Transcriptional responses in the hepatopancreas of Eriocheir sinensis exposed to deltamethrin

Abstract

Deltamethrin is an important pesticide widely used against ectoparasites. Deltamethrin contamination has resulted in a threat to the healthy breeding of the Chinese mitten crab, Eriocheir sinensis. In this study, we investigated transcriptional responses in the hepatopancreas of E. sinensis exposed to deltamethrin. We obtained 99,087,448, 89,086,478, and 100,117,958 raw sequence reads from control 1, control 2, and control 3 groups, and 92,094,972, 92,883,894, and 92,500,828 raw sequence reads from test 1, test 2, and test 3 groups, respectively. After filtering and quality checking of the raw sequence reads, our analysis yielded 79,228,354, 72,336,470, 81,859,826, 77,649,400, 77,194,276, and 75,697,016 clean reads with a mean length of 150 bp from the control and test groups. After deltamethrin treatment, a total of 160 and 167 genes were significantly upregulated and downregulated, respectively. Gene ontology terms “biological process,” “cellular component,” and “molecular function” were enriched with respect to cell killing, cellular process, other organism part, cell part, binding, and catalytic. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes showed that the metabolic pathways were significantly enriched. We found that the CYP450 enzyme system, carboxylesterase, glutathione-S-transferase, and material (including carbohydrate, lipid, protein, and other substances) metabolism played important roles in the metabolism of deltamethrin in the hepatopancreas of E. sinensis. This study revealed differentially expressed genes related to insecticide metabolism and detoxification in E. sinensis for the first time and will help in understanding the toxicity and molecular metabolic mechanisms of deltamethrin in E. sinensis.

Transcriptome analysis of hepatopancreas of Eriocheir sinensis with hepatopancreatic necrosis disease (HPND)

“…The target cDNA fragments were excised from the gel and a PCR reaction was performed to amplify them. After validation using the ABI StepOnePlus Real-Time PCR System (ABI, Foster City, CA, USA) and the Agilent 2100 Bioanalyzer, the cDNA libraries of the corresponding samples were sequenced on a flow cell with the high-throughput mode on an Illumina HiSeq 2500 unit (Illumina, San Diego, CA, USA) [20].…”

“…The DESeq software (version 1.14.0) was used to screen out the DEGs and the fold-change of each transcript was calculated [24]. A two-fold change in expression identified a DEG, and an adjusted p-value < 0.05 was considered statistically significant [20].…”

“…The corrected p-value of 0.05 was used as the threshold to determine statistical significance. When the corrected p-value was < 0.05, the GO term was considered significantly enriched in the DEGs [20].…”

“…The enriched KEGG pathways were identified using the same formula as that used for GO analysis. M is the number of certain genes annotated to the specific pathways; N is the number of genes that were added with KEGG annotation; m represents the number of DEGs included in M; and n is the number of DEGs included in N [20].…”

“…The thermal cycling program was as follows: 95˚C for 30 s, followed by 40 cycles of 95˚C for 5 s, 60˚C for 30 s, and 72˚C for 30 s. Melting curve analysis was performed at the end of qRT-PCR to verify the PCR specificity. The expression levels of DEGs were analyzed using the 2 -ΔΔ CT method [20]. sequencing of the Chinese mitten crab was successful, and the trimmed reads of test and control groups could be used for the further analyses.…”