The Adaptability And Mechanism Of Ahasverus Advena（waltl）to Aflatoxin B₁

The relationship between insects and mycotoxins among ecological factors is an important part of stored grain ecosystem.Knowing the adaptability and mechanism of insect to mycotoxin would helpful for understanding the relationship among ecological factors in grain storage ecosystem.Ahasverus advena（Waltl）can feed on the mycelia and spores grow on the grain,and can survive in the environment contained mycotoxin.The adaptability and mechanism of A.advena to mycotoxin have not been reported.The development durations,survival rates,and feculity of A.advena cultured in the diet contained aflatoxin B₁（AFB₁）at content of 0 mg/kg,2.5 mg/kg,12.5 mg/kg,62.5 mg/kg,250 mg/kg,312.5 mg/kg,500mg/kg,1000 mg/kg,1562.5 mg/kg,2000 mg/kg,4000 mg/kg,8000 mg/kg,and 16000mg/kg were studied.The activities of antioxidant enzyme,detoxification enzyme,digestive enzyme,and amounts of MDA and GSH in larvae and adults of A.advena feed on the diet contained AFB₁ at 0 mg/kg,500 mg/kg,1000 mg/kg,2000 mg/kg,4000 mg/kg,8000 mg/kg,and 16000 mg/kg for 48 h were measured.Transcriptome levels of A.advena larvae and adults cultured in diets contained AFB₁ at 0 mg/kg and 1671 mg/kg were detected.The expression stability of candidate reference genes,expression levels and function of related genes in A.advena at different insect stages cultured in the diets with AFB₁ at concentrations of 0 mg/kg,842 mg/kg,and 1671 mg/kg were analyzed.AFB₁ contents in the maize infested by larvae and adults of A.advena at densities of 15 individuals/kg,45 individuals/kg,and135 individuals/kg were detected in different times.The results are as follows:（1）The developmental duration from egg to adult increased from 20 d with AFB₁ at 0mg/kg to 24 d with AFB₁ at 1562.5 mg/kg and mainly contributed by larvae duration prolonged,and had no significant effect on the egg and pupae durations in the tested content.At a concentration of 2.5 mg/kg,the larvae that experience 3 times ecdysis disappeared,the larvae’s rate that experience 4 times ecdysis decreased from 41%to 31%,the larvae’s rate that experience 5 times ecdysis increased from 39%to 79%.The survival rates of one-day-old,five-day-old,and ten-day-old larvae decreased more than 13%compared to the controls with AFB₁ over 500 mg/kg.AFB₁ at 1000 mg/kg had significant effect on the survival rates of adults A.advena after 60 d exposure,which decreased by 9.4%compared with the controls.The LD₅₀ values of AFB₁ to one-day-old larvae,five-day-old larvae and adults were 1671 mg/kg,2696 mg/kg,and 5768 mg/kg,respectively.The fecundity of adults had decreased over 20%after feeding on diets with AFB₁ more than 500 mg/kg compared to the controls.（2）AFB₁ in diets significantly increased the activities of SOD（7～10%）,CAT（27～33%）,POD（45～62%）,GSH-Px（14～28%）,GST（50～88%）,P450s（54～138%）,Ach E（71～132%）,and trehalase（36～41%）and the levels of MDA（107～125%）in the larvae of A.advena compared with the controls.AFB₁ in diets also significantly increased the activities of SOD（12～16%）,CAT（15～17%）,POD（45～55%）,GR（15～43%）,GSH-Px（48～189%）,GST（102～154%）,P450s（91～213%）,Ach E（49～100%）,and trehalase（14～38%）and the levels of MDA（71～150%）in the adults of A.advena compared with the controls.The levels of GSH decreased for larvae（20～58%）and adults（41～78%）after feeding on the diets contained AFB₁ compared to the controls.In adults,the activity of protease increased（12～28%）with AFB₁ at content of 500 mg/kg and 1000 mg/kg,and decreased by 16%and16%with AFB₁at 8000 mg/kg and 16000 mg/kg compared to the controls.There was no significant change in the activities of amylase and lipase with any of the treated concentrations for larvae and adults of A.advena.（3）In the results of transcriptome sequencing,approximately 2.2 million raw reads and119090 unigenes with the length between 201 bp and 36847 bp,mean length 718 bp,about31.34 Gb were obtained.The unigenes were blast with Nr,Nt,KO,GO,Swiss-prot,Pfam,and KOG databases,and a total of 56224 unigenes had been annotated in at least one database and accounted for 47.2%,and 5492 unigenes had been annotated in all seven databases and accounted for 4.6%.After AFB₁ exposure,there were 2768 and 719differentially expressed genes in larvae and adults of A.advena,respectively,and 82differentially expressed genes in common.GO annotation,KEGG metabolic pathway analysis,and Nr comparison were performed on the differentially expressed genes.Four kinds of unigenes related to AFB₁ metabolism in A.advena were obtained:（a）phase I related gene（P450 gene）;（b）phase II related genes（that responsible for coding various transfer function protein,such as GST）;（c）oxidative metabolism related genes;（d）genes related to cell apoptosis and fatty acid metabolism.The suitable candidate reference genes in different stages of A.advena for qRT-PCR were as follows:RPS27,α-tub,EF1-α,and GAPDH for five-day-old larvae;β-actin,RPS27,GAPDH,andα-tub for ten-day-old larvae;GAPDH,RPS3,RPS27 and G6PDH for pupae;RPS27,GAPDH,RSL40,and EF1-αfor adults;RPS27,GAPDH,β-tub,andα-tub for all samples.RPS27 and GAPDH were most stability among 13 candidate reference genes and suitable reference genes for qRT-PCR in subsequent experiments.RPS27 gene was used as the reference gene for qRT-PCR.The expression levels of Aa CYP304A1,Aa CYP4D2,and Aa GSTs1 were relatively high in pupae and 10-day-old larvae,and were relatively low in five-day-old larvae and adults.For 5-day-old larvae,10-day-old larvae,pupae and adults cultured in the diets with AFB₁ at 842 mg/kg,the expressions levels increased by 1.9 times,1.7 times,5.9 times,and 24.4 times for Aa CYP304A1;3.3 times,2.9 times,3.5 times,and 31.3 times for Aa GSTs1;1.5 times,0.8times,2.0 times,and 26.7 times for Aa CYP4D2,respectively,compared with the controls.After feeding ds304A1,ds GST,and ds4D2 for 3 d,the expression levels of Aa CYP304A1,Aa GSTs1,and Aa CYP4D2 were decreased by 49%,56%,and 36%,compared with the controls.The survival rates of A.advena after feeding on ds RNA cultured in the diet contained AFB₁ at a content of 1000 mg/kg decreased from 57%to 28%,23%,and 40%,respectively,compared with the controls.（4）AFB₁ content decreased significantly in the maize contained 5%broken kernels that infested larvae of A.advena at density of 15 individuals/kg,45 individuals/kg,and 135individuals/kg after 28 d,21 d,and 14 d,AFB₁ content decreased from 38.8μg/kg to 33.5μg/kg,27.1μg/kg,and 24.4μg/kg in 35 days later,respectively;For the maize with 10%broken kernels,AFB₁ content decreased significantly after 35 d,21 d,and 21 d and reduced from 38.3μg/kg to 33.8μg/kg,28.6μg/kg,and 26.7μg/kg in 35 d later,respectively.AFB₁content was lower in the maize with 5%broken kernels that infested adults of A.advena at densities of 15 individuals/kg,45 individuals/kg,and 135 individuals/kg after 21 d,21 d,and 14 d than that of the controls,the content of AFB₁ decreased from 38.8μg/kg to 29.4μg/kg,24.6μg/kg,and 18.7μg/kg in 35 days later,respectively;For the maize with 10%broken kernels,AFB₁ content decreased significantly after 21 d,21 d,and 14 d,AFB₁content decreased from 38.3μg/kg to 30.7μg/kg,26.1μg/kg,and 22.3μg/kg in 35 days later,respectively.The mold quantity decreased from 8.6×10⁶ cfu/g to below 4.1×10⁶ cfu/g in 35days in maize infested larvae and adults of A.advena at density of 135 individuals/kg.In conclusions,the developmental durations and molting times increased,and the survival rates decreased of A.advena after feeding on the diets contained AFB₁.Ahasverus advena had strong adaptability to AFB₁ than other organisms.The adaptive mechanism of A.advena to AFB₁ was related to the increased activity of antioxidant enzyme,detoxification enzyme and digestive enzyme,and the increased expression of Aa CYP304A1,Aa GSTs1,and Aa CYP4D2.AFB₁ content and mold quantity decreased significantly in the maize infested by A.advena.Adults are more effective in decreasing AFB₁ content than larvae.The lower the broken kernels rate was,the more effective in decreasing AFB₁ content.AFB₁ content in maize decreased significantly with the insect density increasing and time going.