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The aim of this study was to assess the impact of treated wastewaters on native wild Prussian carp inhabiting effluent-receiving waters (ERC) receiving municipal and sugar plant treated wastewaters, further downstream waters (DW), and a detached canal unaffected by the WWTP activities. To that end, general fish health status was determined, including plasma biochemical, haematological, oxidative stress and tissue histopathological indices, over three seasons. The greatest tissue alterations were in fall in ERC during sugar beet processing, as hypertrophy of gill epithelial and interlamellar cells, necrosis and lymphocytic infiltration, hyperplasia and hypertrophy of renal tubules, distention of hepatic sinusoids. In fall the lowest leukocytes, lymphocytes and granulocytes (2467 ± 565, 1333 ± 264, 1133 ± 488 cells/μL respectively), as well as highest plasma ALP (52.7 ± 19.39 U/L) were measured. ERC in fall had the highest ammonium (20 mg/L), nitrite (1.48 mg/L), nitrate (13.4 mg/L), and lowest dissolved O 2 (1.23 mg/L). Gill, kidney and liver alterations, and the highest plasma cholesterol (9.1 ± 1.98 mmol/L) were noted in DW fish in fall. Tissue morphology during sugar cane processing seems a consequence of cellular and structural tissue integrity loss. Structural heterogeneity of gills and spleen was enhanced with increasing concentrations of heavy metals and correlated with oxidative stress (SOD 392.5 ± 77.28 U/L). Monogenean infestation was moderate in ERC fish in all seasons compared with DW fish. Prussian carp biological responses to multiple stressors, measured by the effects of WWTP on blood and tissue parameters, reached far downstream and were not of localized nature. This study demonstrated that in aquatic environments impacted with complex contaminants acting synergistically, causal relationships between biological responses and environmental stressors should be interpreted. Integrated histopathological, haematological and biochemical findings are valuable biomarkers for native fish adaptive patterns and monitoring of water quality/pollution of freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Nano-TiO 2 is immunotoxic to fish and reduces the bactericidal function of fish neutrophils. Here, fathead minnows ( Pimephales promelas ) were exposed to low and high environmentally relevant concentration of nano-TiO 2 (2 ng g −1 and 10 μg g −1 body weight, respectively), and were challenged with common fish bacterial pathogens, Aeromonas hydrophila or Edwardsiella ictaluri . Pre-exposure to nano-TiO 2 significantly increased fish mortality during bacterial challenge. Nano-TiO 2 concentrated in the kidney and spleen. Phagocytosis assay demonstrated that nano-TiO 2 has the ability to diminish neutrophil phagocytosis of A. hydrophila . Fish injected with TiO 2 nanoparticles displayed significant histopathology when compared to control fish. The interplay between nanoparticle exposure, immune system, histopathology, and infectious disease pathogenesis in any animal model has not been described before. By modulating fish immune responses and interfering with resistance to bacterial pathogens, manufactured nano-TiO 2 has the potential to affect fish survival in a disease outbreak. [ABSTRACT FROM AUTHOR]

Abstract: Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6′-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases. [Copyright &y& Elsevier]

Abstract: Stimulatory effects of yeast β-1,3–1,6-glucans on neutrophils have long been recognized, but effects of glucans on degranulation of primary granules in fish neutrophils have not been previously reported. Neutrophil function was monitored during in vitro and in vivo application of glucans to non- (NS), acute- (AS) and chronically stressed (CS) fish. β-Glucan proved to be a strong and quick (80%, 2min) stimulant of degranulation. Dietary glucan increased degranulation in NS fish, and prevented a decrease in AS fish. Degranulation in CS fish returned to NS levels 3 days after the glucan diet was fed. Fathead minnows appear to be a useful model to investigate neutrophil degranulation in fish exposed to different environmental conditions and immunomodulators. Use of β-glucans in fish diets prior to AS and during chronic stress can enhance neutrophil function, potentially increasing disease resistance and survival rates after transportation or exposure to poor water quality. [Copyright &y& Elsevier]

The release of nanoparticles to the environment can affect health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers, however the information about use of microRNA in aquatic organisms exposed to nanoparticles (NP) is limited. In silico analysis from publicly available gene expression data was performed. Data selection for the analysis was based on reported biological and pathological outcomes of NP induced toxicity in zebrafish. After identifying relevant genes, we constructed six miRNA-mRNA regulatory networks involved in nanoparticle induced toxicological responses in zebrafish. Based on our prediction and selection criteria we selected six miRNAs that overlapped in constructed networks with remarkable prediction score, and were validated by previous mammalian and zebrafish microRNA profiling studies: dre-miR-124, −144, −148, −155, −19a, −223. The results of this in silico analysis indicate that several highly conserved miRNAs likely have a regulatory role of organismal responses to nanoparticles, and can possibly be used as biomarkers of nanotoxicity in studies using zebrafish as model organism One health approaches. • Six miRNA-mRNA regulation networks after nanoparticle exposure in zebrafish were depicted • Six top predicted miRNAs were selected: dre-miR-124, −144, −148, −155, −19a, −223 • This novel method of identification of validation candidates can assist the delineation of miRNA-mRNA functional networks and support identification of biomarkers of potential nanoparticle toxicity. [ABSTRACT FROM AUTHOR]