Identification Of Cytochrome P450 Family Members Of Gammarus Suifunensis And Inducible Expression Of Major Water Pollutants

As food resources of many fish,freshwater species from genus Gammarus is a biological indicator for water environment.The cytochrome P450 enzyme system is a key detoxification enzyme system in crustaceans,playing an important role in the detoxification and metabolism of pesticides.In this study,a 24-hour semi-static water exchange tonic was used to study the deltamethrin,triazophos and cadmium chloride of G.suifunensis 96 h acute toxicity,and a safe concentration was determined.Based on this,the transcriptomics of G.suifunensis was studied under deltamethrin,triazophos and cadmium chloride stress,and the members of G.suifunensis P450 family were identified using transcriptome data.Finally,in response to the scientific question of whether members of the P450 family of G.suifunensis can be used as biomarkers for polluted water bodies,the stress response of members of the P450 family to deltamethrin,triazophos and cadmium chloride was studied.The main findings are as follows:(1)The results showed that deltamethrin,triazophos,and cadmium chloride had a lethal concentration of 0.034 ?g / L,0.0005 mg / L,and 0.057 mg / L to G.suifunensis at 96 h,respectively,and safe concentrations of at 96 h were 0.0089 ?g / L,0.0001 mg / L,0.0098 mg / L.Studies have shown that deltamethrin,triazophos,and cadmium chloride G.suifunensis are highly toxic.The order of toxicity is deltamethrin > triazophos > cadmium chloride.In view of its sensitivity to deltamethrin,triazophos,and cadmium chloride,G.suifunensis can also be used as an effective indicator organism for contamination with pyrethroid pesticides,organophosphorus pesticides,and heavy metals in the water environment.(2)A total of 20.91 Gb clean data was obtained from the mixed tissue transcripts of G.suifunensis,with 13,420,805 subreads,395,149 ROIs,and 320,562 of full-length non-chimeric reads(FLNC)were obtainedin G.suifunensis.After transcripts clusterand error correction,a total of 12782 high-quailty full-length transcripts were generated,with an average length of 1819 bp and the N50 length of 2128 bp in G.suifunensis.A total of 6456 unigenes were obtained with an average length of 2052 bp,the N50 length of 2322 bp in G.suifunensis.(3)The unigenes were subjected to annotation analysis by matching sequences against Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,and GO databases.the results showed that the Nt database accounted for the annotations(5425,42.44%),followed by KEGG database(4805,37.59%),the smallest was the Nt database(2523,19.73%).According to the Nr database annotations,75.81% of unigene have the genetic similarity with Hyalella azteca,which is consistent with the classification status that the two species belong to the Amphipoda.(4)Analysis of differentially expressed genes in G.suifunensis showed that under the condition of deltamethrin stress,GO had 335 up-regulated genes and 11 down-regulated genes.The enriched genes were related to molecular functional genes,cell component genes and biology Process genes are closely related.Under triazophos stress,GO shrimp was enriched with 12 up-regulated genes and 401 down-regulated genes.There was no cell process entry in the up-regulated genes.Under cadmium chloride stress,there are two up-regulated genes in G.suifunensis,which are not down-regulated genes,and are closely related to molecular functions.(5)The enrichment of KEGG pathway showed that 9 differential genes were enriched in 9 KEGG pathways in G.suifunensis under deltamethrin stress.And these 9 genes are related to the pentose phosphate pathway,cysteine and methionine metabolism,fructose and mannose metabolism,legionellosis,AMPK signaling pathway,oxytocin signaling pathway,glycolysis / gluconeogenesis,apoptosis and Lysosome-related.Under triazophos stress,7 differential genes were enriched in 7 KEGG pathways.And these 7 genes are related to phenylalanine metabolism,viral disease,tyrosine metabolism,thyroid hormone synthesis,protein export,amino sugar and nucleotide sugar metabolism,and protein processing in the endoplasmic reticulum.Under cadmium chloride stress,G.suifunensis were not enriched in the KEGG pathway.(6)Based on the above transcript data,a total of 14 members of the cytochrome P450 family were identified in G.suifunensis,and most of the genes obtained fragments of more than 60% of the full-length sequence.By verifying the 7 cytochrome P450 family members of G.suifunensis,the Gs-CYP2J2,Gs-CYP4c3,and Gs-CYP49a1 genes in G.suifunensis were related to the degree of deltamethrin stress under deltamethrin conditions.Under the conditions of triazophos,the Gs-CYP4c3,Gs-CYP49a1,Gs-CYP3a24,Gs-CYP2J2,Gs-CYP18a1 and Gs-CYP6k1 genes in G.suifunensis were associated with triazophos stress.The Gs-CYP2J2,Gs-CYP18a1,Gs-CYP4c3,Gs-CYP49a1 and Gs-CYP3a24 genes in G.suifunensis under cadmium chloride conditions were related to the degree of cadmium chloride stress.It indicates that these genes may be used as biomarkers of polluted water in G.suifunensis.