Lethal And Sub-lethal Effects Of Chlorpyrifos On Non-target Predator Asian Ladybird Beetle,Harmonia Axyridis

The world population is increasing steadily.Recent population prospects of the UN show that the world population will increase to 9.1 billion in 2050.With such increasing trend in the world population,crop production needs to increase continuously to meet the food requirements of the world.The development and usage of insecticides will also increase to protect the field crops from damaging pests.Bio-control agents or natural enemies are randomly exposed to extensively applied insecticides because they are also present in the same field area along with the target pests.A number of studies have focused on negative effects of pesticides on natural enemies.Chlorpyrifos is a famous pesticide which is applied extensively to control pests of different orders including Diptera,Lepidoptera,Coleoptera and Hemiptera.Harmonia axyridis（Coleoptera:Coccinellidae）is a well-known natural enemy.This predator is considered very important part of different IPM strategies in many parts of world including Europe,Japan,China,Africa,and America.The key purpose of this study was to evaluate the adverse effects of chlorpyrifos on H.axyridis and assessment of its lethal and sublethal effects on different biological parameters including development period,survival rate,lifespan,feeding potential,reproduction,oviposition period and changes in H.axyridis transcriptome library.Results are as following:Acute toxicity determination24 hours old 3^rd instar larvae were used to determine the lethal and sublethal concentrations of chlorpyrifos.Micro applicator was used to treat 3^rd instar larvae with 1μl of pesticide solution.After 72 hours of pesticide treatment,acute toxicity data was recorded.Acute toxicity evaluation of chlorpyrifos revealed that LC₅₀of chlorpyrifos calculated for third instar H.axyridis larvae was 15.90 mg（a.i）L^-1 while LC10 and LC30 concentrations were calculated as 4.62 and 9.59 mg（a.i）L^-1respectively.Acute toxicity results showed that the development period of 3^rd instar larvae decreased at LC₁₀ concentration but it increased after treatment with LC₃₀ while development period of 4^th instar larvae and pupae was significantly prolonged at both concentrations(LC₁₀&LC₃₀).Sublethal effects of chlorpyrifos on H.axyridisChlorpyrifos sublethal concentrations(LC₁₀ and LC₃₀)were applied on 3^rd instar larvae for estimation of sublethal effects.Age-stage life-table technique was used to evaluate the sublethal effects.After pesticide application,development period,fecundity,survival rate,longevity of every individual was recorded after every 24 hours until the last individual dies.The results revealed that pre-oviposition period（TPOP+POP）and mean generation time（T）were significantly increased at both applied concentrations.But at the same time,female fecundity,male and female longevity,population growth parameters including intrinsic rate of increase（r）,finite rate of increase（λ）and net reproductive rate（R₀）were also negatively affected by LC₁₀ and LC₃₀.These results elaborated the adverse effects of chlorpyrifos on vital parameters of life table of H.axyridis,which may lead to a decrease the population growth and predation rate of biological control agents.Change in gene expression under insecticide stressFull genome transcriptome sequencing was carried out for estimation of changes in expression of genes in H.axyridis caused by chlorpyrifos.This technique provides information about the mechanism of a pesticide.H.axyridis larvae were treated with LC₃₀(9.59 mg（a.i）L^-1)of chlorpyrifos and acetone as control.Different DEGs（differentially expressed genes）caused by chlorpyrifos treatment were investigated.Two libraries having61038530 and 65419086 clean reads were formed for insecticide-treated and control group,which were assembled into 143648724 transcripts.All these transcripts were grouped into38722 unigenes.Out of these unigenes,19507 unigenes were found similar to non-redundant（Nr）NCBI proteins while 14089 unigenes were matched to Swiss Prot database.Out of these unigenes,54 genes showing significantly different expression（including 21down and 33 up-regulated genes）were recognized in comparison of control and chlorpyrifos-treated group.8 genes（two down-regulated and six up-regulated）were chosen for validation and q RT-PCR technique was applied for verification.It confirms that may alter different pathways and transcriptome profile of H.axyridis.