| A continuous yield increase of rice is compulsory requirement for world growing population.In order to reduce crop losses due to outbreak of insect pests’,the development and application of different novel insecticides are also increased.However,the hazardous effects of insecticides on human health,environment and natural biological control agents are attracting public attentions.Natural Biological control agents share the same habitat with insect pests,which makes biological control agents easily to be exposed to insecticides used for the management of insect pests.This exposure can be via any direct or indirect route.The potential adverse effect of insecticides on the non-target organisms is an indispensable part of biosafety assessment of insecticide.Many previous studies demonstrated that insecticides have a significant impact on the life-table parameters of biological control agents.Paederus fuscipes(Rove Beetle)is an important biological control agent which can control many destructive insect pests,including various rice insect pests.In rice agro-ecosystem rove beetle is an important predator of brown plant hopper(Nilaparvata lugens).A single adult of rove beetle can consume 9-10 nymphs of brown planthopper per day.It is commonly used as a biological control agent worldwide including China,Malaysia,Thailand and Japan etc.Emamectin benzoate and chlorantraniliprole are two insecticides to control many Coleopteran,Lepidopteran and Dipterous pests on different crops.Emamectin benzoate and chlorantraniliprole are usually used in rice fields for controlling many rice pests such as the rice leaf folder(Cnaphalocrocis medinalis)and the rice stem borer(Chilo suppressalis),but there are little reports about the potential adverse effect research of these two insecticides on P.fuscipes.In the current study,the acute toxicity of emamectin benzoate and chlorantraniliprole against P.fuscipes was evaluated,and the effect of sublethal doses on survival,developmental time,reproduction,body weight,enzymatic activity and transcriptional profile changes of P.fuscipes was measured.The results are as followed.1.Lethal and sublethal effects of emamectin benzoate on P.fuscipesThe results from the present study show that the LC10,LC30 and LC50 of emamectin benzoate to adults of P.fuscipes at 72 h was 1.54(0.39-2.31),2.31(1.03-3.82)and 3.07(1.84-4.54)mg a.i.L-i,respectively,whereas the LC10,LC30 and LC50 of emamectin benzoate to the second instar larvae of P.fuscipes at 72 h was 1.45(0.67-1.92)and 1.77(1.02-2.24)and 2.58(1.95-3.19)mg a.i.L-1,respectively.At the dosage of LC10 and LC30,the second instar developmental time,pre pupal duration,pupal duration and pre-oviposition period was significantly prolonged due to exposure of emamectin benzoate as compared to that of the control.Compared to the control,the LC30 and LC10 significantly reduced the feeding potential of the second instar larvae and the adults emerged from emamectin benzoate treated second-instar larvae.Additionally,the fecundity of the adults emerged from emamectin benzoate treated second-instar larvae also significantly lower than that of control.LC30 significantly reduced the body weight of adults emerged from emamectin benzoate treated larvae as compared to those in control.The fecundity and the feeding potential of the adults directly treated with sublethal dose of emamectin benzoate were significantly reduced in LC30 as compared to LC10 and control.These results revealed that sublethal doses of emamectin benzoate negatively affected biological activities of P.fuscipes such as the development and reproduce.2.Lethal and sublethal effects of chlorantraniliprole on P.fuscipesThe LC10,LC30 and LC50 of chlorantraniliprole to adults of P.fuscipes at 48 h was 93.68(18.57-177.57),137.68(37.66-236.46)and 535.49(344.28-859.95)mg a.i.L-1 respectively,whereas the LC10,LC30 and LC50 of chlorantraniliprole to the second instar larvae of P.fuscipes at 48 h was 21.22(12.04-30.84),56.48(40.99-71.59)and 111.24(89.59-137.19)mg a.i.L-1,respectively.The LC50 of chlorantraniliprole to P.fuscipes is higher than the recommended dose(59.38 mg a.i.L-1)in rice field.Tested sublethal dose(LC30)had significantly prolonged the pre-imaginal developmental duration of P.fuscipes as compared to that of in LC10 and control.Additionally,the LC30 increased the pre-oviposition period of adults emerged from larvae treated with chlorantraniliprole at second instar.The fecundity of adults emerged from the second-instar larvae treated with LC30 and LC10 was unaffected and was the same as control.The body weight of female adults emerged from the second-instar larvae treated with LC3O was significantly lower as compared to control,while male body weight was non-significantly reduced in LC30 compared to control and LC10.Feeding potential of second instar larvae was also reduced due to insecticide,while adult feeding potential was non-significantly affacted.These results revealed that sublethal doses of chlorantraniliprole negatively affected the development and biological activities of P.fuscipes.As emamectin benzoate was found more toxic than chlorantraniliprole,so the sublethal concentrantions of emamectin benzoate LC10 and LC30 were subjected for enzymatic activity assessment and transcriptomic studies.3.The activity of detoxification-related enzymes of P.fuscipes exposed to emamectin benzoateIn different biological processes many enzymes take part in many metabolic processes and detoxification processes of insecticides.CYP450,glutathione S-transferase(GST)and esterase enzymes were the most widely reported detoxification enzymes in the process of detoxification of insecticide,and highly expressed in resistant strain of insect species to different insecticide group.Therefore,we measured the activity of the three detoxification enzymes of P.fuscipes exposed to emamectin benzoate.Results showed that the activity of P450 enzyme was significantly higher in LC30 and LCIO of emamectin benzoate as compared to control,at 72 h after treatment.While in the activity of P450 enzyme at 12h,24,and 48 hours was not affected in LC30 and LC10 as compared to control.Results of GST enzymes activity showed that there was no significant difference between the treatments and control at 12 and 24 h after exposure to emamectin benzoate.The GST activity was significantly higher in LCIO and LC30 of emamectin benzoate at 48 and 72 h as compared to those in control.The results of esterase enzyme activity assessment showed that there was significant increase in esterase activity after 12 and 24 h exposed to LC30 dose of emamectin benzoate but there was no significant cahnge after 48 and 72 h.The esterase enzyme activity was not influenced by LC10.4.Genes inductive to Emamectin benzoate treatmentTranscriptional sequencing was also done to evaluate the gene expression due to exposure of emamectin benzoate to P.fuscipes.Second instar larvae of P.fuscipes was treated with LC30(2.64 mg a.i.L-1)of emamectin benzoate and acetone as control.Alive second instar larvae were collected after 24h exposeing for mRNA sequencing.Differentially expressed genes(DEGs)analysis revealed that 2257 unigenes were significantly regulated,from which 599 up-regulated and 1655 down-regulated genes.After gene function classification(GO)analysis,the metabolic process are the most affected GO item(809 DEGs),then catalytic process(715 DEGs),binding(607 DEGs),organic substances metabolic process(584 DEGs)and signal-organism process(580 DEGs).Among the up-regulated DEGs,most DGEs(171 DEGs)were present in Go item of metabolic process,followed by binding(163 DEGs),catalytic process(150 DEGs),organic substance metabolic process(123 DEGs)and primary metabolic process(122 DEGs),In case of down-regulated GO terms,metabolic process are the Go iterm that contains most DEGs(151 DEGs),then catalytic process(139 DEGs),organic substance metabolic process(125 DEGs)and primary metabolic process(121 DEGs),binding(101 DEGs).Results from comparison of up and down regulated GO terms showed that P.fuscipes mainly depressed the gene expression in response to emamectin benzoate exposure.KEEG pathway analysis showed Signal transduction(1421 DEGs),transport and catabolism(984 DEGs)and translation(862 DEGs)were the three most highly affected pathways to emamectin exposure.Many other pathways containing detoxification related genes were found to be affected by emamectin benzoate e.g."retinol metabolism"(20 DEGs),"linoleic acid metabolism"(5 DEGs),"chemical carcinogenesis"(33 DEGs),"steroid hormone biosynthesis"(17 DEGs),"glutathione metabolism"(20 DEGs),"drug metabolism-other enzymes"(39 DEGs),"drug metabolism-Cytochrome P450"(21 DEGs),"metabolism of xenobiotics by cytochrome P450"(29 DEGs),“pathways of cancer"(19 DEGs)and"insect hormone biosynthesis"(4 DEGs).Subsequently,a number of insecticides-related genes which were differentially regulated such as CYP gene,UGT genes,AChE genes,HPGDS regulating genes and GST regulating genes were identified during DEGs analysis.For transcriptome analysis validation,twelve insecticide related genes from above mentioned pathways were selected and verified through qPCR analysis.Results demonstrated that the results of transcriptome analysis was in accordance with qPCR analysis.Therefore,the results of transcriptome analysis can be a guide way for further molecular research.Present study was designed to assess the adverse effects emamectin benzoate and chlorantraniliprole on P.fuscipes by using different insecticide risk assessment approaches.A set of comprehensive information was provided regarding potential adversities of novel insecticides application on biological control agents.The other aspect of this study was to enlighten the issue of regarding selection and usage of insecticides in the presence of P.fuscipes in different agro-ecosystem.On the other hand,it provides an initial step for further research in evaluating the toxicity of novel insecticides on P.fuscipes with advanced technologies. |
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