Multiple faces of stress in the zebrafish (Danio rerio) brain.

The changing expressions of certain genes as a consequence of exposure to stressors has not been studied in detail in the fish brain. Therefore, a stress trial with zebrafish was conducted, aiming at identifying relevant gene regulation pathways in different regions of the brain. As acute stressors within this trial, feed rewarding, feed restriction, and air exposure have been used. The gene expression data from the experimental fish brains have been analyzed by means of principal component analyses (PCAs), whereby the individual genes have been compiled according to the regulation pathways in the brain. The results did not indicate a mutual response across the treatment and gender groups. To evaluate whether a similar sample structure belonging to a large sample size would have allowed the classification of the gene expression patterns according to the treatments, the data have been bootstrapped and used for building random forest models. These revealed a high accuracy of the classifications, but different genes in the female and male zebrafish were found to have contributed to the classification algorithms the most. These analyses showed that less than eight genes are, in most cases, sufficient for an accurate classification. Moreover, mainly genes belonging to the stress axis, to the isotocin regulation pathways, or to the serotonergic pathways had the strongest influence on the outcome of the classification models.

Effects of different acute stressors on the regulation of appetite genes in the carp (Cyprinus carpio L.) brain.

Our understanding of the timing of stress responses and specific roles of different regulatory pathways that drive stress responses is incomplete. In particular, the regulation of appetite genes as a consequence of exposure to different stressors has not been studied in sufficient detail in fish. Therefore, a stress trial was conducted with koi carp, aiming at identifying typical effects of stress on regulation of appetite genes. The stressors tank manipulation, air exposure and feed rewarding were chosen. The responses to these stressors were evaluated 10, 30 and 60 min after the stressors were applied. Orexigenic and anorexigenic genes were investigated in four different brain regions (telencephalon, hypothalamus, optic tectum and rhombencephalon). The results show that, apart from the typical appetite regulation in the hypothalamus, the different brain regions also display pronounced responses of appetite genes to the different stressors. In addition, several genes in the serotonergic, dopaminergic and gaba-related pathways were investigated. These genes revealed that rearing in pairs of two and opening of the tank lid affected anorexigenic genes, such as cart and cck, which were not changed by air exposure or feed rewarding. Moreover, distress and eustress led to limited, but distinguishable gene expression pattern changes in the investigated brain regions.

The Occurrence of Mycotoxins in Raw Materials and Fish Feeds in Europe and the Potential Effects of Deoxynivalenol (DON) on the Health and Growth of Farmed Fish Species-A Review.

The first part of this study evaluates the occurrence of mycotoxin patterns in feedstuffs and fish feeds. Results were extrapolated from a large data pool derived from wheat (n = 857), corn (n = 725), soybean meal (n = 139) and fish feed (n = 44) samples in European countries and based on sample analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the period between 2012-2019. Deoxynivalenol (DON) was readily present in corn (in 47% of the samples) > wheat (41%) > soybean meal (11%), and in aquafeeds (48%). Co-occurrence of mycotoxins was frequently observed in feedstuffs and aquafeed samples. For example, in corn, multi-mycotoxin occurrence was investigated by Spearman's correlations and odd ratios, and both showed co-occurrence of DON with its acetylated forms (3-AcDON, 15-AcDON) as well as with zearalenone (ZEN). The second part of this study summarizes the existing knowledge on the effects of DON on farmed fish species and evaluates the risk of DON exposure in fish, based on data from in vivo studies. A meta-analytical approach aimed to estimate to which extent DON affects feed intake and growth performance in fish. Corn was identified as the ingredient with the highest risk of contamination with DON and its acetylated forms, which often cannot be detected by commonly used rapid detection methods in feed mills. Periodical state-of-the-art mycotoxin analyses are essential to detect the full spectrum of mycotoxins in fish feeds aimed to prevent detrimental effects on farmed fish and subsequent economic losses for fish farmers. Because levels below the stated regulatory limits can reduce feed intake and growth performance, our results show that the risk of DON contamination is underestimated in the aquaculture industry.

Distress Regulates Different Pathways in the Brain of Common Carp: A Preliminary Study.

In this study, a stress trial was conducted with common carp, one of the most important species in aquaculture worldwide, to identify relevant gene regulation pathways in different areas of the brain. Acute distress due to exposure to air significantly activated the expression of the immediate early gene c-fos in the telencephalon. In addition, evidence for regulation of the two corticotropin-releasing factor (crf) genes in relation to their binding protein (corticotropin-releasing hormone-binding protein, crh-bp) is presented in this preliminary study. Inferences on the effects of due to exposure to air were obtained by using point estimation, which allows the prediction of a single value. This constitutes the best description to date of the previously generally unknown effects of stress in different brain regions in carp. Furthermore, principal component analyses were performed to reveal possible regulation patterns in the different regions of the fish brain. In conclusion, these preliminary studies on gene regulation in the carp brain that has been influenced by exposure to a stressor reveal that a number of genes may be successfully used as markers for exposure to unfavourable conditions.

Preliminary investigation of an idiopathic muscle disease in farmed burbot Lota lota.

The rearing of less established fish species in recirculating aquaculture systems (RASs) is increasing, but may require adaptations of the rearing facilities if health impairments occur. We observed several health issues in burbot Lota lota reared for up to 2 yr in a RAS and used microbiological, histological and molecular-biological methods to identify the causative agents. Minor skin trauma led to the development of ulcers. In addition, several fillets of burbot showed pronounced granulomatous inflammation and calcification with signs of muscle fiber degeneration which resembled a condition called 'sandy flesh disease' in North American walleye. Several infectious agents were able to be excluded as a cause for the disease. Carnobacterium maltaromaticum was isolated in high numbers in some of the affected muscle tissue. However, the role of this bacterium or other causative agents or husbandry conditions remains to be elucidated.

Occurrence of Fungi and Fungal Toxins in Fish Feed During Storage.

Periods of unfavorable storing conditions can lead to changes in the quality of fish feeds, as well as the development of relevant mycotoxins. In the present study, a commercial fish feed was stored under defined conditions for four weeks. The main findings indicate that even storing fish feeds under unsuitable conditions for a short duration leads to a deterioration in quality. Mycotoxin and fungal contamination were subsequently analyzed. These investigations confirmed that different storage conditions can influence the presence of fungi and mycotoxins on fish feed. Notably, ochratoxin A (OTA) was found in samples after warm (25 °C) and humid (>60% relative humidity) treatment. This confirms the importance of this compound as a typical contaminant of fish feed and reveals how fast this mycotoxin can be formed in fish feed during storage.

Risk assessment for mycotoxin contamination in fish feeds in Europe.

Mycotoxins are difficult to monitor continuously, and a tool to assess the risk would help to judge if there is a particular risk due to the inclusion of certain feed ingredients. For this, the toxin contents of 97 commercial fish feeds have been estimated, and the most prominent toxins in fish feed are calculated to be deoxynivalenol, zearalenone, fumonisins and enniatins. These pose a risk to fish well-being, as can be calculated by the Bayesian models for determining the critical concentrations 5% (CC5) for the different toxins. Besides fishmeal, wheat, soybean products and corn are regularly used as fish feed ingredients. The calculated scenarios show that fish are at high risk of toxin contamination if feed ingredients of low quality are chosen for feed production. Due to this, specific maximum allowable levels for several mycotoxins in fish feeds should be established.

Ethoxyquin: a feed additive that poses a risk for aquatic life.

Ethoxyquin (EQ) is an antioxidant that has, to date, been commonly used in feed production. Reports on the detrimental effects of this substance on vertebrates are growing, but effects in aquatic systems have rarely been described. Therefore, the present study was conducted using serial concentrations of EQ ranging from 0.03 to 16.5 mg l-1 to determine effects on 3 types of aquatic organisms. In zebrafish, 5 mg l-1 EQ caused mortality (25%) and a further 62.5% of the embryos showed yolk sac edema as well as deformed bodies or missing eyes. Furthermore, all the investigated EQ concentrations decreased the heart rate of the embryos. The lowest observed effect level was 0.31 mg l-1. In addition to zebrafish, the study also used water fleas Daphnia magna and green algae (Scenedesmus obliquus and Chlorella vulgaris). These treatments revealed that daphnids are also sensitive to EQ, exhibiting detrimental effects with a half-maximal effective concentration (EC50) of 2.65 mg l-1 after 48 h of exposure. The algae appeared to be at least 2 times less sensitive to EQ than fish embryos or daphnids. The results were used to calculate the risk for aquatic life resulting in a maximum tolerable level of 1 µg l-1 for fish embryos and daphnids, with a safety factor of 300. According to current knowledge, this does not exceed environmental concentrations of this substance. However, this study raises further concern about the (until recently) legal maximum tolerable EQ levels in fish feeding and the rather slow pace at which authorization to use EQ as a feed additive for diverse animals in Europe is being suspended.

Toxicity of Ochratoxin to Early Life Stages of Zebrafish (Danio rerio).

Ochratoxin A (OTA) is a known contaminant in fish feed but its effect on fish health remains rather unknown. A study was conducted to investigate the effects of different concentrations of ochratoxin on early life stages of zebrafish (Danio rerio). The tests with ochratoxin A showed a correlation between the exposure to mycotoxin and the amount of damage. The mortality rate and the incidents of embryonal damage was increased by increasing ochratoxin concentrations. The calculations resulted in a lethal concentration for 50% of the embryos (LC50) of 0.29 mg/L and a concentration at which 50% of the animals showed impairment (EC50) of 0.36 mg/L after 96 h of exposure. During the test, reduced heart rates were also observed revealing a clear dose-response relationship. The EC50 determination for this endpoint was 1.26 mg/L after 72 h of exposure. The measurement of oxidative stress was proven to be the most sensitive system to indicate OTA effects on the zebrafish embryos with an EC50 value of 0.067 mg/L after 72 h of exposure. The test validity was given because the control test with 3,4-Dichloroaniline (3,4-D) showed a LC50 value of 2.88 mg after 96 h of exposure which is comparable to the available reference values. According to the current knowledge, these experimental doses did not exceed the environmental concentrations of this ochratoxin A. However, this study raises concerns about the effects of ochratoxin on fish.

Zearalenone (ZEN) and Its Influence on Regulation of Gene Expression in Carp (Cyprinus carpio L.) Liver Tissue.

Zearalenone (ZEN) is a frequently-occurring mycotoxin in both animal and fish feeds. In order to characterize its effects on carp, three groups of fish were fed for 28 days with feeds contaminated with three different levels of ZEN (low: 332 µg kg-1, medium: 621 µg kg-1, and high: 797 µg kg-1 feed). The reversibility of the effects of ZEN was assessed by feeding all of the groups with uncontaminated feed for a further 14 days. Gene expression of immune genes in the liver tissue of the fish was analysed, revealing reduced expressions of immune, antioxidative, and estrogen-related genes after the fish had been exposed to ZEN. However, the expression of vacuole-type H⁺ ATPase increased substantially with ZEN exposure, thus supporting the previously-reported sensitivity of lysosomal functions to ZEN. Feeding the fish with a ZEN-free diet for a further two weeks changed the effects of ZEN on the expression of some genes, including the expressions of the cytokines IL-1ß, IL-8, IL-10, and arginase 2, which were not influenced after four weeks of treatment, but showed lower values after the recovery phase in fish previously treated with ZEN compared with the control group. In summary, this study confirmed the broad effects of ZEN on different essential functions in carp and suggests that the current maximum allowable levels in compound feed are too high to prevent damage to fish.

Physiological responses of carp (Cyprinus carpio L.) to dietary exposure to zearalenone (ZEN).

Zearalenone (ZEN) is a frequent contaminant of animal feeds, but systemic effects on fish and possible metabolic costs have not yet been investigated. In order to fill this gap a feeding trial with juvenile carp was conducted. The fish were fed ZEN-contaminated diets at three concentrations (low: 332µgkg(-1), medium: 621µgkg(-1), and high: 797µgkg(-1) final feed, respectively) for four weeks. Possible reversible effects of ZEN were evaluated by feeding an additional group with the mycotoxin for four weeks period and the uncontaminated diet for further two weeks. After that possible ZEN effects on enzyme activities in kidney, spleen, liver and muscle tissue were investigated to get an organism-wide aspect of ZEN effects. Most organs appeared to (over)compensate ZEN effects during the exposure to this mycotoxin, which caused metabolic costs. Oxygen consumption increased in fish treated with the two higher ZEN concentrations via the diet. The differences between the treatments persisted also after the recovery phase of two weeks. Thus, the present study provided evidence of effects of ZEN on carbohydrate metabolism, lipid peroxidation in organs and metabolic oxygen demand. This is the first evidence for increased metabolic costs in a fish species due to exposure to the mycotoxin ZEN.

Immunomodulation by Zearalenone in Carp (Cyprinus carpio L.).

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feeds, but its effects on fish have not yet been investigated extensively. In order to fill this gap a feeding trial with juvenile carp was conducted. Three groups of fish were fed feeds contaminated with ZEN at three concentrations (low: 332 µg kg(-1), medium: 621 µg kg(-1), and high: 797 µg kg(-1) feed) for four weeks. Possible reversible effects of ZEN were evaluated by feeding additional groups with the ZEN-contaminated feeds for four weeks, followed by the uncontaminated diet for two weeks. Immune function of isolated leukocytes from head kidney and trunk kidney was assessed using the assessment of NO production, the respiratory burst assay, the chemiluminescence assay, and the measurement of arginase activities. These investigations frequently revealed increased immune responses after exposure of fish to low ZEN concentrations and reduced immune responses after exposure to high mycotoxin concentrations. Moreover, the feeding of the uncontaminated diet for further two weeks did not improve the immune responses in most cases. These results indicate that cellular immune functions in ZEN-contaminated carp are influenced which may be relevant for fish health in aquaculture.

Effects of Dietary Exposure to Zearalenone (ZEN) on Carp (Cyprinus carpio L.).

The mycotoxin zearalenone (ZEN) is frequently contaminating animal feeds including feed used in aquaculture. In the present study, the effects of dietary exposure to ZEN on carp (Cyprinus carpio L.) were investigated. ZEN at three different concentrations (low dose: 332 µg kg(-1), medium dose: 621 µg kg(-1) and high dose: 797 µg kg(-1) final feed, respectively) was administered to juvenile carp for four weeks. Additional groups received the mycotoxin for the same time period but were fed with the uncontaminated diet for two more weeks to examine the reversibility of the ZEN effects. No effects on growth were observed during the feeding trial, but effects on haematological parameters occurred. In addition, an influence on white blood cell counts was noted whereby granulocytes and monocytes were affected in fish treated with the medium and high dose ZEN diet. In muscle samples, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected. Furthermore, the genotoxic potential of ZEN was confirmed by analysing formation of micronuclei in erythrocytes. In contrast to previous reports on other fish species, estrogenic effects measured as vitellogenin concentrations in serum samples were not increased by dietary exposure to ZEN. This is probably due to the fact that ZEN is rapidly metabolized in carp.

Acute and subchronic effects on immune responses of carp (Cyprinus carpio L.) after exposure to deoxynivalenol (DON) in feed.

The mycotoxin deoxynivalenol (DON) has been shown to regularly occur at relevant concentrations in feed designed for aquaculture use, but little is known about the consequences of its presence on the organisms that consume the DON-contaminated feed. Previous studies indicated a down-regulation of pro-inflammatory responses in carp (Cyprinus carpio L.) after 4 weeks of feeding DON. The present study examined the time course of innate immune responses of carp to orally administered DON. Changes in mRNA levels of immune genes in different organs (head kidney, trunk kidney, spleen, liver, and intestine) were observed indicating immune-modulating properties of DON. The immune-modulatory effects during the acute phase of DON exposure were characterized by the activation of both pro- and anti-inflammatory cytokines and enzymes in carp. The subchronic responses to DON were characterized by activation of arginases culminating in increased arginase activity in head kidney leukocytes after 26 days of DON treatment. These results suggest profound effects of this mycotoxin on fish in aquaculture.

Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1)) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1) concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1) concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.

Cytotoxic effects of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCHQ) on liver cells are modulated by antioxidants.

The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.

Unraveling the mechanisms involved in zearalenone-mediated toxicity in permanent fish cell cultures.

The world-wide occurrence of zearalenone (ZEN) as a contaminant in feed for farm animals and fish requires the evaluation of toxicity mechanisms of action of ZEN. The present study investigates possible metabolization of ZEN in fish cell lines suggesting that mainly glucuronidation takes place. It demonstrates that concentrations up to 20,000 ng ml(-1) ZEN are capable of influencing cell viability in permanent fish cell cultures in a dose-response manner with different response patterns between the five tested cell lines, whereby lysosomes appeared to be the main target of ZEN. ZEN toxicity is often discussed in the context of oxidative stress. Our study shows a biphasic response of the cell lines when reactive oxygen species (ROS) production is monitored. Damage in cells was observed by measuring lipid peroxidation, DNA strand breaks, and alterations of intracellular glutathione levels. Metabolization of ZEN, especially at concentrations above 7500 ng ml(-1) ZEN, does not prevent cytotoxicity. ZEN as an estrogenic compound may involve processes mediated by binding to estrogen receptors (ER). Since one cell line showed no detectable expression of ER, an ER-mediated pathway seems to be unlikely in these cells. This confirms a lysosomal pathway as a main target of ZEN in fish cells.

In vivo effects of deoxynivalenol (DON) on innate immune responses of carp (Cyprinus carpio L.).

Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can be found in relevant concentrations in animal feeds. The effects of this group of toxins on fish are mostly unknown. The present study shows results from a feeding trial with carp (Cyprinus carpio L.) using three different concentrations of DON (352µgkg(-1), 619µgkg(-1), and 953µgkg(-1) final feed, respectively) which are comparable to levels found in commercial fish feeds. Effects on growth and mass of fish were not observed during this 6weeks lasting experiment. Only marginal DON concentrations were found in muscle and plasma samples. Blood parameters were not influenced although smaller erythrocytes occurred in fish treated with 352µgkg(-1) DON. Analysis of antioxidative enzymes in erythrocytes showed increased superoxid dismutase and catalase activities in fish fed the low-dose feed. Immunosuppressive effects of DON were confirmed whereby cytotoxic effects on immune cells only partly explained the impairment of innate immune responses. Exact polarization of the immune system into pro-inflammatory or anti-inflammatory responses due to DON exposure should be clarified in further experiments, especially since the current results raise concern about impaired immune function in fish raised in aquaculture.

Organ damage and hepatic lipid accumulation in carp (Cyprinus carpio L.) after feed-borne exposure to the mycotoxin, deoxynivalenol (DON).

Deoxynivalenol (DON) frequently contaminates animal feed, including fish feed used in aquaculture. This study intends to further investigate the effects of DON on carp (Cyprinus carpio L.) at concentrations representative for commercial fish feeds. Experimental feeding with 352, 619 or 953 µg DON kg(-1) feed resulted in unaltered growth performance of fish during six weeks of experimentation, but increased lipid peroxidation was observed in liver, head kidney and spleen after feeding of fish with the highest DON concentration. These effects of DON were mostly reversible by two weeks of feeding the uncontaminated control diet. Histopathological scoring revealed increased liver damage in DON-treated fish, which persisted even after the recovery phase. At the highest DON concentration, significantly more fat, and consequently, increased energy content, was found in whole fish body homogenates. This suggests that DON affects nutrient metabolism in carp. Changes of lactate dehydrogenase (LDH) activity in kidneys and muscle and high lactate levels in serum indicate an effect of DON on anaerobic metabolism. Serum albumin was reduced by feeding the medium and a high dosage of DON, probably due to the ribotoxic action of DON. Thus, the present study provides evidence of the effects of DON on liver function and metabolism.

Occurrence of deoxynivalenol and zearalenone in commercial fish feed: an initial study.

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 µg kg(-1) feed was found in one feed whereas average values of 289 µg kg(-1) feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 µg kg(-1) feed.