| The production of crops needs to increase continuously to fulfill food requirements of growing population around the world.In order to overcome the crop loss due to insect pest‘s outbreak,the development and application of novel insecticides will also be increased.However,the harmful effects of insecticides on the environment,human health and biological control agents are the burning issue of the world.Biological control agents are also inhabited in same crop area of insecticides target pests,which is frequently exposed to insecticides via different routes directly or indirectly.The potential adverse effects of these insecticides on biological agents are drawing the intensive attention of the public and scientists.There have been a large number of reports on the biological impacts of pesticides on biological control agents.Chlorantraniliprole and sulfoxaflor are two widely used pesticides to control Coleoptera,Lepidoptera,Diptera and Hemiptera insect pests in different agriculture cropping systems.However,Harmonia axyridis(Coleoptera: Coccinellidae)is one of the most important biological control agents to control a wide range of destructive insect pests,including various aphids species,coccids,aleyrodids,whiteflies,aphids,mealybugs,scales,mites and psyllids species.It is commonly used as a biological control alone as well as an important component of IPM programs in several countries of the world including China,Japan,America,Africa and Europe,etc.In the current study,we examined the accurate toxicity of chlorantraniliprole and sulfoxaflor insecticides on H.axyridis and evaluated the effects of chlorantraniliprole and sulfoxaflor even at the sublethal dosage on the survival,developmental time,lifespan,reproduction,body weight,enzymatic activities and variation in transcriptome profile of H.axyridis.The results are as followed.1.The acute toxicity bioassaysThe lethal and sublethal concentrations range of both insecticides determined on H.axyridis 2nd instar larvae(? 24 h-old).The subjected larvae were treated topically with 1?l of the particular solution on ventral abdominal region using micro-applicator.The acute toxicity data were recorded at 72 h post-treatment.The acute toxicity results revealed that LC50 value of chlorantraniliprole for second instar H.axyridis larvae was estimated 36.67 mg a.i./L while the LC10 and LC30 values were assessed to be 2.42 and 12.06 mg a.i./L respectively.Sulfoxaflor acute toxicity results indicated that LC50 value was 53.42 mg a.i./L and the estimated concentrations of sulfoxaflor causing 30% and 10% mortality were 21.30 mg a.i./L and 5.65 mg a.i./L respectively.These two insecticides are considered harmful to H.axyridis because the LC50 values of both insecticides were found lower than a wide range of recommended field doses,which are used to control numerous target pests in the fields.Therefore,it was needed to evaluate sublethal effects of both insecticides at lower doses.2.The sublethal effects of chlorantraniliprole and sulfoxaflor on H.axyridis life-table parametersThe sublethal concentrations(LC30 and LC10)of chlorantraniliprole and sulfoxaflor adopted for sublethal effect bioassays on H.axyridis life-table parameters.The sublethal effects evaluated according to the age-stage two-sex life-table procedure.In this bioassay,a group of 100 H.axyridis second instar larvae(? 24 h)was treated topically with LC10 and LC30 of insecticide and control(only acetone)using the topical method as described above.After treatment,the survival rate,developmental time,fecundity and longevity of each individual at all life stages were recorded daily until the death of the last individual.In addition,the body weight of second instar larvae was measured at 24 h post-treatment,while other developmental stages were weighed upon fresh emergence.The results of chlorantraniliprole sublethal effects on H.axyridis life table parameters indicated that the development time of second,fourth instar larvae and pupa was significantly prolonged in populations treated with LC10(2.42 mg a.i./L)and LC30(12.06 mg a.i./L).Similarly,the preoviposition period(POP)was significantly prolonged,while adult longevity and fecundity were significantly reduced following treatment compared to control.Additionally,the long-term growth parameters including the net reproductive rate(R0),as well as the intrinsic(r)and finite rate of increase(?)were significantly decreased in groups treated with the chlorantraniliprole.In the same way,the sublethal effects of sulfoxaflor insecticide were examined on life-table parameters and body weight of second instar H.axyridis larvae.The sublethal results indicated that the pre-adult developmental time,mean generation time(T)and total pre-ovipositional period(TPOP)were significantly prolonged in sulfoxaflor sublethal concentrations LC10(5.65 mg a.i./L)and LC30(21.30 mg a.i./L)treated populations in comparison with control.In addition,the adult longevity and fecundity were significantly reduced in treated cohorts.Meanwhile,the population growth parameters were also adversely affected by LC10,and LC30 such as the intrinsic rate of increase(r)was decreased to 0.10 d-1 in LC30 treated population,which was followed by LC10(0.11 d-1),where these values were significantly lower than control(0.12 d-1).The similar tendency was found in the finite rate of increase(?),and net reproductive rate(R0)in sulfoxaflor treated population.Additionally,the body weight in different life stages of H.axyridis was found significantly decreased in sulfoxaflor-treated populations.These sublethal results revealed that both chlorantraniliprole and sulfoxaflor has adversely affected the vital life-table parameters of H.axyridis,which may lead to decrease the population growth and predation rate of biological control agents.H.axyridis might be engaged in detoxification process under insecticide stress,which leads to adverse effects on its biological,developmental and reproduction processes.Therefore,further investigation was necessary to evaluate detoxification enzymes response of H.axyridis under pesticide exposure.3.The detoxification-related enzymes induction under sulfoxaflor pressureThe activities of detoxification-related enzymes such as glutathione-S-transferase(GST),cytochrome P450(P450)and carboxylesterases(Car Es)to sublethal concentration of sulfoxaflor were determined.During this study,second-instar H.axyridis larvae were exposed to LC10(5.65 mg a.i./L)and LC30(21.30 mg a.i./L)of sulfoxaflor along with their control(acetone)as described above.Only live larvae collected at 24 h,48 h and 72 h post-treatment for enzymes activity bioassays.The results showed that GST activity was significantly raised in LC10 and LC30 treatment of sulfoxaflor at 24 h,48 h,and 72 h in comparison to control.Similarly,increase tendency in P450 and Car E enzymes activities were also observed in sulfoxaflor treated populations as compared to control after 72 h of treatments.Therefore,GST enzyme was mainly involved in sulfoxaflor detoxification mechanism in H.axyridis,while P450 and Car E enzymes are partially involved.4.Genes variation and expression under insecticide pressureThe genome-wide transcriptome sequencing was conducted to evaluate gene expression pattern of H.axyridis induced by sulfoxaflor exposure,which was necessary to understand the action mechanisms of this insecticide in H.axyridis.Therefore,secondinstar H.axyridis larvae were exposed to LC30(21.29 mg a.i./L)of sulfoxaflor and control as described above.Only live larvae were collected at 24 h for high-throughput RNA sequencing.The differentially expressed genes(DEGs)induced by sulfoxaflor exposure were analyzed.Two libraries were generated with 50,702,976 and 47,005,096 clean read for sulfoxaflor-treated larvae and control respectively,which grouped into 112,497 transcripts.These transcripts further assembled into 52,229 unigenes,of which 33,820 and 25,175 unigenes were matched to proteins in the NCBI non-redundant protein sequences(Nr)and Swiss Prot databases,respectively.From these unigenes,794 significant differentially expressed genes,including 446 up-and 348 down-regulated genes were identified in the comparison between sulfoxaflor-treated and control.Consequently,a number of the insecticide-related differentially expressed genes such as P450 s,GSTs,USTs,ESTs,and others were identified from DEGs analysis.In addition,the gene ontology(GO)analysis indicated that the majority of Go terms were significantly down-regulated in sulfoxaflor-treated larvae.Furthermore,these insecticiderelated DEGs were also involved in some critical KEGG pathways such as retinol metabolism,drug metabolism,metabolism of xenobiotic and prolactin signaling pathways.For transcriptome validation,ten insecticide-related genes were selected and verified through quantitative real-time PCR(q RT-PCR)analysis.These results demonstrate that sulfoxaflor can induce variation in H.axyridis transcriptome profile and pathways,which established an initial step for further molecular investigations.In conclusion,the present study demonstrates the adverse effects of chlorantraniliprole and sulfoxaflor on H.axyridis by using different insecticide risk assessment approaches.This study provides comprehensive information regarding the potential adverse effects of these novel insecticides application on biological control agents.On the one hand,these findings offer important issues in regarding the selection and use of insecticides in the presence of H.axyridis in both IPM and chemical control in different cropping systems.On the other hand,it provides an initial step for further research in evaluating the toxicity of novel insecticides on biological control agents with advanced technologies. |
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