Effects of hydroxy-selenomethionine on performance, innate immune system and antioxidant defense of tambaqui (Colossoma macropomum) exposed to a physical stressor.

Selenium (Se) is a mineral with natural antioxidant properties that constitutes a number of enzymes with a fundamental role in the immunity and antioxidant systems and may confer a protective role against oxidative stress in fish following exposure to physical stressors. Adopting an integrated approach, this study investigated simultaneously the role of hydroxy-selenomethionine (OH-SeMet) supplementation in performance, hematological parameters, innate immune, antioxidant capacity and tissue Se retention of tambaqui (Colossoma macropomum) and the possible protective role of dietary selenium when fish are exposed to a physical stressor (transport). Juvenile specimens (15.71 ± 1.90 g) were fed one of five diets: a basal unsupplemented diet (0.0 mg kg-1 Se) or diets supplemented with OH-SeMet to provide 0.3, 0.6, 0.9 and 1.2 mg kg-1 Se of diet for 75 days prior to subjection of fish to transport stress. Dietary supplementation with Se in the form of OH-SeMet for 75 days did not affect the production performance of juvenile tambaqui, but increased innate immunity parameters (oxidative burst) from the Se inclusion level of 0.6 mg kg-1 and induced the activation of the antioxidant defense system (GPX, GSH and GST) especially at the Se inclusion level of 0.9 mg kg-1. In addition, the Se content in the fillet rose significantly, as the OH-SeMet contents in the diet were increased. The stress caused by transport resulted in alterations in hematological parameters, blood protein profile and immune and enzymatic responses in the species. However, Se supplementation at 0.9 mg kg-1 had a positive effect, increasing innate immunity and activating antioxidant defenses (CAT and GPx, especially) after this physical stressor was applied. These results demonstrate that, when submitted to transport stress, juvenile tambaqui use Se stored in the muscle and dietary supplementation with OH-SeMet at 0.9 mg kg-1 improves the innate immunity and antioxidant system parameters of fish after transport. These findings reinforce the need for supplementing hydroxy-selenomethionine in commercial diets for tambaqui to ensure tissue Se reserves as a contingency in cases of stress.

Modulation of the innate immune response, antioxidant system and oxidative stress during acute and chronic stress in pacu (Piaractus mesopotamicus).

Stress is an energy-demanding process, as well as the responses of the innate immune system, that impose a metabolic overload on cellular energy production, which can affect the cellular redox balance, causing oxidative damage. We evaluated the role of stress in the modulation of innate immune and oxidative/antioxidant mechanisms in juvenile pacu exposed to acute and chronic stressors. The experimental period lasted 30 days, and fish (113.7 ± 35.1 g) were fed commercial feed. During this period, half of the fish were not manipulated (Condition A), and the other half were chased with a dip net for 5 min twice a day (Condition C). After the 30-day period, fish from both groups were sampled (baseline sampling), and the remainders (not sampled) were air exposed for 3 min (acute stressor), returned to the tanks, and were sampled again 30 min, 3 h, 6 h, and 24 h after air exposure. We evaluated biomarkers of stress (circulating cortisol and glucose), the innate immune system (respiratory burst activity/RBA, hemolytic activity of the complement system (HA-AP) and serum concentration of lysozyme), oxidative damage (lipid peroxidation/LPO), and antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH-Px). Our results showed that stress, acutely or chronically, caused a transient reduction of RAL and activated the HA-AP. Acutely, stress increased the lysozyme concentration. Furthermore, both conditions caused oxidative stress in the liver, and differently they modulated the antioxidant system, enhancing SOD activity and impairing CAT and GSH-Px activity.

Development of a SNP linkage map and genome-wide association study for resistance to Aeromonas hydrophila in pacu (Piaractus mesopotamicus).

BACKGROUND: Pacu (Piaractus mesopotamicus) is one of the most important Neotropical aquaculture species from South America. Disease outbreaks caused by Aeromonas hydrophila infection have been considered significant contributors to the declining levels of pacu production. The current implementation of genomic selection for disease resistance has been adopted as a powerful strategy for improvement in fish species. This study aimed to investigate the genetic architecture of resistance to A. hydrophila in pacu via Genome-Wide Association Study (GWAS), the identification of suggestive Quantitative Trait Loci (QTLs) and putative genes associated with this trait. The genetic data were obtained from 381 juvenile individuals belonging to 14 full-sibling families. An experimental challenge was performed to gain access to the levels of genetic variation for resistance against the bacteria using the following trait definitions: binary test survival (TS) and time of death (TD). RESULTS: The analyses of genetic parameters estimated moderate heritability (h2) for both resistance traits: 0.20 (± 0.09) for TS and 0.35 (± 0.15) for TD. A linkage map for pacu was developed to enable the GWAS, resulting in 27 linkage groups (LGs) with 17,453 mapped Single Nucleotide Polymorphisms (SNPs). The length of the LGs varied from 79.95 (LG14) to 137.01 (LG1) cM, with a total map length of 2755.60 cM. GWAS identified 22 putative QTLs associated to A. hydrophila resistance. They were distributed into 17 LGs, and were considered suggestive genomic regions explaining > 1% of the additive genetic variance (AGV) for the trait. Several candidate genes related to immune response were located close to the suggestive QTLs, such as tbk1, trim16, Il12rb2 and lyz2. CONCLUSION: This study describes the development of the first medium density linkage map for pacu, which will be used as a framework to study relevant traits to the production of this species. In addition, the resistance to A. hydrophila was found to be moderately heritable but with a polygenic architecture suggesting that genomic selection, instead of marker assisted selection, might be useful for efficiently improving resistance to one of the most problematic diseases that affects the South American aquaculture.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus.

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

Morphological and immunohistochemical comparison of the pituitary gland between a tropical Paracheirodon axelrodi and a subtropical Aphyocharax anisitsi characids (Characiformes: Characidae)

Cardinal tetra Paracheirodon axelrodi and bloodfin tetra Aphyocharax anisitsi are two species of characids with high trade value as ornamental fish in South America. Although both species inhabit middle water layers, cardinal neon exhibits a tropical distribution and bloodfin tetra a subtropical one. Generally, these species are difficult to grow, so it becomes essential to know some key components of the neuroendocrine system to achieve their reproduction in captivity. Considering the importance of deepening the knowledge of the reproductive physiology through functional morphology, for the first time in this work we performed an anatomical, morphological and immunohistochemical analysis of the pituitary gland of these two species. In both species, a leptobasic type pituitary is found in the ventral zone of the hypothalamus and it is characterized by a neurohypophysis which has a well-developed pituitary stalk and a globular adenohypophysis. The pituitary components, characterized by histochemistry and immunohistochemistry, shows a distribution pattern of cells types similar to other teleost species, with only slight differences in the distribution of βFSH and βLH for P. axelrodi.(AU)

El cardenal tetra Paracheirodon axelrodi y el tetra Aphyocharax anisitsi son dos especies de carácidos con alto valor comercial como peces ornamentales en América del Sur. Aunque ambas especies habitan en las capas medias de agua, el neón cardenal exhibe una distribución tropical, mientras que el tetra cola roja una distribución subtropical. En general estas especies son difíciles de cultivar, por lo que es esencial conocer algunos componentes clave de los sistemas neuroendocrinos para lograr su reproducción en cautiverio. Considerando la importancia de profundizar en el conocimiento de la fisiología reproductiva a través de la morfología funcional, en este trabajo realizamos, por primera vez, un análisis anatómico, morfológico e inmunohistoquímico de la glándula pituitaria de estas dos especies. En ambas especies, la hipófisis, del tipo leptobásica, se encontró en la zona ventral del hipotálamo y se caracteriza por una neurohipófisis con un tallo hipofisario bien desarrollado y una adenohipófisis globular. Los componentes hipofisarios, caracterizados por la histoquímica y la inmunohistoquímica, mostraron un patrón de distribución de tipos de células similares a otras especies de teleósteos, con solo pequeñas diferencias en la distribución de βFSH y βLH para P. axelrodi.(AU)

ß-glucan modulates cortisol levels in stressed pacu (Piaractus mesopotamicus) inoculated with heat-killed Aeromonas hydrophila.

In this study, we show that ß-glucan can modulate cortisol release in fish. We simulated a common situation in aquaculture: the transport of fish followed by contact with an opportunistic pathogen and observed what effect glucan had on the immune and stress response in these conditions. Pacu (Piaractus mesopotamicus) were fed with a diet containing ß-glucan (0.1%) for 15 days prior to transport followed by an injection with heat-killed Aeromonas hydrophila. We sampled fish before transport, at arrival and at 3 and 24 h after bacterial injection. ß-Glucans are used in aquaculture and have a known immunostimulatory effect, which was observed in this study. The results showed that ß-glucan modulated the plasma cortisol levels differently by increasing these levels up to 24 h after transport and preventing the increase caused by bacterial inoculum injection. In addition, ß-glucan enhanced the activity of the complement system at 24 h and reduced the monocytes and lymphocytes number in peripheral blood at 3 and 24 h after bacterial inoculation. Our results suggest that ß-glucan modulated a bidirectional interaction between the stress and the immune responses. The modulation of cortisol levels and the immunostimulation by ß-glucan at different moments in our study suggest the compound has a protective effect by avoiding higher levels of the hormone and improving resistance against bacterial infection in pacu. These results add evidence to support the use of ß-glucan as an immunomodulator in the aquaculture industry.

Modulation of stress and innate immune response by corticosteroids in pacu (Piaractus mesopotamicus).

Understanding how stress and corticosteroid modulates the innate immune response is one of the keys to improving productivity and reducing losses in intensive aquaculture. Thus, we investigated the effects of dietary corticosteroids (7 days; long-term exposure) and transport (4 h; short-term stress) on stress and innate immune response in pacu. For this end, fish were fed with diets containing dexamethasone (100 mg kg-1) or hydrocortisone (200 mg kg-1), followed by transport, and then were intraperitoneally inoculated with heat-killed Aeromonas hydrophila or PBS (sham-inoculation). Fish were sampled after a 7-day feeding period, immediately post-transport and 24 h post-transport and inoculation. The dietary treatment of corticosteroids decreased resting cortisol levels by inhibiting the production of cortisol on the hypothalamus pituitary interrenal-axis. Further, both corticosteroids reduced hematocrit, red blood cells, haemoglobin and hemolytic activity of the complement, while they increased glucose levels and serum lysozyme concentrations. The transport increased cortisol and glucose levels and reduced the humoral immune defenses such as serum lysozyme concentration and hemolytic activity of the complement system. Interestingly, the hemolytic activity of the complement system increased sharply in fish fed with corticosteroids immediately post-transport, when they had their HPI-axis partially suppressed by the corticosteroids. This finding suggests a stimulatory effect of the catecholamines released during the transport on the activity of the complement system. Our results are highly valuable to understanding the stress and innate immune responses to long-term exposure to corticosteroids and short-term stress in fish and may provide insights into how corticosteroids modulate the innate immune system.

Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila.

The pathogenesis of sepsis involves complex systems and multiple interrelationships between the host and pathogen producing high mortality rates in various animal species. In this study, hematological disturbances, innate immunity and survival during the septic process in Piaractus mesopotamicus inoculated with Aeromonas hydrophila were studied. For this aim, fish blood samples were taken from control and infected groups 1, 3, 6, and 9 h post-inoculation (HPI). Leukogram showed reduction in the number of leukocytes and thrombocytes, followed by cessation of leukocyte chemotaxis 6 HPI and severe morphological changes in leukocytes and erythrocytes. At 3 HPI production of reactive oxygen species increased and at 6 HPI decreased. There was no change in serum lysozyme concentration and lytic activity of the complement system, despite the progressive increase in serum lytic activity and bacterial agglutination. Finally, the changes in clinical signs due to aeromonosis and increasing septicemia resulted in a reduction in survival to 57.14% after 36 HPI. It was possible concluded that these hematological and immune are crucial event in the worsening of sepsis in P. mesopotamicus, and these findings are utility for diagnosing and understanding the pathophysiology sepsis in pacu induced by A. hydrophila.

Distinct ß-glucan molecules modulates differently the circulating cortisol levels and innate immune responses in matrinxã (Brycon amazonicus).

This study investigated the effects of two ß-glucan molecules with different purities and isolated by different biotechnological processes on the immune response of matrinxã (Brycon amazonicus) prior and after challenge with Aeromonas hydrophila. In this sense, we evaluated serum cortisol and plasma glucose levels, the number of leukocytes (lymphocytes, neutrophils and monocytes), as well as the respiratory activity of leukocytes prior to, 6 and 24 h post infection (hpi). During 15 days, fish were fed with diets containing 0.1% of two ß-glucans (ß-G 1 and ß-G 2, with 71 and 62% of purity, respectively) and then submitted to challenge. Results were compared with a positive control group fed with a ß-glucan-free diet. A negative control group, also fed with ß-glucan-free diet but inoculated with PBS, was established to evaluate the effect of handling during injection. Our results showed that different ß-glucans affected differently the biological responses of matrinxã. The ßG 2 modulated the cortisol profile prior to and after the acute infection with A. hydrophila, and increased the mobilization and activity of leukocytes. The infection promoted lymphopenia at 6 hpi and both ß-glucans increased the circulating lymphocyte population 24 hpi. Moreover, the ß-G 2 prevented the infection-induced neutrophilia at 6 and 24 hpi. Finally, the ß-G 2 caused a marked increase in the circulating monocytes prior to infection, and a reduction at 6 hpi that was reversed at 24 hpi. In summary, our study demonstrates that ß-G 2 was more efficient on the induction of the cell-mediate immunity in matrinxã.

Development of an indirect ELISA assay to evaluation of the adaptive immune response of pacu (Piaractus mesopotamicus).

The pacu is one of the most important species for Brazilian fish farming and is considered emerging in the global aquaculture. Despite its importance, no effective tool for evaluation of the adaptive immune response of this species has been developed. Therefore, this study aimed the development and standardization of indirect ELISA for the measurement of pacu antigen-specific antibodies using polyclonal rabbit anti-pacu IgM used as detector antibody. For this purpose was isolated and purificated of pacu IgM using mannose-binding protein affinity chromatography and produced specific polyclonal antibodies against heavy and light chains pacu IgM, that showed a molecular weight of 72 kDa and 26 kDa, respectively. Polyclonal antibodies obtained demonstrated specificity with heavy and light Ig chains of pacu serum in western blotting. These polyclonal antibodies allowed the development of an indirect ELISA assay of high sensitivity and specificity for the detection and quantification of pacu IgM antibodies immunized with bovine IgG. In conclusion, this approach has great potential to improve the monitoring of vaccine-induced immune responses and help develop immunodiagnostic and epidemiological studies of infectious diseases in pacu systems.

Characterization and gene expression analysis of pacu (Piaractus mesopotamicus) inducible nitric oxide synthase (iNOS) following Aeromonas dhakensis infection.

Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red-bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48 h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu.

Glucan-MOS® improved growth and innate immunity in pacu stressed and experimentally infected with Aeromonas hydrophila.

We tested the efficacy of a commercial product (Glucan-MOS®) derived from yeast Saccharomyces cerevisiae, containing two combined products, ß-1,3-1,6 glucans and mannans on the growth, feed efficiency, stress and innate immune responses of juvenile pacu (Piaractus mesopotamicus) after a stressful handling and bacterial inoculation. For this, we evaluated the serum cortisol and plasma glucose levels, the respiratory activity of leukocytes, the serum lysozyme levels, as well as the number of circulating erythrocytes and leukocytes of fish fed during 30 days with diets containing increased levels of Glucan-MOS (0.0, 0.1, 0.2, 0.4 and 0.8%). The supplementation of 0.1% improved weight gain, feed conversion and the protein efficiency ratio compared to a control diet. The 0.2 and 0.4% Glucan-MOS® diets were sufficient to increase the respiratory burst of leukocytes and lysozyme activity, the number of thrombocytes, neutrophils and monocytes in the blood after a stressful handling and bacterial challenge, and minimized stress response as shown by decreased cortisol and glucose levels when compared to the control. The results of this work reinforce the benefits of the adoption of feeding strategies including combination of both ß-1,3-1,6 glucans and mannans as a dietary supplement in periods prior to intensive management. The 30-day period was sufficient to stimulate growth performance, improve nutrient utilization, minimize stress response and modulate innate immunity responses.

Long-term organic selenium supplementation overcomes the trade-off between immune and antioxidant systems in pacu (Piaractus mesopotamicus).

Selenium (Se) is an essential nutrient for antioxidant defenses in fish because of its role in preventing immunosuppression caused by oxidative stress. In this study it was demonstrated the relation between the oxidative stress and immune status after a long Se supplementation period, as a result of the evaluation of immunological, hematological and antioxidant responses, as well as growth performance of pacu fed diets supplemented with different concentrations of organic selenium (0, 0.3, 0.6, 0.9, and 1.8 mg Se-yeast/kg, but the final analyzed selenium concentrations were 0.72, 0.94, 1.15, 1.57 and 2.51 mg/kg, respectively) for 65 days. Dietary Se supplementation at 1.15 mg Se-yeast/kg (analyzed value) restored the production of antioxidant enzymes (glutathione peroxidase (GPx) and glutathione S-transferase (GST)), and consequently allowed the increased of some immunological parameters (leukocyte respiratory burst activity and lysozyme activity), hematological parameters (red blood cell count (RBC), hematocrit (HTC), mean corpuscular volume (MCV), and white blood cell count (WBC)). Se supplementation in pacu diets at 1.15 mg Se-yeast/kg for 65 days improved immune response and antioxidant defenses, suggesting that oxidative stress impairs immune system response to prevent excessive reactive oxygen species in cells and indicating the occurrence of a physiological trade-off between immune and antioxidant systems. Higher Se levels, such as 1.57 mg Se-yeast/kg increased the leukocyte respiratory burst activity, the WBC and thrombocyte counts, the RBC and HTC, and the GST and GPx enzymes. However, 2.51 mg Se-yeast/kg decreased the lysozyme levels, the WBC and thrombocyte counts, the RBC, HTC and MCV, and the GST and GPx enzymes. Those findings are important to future studies because showed the negative effect of oxidative stress on immunity, and may help to prevent any inhibition of the expected immune response after immunomodulators administration and vaccination. Also it was possible to meet the dietary selenium requirement of pacu, that was estimated to be 1.56 mg/kg.

The immune system is limited by oxidative stress: Dietary selenium promotes optimal antioxidative status and greatest immune defense in pacu Piaractus mesopotamicus.

Reactive oxygen species (ROS) are reactive molecules containing oxygen, that form as byproducts of aerobic metabolism, including immune system processes. Too much ROS may cause oxidative stress. In this study, we examined whether it can also limit the production of immune system compounds. To assess the relationship between antioxidant status and immunity we evaluated the effect of dietary supplementation with organic selenium, given at various levels for 10 days, on the antioxidant and immune system of the pacu fish (Piaractus mesopotamicus). Fish fed a diet containing 0.6 mg Se-yeast kg(-1) showed significant improvement in antioxidant status, as well as in hematological and immunological profiles. Specifically, they had the highest counts for catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), red blood cells, and thrombocytes; the highest leukocyte count (particularly for monocytes); and the highest serum lysozyme activity. There was also a positive correlation between GPx and lysozyme in this group of fish. These findings indicate that short-term supplementation with 0.6 mg Se-yeast kg(-1) reestablished the antioxidative status, allowing the production of innate components which can boost immunity without the risk of oxidative stress. This study shows a relationship between oxidative stress and immunity, and, from a practical perspective, shows that improving immunity and health in pacu through the administration of selenium could improve their growth performance.

Aloe vera bathing improved physical and humoral protection in breeding stock after induced spawning in matrinxã (Brycon amazonicus).

In this study, we show that induced spawning causes stress, an intense loss of epithelia and immunosuppression, decreasing physical and humoral protection in fish, effects that were prevented or improved in fish bathed with Aloe vera. A. vera has several medicinal properties, including wound healing and immunostimulatory effects, which we observed in this study. Fish bathed with A. vera had a higher number of epidermal goblet cells and, in general, an improved wound healing rate compared with the control after induced spawning. These effects might be related to (1) the stimulation of leukocyte activity, represented here by the increased leukocyte respiratory activity triggered by A. vera (leukocytes are recognized as playing an important role in wound repair); (2) the antimicrobial properties of A. vera, which decrease wound infection and accelerate the healing process; and (3) several mechanisms that explain the healing effect of A. vera (increased collagen synthesis, rate of epithelialization, and anti-inflammatory and moisturizing effects). Our results also suggest that caution is necessary during the induced spawning process, especially during stripping, and A. vera bathing is recommended after intensive aquaculture operations.

Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus argenteus from a polluted river in southeast Brazil.

The fish gill is in direct and standing contact with the immediate external environment and, therefore, is highly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two different points in São Francisco river. The first point is located near Três Marias dam, while the second is placed downstream the Abaeté river. Chemical approaches showed the presence of metals contamination in the first point. Thus, the main goal of this study was to investigate the possible toxic effects of these contaminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish were obtained in order to calculate the condition factor as an organismal biomarker. The histological changes in gills and alterations in mucous and rodlet cells occurrence were detected microscopically and evaluated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were also assessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals at the two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cell hyperplasia, lamellar fusion, lamellar edema and inflammatory foci varied according to the point. Additionally, mucous and rodlet cells and MPO and EPO activities showed variability according to the environmental conditions. Furthermore, with exception of lamellar hyperplasia and eosinophil peroxidase activity, all others parameters showed sex-variation responses. At the first point, male fish showed a chronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence of inflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an opposite pattern of response to the same environmental conditions. Therefore, we suggest the use of such biomarkers in future monitoring of aquatic systems, taking into account the sex-variation responses.

Generation of reactive oxygen species by leukocytes of Prochilodus lineatus.

Prochilodus lineatus (curimbatá), from the Procholodontidae family, is a Brazilian freshwater fish, which is important commercially, nutritionally and ecologically. It is encountered in the Rio da Prata Bay in Southern South America. Studies on the immune system of this fish are scarce, but the physiological mechanisms of the species are analogous to those of other vertebrates. Thus, this work discusses the present study, which correlates P. lineatus leukocytes and the generation of reactive oxygen species after modulatory stimuli. Leukocytes were characterized by light and electron transmission microscopy and investigated by the generation of H2O2 and O2 (-), using phenol red, flow-cytometry and electron transmission histochemistry. The study determined that monocytes and neutrophils are the main cells responsible for generating O2 after stimulation with phorbol myristate acetate. Superoxide dismutase successfully inhibited the generation of reactive oxygen species in neutrophils and monocytes, but stimulated generation when in association with phorbol myristate acetate. Fish leukocyte samples from P. lineatus showed cross-reactivity with antibodies directed against human NADPH-oxidase antibody subunits (p47(phox) and p67(phox)). Thus, catalase enhanced the presence of p47(phox). Neutrophil mitochondria were shown to be generators of H2O2 (charged by cerium precipitate), being enlarged and changing their format. The present study contributes to a better understanding of the respiratory burst pathways in this species and suggests mitochondria as the organelle responsible for generation of reactive oxygen species.