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Publikation Global transcriptome analysis reveals relevant effects at environmental concentrations of cypermethrin in honey bees (Apis mellifera)(Elsevier, 04.12.2019) Fent, Karl; Christen, Verena01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Biopesticide spinosad induces transcriptional alterations in genes associated with energy production in honey bees (Apis mellifera) at sublethal concentrations(Elsevier, 15.10.2019) Christen, Verena; Krebs, Jana; Bünter, Ivan; Fent, Karl01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Fungicides chlorothanolin, azoxystrobin and folpet induce transcriptional alterations in genes encoding enzymes involved in oxidative phosphorylation and metabolism in honey bees (Apis mellifera) at sublethal concentrations(Elsevier, 05.09.2019) Christen, Verena; Krebs, Jana; Fent, KarlFungicides are highly used for plant protection but their molecular and chronic effects are poorly known. Here, we analyse transcriptional effects in the brain of honey bees of three frequently applied fungicides, azoxystrobin, chlorothanolin and folpet, after oral exposure for 24, 48 and 72 h. Among transcripts assessed were genes encoding proteins for immune and hormone system regulation, oxidative phosphorylation, metabolism, and acetylcholine receptor alpha 1. Azoxystrobin and folpet induced minor alterations, including down-regulation of hbg-3 by azoxystrobin and induction of ndufb-7 by folpet. Chlorothanolin induced strong transcriptional down-regulation of genes encoding enzymes related to oxidative phosphorylation and metabolism, including cyp9q1, cyp9q2 and cyp9q3, acetylcholine receptor alpha 1 and hbg-3 and ilp-1, which are linked to hormonal regulation and behavioural transition of honey bees. Exposures to chlorothanolin in different seasonal times showed different responsiveness; responses were faster and often stronger in April than in June. Chlorothanolin caused the strongest effects and affected transcriptional abundance of genes related to energy production, metabolism and the endocrine system. Disturbed energy production may reduce foraging activity and hormonal dysregulation, such as the transition of nurse bees to foragers. Further analyses are needed to further substantiate potential adverse effects of chlorothanolin in bees on the physiological level.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation A vitellogenin antibody in honey bees (Apis mellifera ): Characterization and application as potential biomarker for insecticide exposure(Wiley, 02/2019) Christen, Verena; Fent, Karl; Hettich, TimmThe insect yolk precursor vitellogenin is a lipoglycoprotein synthesized and stored in the fat body and secreted into the hemolymph. In honey bees, vitellogenin displays crucial functions in hormone signaling, behavioral transition of nurse bees to foragers, stress resistance, and longevity in workers. Plant protection products such as neonicotinoids, pyrethroids, and organophosphates alter the transcriptional expression of vitellogenin. To assess plant protection product‐induced alterations on the protein level, we developed a rabbit polyclonal vitellogenin antibody. After characterization, we assessed its specificity and vitellogenin levels in different tissues of worker bees. The vitellogenin antibody recognized full‐length 180‐kDa vitellogenin and the lighter fragment of 150 kDa in fat body, hemolymph, and brain. In hemolymph, a band of approximately 75 kDa was detected. Subsequent mass spectrometric analysis (liquid chromatography–mass spectrometry) confirmed the 180‐ and 150‐kDa bands as vitellogenin. Subsequently, we evaluated vitellogenin expression in brain, fat body, and hemolymph on 24‐h exposure of bees to 3 ng/bee to the neonicotinoid clothianidin. Full‐length vitellogenin was upregulated 3‐fold in the fat body, and the 150‐kDa fragment was upregulated in the brain of exposed honey bees, whereas no alteration occurred in the hemolymph. Upregulation of the vitellogenin protein by the neonicotinoid clothianidin is in line with the previously shown induction of its transcript. We conclude that vitellogenin might serve as a potential biomarker for neonicotinoid and other pesticide exposure in bees. Environ Toxicol Chem 2019;00:1–10. © 2019 SETAC01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Transcriptional and physiological effects of the pyrethroid deltamethrin and the organophosphate dimethoate in the brain of honey bees (Apis mellifera)(Elsevier, 01/2019) Christen, Verena; Joho, Yvonne; Vogel, Maren; Fent, KarlThe pyrethroid deltamethrin and the organophosphate insecticide dimethoate are widely used in agriculture and in urban areas. Both plant protection products (PPPs) unintendedly result in adverse effects in pollinators. Currently, the sublethal effects of both compounds are poorly known, particularly on the molecular and biochemical level. Here we analysed effects of deltamethrin and dimethoate at environmental and sublethal concentrations in honey bee workers by focusing on transcriptional changes of target genes in the brain. In addition, expression of vitellogenin protein and activity of acetylcholinesterase were assessed upon dimethoate exposure to assess physiological effects. Deltamethrin resulted in induction of the cyp9q2 transcript at 0.53 ng/bee, while dimethoate led to induction of vitellogenin on the mRNA and protein level at 2 ng/bee. Transcripts of additional cytochrome P450-dependent monooxygenases (cyps) and genes related to immune system regulation were not differentially expressed upon PPP exposure. Dimethoate but not deltamethrin led to a strong and concentration-related inhibition of the acetylcholinesterase at 2 and 20 ng/bee. Our data demonstrate that deltamethrin and dimethoate exhibit transcriptional effects at environmental concentrations in the brain of honey bees. Dimethoate also strongly affected physiological traits, which may translate to adverse effects in forager bees.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Endocrine disruption and chronic effects of plant protection products in bees: Can we better protect our pollinators?(Elsevier, 09/2018) Christen, Verena; Kunz, Petra Y.; Fent, KarlExposure to plant protection products (PPPs) is one of the causes for the population decline of pollinators. In addition to direct exposure, pollinators are exposed to PPPs by pollen, nectar and honey that often contain residues of multiple PPPs. While in legislation PPPs are regarded mainly for their acute toxicity in bees, other effects such as neurotoxicity, immunotoxicity, behavioural changes, stress responses and chronic effects that may harm different physiologically and ecologically relevant traits are much less or not regarded. Despite the fact that endocrine disruption by PPPs is among key effects weakening survival and thriving of populations, pollinators have been poorly investigated in this regard. Here we summarize known endocrine disruptive effects of PPPs in bees and compare them to other chronic effects. Endocrine disruption in honey bees comprise negative effects on reproductive success of queens and drones and behavioural transition of nurse bees to foragers. Among identified PPPs are insecticides, including neonicotinoids, fipronil, chlorantraniliprole and azadirachtin. So far, there exists no OECD guideline to investigate possible endocrine effects of PPPs. Admittedly, investigation of effects on reproduction success of queens and drones is rarely possible under laboratory conditions. But the behavioural transition of nurse bees to foragers could be a possible endpoint to analyse endocrine effects of PPPs under laboratory conditions. We identified some genes, including vitellogenin, which regulate this transition and which may be used as biomarkers for endocrine disruptive PPPs. We plea for a better implementation of the adverse outcome pathway concept into bee's research and propose a procedure for extending and complementing current assessments, including OECD guidelines, with additional physiological and molecular endpoints. Consequently, assessing potential endocrine disruption in pollinators should receive much more relevance.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Global Transcriptomic Effects of Environmentally Relevant Concentrations of the Neonicotinoids Clothianidin, Imidacloprid, and Thiamethoxam in the Brain of Honey Bees ( Apis mellifera).(American Chemical Society, 06/2018) Christen, Verena; Schirrmann, Melanie; Frey, Juerg E.; Fent, KarlNeonicotinoids are implicated in the decline of honey bees, but the molecular basis underlying adverse effects is poorly known. Here we describe global transcriptomic profiles in the brain of honey bee workers exposed for 48 h at one environmentally realistic and one sublethal concentration of 0.3 and 3.0 ng/bee clothianidin and imidacloprid, respectively, and 0.1 and 1.0 ng/bee thiamethoxam (1-30 ng/mL sucrose solution) by high-throughput RNA-sequencing (RNA-seq). All neonicotinoids led to significant alteration (mainly down-regulation) of gene expression, generally with a concentration-dependent effect. Among many others, genes related to metabolism and detoxification were differently expressed. Gene ontology (GO) enrichment analysis of biological processes revealed catabolic carbohydrate metabolism (regulation of enzyme activities such as amylase), lipid metabolism, and transport mechanisms as shared terms between all neonicotinoids at high concentrations. KEGG pathway analysis indicated that at least two neonicotinoids induced changes in expression of various metabolic pathways: pentose phosphate pathways, starch and sucrose metabolism, and sulfur metabolism, in which glucose 1-dehydrogenase and alpha-amylase were down-regulated and 3'(2'), 5'-bisphosphate nucleotidase was up-regulated. RT-qPCR analysis confirmed the down-regulation of major royal jelly proteins, hbg3, and cyp9e2 found by RNA-seq. Our study highlights the comparative molecular effects of neonicotinoid exposure to bees. Further studies should link these effects with physiological outcomes for a better understanding of effects of neonicotinoids01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Developmental neurotoxicity of different pesticides in PC-12 cells in vitro(Elsevier, 2017) Christen, Verena; Fent, Karl; Rusconi, Manuel; Crettaz, PierreThe detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5 days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Cytotoxicity and molecular effects of biocidal disinfectants (quaternary ammonia, glutaraldehyde, poly(hexamethylene biguanide) hydrochloride PHMB) and their mixtures in vitro and in zebrafish eleuthero-embryos(Elsevier, 2017) Christen, Verena; Faltermann, Susanne; Fent, Karl; Brun, NadjaFrequently used biocidal disinfectants, including quaternary ammonium compounds (QAC), glutaraldehyde and poly(hexamethylene biguanide) hydrochloride (PHMB), occur in the aquatic environment but their potential effects in fish are poorly known, in particular when occurring as mixtures. To investigate their joint activity, we assessed the cytotoxicity of three QACs (BAC, barquat and benzalkonium chloride), glutaraldehyde andPHMB by the MTT assay individually, followed by assessing binary and ternary mixtures in zebrafish liver cells (ZFL) and human liver cells (Huh7). We also analysed molecular effects by quantitative PCR in vitro and in zebrafish eleuthero-embryos employing a targeted gene expression approach. QACs displayed strong cytotoxicity in both cell lines with EC50 values in the low μg/ml range, while glutaraldehyde and PHMB were less cytotoxic. Most of the binary and both ternary mixtures showed synergistic activity at all equi-effective concentrations. A mixture containing all five compounds mixed at their no observed effect concentrations showed strong cytotoxicity, suggesting a synergistic interaction. Additionally, we determined transcriptional alterations of target genes related to endoplasmatic reticulum (ER) stress, general stress, inflammatory action and apoptosis. Induction of ER stress genes occurred at non-cytotoxic concentrations of barquat, glutaraldehyde and BAC in ZFL cells. Barquat and BAC induced tumor necrosis factor alpha (tnf-α). Similar transcriptional alterations were found in vivo upon exposure of zebrafish eleuthero-embryos for 120 h. Glutaraldehyde led to induction of ER stress genes and tnf-α, while BAC additionally induced genes indicative of apoptosis, which was also the case with benzalkonium chloride at the highest concentration. We demonstrated strong cytotoxicity of QACs, and synergistic activity of binary, ternary and quintuple mixtures. Barquat and BAC let to induction of ER stress and inflammation in vitro, and BAC and glutaraldehyde at non-toxic concentrations in vivo, while benzalkonium chloride induced expression of tnf-α only.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Anti-Inflammatory Activity of Cyanobacterial Serine Protease Inhibitors Aeruginosin 828A and Cyanopeptolin 1020 in Human Hepatoma Cell Line Huh7 and Effects in Zebrafish (Danio rerio)(MDPI, 14.07.2016) Faltermann, Susanne; Hutter, Simon; Christen, Verena; Hettich, Timm; Fent, KarlIntensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L−1 aeruginosin 828A, and 100 µmol·L−1 cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A.01A - Beitrag in wissenschaftlicher Zeitschrift