Expression Of Mitochondrial Associated Genes In Liposcelis Tricolor Under Allyl Isothiocyanate Fumigation

Liposcelis tricolor?Psocoptera:Liposcelididae?also named psocids or booklice,and usually occurred in indoors and granaries.Psocids are found worldwide and have proven difficult to control because they do not respond to management tactics that have been developed for other stored-product pests.Because the wide range of food,serious overlapping of generations,rapid development of high levels of resistance to various insecticides,the psocids are becoming one of the major storage pests that threaten the global food security and safety.The new technology for the prevention and control of stored grain pests has always been a hot spot for researchers.Currently,fumigants,grain protectant and modified atmosphere are the main measures to control stored grain pests.Psocids often commix occurred at the same time in vast numbers infesting stored cereal grains,and they also have developed high level resistance to various insecticides,as well as phosphine.Furthermore,aluminium phosphide which can generate the phosphine will soon be restricted or prohibited.Therefore,it is urgent to find a new type of green fumigant that can replace phosphine.Allyl isothiocyanate?AITC?is a secondary metabolite derived from cruciferous plants such as horseradish.AITC has good fumigation effect on stored grain pests and it has good application prospects in the future.Recent studies indicated that mitochondrial functions are associated with the growth and development,insecticide resistance,longevity and environmental adaptation of insects.For example,after fumigation with AITC,the mitochondrial functions of complexes I and IV are greatly decreased,and which resulted in insufficient energy supply and accumulation of reactive oxygen species?ROS?,and which caused tissue damage in insects.In order to further evaluate the control effect of AITC and explore its mechanism in psocids,we took L.trcolor as the research object,and series of studies were carried out,i.e.,the determination of the fumigating activity of AITC on the Liposcelis species,sequencing and analyzing of mitochondrial genome of L.tricolor,expression pattern of mitochondrial genes under the treatment of AITC,and analysis of gene expression profiles after AITC exposure.Meanwhile,functional verification of the cox1 and cox3 was performed using RNA interference technology.The main findings are as follows:1.AITC has a good fumigating activity against the booklice.In this thesis,the fumigating activities of AITC against five Liposcelis species are investigated.The LC₅₀ of AITC is ranging from 0.5?L/L to 1.4?L/L.The sensitivities of booklice to AITC were as follows:L.entomophila?0.595?L/L?>L.tricolor?0.851?L/L?>L.decolor?1.098?L/L?>L.yangi?1.098?L/L?>L.bostrychophila?1.368?L/L?.Among them,the males were more sensitive to AITC than females.For example,the LC₅₀ of AITC?0.853?L/L?for L.tricolor female is a liitle greater than males?0.586?L/L?.The LC₅₀ of AITC?1.178?L/L?in L.yangi females is much larger than that in males?0.617?L/L?.In addition,the sensitivity of different populations of L.bostrychophila to AITC is also different.Shangqiu and Chongqing population are more sensitive to AITC than Guangdong and Huaibei population.This significant difference may be due to the different prevention and control measures in different regions.2.L.tricolor has multipartite mitochondrial?mt?genomes.The mt genome of L.tricolor has been fragmented into three or more circular mitochondrial chromosomes.Totally,thirteen mitochondrial protein coding genes,two ribosomal RNA genes and eight tRNA genes were identified.Among these genes,atp6,atp8,trnR,trnV,trnT,trnW are distributed on a smaller mt chromosome?Lt mt chromosome I?;trnY,trnK,trnN,trnS,nad5,cox2,cox3,nad1 are distributed on a larger chromosome?Lt mt chromosome II?,and cox1,nad3,cytb,nad2 are distributed on the third chromosome?Lt mt chromosome III?.The total length of the mitochondrial protein-coding genes is 10458 bp with a 75.87%of AT content.The presence of 98 bp non-coding regions on three chromosomes may be a potential AT control region.Phylogenetic analysis indicated that Liposcelis species and Phthiraptera were sister groups,and Psocoptera is a paraphyletic group.3.In this thesis,the differential expression patterns of seven mitochondrial coding genes?cox1,cox3,nad1,nad4,nad5,atp6 and cytb?in L.tricolor under different gender and developmental stages,as well as different treatments were investigated.The results show that the expression level of mitochondrial genes in females is higher than that in males,but there was no significant difference.The expression levels of most mitochondrial genes?nad1,nad4,nad5 and cox1?are significantly higher in the egg stage than that in the nymphal and adult stages?P<0.05?.Most mitochondrial genes show an up-regulated trend after short-term temperature?40?,0??stress,but there is no significant changes.After the treatment with AITC,the expression of mitochondrial genes is down-regulated.Most genes?cox1,cox3,nad1,nad4 and nad5?are significantly down-regulated under low dose treatment,while atp6 and cytb are significantly down-regulated under high dose treatment.4.A fast and efficient RNAi technology system for booklice was constructed.Here,freezer dryer?Labogene?was used to make the liquid of dsRNA into the powder,and then mixed the dsRNA with psocids dry diets in a proportion of 1:10.Through this feeding method,the target genes can be effectively silenced in booklice.Functional verification of the mitochondrial coding genes cox1 and cox3 in complex IV was conducted by using this RNAi technology.The results show that after 24hours of feeding dscox1 and dscox3,the silencing efficiency of the cox1 and cox3 could reach as high as 66%and 87%,respectively.Meanwhile,silencing of the mitochondrial-encoding genes led to a dramatic increase in the mortality rates of the L.tricolor adults?20.68%and 20.65%,respectively?.Therefore,the mitochondrial coding gene can be used as a potential lethal target gene for controlling booklice.5.Based on digital gene expression profiling technique,the expression patterns of differentially expressed genes in L.tricolor treated with AITC were analyzed.A total of 7174 differential transcripts are obtained,of which 4724 are up-regulated and 2450 are down-regulated.GO annotations of 1768differential genes are assigned to 47 categories,mainly in the catalytic activity and metabolic process.2983 differential gene are assigned to 44 categories in KEGG Pathway analyses,mainly in Global and overview maps and Signal transduction.Furthermore,fifty differentially expressed genes could be annotated to the oxidative phosphorylation pathway,in which 7 genes are significantly up-regulated and 43 genes are significantly down-regulated.In conclusion,AITC has good toxic effect against booklice,and it can be potential used as a green fumigation agent for substituting the phosphine.Our results suggested that the target of AITC in L.tricolor is mainly in mitochondria,which can significantly down-regulate the expression of mitochondrial protein-coding genes and inhibit the function of mitochondrial respiratory chain.In the oxidative phosphorylation pathway,the mitochondrial complex-related protein gene changes significantly,leading to its functional disorder and then a lethal effect.Meanwhile,this thesis constructed a rapid and efficient RNAi technology system in booklice for the first time,and which will absolutely provide new strategy for the future research on the function of the target genes in psocids.