Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales: Entomophthoraceae), a new species infecting pear psyllid Cacopsylla pyri L. (Hemiptera: Psyllidae).

The new species Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales) is described. The fungus was found on infected pear psyllids Cacopsylla pyri (Hemiptera: Psyllidae) in a pear orchard in Zealand, Denmark. Morphological structures (conidia, rhizoids, cystidia) were described on the designated type host C. pyri. In addition, conidia from an in vitro culture were described. Pandora cacopsyllae differs from other Pandora species by a) C. pyri is the natural host; b) conidia are different from other Pandora species infecting Psylloidea; c) ITS differs from other Pandora species infecting Hemiptera. The fungus has a high potential for future use in biological control of Cacopsylla pest species as well as other psyllids.

Modification and quantification of in vivo EROD live-imaging with zebrafish (Danio rerio) embryos to detect both induction and inhibition of CYP1A.

The visualization of specific activation of the aryl hydrocarbon receptor (AhR) directly in the zebrafish embryo (Danio rerio) via live-imaging is a reliable tool to investigate the presence of dioxin-like substances in environmental samples. The co-existence of inducers and inhibitors of cytochrome P450-dependent monooxygenases (CYP1A) is typical of complex environmental mixtures and requires modifications of the in vivo EROD assay: For this end, zebrafish embryos were used to evaluate the EROD-modifying potentials of common single-compound exposures as well as binary mixtures with the PAH-type Ah-receptor agonist ß-naphthoflavone. For chemical testing, chlorpyrifos and Aroclor 1254 were selected; ß-naphthoflavone served as maximum EROD induction control. Chlorpyrifos (≤EC10) could be documented to be a strong CYP1A inhibitor causing characteristic edema-related toxicity. Aroclor 1254 resulted in inhibition of CYP1A catalytic activity in a concentration- and specific time-dependent manner. Next to a fast CYP1A induction, CYP1A inhibition could also be detected after 3h short-term exposure of zebrafish embryos to chlorpyrifos. This communication also describes techniques for the quantification of fluorescence signals via densitometry as a basis for subsequent statistical assessment. The co-exposure approach with zebrafish embryos accounts for the nature of potential interaction between CYP1A inducers and inhibitors and thus pays tribute to the complexity of environmental mixtures. The co-exposure EROD live-imaging assay thus facilitates a better understanding of mixture effects and allows a better assessment and interpretation of (embryo) toxic potentials.

In vivo EROD assays with the zebrafish (Danio rerio) as rapid screening tools for the detection of dioxin-like activity.

The present study compares two alternative in vivo approaches for the measurement of ethoxyresorufin-O-deethylase (EROD) activity in zebrafish (Danio rerio) following exposure to acetonic model sediment extracts: (1) the live-imaging EROD assay for the direct detection of EROD induction in individual livers via epifluorescence, and (2) the fish embryo EROD assay in subcellular fractions derived from entire zebrafish embryos after in vivo exposure. For toxicity assessment, each sediment extract was tested with the standard fish embryo test (FET). Upon completion of a functioning liver after 72h, the embryos gave a distinct fluorescent signal in the liver, and a corresponding EROD activity could be detected in the fish embryo EROD assay. The exposure time in the live-imaging EROD assay was reduced to 3h, which resulted in a stronger, less variable and more sensitive EROD response. Overall, the live-imaging and the fish embryo EROD assays showed the same tendencies and gave comparable results, e.g. a concentration-dependent increase in EROD activity at concentrations one order of magnitude below concentrations producing macroscopically visible abnormalities. At higher concentrations, however, a decrease of EROD activity was observed in either test. Both tests ranked the three model sediment extracts in the same order. Results indicate that both test systems complement each other and together provide a rapid and reliable in vivo tool to investigate the presence of dioxin-like substances in environmental samples.

Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos?

Earlier studies have shown that perfluorooctane sulfonate (PFOS) increases the toxicity of other chemicals by enhancing their uptake by cells and tissues. The present study aimed at testing whether the underlying mechanism of enhanced uptake of chemicals by zebrafish (Danio rerio) embryos in the presence of PFOS is by interference of this compound with the cellular efflux transporter Abcb4. Modifications of uptake/clearance and toxicity of two Abcb4 substrates, the fluorescent dye rhodamine B (RhB) and vinblastine, by PFOS were evaluated using 24 and 48h post-fertilization (hpf) embryos. Upon 90min exposure of 24hpf embryos to 1µM RhB and different PFOS concentrations (3-300µM) accumulation of RhB in zebrafish was increased by up to 11.9-fold compared to controls, whereas RhB increases in verapamil treatments were 1.7-fold. Co-administration of PFOS and vinblastine in exposures from 0 to 48hpf resulted in higher vinblastine-caused mortalities in zebrafish embryos indicating increased uptake of this compound. Interference of PFOS with zebrafish Abcb4 activity was further studied using recombinant protein obtained with the baculovirus expression system. PFOS lead to a concentration-dependent decrease of the verapamil-stimulated Abcb4 ATPase activity; at higher PFOS concentrations (250, 500µM), also the basal ATPase activity was lowered indicating PFOS to be an Abcb4 inhibitor. In exposures of 48hpf embryos to a very high RhB concentration (200µM), accumulation of RhB in embryo tissue and adsorption to the chorion were increased in the presence of 50 or 100µM PFOS. In conclusion, the results indicate that PFOS acts as inhibitor of zebrafish Abcb4; however, the exceptionally large PFOS-caused effect amplitude of RhB accumulation in the 1µM RhB experiments and the clear PFOS effects in the experiments with 200µM RhB suggest that an additional mechanism appears to be responsible for the potential of PFOS to enhance uptake of Abcb4 substrates.

Acetylcholinesterase in zebrafish embryos as a tool to identify neurotoxic effects in sediments.

In order to clarify the suitability of zebrafish (Danio rerio) embryos for the detection of neurotoxic compounds, the acetylcholinesterase assay was adapted and validated with a series of priority pollutants listed as relevant for the European water policy (Aroclor 1254, 2,3-benzofuran, bisphenol A, chlorpyrifos, paraoxon-methyl, quinoline, and methyl mercury chloride) as well as acetonic extracts from three sediments of known contamination. The acute toxicities of the model substances and the sediment extracts were determined by means of the fish embryo test as specified in OECD TG 236, and concentrations as low as the effective concentration at 10% inhibition (EC10) were used as the highest test concentration in the acetylcholinesterase test in order to avoid nonspecific systemic effects mimicking neurotoxicity. Among the model compounds, only the known acetylcholinesterase inhibitors paraoxon-methyl and chlorpyrifos produced a strong inhibition to about 20 and 33%, respectively, of the negative controls. For the sediment extracts, a reduction of acetylcholinesterase activity to about 60% could only be shown for the Vering Canal sediment extracts; this could be correlated to high contents of acetylcholinesterase-inhibiting polycyclic aromatic hydrocarbons (PAHs) as identified by chemical analyses. Co-incubation of the Vering Canal sediment extracts with chlorpyrifos at EC10 concentrations each did not significantly increase the inhibitory effect of chlorpyrifos, indicating that the mode of action of acetylcholinesterase inhibition by the sediment-borne PAHs is different to that of the typical acetylcholinesterase blocker chlorpyrifos. Overall, the study documents that zebrafish embryos represent a suitable model not only to reveal acetylcholinesterase inhibition, but also to investigate various modes of neurotoxic action.

The fish embryo test (FET): origin, applications, and future.

Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.