid damage. Moreover, 300 mg/kg GPF exerted its hepatoprotective effects by improving enzymatic and non-enzymatic antioxidant defense system. In summary, this supplementation can be an interesting approach to prevent P. aeruginosa-induced liver damage.

Oxidative stress mediated the inhibition of cerebral creatine kinase activity in silver catfish fed with aflatoxin B1-contaminated diet.

Aflatoxin B1 (AFB1) is an environmental toxicant and neurotoxic compound that induces the production of free radicals, causing oxidative stress. Creatine kinase (CK) is a central controller of energy metabolism in tissues with a large and fluctuating energy demand, and it is highly susceptible to inactivation by free radicals and oxidative damage. Thus, the aim of this study was to evaluate whether a diet for freshwater silver catfish (Rhamdia quelen) containing AFB1 inhibits cerebral CK activity, as well as the involvement of the oxidative stress on this inhibition. Brain CK activity was lower on days 14 and 21 post-feeding in animals that received AFB1-contaminated diet compared to the control group (basal diet), similarly to the brain sodium-potassium pump (Na+, K+-ATPase) activity. On the other hand, lipid peroxidation and protein carbonylation levels were higher on days 14 and 21 post-feeding in animals fed with AFB1-contaminated feed compared to the control group, while the antioxidant capacity against peroxyl radicals and thiol content was lower. Based on these evidences, the data demonstrated that diet containing AFB1 severely affects CK activity, an essential enzyme that plays an important role in brain energy homeostasis. Also, the impairment of energetic homeostasis linked with the use and generation of ATP via inhibition of CK activity elicited an inhibition of enzymes ATP-dependent, such as Na+, K+-ATPase. Moreover, the inhibition of brain CK activity appears to be mediated by the oxidation of lipids, proteins, and thiol group, as well as by a reduction in the antioxidant capacity.

Involvement of HPI-axis in anesthesia with Lippia alba essential oil citral and linalool chemotypes: gene expression in the secondary responses in silver catfish.

In teleost fish, stress initiates a hormone cascade along the hypothalamus-pituitary-interrenal (HPI) axis to provoke several physiological reactions in order to maintain homeostasis. In aquaculture, a number of factors induce stress in fish, such as handling and transport, and in order to reduce the consequences of this, the use of anesthetics has been an interesting alternative. Essential oil (EO) of Lippia alba is considered to be a good anesthetic; however, its distinct chemotypes have different side effects. Therefore, the present study aimed to investigate, in detail, the expression of genes involved with the HPI axis and the effects of anesthesia with the EOs of two chemotypes of L. alba (citral EO-C and linalool EO-L) on this expression in silver catfish, Rhamdia quelen. Anesthesia with the EO-C is stressful for silver catfish because there was an upregulation of the genes directly related to stress: slc6a2, crh, hsd20b, hspa12a, and hsp90. In this study, it was also possible to observe the importance of the hsd11b2 gene in the response to stress by handling. The use of EO-C as anesthetics for fish is not recommended, but, the use of OE-L is indicated for silver catfish as it does not cause major changes in the HPI axis.

Disturbance of energetic homeostasis and oxidative damage provoked by trichlorfon as relevant toxicological mechanisms using silver catfish as experimental model.

Recent evidences have suggested the involvement of phosphoryl transfer, catalyzed by creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK), to metabolic alterations and impairment of bioenergetics homeostasis linked to adenosine triphosphate (ATP) production, and utilization during exposure to pesticides. It is recognized that sublethal concentrations of trichlorfon alter hepatic and branchial metabolism, but the pathways involved in this process remains unknown. Thus, the aim of this study was to evaluate whether phosphoryl transfer network can be a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon using silver catfish Rhamdia quelen as experimental model. Hepatic and branchial CK (cytosolic and mitochondrial isoforms) and PK activities were inhibited after 48 h of exposure to 11 and 22 mg/L trichlorfon compared to control group, while AK activity did not differ between groups. In addition, sodium-potassium pump (Na+, K+-ATPase) activity was lower after 48 h of exposure to 22 mg/L trichlorfon compared to control group. Thiobarbituric acid reactive substances (TBARS) were higher in liver samples after 24 h of exposure to 22 mg/L trichlorfon compared to control group, as well as after 48 h of exposure to 11 and 22 mg/L trichlorfon in liver and gills. Finally, hepatic and branchial non-protein thiol (NPSH) levels were lower after 48 h of exposure to 11 and 22 mg/L trichlorfon. All evaluated parameters did not recover after 48 h in clean water. Based on these evidence, the impairment of phosphoryl transfer network can be considered a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon. Moreover, alterations on CK and PK activities provoke an impairment on Na+, K+-ATPase activity, which can be mediated by lipid oxidative damage and reduction of NPSH content.

Caffeine modulates brain purinergic signaling in Nile tilapia (Oreochromis niloticus) under hypoxia conditions: improvement of immune and inflammatory responses.

Purinergic signaling is linked to neurodegenerative and proinflammatory damage during pathological conditions such as hypoxia, but involvement of this pathway in brain damage in fish exposed to environmental hypoxia remains unknown, and we propose dietary supplementation with caffeine in order to improve the immune response. Therefore, the aim of the study was to evaluate whether the enzymatic purinergic signaling pathway is associated with inflammatory brain damage in Nile tilapia (Oreochromis niloticus) exposed to environmental hypoxia and whether dietary supplementation with caffeine (5% and 8%) can prevent these changes in purinergic signaling. Animals were randomly divided into six groups (A-F, n = 6 per group, in triplicate), as follows: groups A-C were submitted to normoxia, while groups D-F were submitted to hypoxia. Groups A and D received the basal diet, while groups B and D and groups C and F received a diet containing 5% and 8% caffeine, respectively, and fed with their respective diets for 21 days. After 21 days, aeration was disconnected (groups D-F) and the dissolved oxygen levels were maintained as follows: group A (6.55 ± 0.23 mg/L), group B (6.51 ± 0.24 mg/L), group C (6.58 ± 0.22 mg/L), group D (1.23 ± 0.11 mg/L), group E (1.20 ± 0.15 mg/L), and group F (1.18 ± 0.13 mg/L). Cerebral triphosphate diphosphohydrolase (NTPDase) using adenosine triphosphate (ATP) as a substrate and 5'-nucleotidase activities decreased in fish exposed to 72 h of hypoxia compared with the normoxia group, while adenosine deaminase (ADA) activity and levels of nitric oxide (NOx) metabolites were higher. Dietary supplementation with 5% and 8% caffeine prevented all alterations elicited by hypoxia, with the exception of ADA activity in the case of 5% caffeine. Based on this evidence, our findings reveal that nucleotide/nucleoside hydrolysis is modified in the brains of fish exposed to 72 h of hypoxia, contributing to inflammatory damage, which apparently is mediated by excessive ATP content in the extracellular medium and by excessive NOx production. Also, the use of a diet containing 5% and 8% caffeine prevented these alterations (except 5% of dietary caffeine on ADA activity) and can be considered an interesting approach to preventing the impairment of immune and inflammatory responses elicited by hypoxia, principally the inclusion of 8% caffeine.

Organophosphate pesticide trichlorfon induced neurotoxic effects in freshwater silver catfish Rhamdia quelen via disruption of blood-brain barrier: Implications on oxidative status, cell viability and brain neurotransmitters.

The aim of this study was to evaluate whether rupture on blood-brain barrier (BBB) can be a pathway for trichlorfon-induced neurotoxic effects, and to investigate its implications on oxidative status, cell viability and brain neurotransmitters in silver catfish (Rhamdia quelen). The BBB permeability was increased in fish exposed for 24 h to 22 mg/L of trichlorfon compared to the control group, as well as in those exposed to 11 and 22 mg/L of trichlorfon for 48 h. Compared to the control group, brain reactive oxygen species and lipid peroxide levels were higher when exposed to 22 mg/L of trichlorfon and 11 and 22 mg/L of trichlorfon after 24 h and 48 h, respectively, while the antioxidant capacity against peroxyl radical levels was lower. Exposure to 22 mg/L of trichlorfon for 24 h reduced brain cell viability compared to the control group, together with 11 and 22 mg/L of trichlorfon for 48 h. Also, brain AChE, Na+ and K+-ATPase activities were reduced in those fish exposed to trichlorfon compared to the control group. Thus, the rupture of BBB can be considered an important pathway involved in trichlorfon-induced neurotoxic effects, which contributes to brain oxidative damage and important changes on brain neurotransmitters.

Plant essential oils against bacteria isolated from fish: an in vitro screening and in vivo efficacy of Lippia origanoides / Óleos essenciais de plantas contra bactérias isoladas de peixes: uma triagem in vitro e eficácia in vivo de Lippia origanoides

ABSTRACT: The use of natural products, such as essential oils (EOs), is a potential novel approach to treat fish bacterial infections with a lower risk of developing resistance. There has been a number of studies reporting the activity of EOs as those obtained from the species Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides, and Melaleuca alternifolia against bacteria. However, there are few studies investigating the effect of these EOs against fish bacteria. Therefore, the aim of this study was to evaluate the in vitro antibacterial activity of EOs against the following fish bacteria, Aeromonas hydrophila, Citrobacter freundii, and Raoultella ornithinolytica. Additionally, the in vivo antibacterial activity of the EO L. origanoides was evaluated against experimentally induced A. hydrophila infection of silver catfish (Rhamdia quelen). The EO of L. origanoides was chosen as it showed the highest in vitro antibacterial activity, with minimum inhibitory concentrations ranging from 0.2 to 0.8 mg mL-1. This EO also presented a therapeutic success of 58.33%, on a 30 day A. hydrophila infection. Therefore, we suggested that the EO of L. origanoides may be a viable alternative as a treatment for A. hydrophila infection.

RESUMO: O uso de produtos naturais, como óleos essenciais (OEs), é uma nova abordagem potencial para o tratamento de infecções bacterianas em peixes com um baixo risco de desenvolvimento de resistência. Existem estudos reportando a atividade de OEs como aqueles obtidos das espécies Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides e Melaleuca alternifolia contra bactérias. Porém, existem poucos estudos investigando o efeito desses OEs supracitados contra bactérias isoladas de peixes. Portanto, o objetivo desse estudo foi avaliar a atividade antibacteriana in vitro desses OEs contra bactérias isoladas de peixes (Aeromonas hydrophila, Citrobacter freundii e Raoultella ornithinolytica). Assim como avaliar a atividade antibacteriana in vivo do OE de L. origanoides contra uma infecção por A. hydrophila experimentalmente induzida em jundiá (Rhamdia quelen). O OE de L. origanoides foi escolhido porque apresentou a melhor atividade antibacteriana in vitro, com concentrações inibitórias mínimas variando de 0,2 a 0,8 mg mL-1. Esse OE apresentou sucesso terapêutico de 58,33% no dia 30 pós-infecção. Portanto, sugerimos que o OE de L. origanoides possa ser uma alternativa viável no tratamento da infecção por A. hydrophila em peixes.

Serum and hepatic oxidative damage induced by a diet contaminated with fungal mycotoxin in freshwater silver catfish Rhamdia quelen: Involvement on disease pathogenesis.

It has been recognized that oxidative stress is implicated in the initiation and progression of diseases due to the excessive formation of free radicals and impairment of the antioxidant defense system, contributing to the mortality of affected animals. The occurrence of a disequilibrium between the antioxidant/oxidant status in serum and liver of freshwater fish fed with aflatoxin B1 (AFB1) remains poorly understood and limited to only a few oxidant variables. Thus, the aim of this study was to evaluate whether an AFB1-contaminated diet causes disturbance on the antioxidant and oxidant status in silver catfish (Rhamdia quelen) of freshwater. Serum and hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), and lipid hydroperoxide increased on days 14 and 21 post-feeding in animals that received AFB1 contaminated diet compared to the control group (basal diet), while protein carbonylation levels increased on day 21 post-feeding. On the other hand, serum and hepatic antioxidant capacity against peroxyl radical and vitamin C levels, as well as glutathione peroxidase and catalase activities were lower on days 14 and 21 post-feeding in animals that received AFB1 contaminated diet compared to the control group. No difference was observed between groups regarding the superoxide dismutase activity and glutathione levels. Based on these evidences, an AFB1-contaminated diet causes a disturbance on serum and hepatic antioxidant/oxidant system due to lipid and protein damage elicited by excessive ROS and NOx production. Also, the antioxidant defense system was unable to avoid or minimize ROS and NOx deleterious effects, and consequently, the oxidative damage. In summary, this disturbance can contribute to understand the pathophysiology and mortality of fish after the consumption of AFB1-contaminated diets.

Purinergic signalling displays a pro-inflammatory profile in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by fungal mycotoxin: Involvement on disease pathogenesis.

The spleen is an important secondary lymphatic organ that plays a key role in the immune and inflammatory responses of teleost fish. The purinergic signalling has been associated to these types of responses under pathological conditions by the regulation of extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado), where both exert potent pro-inflammatory and anti-inflammatory profiles, respectively. The exact pathway involved on the immunotoxic effects of aflatoxin B1 (AFB1) in fish fed with diets containing this mycotoxin remains poorly understood. Thus, the aim of this study was to evaluate whether purinergic signalling exerts anti or pro-inflammatory effects in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by AFB1. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) decreased in spleen and splenic lymphocytes of fish fed with an AFB1-contaminated diet on day 21 post-feeding compared to fish fed with a basal diet; while adenosine deaminase (ADA) activity increased. No differences were observed between groups or over time regarding NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. In summary, the purinergic signalling can be a pathway involved in the impairment of the immune and inflammatory responses in fish fed with an AFB1-contaminated diet, contributing to the immunotoxic effects of AFB1 in spleens of fish.

Aflatoxin B1-contaminated diet disrupts the blood-brain barrier and affects fish behavior: Involvement of neurotransmitters in brain synaptosomes.

It is known that the cytotoxic effects of aflatoxin B1 (AFB1) in endothelial cells of the blood-brain barrier (BBB) are associated with behavioral dysfunction. However, the effects of a diet contaminated with AFB1 on the behavior of silver catfish remain unknown. Thus, the aim of this study was to evaluate whether an AFB1-contaminated diet (1177 ppb kg feed-1) impaired silver catfish behavior, as well as whether disruption of the BBB and alteration of neurotransmitters in brain synaptosomes are involved. Fish fed a diet contaminated with AFB1 presented a behavioral impairment linked with hyperlocomotion on days 14 and 21 compared with the control group (basal diet). Neurotransmitter levels were also affected on days 14 and 21. The permeability of the BBB to Evans blue dye increased in the intoxicated animals compared with the control group, which suggests that the BBB was disrupted. Moreover, acetylcholinesterase (AChE) activity in brain synaptosomes was increased in fish fed a diet contaminated with AFB1, while activity of the sodium-potassium pump (Na+, K+-ATPase) was decreased. Based on this evidence, the present study shows that silver catfish fed a diet containing AFB1 exhibit behavioral impairments related to hyperlocomotion. This diet caused a disruption of the BBB and brain lesions, which may contribute to the behavioral changes. Also, the alterations in the activities of AChE and Na+, K+-ATPase in brain synaptosomes may directly contribute to this behavior, since they may promote synapse dysfunction. In addition, the hyperlocomotion may be considered an important macroscopic marker indicating possible AFB1 intoxication.

Changes in the cerebral phosphotransfer network impair energetic homeostasis in an aflatoxin B1-contaminated diet.

The phosphotransfer network system, through the enzymes creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), contributes to efficient intracellular energetic communication between cellular adenosine triphosphate (ATP) consumption and production in tissues with high energetic demand, such as cerebral tissue. Thus, the aim of this study was to evaluate whether aflatoxin B1 (AFB1) intoxication in diet negatively affects the cerebral phosphotransfer network related to impairment of cerebral ATP levels in silver catfish (Rhamdia quelen). Brain cytosolic CK activity decreased in animals fed with a diet contaminated with AFB1 on days 14 and 21 post-feeding, while mitochondrial CK activity increased, when compared to the control group (basal diet). Also, cerebral AK and PK activity decreased in animals fed with a diet contaminated with AFB1 on days 14 and 21 post-feeding, similarly to the results observed for cerebral ATP levels. Based on this evidence, inhibition of cerebral cytosolic CK activity is compensated by stimulation of mitochondrial CK activity in an attempt to prevent impairment of communication between sites of ATP generation and ATP utilization. The inhibition of cerebral AK and PK activity leads to impairment of cerebral energy homeostasis, decreasing the brain's ATP availability. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes to cerebral energetic imbalance, which may contribute to disease pathophysiology.

Efficacy of dietary curcumin supplementation as bactericidal for silver catfish against Streptococcus agalactiae.

The antibiotics were frequently used for combating bacterial infections in aquaculture, but this treatment causes antibiotic resistance, negative impact on the environment and on health, and accumulation of residual in edible tissues. Several evidences have considered the dietary supplementation with natural products an interesting alternative to antibiotics, as the use of curcumin, a polyphenol with anti-inflammatory, antioxidant and antimicrobial properties. Thus, the aim of this study was to evaluate whether fish fed with a diet containing 150 mg curcumin/kg feed is able to enhance the resistance of silver catfish to Streptococcus agalactiae infection. Our results demonstrated that curcumin dietary supplement exerts potent bactericidal action against S. agalactiae, presenting 100% of therapeutic efficacy when compared to infected and non-supplemented animals. Also, the treatment prevented the occurrence of clinical signs of disease, as erratic swimming, corneal opacity, skin lesions in the fin and tail, and loss of appetite. In summary, curcumin can be a promising dietary supplement for improving disease resistance.