Eye fluke infection changes diet composition in juvenile European perch (Perca fluviatilis).

Intraspecific diet specialization, usually driven by resource availability, competition and predation, is common in natural populations. However, the role of parasites on diet specialization of their hosts has rarely been studied. Eye flukes can impair vision ability of their hosts and have been associated with alterations of fish feeding behavior. Here it was assessed whether European perch (Perca fluviatilis) alter their diet composition as a consequence of infection with eye flukes. Young-of-the-year (YOY) perch from temperate Lake Müggelsee (Berlin, Germany) were sampled in two years, eye flukes counted and fish diet was evaluated using both stomach content and stable isotope analyses. Perch diet was dominated by zooplankton and benthic macroinvertebrates. Both methods indicated that with increasing eye fluke infection intensity fish had a more selective diet, feeding mainly on the benthic macroinvertebrate Dikerogammarus villosus, while less intensively infected fish appeared to be generalist feeders showing no preference for any particular prey type. Our results show that infection with eye flukes can indirectly affect interaction of the host with lower trophic levels by altering the diet composition and highlight the underestimated role of parasites in food web studies.

mRNA expression of antioxidant and biotransformation enzymes in zebrafish (Danio rerio) embryos after exposure to the tricyclic antidepressant amitriptyline.

Now-a-days, the occurrence of antidepressant residues in surface waters has become a major concern. Amitriptyline (AMI) has been described to treat depression and other disorders for decades. However, little is known about its effect on non-target organisms. The aim of this study was to assess the potential impact of AMI on the mRNA expression of antioxidant and detoxification enzymes during the early embryonic development of zebrafish (Danio rerio). Fertilized D. rerio embryos were exposed to AMI at concentrations of 300 ng/L and 30 µg/L and sampled 24, 48, 96, and 144 h post fertilization (hpf) to assess the mRNA expressions of cytochrome P450 1A1, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, and catalase. The time courses of the mRNA expressions of antioxidant and detoxification enzymes revealed characteristic changes during embryonic development causing generally transient changes post hatching; however, AMI did not cause any significant impact except in the case of CAT after 144 h, which was significantly upregulated by the AMI concentration of 30 µg/L. The results suggest that the antidepressant AMI causes only moderate to minor impacts on antioxidant and detoxification enzymes during early embryonic development of the non-target organism D. rerio and that CAT is the only biomarker affected by AMI.

Endocrine disruption by environmental gestagens in amphibians - A short review supported by new in vitro data using gonads of Xenopus laevis.

Endocrine disruption caused by various anthropogenic compounds is of persisting concern, especially for aquatic wildlife, because surface waters are the main sink of these so-called endocrine disruptors (ED). In the past, research focused on (anti)estrogenic, (anti)androgenic, and (anti)thyroidal substances, affecting primarily reproduction and development in vertebrates; however, other endocrine systems might be also targeted by ED. Environmental gestagens, including natural progestogens (e.g. progesterone (P4)) and synthetic progestins used for contraception, are supposed to affect vertebrate reproduction via progesterone receptors. In the present paper, we review the current knowledge about gestagenic effects in amphibians, focussing on reproduction and the thyroid system. In addition, we support the literature data with results of recent in vitro experiments, demonstrating direct impacts of the gestagens levonorgestrel (LNG) and P4 on sexually differentiated gonads of larval Xenopus laevis. The results showed a higher susceptibility of female over male gonads to gestagenic ED. Only in female gonads LNG, but not P4, had direct inhibitory effects on gene expression of steroidogenic acute regulatory protein and P450 side chain cleavage enzyme, whereas aromatase expression decreased in reaction to both gestagens. Surprisingly, beyond the expected ED effects of gestagens on reproductive physiology in amphibians, LNG drastically disrupted the thyroid system, which resembles direct effects on thyroid glands and pituitary along the pituitary-thyroid axis disturbing metamorphic development. In amphibians, environmental gestagens not only affect the reproductive system but at least LNG can impact also development by disruption of the thyroid system.

The synthetic gestagen levonorgestrel directly affects gene expression in thyroid and pituitary glands of Xenopus laevis tadpoles.

The synthetic gestagen levonorgestrel (LNG) was previously shown to perturb thyroid hormone-dependent metamorphosis in Xenopus laevis. However, so far the mechanisms underlying the anti-metamorphic effects of LNG remained unknown. Therefore, a series of in vivo and ex vivo experiments was performed to identify potential target sites of LNG action along the pituitary-thyroid axis of X. laevis tadpoles. Prometamorphic tadpoles were treated in vivo with LNG (0.01-10nM) for 72h and brain-pituitary and thyroid tissue was analyzed for marker gene expression. While no treatment-related changes were observed in brain-pituitary tissue, LNG treatment readily affected thyroidal gene expression in tadpoles including decreased slc5a5 and iyd mRNA expression and a strong induction of dio2 and dio3 expression. When using an ex vivo organ explant culture approach, direct effects of LNG on both pituitary and thyroid gland gene expression were detecTable Specifically, treatment of pituitary explants with 10nM LNG strongly stimulated dio2 expression and concurrently suppressed tshb expression. In thyroid glands, ex vivo LNG treatment induced dio2 and dio3 mRNA expression in a thyrotropin-independent manner. When thyroid explants were cultured in thyrotropin-containing media, LNG caused similar gene expression changes as seen after 72h in vivo treatment including a very strong repression of thyrotropin-induced slc5a5 expression. Concerning the anti-thyroidal activity of LNG as seen under in vivo conditions, our ex vivo data provide clear evidence that LNG directly affects expression of genes important for thyroidal iodide handling as well as genes involved in negative feedback regulation of pituitary tshb expression.

Effects of the pharmaceuticals diclofenac and metoprolol on gene expression levels of enzymes of biotransformation, excretion pathways and estrogenicity in primary hepatocytes of Nile tilapia (Oreochromis niloticus).

The expression levels of key enzymes of the xenobiotic metabolism and excretion pathways concerning biotransformation phases I (cytochrome P4501A), II (glutathione S-transferase) and III (multidrug resistance protein) and of the estrogenic biomarker vitellogenin (vtg) were investigated in primary hepatocytes isolated from male Nile tilapia (Oreochromis niloticus) after exposure to diclofenac and metoprolol, two pharmaceuticals prevalent in the aquatic environment worldwide. The lowest test concentration (4×10(-9) M) was chosen to reflect an environmentally relevant exposure situation. Furthermore concentration dependent effects were investigated. Therefore a series of concentrations higher than the environmentally relevant range were used (10- and 100-fold). Diclofenac significantly induced all chosen biomarkers already at the environmentally relevant concentration indicating that biotransformation and elimination occur via the pathways under investigation. Estrogenic potential of this substance was demonstrated by VTG up-regulation as well. Metoprolol was either less effective than diclofenac or metabolized using different pathways. Key enzymes of the xenobiotic metabolism were less (CYP1A, GST) or not (MDRP) induced and a mild increase in vtg mRNA was detected only for 4×10(-8) M. No concentration-dependency for metoprolol was found.

Biochemical and histopathological responses of Wistar rats to oral intake of microcystins and cyanobacterial biomass.

OBJECTIVES: Cyanobacteria are producers of potent and environmentally abundant microcystins, representing an emerging global health issue. In the present study, we investigated the impact of pure microcystins and cyanobacterial biomass on laboratory rats (Wistar albino rats, males, 30 days old) under different exposure scenarios. METHODS: The rats were fed diets containing fish meat with microcystins in various concentrations and forms (cyanobacterial biomass and isolated microcystins) for 28 days. RESULTS: Although considerable amounts of microcystins (MCs) were administered to the rats, all levels of MCs in the liver were close to the detection limit (3-5 ng/g fresh weight) using liquid chromatography - tandem mass spectrometry. Only rats exposed to cyanobacterial biomass had clearly higher hepatic and splenic somatic indexes while markers of oxidative stress (glutathione-S-transferase, glutathione reductase, lipid peroxidatio) were significantly increased in the group exposed to the high dose of MCs. Most of the analysed biochemical parameters did not show clear differences among groups. Levels of bilirubin and lipases were significantly increased only after exposure to cyanobacterial biomass and MCs, respectively. Considering microscopic findings in the liver, kidney, thymus, spleen and brain, histopathology was dominated by alterations in the hepatic parenchyma and renal cortical tubular system. CONCLUSIONS: The present study demonstrates that oral exposure to MCs and cyanobacterial biomass may induce biochemical and detoxification responses associated with damage to liver and kidneys and in the laboratory rat.

Concentrations of microcystins in tissues of several fish species from freshwater reservoirs and ponds.

The aim of this study is to summarise the determination of concentrations of microcystins (MCs) in muscle and liver of freshwater fish species caught in stagnant waters of the Czech Republic. Within the years 2007-2009, 351 muscle samples and 291 liver samples of 16 freshwater fish species derived from four fishponds, and four water reservoirs were analysed. MCs were detected in 53 liver samples. The highest concentrations of microcystins were determined in liver samples of carnivorous fish species; 50.3 ng/g of fresh weight (FW) in perch (Perca fluviatilis) and 22.7 ng/g FW in pikeperch (Sander lucioperca). MCs in liver were detected in other five fish species; asp (Aspius aspius), pike (Esox lucius), common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella) and European eel (Anguilla anguilla). Concentrations of MCs in liver of nine fish species (European bream, whitefish, tench, silver carp, European catfish, roach, chub, crucian carp and rudd) were below the detection limit of 1.2-5.4 ng/g FW for different MC congeners. However, the concentrations of MCs in all muscle samples were below the detection limit. The assessment of MCs concentrations might be influenced by the detection method used. Due to the concentrations of MCs being below the detection limit in muscle samples of all fish species analysed, it seems that there might be a low potential threat for human health in case of fish muscle consumption.

Physiological responses of Xenopus laevis tadpoles exposed to cyanobacterial biomass containing microcystin-LR.

Cyanobacteria are the primary biomass producers and some species synthesize remarkable amounts of secondary metabolites, the so-called cyanotoxins. Several reports deal with the most common cyanotoxins, microcystins (MCs), and their effects on fishes but only a few studies investigated a natural exposure to MCs and limited information is available concerning the further aquatic vertebrate class, amphibians. In the present study, Xenopus laevis tadpoles at stage 52 (Nieuwkoop and Faber, 1994) were exposed for 1, 3, 7, and 21 days to diets containing lyophilized cyanobacterial biomass without and with microcystin-LR (MC-LR) at concentrations of 42.8 and 187.0 µg MC-LR/g diet, respectively, to determine impacts on MC-LR bioaccumulation, development, stress, and biotransformation. The fate of MC-LR present in diet and water was determined in whole body using liquid chromatography with tandem mass spectrometry detection. Effects on development were assessed by recording mortality, weight and developmental stage. In parallel, mRNA levels of hypophyseal thyroid stimulating hormone (TSH) associated with metamorphosis and of gonadotropins, luteinizing hormone and follicle stimulating hormone, triggering sexual differentiation, were assessed. Concerning stress, corticosteroid levels and mRNA expression of heat shock protein 70 (HSP70) as stress biomarkers were examined. Furthermore, mRNA expression of biotransformation enzymes of all three phases as well as biomarkers for oxidative stress were determined. Surprisingly, exposure to cyanobacterial biomass containing MC-LR supplied via diet as natural exposure neither resulted in measurable bioaccumulation of MC-LR nor affected dramatically development. Only minor to negligible physiological impacts on development, stress, and biotransformation mechanisms were found suggesting that X. laevis tadpoles seem to have some mechanisms to be able to cope quite well with diets containing lyophilized cyanobacterial biomass even with considerable amounts of MC-LR.

The effect of peroral administration of toxic cyanobacteria on laboratory rats (Rattus norvegicus var. alba).

OBJECTIVES: The toxic cyanobacteria are a serious problem for water supply systems, recreation, and agriculture. Cyanobacteria produce numerous bioactive compounds including microcystins - the most studied cyanobacterial hepatotoxins. Only rare studies addressed realistic situation, i.e. impact of MCs accumulated in the fish tissues on the overall physiology. The aim of the present study was to provide a model simulation of the simple food chain for evaluation of impacts of cyanobacteria on the rat physiology under different exposure scenario. METHODS: Experimental rats were fed with food with fish meat, which contained external additions of isolated microcystins as well as toxic cyanobacteria Microcystis, nontoxic cyanobacteria Arthrospira and green alga Chlorella. Subgroups of the animals were also challenged with a model antigen KLH to investigated immune-related parameters. We studied parameters of oxidative stress in the liver as levels of lipid peroxidation and glutathion levels. Series of hematological, biochemical and immunological parameters were also investigated. RESULTS: Although considerable amounts of microcystins were administered to rats, all levels of MCs were under the detection limit (1 ng/g fresh weight) in the rat tissues using tandem LC/MS. Only some conjugates of microcystins with cystein and glutathion were detected in the rat liver exposed to Microcystis biomass (values were around the detection limit). Statistically significant depletion of body and liver weight was observed in groups with microcystin addition in comparison with all other groups. Rats exposed to MCs had stimulated immune system (showed higher antibody answer on administered antigen). Also modulation of some lymphocyte subpopulations was recorded with the most interesting observation of stimulated NK cell numbers in groups exposed to isolated toxins (but not to biomass containing the same toxin amount). CONCLUSIONS: Our study demonstrates that oral exposure to microcystins in the diet may induce some detoxification responses and modulation of some hematological and immunological parameters.

Impact of microcystin containing diets on physiological performance of Nile tilapia (Oreochromis niloticus) concerning detoxification.

Nile tilapia (Oreochromis niloticus) were fed by diets supplemented with cyanobacteria containing in part the cyanotoxin microcystin-LR (MC-LR) to determine the potential impacts on detoxification. Four different diets were prepared based on a commercial diet: (1) control, (2) MC-5% (containing 5% dried Microcystis sp. biomass with 4.92 µg MC-LR g(-1) diet), (3) MC-20% (containing 20% dried Microcystis sp. biomass with 19.54 µg MC-LR g(-1) diet), and (4) Arthr-20% (containing 20% dried Arthrospira sp. biomass without MC-LR). Blood and liver samples were taken after one, 7, and 28 days and protein has been determined in plasma and liver. In the liver, impacts on detoxification were measured by glutathione-S-transferase (GST) activities and gene expression of multi drug resistance protein (MDRP). Plasma protein did not change between all four diets at any sampling time whereas liver protein was significantly elevated already after one day in Arthr-20% and after 28 days in both, MC-20% and Arthr-20%. Biochemical measurements of GST activities revealed no significant impact at any sampling time. In order to characterize the potential effect of MC-LR on MDRP, RT-qPCR method was established. However, as for GST activities no significant changes in MDRP gene expression have been observed. Thus, in summary, oral exposure of MC-LR containing cyanobacteria to Nile tilapia via feed ingestion did not impact significantly detoxification in liver concerning GST activities and MDRP expression despite biochemical composition concerning liver protein was significantly elevated by the diets containing 20% cyanobacteria biomass, regardless whether they contained MC-LR or not.

Impact of microcystin containing diets on physiological performance of Nile tilapia (Oreochromis niloticus) concerning stress and growth.

Diets containing Microcystis with considerable amounts of the cyanotoxin microcystin-LR (MC-LR) were fed to determine their impact on the physiological performance of the omnivorous Nile tilapia (Oreochromis niloticus) with regard to stress and growth performance. Four different diets were prepared based on a commercial diet (control, MC-5% [containing 5% dried Microcystis biomass], MC-20% [containing 20% dried Microcystis biomass], and Arthrospira-20% [containing 20% dried Arthrospira sp. biomass without toxin]) and fed to female Nile tilapia. Blood and tissue samples were taken after 1, 7, and 28 d, and MC-LR was quantified in gills, muscle, and liver by using high-performance liquid chromatography (HPLC). Only in the liver were moderate concentrations of MC-LR detected. The stress hormone cortisol and glucose were analyzed from plasma, suggesting that all modified diets caused only minor to moderate stress, which was confirmed by analyses of hepatic glycogen. In addition, the effects of the different diets on growth performance were investigated by determining gene expression of hypophyseal growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I). For all diets, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) demonstrated no significant effect on gene expression of the major endocrine hormones of the growth axis, whereas classical growth data, including growth and feed conversion ratio, displayed slight inhibitory effects of all modified diets independent of their MC-LR content. However, no significant change was found in condition or hepatosomatic index among the various diets, so it seems feasible that dried cyanobacterial biomass might be even used as a component in fish diet for Nile tilapia, which requires further research in more detail.