Whole-Organism Transcriptomic Analysis Provides Mechanistic Insight into the Acute Toxicity of Emamectin Benzoate in Daphnia magna

Abstract

Emamectin benzoate (EMB) is an antisea lice chemical widely used in the aquaculture that may also unintentionally affect nontarget crustaceans in the environment. Although the adverse effects of this compound are well documented in various species, the full modes of action (MoAs) are still not well characterized. The current study was therefore conducted to characterize the MoAs of EMB and link perturbations of key toxicological pathways to adverse effects in the model freshwater crustacean Daphnia magna. Effects on molting and survival were determined after 48 h exposure to EMB, whereas global transcriptional changes and the ecdysone receptor (EcR) binding potency was determined to characterize the MoA. The results showed that the molting frequency and survival of D. magna decreased in a concentration-dependent manner, and the observed changes could not be attributed to direct interactions with the EcR. Major MoAs such as activation of glutamate-gated chloride channels and gamma-aminobutyric acid signaling, disruption of neuroendocrine regulation of molting, perturbation of energy homeostasis, suppression of DNA repair and induction of programmed cell death were observed by transcriptional analysis and successfully linked to the adverse effects. This study has demonstrated that acute exposure to intermediate and high pM levels of EMB may pose hazards to nontarget crustaceans in the aquatic environment.

Gamma radiation induces dose-dependent oxidative stress and transcriptional alterations in the freshwater crustacean Daphnia magna

“…After 24 hrs exposure to gamma radiation, six daphnids were pooled for each replicate (n=5), sampled in RNALater (Sigma-Aldrich) and stored at -80°C until use. Total RNA was isolated using the ZR Tissue & Insect RNA MicroPrep kit in combination with on-column DNase I treatment (Zymo Research Corp., Irvine, CA) as previously described (Song et al, 2016). The purity (260/280>1.8, yield > 100 ng) and integrity (clear RNA peaks, flat baselines) of RNA were assessed using Nanodrop ND-1000 (Nanodrop Technologies, Wilminton, DE) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively.…”

“…Transcriptomic analysis was performed using Agilent custom 60,000-feature D. magna oligonucleotide microarrays and 50 ng input RNA according to Agilent's standard protocol "One-Color Microarray-Based Gene Expression Analysis, version 6.5", with modifications (Song et al, 2016). Raw microarray data (signal intensity) was extracted from scanned images using the Feature Extraction software v10.7 (Agilent), and data corrected for baseline variance (normexp method), inter-array variance (quantile method), filtered for low expression probes and technical replicate probes merged using the Bioconductor package LIMMA (Smyth, 2005) in the R statistical environment v3.1.2, as previously described (Jensen et al, 2016).…”

“…A selection of 13 target genes considered relevant to potential MoAs of gamma radiation was further verified using quantitative real-time reverse transcription polymerase chain reaction (qRTPCR) essentially as described by Song et al (2016). The qRTPCR analysis was conducted on a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Oslo, Norway) using the same RNA as used in the microarray analysis (n=5).…”

“…A general activation of genes associated with Calcium signaling pathways such as Cadependent events, Calmodulin induced events and CaM pathway were observed in daphnids exposed to 42.9 and 106 mGy/h. Calmodulin (CaM), the ubiquitously expressed and highly conserved protein that is essential for numerous cellular processes and is the key mediator of Ca 2+ signals (Altshuler et al, 2015;Song et al, 2016), was significantly down-regulated by 0.41, 1.1, 4.3 and 10.7 mGy/h and up-regulated at mGy/h (qPCR). Cells tightly regulate their cytoplasmic calcium concentrations, as Ca 2+ ions are used in a several concentrationdependent processes, which in crustaceans can be directly related to molting, mTOR signaling and intracellular calcium influx (Altshuler et al, 2015).…”

The Role of Omics in the Application of Adverse Outcome Pathways for Chemical Risk Assessment

“…Within the AOP framework, omics datasets also enable a more precise definition of the MIE and the selection of biomarkers relevant for assessing effects and/or exposure. Omics data provide both gene- and pathway-level read-outs (eg, changes in individual gene expression levels or statistical indices for the differential regulation of sets of genes within a specific biological pathway) that can be used for selecting measurable and relevant biomarkers ( Gatzidou et al , 2007 ; Song et al , 2016 ). In addition, by building upon the omics discoveries made by the broader research community (eg, The modENCODE Consortium et al , 2010), a carefully chosen suite of model test species that represent major evolutionary lineages of animal diversity could allow researchers to gain knowledge for accurately predicting chemical susceptibility in a range of organisms.…”