Sublethal Effect Of Deltamethrin On Habrobracon Hebetor And The Function Of Reproductive Related Genes

Ephestia elutella（Hübner）is a major storage pest on tobacco leaves,while Habrobracon hebetor（Say）is an important parasitoid natural enemy of lepidopteran pests,such as E.elutella,and plays an essential role in the biological control of storage pests.However,the successful control of storage pests,such as E.elutella,relying on H.hebetor is rare.Therefore,coordination of deltamethrin,a common protective agent used in tobacco storage,and H.hebetor is an effective control measure for successful control of E.elutella.But the direct and indirect lethal and sublethal effects of deltamethrin on H.hebetor are essential before integrated control of E.elutella.Therefore,based on the optimization of the rearing system of E.elutella as host.The indirect effects of sublethal dose of deltamethrin on the exposure E.elutella were evaluated by two sex life table theory.The direct effects of sublethal dose of deltamethrin on the fitness of contemporary and next generation parasitoid wasps were evaluated by the same method.The transcriptome of adult H.hebetor treated with sublethal dose of deltamethrin was measured.Then,the Vg,VgR and EcR were cloned and their functions were verified.This study was aimed to investigate the sublethal effect of deltamethrin on the parasitoid wasp and its reproductive response to deltamethrin.At the same time,the results provided a scientific basis for coordinating the control of storage pests with H.hebetor and deltamethrin.The main results as following:1.Effects of different diets on growth,development and reproduction of Ephestia elutellaIn order to determine the life table parameters of E.elutella under the conditions of artificial diet and tobacco leaves,the rearing system of E.elutella was optimized to provide sufficient host for rearing the natural enemy of H.hebetor.The growth and development,reproduction,population growth potential and mass rearing system of E.elutella were evaluated by age-stage two sex life table theory（hereinafter the same）.The results showed that there were significant differences in the development period of E.elutella reared on two diets,and the survival rate of E.elutella reared on artificial diets（88%）was significantly higher than that of tobacco leaves（73%）.In the artificial diet,the preoviposition（APOP）,total preoviposition（TPOP）,fecundity,number of spawning days（O_d）and daily spawning amount（E_d）of E.elutella were significantly better than those rearing on tobacco leaves.In the same diet,the intrinsic growth rate（r）and the finite rate of increase（λ）of single feeding were significantly higher than those of group rearing.The population parameters r,λand R₀ were all higher and the average generation period（T）was shorter when E.elutella was fed on artificial diets,including both reared in single or group.The population prediction results showed that the artificial feed could raise a large number of E.elutella larvae.When 10,000 larvae of the last instar of E.elutella were harvested daily on two diets,the daily cost savings of the artificial feed system was 74.94 yuan.Therefore,artificial diet is more suitable for rearing E.elutella than tobacco leaves.The results provide theoretical and practical basis for mass rearing of E.elutella.2.Indirect effects of sublethal dose of deltamethrin on fitness of Habrobracon hebetorIn order to determine the indirect effect of sublethal dose of deltamethrin on fitness of H.hebetor,adult H.hebetor was placed into 5th instar larvae of E.elutella infected with sublethal dose of deltamethrin(control,LC₅,LC₁₀).The growth,development,reproduction,paralysis rate,population growth potential and variability of H.hebetor under two treatments and control were analyzed by using two sex life table theory.The toxicity test results showed that LC₅ and LC₁₀ were 51.90 mg·L^-1 and 79.08 mg·L^-1,respectively.The survival rate of H.hebetor was significantly decreased with the increase of deltamethrin concentration.And the survival rate of LC₁₀ treatment（40.98%）was significantly lower than that of control（75.92%）.The intrinsic growth rate（r）,the finite growth rate（λ）and net growth rate（R₀）decreased with the increase of sublethal dose of deltamethrin and there was no significant difference with control.The sublethal dose of deltamethrin significantly inhibited net paralysis rate（C₀）and conversion rate（Q_p）.In addition,the population growth potential,paralysis potential and variability were significantly affected by sublethal dose of deltamethrin.Indirect lethal effect of deltamethrin sublethal dose(LC₅ and LC₁₀)was confirmed on H.hebetor.Therefore,careful consideration should be taken when using H.hebetor and deltamethrin together to control storage pests such as E.elutella to ensure the safety of natural enemies.3.Direct effect of deltamethrin sublethal concentration on the pupae of Habrobracon hebetorIn order to investigate the effects of deltamethrin on biological characteristics of H.hebetor and to determine the effects of sublethal dose of deltamethrin on parental（F₀）and offspring（F₁）populations of the wasp,the sublethal concentration of deltamethrin on pupa stage of H.hebetor was determined by dipping method,and LC₁₀,LC₂₅ and LC₅₀ of deltamethrin on pupa stage of H.hebetor were calculated by PROBIT-MSChart.The effects of sublethal concentration of deltamethrin on reproductive fitness of F₀ generation,growth and development,reproduction,paralysis rate and population growth potential of F₁ generation were also evaluated by two sex life table theory.According to the results of laboratory bioassay,LC₁₀,LC₂₅ and LC₅₀ of deltamethrin on pupa stage of H.hebetor were 112.61,191.27 and 344.56 mg.L^-1,respectively.There was no significant difference in the life span of adult of F₀ generation treated with deltamethrin sublethal concentration（27.5 d in treatment and 29.83 d in control）.The average fecundity of LC₅₀（441.55 eggs）was significantly lower than control（535.87 eggs）,but the number of spawning days was not significantly different.The average daily egg production of LC₅₀（21.18 eggs/d）was significantly lower than control（23.49 eggs/d）and LC₂₅（22.27 eggs/d）,but there was no significantly difference from LC₁₀（21.41 eggs/d）.In addition,the daily and total paralysis rates were significantly affected by the sublethal concentration of deltamethrin.These sublethal concentrations had significant effects on the developmental period and survival rate of the preadult of F₁ generation.But remaining parameters had no significant difference.The intrinsic growth rate（r）,the finite rate of increase（λ）,net proliferation rate（R₀）,mean generation period（T）,net paralysis rate（C₀）,circumscribed paralysis rate（ω）and stable paralysis rate（ψ）were not affected by sublethal concentrations of deltamethrin.The population growth trend and paralysis rate trend were similar in the three treatments and control.Sublethal dose of deltamethrin inhibited fecundity and paralysis rate of F₀ generation of H.hebetor,and then affected the population and damage control ability of this offspring.The sublethal dose of deltamethrin significantly decreased the survival rate of F₁ generation,but had no significant effect on remaining indexes,suggesting that it was feasible to apply deltamethrin for emergency control of E.elutella when the wasp H.hebetor was in the pupal stage.4.Direct effect of deltamethrin sublethal concentration on the female of Habrobracon hebetorIn order to investigate the effects of deltamethrin on biological characteristics of H.hebetor and to determine the effects of sublethal dose of deltamethrin on parental（F₀）and offspring（F₁）populations of the wasp.Sublethal dose of deltamethrin on adult wasp was determined by test-tube drug film method.LC₂₅ and LC₅₀ values of deltamethrin on adult wasp were calculated by PROBIT-MSChart software.The effects of sublethal dose of deltamethrin on reproductive fitness of F₀ generation,growth and development,reproduction,paralysis rate and population growth potential of F₁generation were evaluated by two sex life table theory.According to the results of laboratory bioassay,LC₂₅ and LC₅₀ of deltamethrin for 24 h treatment was 178.45 and251.91 mg.L^-1,respectively.With the increase of sublethal concentration,the average fecundity of LC₂₅（358.43 eggs）and LC₅₀（242.04 eggs）decreased as compared to control（511.41 eggs）by 29.91%and 52.67%,respectively.In addition,the total paralysis rate of H.hebetor decreased significantly as LC₂₅（158.82）and LC₅₀（122.13）decreased with the increase of sublethal dose when comparing control group（195.32）by 18.69%and 37.47%,respectively.These sublethal concentrations had no significant effects on pupal stage and female wasp life except for F₁ generation.The intrinsic growth rate（r）,Finite rate of increase（λ）,net proliferation rate（R₀）,mean generation period（T）,net paralysis rate（C₀）,circumscribed paralysis rate（ω）,stable paralysis rate（ψ）were not significantly affected by sublethal concentration of deltamethrin.The trends in population growth and paralysis rate were similar in the two treatment and control groups,but the actual（no common logarithm was taken）population size and paralysis potential were affected by the sublethal dose of deltamethrin.Sublethal concentration of deltamethrin inhibited fecundity and paralysis rate of F0 generation,thus affecting the population size and damage control potential of the parasitoid wasp,but had no significant effect on population parameters and paralysis rate parameters of F₁ generation.The results suggest that deltamethrin should be applied with caution when the wasp H.hebetor during adult stage.5.Transcriptome analysis of Habrobracon hebetor under deltamethrin sublethal concentration stressIn order to further explore the effects of deltamethrin sublethal concentration stress on the development and reproduction of H.hebetor,transcriptome analysis was performed to obtain the key pathways and genes affecting the reproduction of H.hebetor under deltamethrin stress.The 76.44 Gb data of each treatment were sequenced.After assembly and redundancy removal,29,510 unigenes were obtained.The total length,maximum length,minimum length,N50 and GC content were 29,743,152 bp,13,233 bp,297 bp,2,016 bp and 43.62%,respectively.These unigenes were compared and annotated with seven functional databases.The most matched species was Diachasma alloeum（56.46%）by NR annotation and sequence homology comparison.A total of 591 unigenes were differentially expressed under deltamethrin sublethal concentration LC₁₀,including 519 up-regulated and 72 down-regulated expressions.Under the stress of deltamethrin sublethal concentration LC₂₅,the number of differentially expressed unigenes increased to 1,938,of which 1,104 were up-regulated and 834 down-regulated.The GO functional enrichment analysis of the differential genes under deltamethrin stress showed that the functional group significantly enriched in ACK vs ALC10 and ACK vs ALC25 was serine-type endopeptidase activity,extracellular region,fatty acid biosynthetic process,odorant binding,carbohydrate metabolic process,integral component of membrane,iron ion binding,transmembrane transporter activity,zinc ion binding and RNA binding.Functional enrichment of KEGG indicated that most of the differentially expressed genes in ACK vs ALC10 and ACK vs ALC25 were in protein digestion and absorption,Toll and Imd signaling pathway,PI3K-Akt signaling pathway,focal adhesion,ECM-receptor interaction and fat digestion and absorption.6.Cloning and functional research of HhVg,HhVgR and HhEcR genes of Habrobracon hebetorThe HhVg,HhVgR and HhEcR genes of H.hebetor were cloned based on the transcriptome data.The HhVg have a cDNA full length sequence of 5,545 bp,ORF length of 5,499 bp and encoding 1,832 amino acids.The HhVgR have a cDNA full length sequence of 5,670 bp,ORF length of 5,163 bp and encoding 1,720 amino acids.The HhEcR have a cDNA full length sequence of 2,058 bp,ORF length of 1,884 bp and encoding 627 amino acids.The expression of HhVg and HhVgR were higher in the 6th and 7th day of pupa and within 2 hours of emergence,and increased sharply at 6 h to24 h after emergence.The expression of HhVg and HhVgR were significantly higher in female than male.However,the HhEcR gene was highly expressed in 3-day-old and 5-day-old male pupae and 24 h emergence males.Gene function was studied by RNAi,and the results showed that the expression levels of corresponding target genes were significantly inhibited after injected dsVg,dsVgR or dsEcR alone.In addition,the ovaries injected with dsVg showed a decrease in the number of ovular chambers,a smaller ovular chamber and less yolk deposition.Similarly,dsVgR and dsEcR injected were also reduced the number of ovular cells and deposited less or no yolk.