Transcriptome Analysis of Thiram-Treated Zebrafish (Danio rerio) Embryos Reveals Disruption of Reproduction Signaling Pathways

Abstract

Thiram, a dithiocarbamate fungicide, is used for the treatment of various fungal infections affecting crops and ornamentals. However, thiram-associated toxicity has been reported in animals, including fish, and the underlying molecular mechanisms are unclear. Herein, we employed zebrafish (ZF) to gain further insights into thiram toxicity-associated molecular mechanisms. We studied developmental abnormalities and performed whole-transcriptome analysis of ZF embryos exposed to thiram for 96 h. Embryos exposed to 4.0 μg/L thiram exhibited several phenotypic abnormalities, including bradycardia, spinal curvature, hatching arrest, and growth retardation. Whole-transcriptome analysis revealed 1754 differentially expressed genes (DEGs), with 512 upregulated and 1242 downregulated DEGs. The majority of biological processes affected by thiram were metabolic. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis yielded terms related to reproduction, such as steroid biosynthesis and steroid hormone biosynthesis. Quantitative real-time polymerase chain reaction validation results were in line with sequencing data for ten DEGs. The study results improve our current understanding of the effects of thiram exposure in ZF.

Transcriptome Profiling of Etridiazole-Exposed Zebrafish (Danio rerio) Embryos Reveals Pathways Associated with Cardiac and Ocular Toxicities

“…Given that protein synthesis components, such as tRNAs and aminoacyl synthases, were found to be downregulated in ZF that displayed growth retardation following pesticide treatment in earlier studies [14,15], protein biosynthesis disruption may explain the growth retardation observed in ETZF (Figure 3). Notably, aminoacyl-tRNA biosynthesis, a critical mechanism that connects amino acids to their corresponding transfer RNA (tRNA) molecules to ensure proper protein synthesis, was one of the ten most enriched KEGG pathways in ETZF (Figure 6), suggesting abnormal protein synthesis in ETZF.…”

“…Pesticides have complex chemical profiles and are known to interfere with several biological functions in organisms [16], making it difficult and time-consuming to unravel the molecular mechanisms of off-target pesticide toxicity using standard approaches. Next-generation high-throughput sequencing technology, on the other hand, has been successful in demonstrating pesticide toxicity at the molecular level in off-targets, particularly ZF [14,15]. Therefore, we employed transcriptome analysis, in combination with developmental toxicity assessment, to understand the molecular mechanisms associated with EDZ toxicity in the ZF model.…”

“…For transcriptome analysis, three samples containing 30-40 embryos/larvae under each test condition were frozen (liquid nitrogen) and stored. The experimental protocols for RNA extraction, quantification, and cDNA library preparation were based on our previous studies [14,15]. Paired-end sequencing (2 × 100 bp) was carried out on the NovaSeq platform (Illumina, Inc., San Diego, CA, USA).…”

“…Three samples of UTZF and ETZF, each containing 30-40 embryos/larvae, were used for qPCR-based validation of the transcriptome data. The experimental techniques for RNA extraction, cDNA preparation, and qPCR were based on our previous study [14]. Under each test condition, RNA was extracted from three biological replicates, and 1000 ng of RNA was used for cDNA conversion following the manufacturer's protocol (ReverTra Ace™ qPCR RT master mix with gDNA remover (Toyobo, Osaka, Japan)).…”

“…Understanding the toxicity mechanisms of pesticides using traditional methods is time-consuming and less informative [9][10][11][12]. Next-generation sequencing is becoming increasingly popular due to its ability to perform high-throughput analysis of complex biological processes [13], which has successfully identified the molecular signaling pathways underlying pesticide-related developmental toxicity in ZF [14,15].…”