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Transcriptomic analysis of intestinal tissue of earthworm exposed to Tri-n-butyl phosphate (TnBP)

ABSTRACT: Purpose: The objective of this study was to investigate the TnBP-induced intestinal toxicity mechanism on earthworm Eisenia fetida. Methods: The intestinal tissues of nine earthworms under control and 10 mg/kg TnBP treatments were separately pooled into three replicates (three earthworms per replication) to obtain sufficient amounts of RNA for transcriptomic assessments, using BGISEQ-500. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 10,401 contig sequences, mapped to 313 KEGG pathways,were classified. Scatter plots of DEGs based on the results showed that 3786 genes were significantly up regulated and 1084 genes were significantly downregulated at a P-value cut off of 0.01 . Pathway enrichment analysis of DEGs was performed to identify the potential biological functions for the interaction of genes based on the KEGG database. Pathways of the Digestive system were severely affected in the transcriptome dataset for the pathways of Protein digestion and absorption, Salivary secretion, Gastric acid secretion, and Bile secretion disruption. This is in accordance with the intestinal injury results of the digestive tract degradation and the alteration of digestive activities.

ORGANISM(S): Eisenia fetida

PROVIDER: GSE147869 | GEO | 2020/04/02

REPOSITORIES: GEO

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Project description:The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are ecologically relevant but provide little mechanistic insight into the toxicity pathways, especially at the molecular level. To better understand toxicological modes of action and to facilitate the development of molecular biomarkers, we have obtained 30,245 unique EST sequences from E. fetida and have designed a novel microarray with 15,119 60-mer oligonucleotide probes. These probes target the unique non-redundant EST sequences identified in E. fetida. Using this array we have profiled gene expression of E. fetida after exposure to CL-20, a cage cyclic nitramine previously found exhibiting reversible neurotoxicity to worms. Worms were exposed for 6 days to CL-20. Half of the exposed worms were allowed to recover in a clean environment for 7 days. Electrophysiological analysis showed that the conduction velocity of worm medial giant nerve fiber was significantly decreased after 6-d exposure to CL-20, and that giant nerve fiber function was restored at the end of the 7-d recovery period. Total RNA samples isolated from four treatment groups (6 replicates per group), i.e., 6-d control, 6-d exposed, 13-d control and 6-d exposed with 7-d recovery, were analyzed using the new 15K oligo array. Bioinformatics and statistical analyses have identified specific neurological pathways affected by CL-20 and recovery of these pathways after CL-20 removal. These results provide significant insights on the CL-20 toxic mode of action and how earthworms can recover from chemical stressors. Adult earthworms (E. fetida) were exposed on filter paper to CL-20 (0.2 ug/cm2) for 6 days with or without 7-day recovery (4 treatment groups in total). Each treatment group had 9 replicate worms, six of which were used for gene expression analysis. Worms were measured for their medial giant nerve fiber conduction velocity using a non-invasive electrophysiological technique immediately before takedown. At the termination of the 6-d or 13-d experiment, worms were snap-frozen and fixed in RNAlater-ICE. Total RNA was isolated from the fixed worms. A total of 24 worm RNA samples were hybridized to three 8x15K custom-designed Agilent oligo arrays using Agilentâs one-color Low RNA Input Linear Amplification Kit. The array contains 15,208 non-redundant 60-mer probes, each targeting a unique E. fetida transcript. After hybridization and scanning, gene expression data were acquired using GenePix Pro 6.0.

Project description:Background: Earthworms are essential for a healthy soil ecosystem, performing key functions such as bioturbation. The soil ecosystem can be challenged by natural toxins such as isothiocyanates (ITC), secondary metabolites produced by many commercial crops. This study investigated the effects of the natural toxin, 2-phenylethyl isothiocyanate on the earthworm Eisenia andrei using an ecotoxicogenomics approach that combined standardised ecotoxicological testing with gene expression analyses. Results and discussion: Exposure to 2-Phenylethyl ITC reduced both survival and reproduction of E. andrei in a dose-dependent manner, with an EC50 (reproduction) value of 556 nmol/g soil. Cross-species comparative genomic hybridisation validated the applicability of an existing 4x44k E. fetida microarray to E. andrei. Gene expression profiles revealed the importance of metallothionein (mt) as an early warning signal when E. andrei is exposed to low concentrations of 2-phenylethyl ITC. Allignment of these mt genes with the mt-2 gene of Lumbricus rubellus showed that there must at least have 2 mt genes in the Eisenia sp. genome. At high exposure concentrations of 2-phenylethyl ITC gene expression was mainly affected by inhibiting chitinase activities, inducing an oxidative stress response, and stimulating energy metabolism. KEGG pathway analysis, furthermore, implied that the high concentration may cause impaired light sensitivity, angiogenesis (e.g. wound healing), olfactory perception (olfaction), learning and memory. Conclusions: Increased levels of isothiocyanates may be found in the field in the near future. Risks of such compounds should be carefully investigated before allowing them to enter the soil on a large scale. This is the first study to investigate the effects of an isothiocyanate on an earthworm both on the ecotoxicological and genomic level. Three-exposure experiment; Acetone control vs.EC10 vs.EC50 exposed animals. Biological replicates: 4 replicates for each exposure. Technical replicates: 2 replicates for each exposure

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Transcriptomic analysis of intestinal tissue of earthworm exposed to Tri-n-butyl phosphate (TnBP)

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