Mechanisms Of Allyl Isothiocyanate Fumigation On Mitochondria In Sitophilus Zeamais Mostch

Stored-product pests are serious problems for the storage of agricultural products and they could cause significant damages for the quality and quantity of agricultural products.Many researchers have focused on the management of stored-product pests. Sitophilus zeamais Motsch, a representative stored-product pest, has caused serious damage for agricultural products storage worldwidely. Phosphine is an important fumigants applied worldwidely. However, irreasonable use for longtime have induced drawbacks to become more and more serious. Resistance of pests against phosphine has resitricted its further application, and discovery and development of novel biorational fumigant have become more and more urgent. Ally isothiocyanate(AITC), isolated from crucifer, was a hydrolysis product of glucosinolate and has been proved with significant fumigation activities againt product-stored pests and the potential to be developed as a botanical fumigant. With Sitophilus zeamais Motsch as the target organism, this study has confirmed the fumigation activity of AITC against S.zeamais and completed the symptom and histopathology observation on the insect after fumigation by AITC. Then with calcium phosphide as a positive control, we performed the research on effects on mitochondrial electron transport chain by AITC fumigation. The transcriptomic alterations in the insects in response to AITC fumigation were also analyzed. Finally, functions of mitochondrial genes nd5, nd6, cox1 and cox5 played in AITC fumigation mechanisms were identified with RNA interference.Following results were obtained:1 Fumigation activity of AITC against S.zeamais was confirmed indoors and pilot scale test. The symptom and histopathology observation on the insect after fumigation by AITC was completed as well. Following results were obtained:(1) AITC showed significant fumigation activity against S.zeamais with a LC50 value of 0.74 ?g/ml(72h), and the pilot scale test showed an effective control of AITC, which indicated AITC with potentials to be developed as a biofumigant;(2) After AITC fumigation, S.zeamais insects would suffer four stage: excitement, convulsion, paralysis and death.(3) Transparante electron microspectre observation showed that mitochondria in Sitophilus zeamais would be damaged andvacuolization induced by AITC fumigation.2 Comparative effects of AITC and phosphine against mitochondria in S.zeamais were processed and following results were obtained:(1) at the same lethal concentration, similar inhibitory effects of AITC and phosphine on electron transport chain complex I and IV were determined;(2) similar inhibitory effects on complex IV with a time response effect and a dose response effect by AITC and phosphine fumigation were obtained, at concentrations of LC25, LC50 and LC75 AITC could induce the decrease of 20.04%, 37.47% and 54.32%,respectively, while the decrease caused by phosphine would be 22.61%, 50.65% and 69.04%respectively. While there were several differences on complex I that inhibition effect by AITC showed a dose response effect, decrease ratios of 14.25%, 17.17% and 37.68% would be determined after AITC fumigation at LC25, LC50 and LC75, as that by phosphine didn't;(3)inhibition effects on mitochondria electron transport chain would induce ROS stimulation in insects and affect enzymes relevant to ROS, then the oxgen damage would induce mitochondrial membrane lipid peroxidation.3 Transcriptomic alterations in S.zeamais in response to AITC fumigation were determined and analyzed with RNA-seq with following results:(1) 31,041 unigenes were assemblied after transcriptomc sequenced of S.zeamais, and 388 differently expressed transcripts including 117 up-regulated transcripts and 271 down-regulated transcripts in response to AITC fumigation were obtained;(2) according to the GO enrichment analysis, the differently expressed transcripts were enriched into 38 GO terms and the majority were“Binding”, “Cell process” and metabolism, while KEGG analysis indicated that the differently expressed transcripts were main enriched into “Folding, sorting and degradation”,“Transport and catabolism”, “Energy metabolism” and “Carbonhydrate metabolism”;(3)qRT-PCR results showed that expression levels of cat and cuznsod were affected by AITC and conneted with previous studies on ROS stimulation by AITC.4 Functions of gene nd5, nd6, cox1 and cox 5 played in AITC lethal mechanism were identified with RNA interference and following results were obtained:(1) after RNAi treatment, lifespans of the insects were shortened, amoung which the shortened effect of insects treated with cox1 RNAi was the most serious, while lifespan of insects treated with nd6 RNAi wasn't significant different with control insect;(2) AITC showed less significant fumigation activity against S.zeamais treated with cox1 RNAi with a derease ratio of 19.07%,while more toxicity against insects treated with nd6 RNAi with a increase ratio of 25.53%;(3)AITC fumigation could induce mitochondrial dependent ROS stimulation, while different effects by AITC on that ROS in insects treated with RNAi were obtained as well.After AITC fumigation, ROS in insects treated with cox1 RNAi was less than that in non-RNAi treatedinsects, while that in insects treated with nd6 RNAi was more significant than that in non-RNAi treated insects.In conclusion, this study proposed the fumigation mechanism of AITC against S.zeamais as following: AITC binded and inhibited mitochondrial electron transport chain complexes I and IV, then oxygen prepared for respirotary would be reducted unenough with free ROS generation. As CAT and POD would be inbibted by AITC, while SOD would be activated,more free ROS would be accumulated and mitochondrial membrane lipid would be peroxidation until insect death.