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Research Collection Journal Article The neonicotinoid thiacloprid causes transcriptional alteration of genes associated with mitochondria at environmental concentrations in honey bees Author(s): Fent, Karl; Schmid, Michael; Hettich, Timm; Schmid, Simon Publication Date: 2020-11 Permanent Link: https://doi.org/10.3929/ethz-b-000432839 Originally published in: Environmental Pollution 266, http://doi.org/10.1016/j.envpol.2020.115297 Rights / License: Creative Commons Attribution 4.0 International This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Environmental Pollution 266 (2020) 115297 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol The neonicotinoid thiacloprid causes transcriptional alteration of genes associated with mitochondria at environmental concentrations in honey bees* * Karl Fent a, b, , Michael Schmid c, Timm Hettich a, Simon Schmid a a University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Hofackerstrasse 30, CHe4132, Muttenz, Switzerland b Swiss Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and Pollution Dynamics, Department of Environmental Systems Science, CHe8092, Zürich, Switzerland c Genexa AG, Dienerstrasse 7, CH-8004, Zürich, Switzerland article info abstract Article history: Thiacloprid is widely used in agriculture and may affect pollinators. However, its molecular effects are Received 16 June 2020 poorly known. Here, we report the global gene expression profile in the brain of honey bee foragers Received in revised form assessed by RNA-sequencing. Bees were exposed for 72 h to nominal concentrations of 25 and 250 ng/ 13 July 2020 bee via sucrose solution. Determined residue concentrations by LC-MS/MS were 0.59 and 5.49 ng/bee, Accepted 16 July 2020 respectively. Thiacloprid exposure led to 5 and 71 differentially expressed genes (DEGs), respectively. Available online 11 August 2020 Nuclear genes encoding mitochondrial ribosomal proteins and enzymes involved in oxidative phos- phorylation, as well as metabolism enzymes and transporters were altered at 5.49 ng/bee. Kyoto Encylopedia of Genes and Genomes (KEGG) analysis revealed that mitochondrial ribosome proteins, mitochondrial oxidative phosphorylation, pyrimidine, nicotinate and nicotinamide metabolism and additional metabolic pathways were altered. Among 21 genes assessed by RT-qPCR, the transcript of farnesol dehydrogenase involved in juvenile hormone III synthesis was significantly down-regulated. Transcripts of cyp6a14-like and apolipophorin-II like protein, cytochrome oxidase (cox17) and the non- coding RNA (LOC102654625) were significantly up-regulated at 5.49 ng/bee. Our findings indicate that thiacloprid causes transcriptional changes of genes prominently associated with mitochondria, partic- ularly oxidative phosphorylation. This highlight potential effects of this neonicotinoid on energy meta- bolism, which may compromise bee foraging and thriving populations at environmentally relevant concentrations. © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction (Sanchez-Bayo and Goka, 2014). Insects are exposed to numerous agricultural pesticides (David The documented decline of insects (Hallmann et al., 2017) and et al., 2016) including neonicotinoids (Mullin et al., 2010; arthropods biomass and diversity (Seibold et al., 2019) are of major Sanchez-Bayo and Goka, 2014). Direct exposure of bees may cause concern, which also holds for bees (Lee et al., 2015; Ollerton et al., acute toxicity but also chronic effects to the nervous system 2014). Bee decline compromises pollination (Cameron et al., 2011; (Decourtye et al., 2004), immune system (Di Prisco et al., 2013)or Goulson et al., 2015; Grab et al., 2019) and has many causes on energy allocation (Christen et al., 2019a). Their neurotoxicity (Goulson et al., 2015) including loss of plant biodiversity associated may compromise memory and orientation as demonstrated for with intensified farming, impacts of pathogens and pesticides thiacloprid that reduced homing success of honey bees (Tison et al., 2016). Neonicotinoid insecticides may also harm bee populations (Henry et al., 2012; Rundlof€ et al., 2015; Tsvetkov et al., 2017). The neonicotinoid thiacloprid has now been banned in the Eu- * This paper has been recommended for acceptance by Christian Sonne. ropean Union as the highly toxic imidacloprid, thiamethoxam, * Corresponding author. University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Hofackerstrasse 30, CHe4132, Muttenz, clothianidin in outdoor applications due to their high risks for bees. Switzerland. However, it still finds application indoors (greenhouses). E-mail address: [email protected] (K. Fent). https://doi.org/10.1016/j.envpol.2020.115297 0269-7491/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2 K. Fent et al. / Environmental Pollution 266 (2020) 115297 Thiacloprid binds to and activates acetylcholine receptors in the similar as in nectar, and the higher concentration about five times nervous system of bees (Manjon et al., 2018). The relatively lower above highest reported concentrations (Ellis et al., 2017). toxicity of thiacloprid compared to the highly toxic neonicotinoids In our report, we give nominal concentrations and report is suggested to be based on ready metabolism by cytochrome P450 effective exposure concentrations in parenthesis. Thiacloprid con- dependent monooxygenases (cyp). Thiacloprid is detoxified in centrations were assessed by trace analytical chemistry in solvent honey and bumble bees by cyp9q3, and to a lesser extent, by control bees and thiacloprid exposed bees after experimental cyp9q2 (Iwasa et al., 2004; Manjon et al., 2018). Efficient meta- exposure as described in the SM. The nominal concentrations were bolism may also convey tolerance to this insecticide as shown in the 1.47% and 14.7%, respectively, of the LD50 value of 1700 ng/bee solitary bee Osmia bicornis, where cyp9bu is important for thia- (Sanchez-Bayo and Goka, 2014) and were selected based on re- cloprid detoxification (Beadle et al., 2019). Over-expression of cyp ported residues in nectar and bees from thiacloprid treated fields enzyme transcripts were found in thiacloprid-treated honey bees (Ellis et al., 2017). (Alptekin et al., 2016) or insects resistant to this insecticide We exposed bees for 72 h to investigate effects of chronic (Karunker et al., 2008). exposure. Gene expression was assessed in the brain at 72 h. Of ten Thiacloprid induces sublethal effects in bees including altered bees per flask, three bees were stored at À20 C for RNA-extraction behaviour in the field that affected foraging, homing success and and the rest at À80 C for chemical residue analysis and backup. navigation (Fischer et al., 2014; Tison et al., 2016). Furthermore, Brains of three bees per bottle were dissected and pooled to obtain adverse effects to the immune system (Brandt et al., 2016)were one RNA sample per replicate, which yielded five biological repli- demonstrated and colony development of bumble bees was cates per exposure group (total of 15 samples). RNA isolation is impacted under field conditions (Ellis et al., 2017). described in the SM. One bee per bottle was used for chemical Currently, the molecular effects of thiacloprid are poorly known. residue analysis. Metabolomic analyses in the head of thiacloprid treated bees fed with 120 ng/bee revealed 115 metabolites, which belonged to a 2.2. RNA-sequencing wide range of pathways associated with oxidative stress and detoxification (Shi et al., 2018). Investigation into molecular effects Library preparation and sequencing was performed at Func- of highly toxic neonicotinoids in the brain of honey bees by RNA- tional Genomics Center Zurich, ETH and University of Zürich, sequencing showed that metabolic and other pathways were Switzerland. The library preparation was performed using the affected at environmentally relevant concentrations (Christen et al., Illumina TruSeq stranded mRNA kit, which includes a poly-A 2018a). Furthermore, clothianidin and imidacloprid affected bio- enrichment step and is strand specific via dUTP incorporation logical processes including mitochondrial function in bumble bees during the second strand synthesis. The samples were barcoded (Colgan et al., 2019). with 15 different Illumina TruSeq index pairs. Barcoded libraries Sequencing of the whole genome of honey bees (Wallberg et al., were sequenced on an Illumina NovaSeq6000 as single end, 100 bp 2014) allows now to perform better global transcriptome analyses reads (SE100bp). The libraries were then de-multiplexed based on by RNA-sequencing. The aim of our study was to evaluate molecular their indices. effects and the modes of action of thiacloprid in the brain of honey Quality checking of raw data and trimming, mapping of trim- bees by means of RNA-sequencing and quantitative RT-PCR and med data, transcriptome assembly and transcript abundance esti- thereby providing novel insight into the mechanisms mediating mation, analysis for differential expression and ratio of fold-change thiacloprid toxicity. Our current study provides several advance- for differentially expressed genes (DEGs) between