Study On The Sublethal Effect Of Chlorpyrifos On The Predatory Natural Enemy,Eocanthecona Furcellata In Litchi Orchards

Eocanthecona furcellata（Wolff）,a common predatory natural enemy,is widely used in biological control of pests and plays an important role in the ecosystem of orchards.Although the occurrence of Eocanthecona furcellata in litchi orchards is not significant in number,it is regarded as one of the important natural enemies in the litchi orchard because of its prolonged presence,which can provide continuous biological control effect for the orchard.Currently,the pest control in lychee orchards still mainly relies on chemical pesticides.There are a total of 55 registered effective insecticide products for litchi,mainly organophosphorus and pyrethroid agents.Among them,15 products contain chlorpyrifos as the active ingredient,accounting for27.27%of the total registered products.Chlorpyrifos has a strong killing effect on various pests and is relatively low in cost,making it commonly used in lychee orchards.However,researches have shown that during the process of chemical control of pests,the use of insecticides may induce sublethal effects on predatory natural enemies,impacting their growth and development,fecundity,lifespan,behavior,and physiology.In view of this,upon the successful establishment of an experimental population of E.furcellata,the contact toxicity of chlorpyrifos on E.furcellata was determined,and the female adults were treated with LC₃₀-chlorpyrifos to study the effects on their biological characteristics,predatory behavior,physiology,and genetic expression.These results will help us to comprehensively understand the effects of chlorpyrifos on predatory natural enemies in pest control,and provide reference for the scientific application of pesticides in pest management of lychee orchards.The main results are as follows:（1）The toxicity of chlorpyrifos to the 2-day-old female adults of E.furcellata was determined by using the film coating method.At 48 hours after treatment,the LC₃₀and LC₅₀values of chlorpyrifos against E.furcellata females were calculated as 1.07 mg/L and 2.00 mg/L,respectively.Chlorpyrifos has high toxicity to E.furcellata.（2）The 2-day-old female adults of E.furcellata were treated with the LC₃₀concentration of chlorpyrifos.The pre-oviposition duration and oviposition duration of the F₀generation females treated with chlorpyrifos were 8.47±0.46 d、8.53±0.60 d,respectively,and the longevity and average number of eggs laid per female in a single oviposition event were 19.72±1.19d、24.93±3.61 grains,which showed no significant differences compared with the control group.Also,the duration of egg,and 1st-5th instar nymphs of F₁generation showed no significant differences between the chlorpyrifos-treated group and the control group,and the nymphal duration（18.05±0.47 d）was significantly longer than that of the control group.The survival rates of 2nd-5th instar nymphs of F₁generation exhibited slight decreases compared with the control group,but the differences were not significant.The pre-oviposition period,oviposition duration,and mean number of eggs laid per female of F₁generation female adults were 8.67±0.57 d,8.20±0.37 d,and 33.47±3.62grains,respectively,which also showed no significant differences compared with the control group.The mean generation time（T）of F₁generation in the chlorpyrifos-treated group was significantly extended,but the intrinsic rate of increase（r_m）,finite rate of increase（λ）,net reproductive rate（R₀）,and the population doubling time（D_t）were not significantly different from the control group.The sublethal concentration of chlorpyrifos(LC₃₀)had no significant effect on the fecundity of F₀and F₁generation female adults,the population survival rate of F₁generation,and the developmental period of eggs and1st-5th instar nymphs of E.furcellata,but significantly prolonged the mean generation time in the population life table parameters.（3）After treatment with the sublethal concentration LC₃₀of chlorpyrifos,the predation functional response type of the female adults of E.furcellata remained unchanged and continued to exhibit a Holling type II.Compared with the control group,E.furcellata treated with chlorpyrifos exhibited significantly lower prey handling time（T_h）（Chlorpyrifos-treated group:T_h=0.2468 d,control group:T_h=0.2906 d）,higher predation capacity（a/T_h）,and higher daily maximum predation（1/T_h）.And the searching efficiency of E.furcellata treated with chlorpyrifos increased by 12.61–5.88%when the density of T.molitor was 2-12 individual/box.In the predator density interference bioassay,the searching constant（Q）value and the disturbance coefficient（m）value of E.furcellata female adults in the chlorpyrifos-treated group were significantly higher than that in the control group,which indicated that chlorpyrifos had a synergistic effect on the mutual interference among individuals of E.furcellata female adluts.The sublethal concentration of chlorpyrifos(LC₃₀)did not change the predation functional response type of E.furcellata.However,it stimulated predation behavior and enhanced intraspecific predation interference effects.（4）Compared with the control group,the activity of peroxidase（POD）in the female adults of E.furcellata treated with LC₃₀-chlorpyrifos significantly decreased at 12 h after treatment.However,there were no significant changes in the activity of superoxide dismutase（SOD）,catalase（CAT）and alkaline phosphatase（AKP）at 24–60 h after treatment between the chlorpyrifos-treated group and the control group.Compared with the control group,the activities of glutathione S-transferase（GST）were significantly decreased at 12–60 h after treatment,and there were no significant differences in the activities of carboxylesterase（Car E）,acetylcholinesterase（ACh E）and cytochrome P450（CYP450）.POD and GST may be involved in the stress response of E.furcellata female adults to sublethal concentrations of chlorpyrifos.（5）High-throughput RNA-seq was employed to investigate the effect of chlorpyrifos at sublethal concentration LC₃₀on the transcriptome profiles of female adults of E.furcellata.The differentially expressed genes（DEGs）were subsequently screened,and the reliability of the transcriptome data were verified by RT-q PCR.RNA-seq results showed that 564 genes were significantly differentially expressed in E.furcellata exposed to chlorpyrifos,of which 260 genes were up-regulated and 304 genes were downregulated.GO function annotation results showed that the enriched DEGs are mainly linked to cellular process,metabolic process,developmental process process,reproduction,behavior,detoxification,binding,catalytic activity,antioxidant activity activity,and transcription factor activity,etc.KEGG pathway enrichment analysis showed that the most enriched pathway for DEGs was protein processing in the endoplasmic reticulum.Combining GO functional annotation and KEGG metabolic pathway analysis,this study indicated that a large number of DEGs were mainly involved in cellular processes,metabolic processes,catalytic activity,and protein processing metabolism.The sublethal concentration of chlorpyrifos has exerted a certain influence on the physiological metabolism and molecular level of E.furcellata.In conclusion,the sublethal effects of chlorpyrifos at concentration LC₃₀on the biological characters and predatory behavior of E.furcellata were clarified in this study.The changes in detoxifying and protective enzyme activities after exposure to chlorpyrifos were revealed.The differentially expressed genes related to chlorpyrifos stress were screened out.The safety of chlorpyrifos on E.furcellata was evaluated comprehensively.The results provide a theoretical basis for further elucidating the mechanism of the sublethal effect of chlorpyrifos on the growth,development and predation behavior of predatory natural enemies,and also provide a reference for the protection of predatory natural enemies in agro-ecosystem.This study has important theoretical and practical value.