Assessment Of Toxicity Of Imidacloprid And Thiacloprid To Non-target Soil-arthropod Folsomia Candida(Collembola)

Neonicotinoids were developed in the 1980s,and imidacloprid was the first neonicotinoid to be used commercially in US in 1991.They are the most widely used insecticides in the world.They affect a specific neural pathway in the central nervous system of insects by acting on nicotinic acetylcholine receptors?nAChR?on the postsynaptic membrane.The unique mode of action makes them highly effective for controlling sucking pests like whiteflies,mites and aphids.Imidacloprid and thiacloprid are two widely used neonicotinoids in different agricultural cropping systems.These two representative neonicotinoids were chosen in this study due to their frequent and wide use in rice fields of China.Folsomia candida is a soil-dwelling arthropod that plays a vital role in the ecosystem by facilitating decomposition of organic matter and nutrient recycling.They are common and widespread arthropod that occurs in soils throughout the world inhabiting rich soil and leaf litter layers.In the present study,we examined the lethal and chronic toxicity of imidacloprid and thiacloprid on F.candida,and also evaluated their sublethal effects on the survival,reproduction,detoxification enzyme activities and transcriptomic profiles of F.candida.These were achieved by investigating the toxicity of imidacloprid and thiacloprid upon soil treatment to F.candida.The results are as followed.1.Lethal toxicity of imidacloprid and thiacloprid on F.candida.The lethal toxicity of both neonicotinoids was determined on 9-12 days old F.candida juveniles.The toxicity data were recorded after 14 days post-treatment.Both neonicotinoids were lethal to F.candida.The LC₅₀ were 2.17 and 17.63 mg a.i.kg^-1 soil dry weights for imidacloprid and thiacloprid,respectively.2.Chronic toxicity of imidacloprid and thiacloprid on F.candida.The chronic toxicity of both neonicotinoids was determined on 9-12 days old F.candida juveniles.The toxicity data were recorded after 28 days post-treatment.The LC₅₀,LC₃₀,LC₁₀ were 0.42,0.19,0.022 mg a.i.kg^-1 soil dry weight and 1.32,0.27,0.022 mg a.i.kg^-1 soil dry weight for imidacloprid and thiacloprid,respectively.Furthermore,the EC₅₀,EC₃₀,EC₁₀ for effects on the reproduction were 0.42,0.053,0.0019 mg a.i.kg^-1 soil dry weight and 0.34,0.08,0.0082 mg a.i.kg^-1 soil dry weight for imidacloprid and thiacloprid,respectively.These results revealed that both neonicotinoids are harmful to F.candida.3.Sublethal effects of imidacloprid and thiacloprid on the detoxified enzyme activities.The sublethal effects of effective dosage of these neonicotinoids on activities of detoxified enzymes such as cytochrome P450?CYP450?,carboxylesterases?CarEs?and glutathione-S-transferases?GSTs?were analyzed.In this study,20 days old F.candida adults were exposed to the dosage of EC₁₀ and EC₃₀ of these neonicotinoids along with their acetone as control.Only live adults collected at 24,48 and 72 h post-treatment for enzyme activity bioassays.The results indicated significant decrease of GST activities in EC₁₀ and EC₃₀ treatments of imidacloprid and thiacloprid at 24,48,and 72 h in comparison to control.The lowest GST activity was 9.50?mol?min^-1?mg^-11 protein and5.75?mol?min^-1?mg^-11 protein detected in the 72 h treated organisms at EC₃₀ of imidacloprid and thiacloprid,respectively.Similar decrease tendencies in CYP450 and CarE enzyme activities were only observed in 72 h post-treatment.These findings suggest that alterations of these detoxified enzyme activities were involved in the response of F.candida to the neonicotinoids exposure,so that the detoxified enzyme activities could be used as biomarkers in evaluation of sublethal effects of neonicotinoids on F.candida.4.Sub-lethal effects of imidacloprid and thiacloprid on the transcriptomic profiles.The transcriptome sequencing was conducted to evaluate the induced or inhibited gene expressive patterns of F.candida by imidacloprid and thiacloprid exposure.20 days old F.candida adults were exposed to the dosage of EC₃₀ of imidacloprid or thiacloprid and acetone was used as control.Only live adults were collected after 72 h post-treatment for high-throughput RNA sequencing.A reference transcriptome was built by de novo assembly and functionally annotated.Overall,21,815 unigenes could be successfully annotated in databases.Among these unigenes,21,294 unigenes in NCBI NR database,17,490 unigenes in Swissprot,14,549 unigenes in GO,11,550 unigenes in KOG database and 3,315 unigenes in KEGG database.Among these unigenes,1,845 significant differentially expressed genes?DEGs?,including 960 up-and 885 down-regulated genes were identified in the comparison between imidacloprid treatment and control.Similarly,1,408 significant DEGs,including893 up-and 515 down-regulated genes were identified in the comparison between thiacloprid treatment and control.Among these DEGs,a total of one down-regulated AChE gene,7 CYP450 genes?1 up-and 6 down-regulated?,5 ESTs genes?all down-regulated?,2 GSTs genes?all down-regulated?,and 7UDP-glucuronosyltransferases?UGTs?genes?1 up-and 6 down-regulated?in imidacloprid treatment and 11 CYP450s genes?all down-regulated?,8 ESTs genes?2 up-and 6 down-regulated?,11 GSTs genes?1 up-and 10 down-regulated?,and 4 UGTs genes?all down-regulated?in thiacloprid treatment were identified.In addition,the gene ontology?GO?analysis indicated that the genes in pathways of cell,cell part,cellular process,binding and catalytic activity were the most dominant and significantly down-regulated in both treatments.Furthermore,KEGG enrichment showed six genes in pathways of metabolism of xenobiotics by cytochrome,steroid hormone biosynthesis,drug metabolism-cytochrome P450,retinol metabolism,linoleic acid metabolism and tyrosine metabolism were down-regulated.Further quantitative real-time PCR?qPCR?analysis validated transcriptomic data.In conclusion,the present study used multi-biomarker approach to provide comprehensive information on the toxicity of imidacloprid and thiacloprid on F.candida,and also provides an initial step for further researches in evaluating the toxicity of novel insecticides on F.candida.