Effects Of Allyl Isothiocyanate On The Biological Functions Of The Core Subunits Of Cytochrome C Oxidase In Sitophilus Zeamais

Sitophilus zeamais Motschulsky is a serious early-stage storage pest of the Coleoptera Curculionidae.With the main fumigant methyl bromide being banned and the intensification of phosphine resistant,"bio fumigation" is considered to be a potent approach to storage pest management.AITC is a botanical fumigant with great activity on storage pest such as S.zeamais.At present,Cytochrome C oxidase(COX)is regarded as the target of AITC on S.zeamais,however,the interaction mechanism and binding sites between AITC and COX are still worthy of further study.RNA interference(RNAi)possessing a post-transcriptional gene silencing mechanism can be used to verify drug targets.In this paper,RNAi was used to explore the effects of AITC on the biological function of COX core subunits,and the following results were obtained:1.Time and dosage expressions of COX core subunits of S.zeamais.Our study showed that COXⅡ,COXⅢ expression gradually increased from 2-day-old eggs to 20-day-old larvae and peaked in the 20-day-old larvae,after that,the expression began to decline.For COX I,the relatively expression level continuously climbed from 2-day-old eggs to 1-day-old pupae and peaked in 1-day-old pupae before dropping to the minimum in 1-day-old adults.The expression patterns of COXⅠ,COXⅡ and COXⅢ were exactly similar in four tested tissues including the head,gut,epidermis and fat body.The highest level of expression for COXⅠ,COXⅡ and COXⅢ was in the gut,while the head was indistinctive from the epidermis and the fat body both in COXI and COXII expression.2.Interfering fragments dsRNACOXⅠ,dsRNACOXⅡ,and dsRNACOXⅢ were synthesized which were 493 bp,499 bp,and 411 bp in length,respectively.Microinjection of 18-day-old S.zeamais larvae were performed to detect the gene expression levels of COXⅠ,COXⅡ,and COXⅢ,and statistics on morality and phenotype changes were collected.For the interference efficiency of dsRNACOXⅠ,dsRNACOXⅡ,and dsRNACOXⅢ,there was no significant dose effect;but there was a stronge positive correlation between time and the interference efficiency of dsRNACOXⅠ and dsRNACOXⅡ while the interference efficiency of dsRNACOXⅢ showed a negative correlation with time.We first evaluated the impact of COXⅠ,COXⅡ and COXⅢ on the mortality caused by dsRNA at 24 h,48 h and 72 h after dsRNA injection.Specifically,mortality increased from 23.33%to 42.22%to 75.55%with dsRNACOXⅠ injection,from 22.45%to 53.87%to 71.88%with dsRNACOXⅡ injection,and from 14.44%to 61.11%to 82.22%with dsRNACOXⅢ injection.The phenotypes after injection of 100 ng dsRNACOXⅠ,50 ng dsRNACOXⅡ,and 50 ng dsRNACOX Ⅲ showed blackened body,mouth and wings.3.Changes in COX activity and in mitochondrial ultra-structures after dsRNA injection.At 36 h after injection of 100 ng dsRNACOXⅠ,the COX activity was significantly lower than the negative control,and the enzyme activity at 48 h was significantly higher than the negative control.After 36 h of injection of 0.75%NaCl,the outline of mitochondria was clearly visible.After injection of 100 ng dsRNACOXⅠ,the edges of mitochondria appeared fuzzy and vacuolated.After injection of 50 ng dsRNACOXⅡ,mitochondria broke down and decomposed.After injection of 50 ng dsRNACOXⅢ,mitochondria appeared blurry edges and vacuoles.4.Detection of the sensitivity to AITC fumigation after RNAi.After treatment for 24 h,the toxic regression equation of AITC against adult S.zeamais was y=5.05x-4.425,and the 20%lethal concentration was found to be 3.997 μL/L.After injection of 100 ng dsRNACOXⅠ,the sensitivity of the test insects to AITC increased significantly at 24 and 48 h,and there was no significant changes at 72 h.After injection of 50 ng dsRNACOXⅡ,the sensitivity of the test insects to AITC increased significantly at 24 h,and there was no significant changes at 48 h and 72 h.After injection of 50 ng dsRNACOXⅢ,the sensitivity of the test insects to AITC decreased significantly at 24 h,and there was no significant changes at 48 h and 72h.In conclusion,we speculate that AITC is likely to act on the core subunits COXⅠ and COXⅡ of S.zeamais,especially COXⅠ,but the specific binding sites worth further studied.The results of this study provide basic data for the efficient utilization of dsRNACOX of core subunits and the application of dsRNACOX core subunits combined with AITC,and also lay a foundation for the research and development of this kind of small molecular nucleic acid pesticide.