Apolipoprotein D In Adaptation To Feeding-related Oxidative Stress In Spider Mites

Spider mites are destructive pests,with small body size and high fecundity,and are typical r-strategic organisms.The host range of spider mites is wide,reaching more than 3,800 plant species,while the mechanism of its wide host adaptability is still not fully understood.In the long-term interaction and co-evolution of plants with herbivorous insects(mites),a wide range of defense mechanisms have evolved to cope with insects(mites)feeding.However,as sessile organisms,plants rely heavily on chemical substances for defense.Reactive Oxygen Species(ROS)participates in a variety of important functions in plants.When plants are infested by insects(mites),ROS quickly accumulates in cells,forming oxidative bursts,and causing oxidative stress on insects(mites).At the same time,insects(mites)also cause oxidative stress when they metabolize the defensive secondary metabolites of plants.ROS is highly active and can induce oxidation of DNA and protein,and simultaneously attack polyunsaturated fatty acids to form lipid peroxides and initiate chain reactions.Apolipoprotein D(ApoD)is a small secreted glycoprotein which is closely related to the control of lipid peroxidation levels and can reduce the oxidative damage caused by lipid peroxides.It is still unclear whether the evolution of ApoD in spider mites involved in the adaptation to oxidative stress.Accordingly,this dissertation studied the protective effect of ApoD on spider mites during the feeding process.The research results have important biological significance for understanding the mechanism of spider mites’ wide-ranging host adaptation.The main results are as follows:1.Super expansion of ApoD in spider mitesBased on the genome information of 3 spider mites species(T.urticae,P.citri and P.ulmi),mammals(human and mouse)and various insect species(Drosophila melanogaster,Acyrthosiphon pisum,Bombyx mori,Spodoptera litura,Bemisia tabaci,Nilaparvata lugens,Aedes albopictus,and Leptinotarsa decemlineata),HMMER and Blast were used to identify ApoD genes at the genome-wide level.The results showed that there is only one ApoD gene in the human and mouse genomes.Dipteran D.melanogaster and A.albopictus have 2 and 7 ApoD genes,respectively;Lepidopteran B.mori and S.litura have 6 and 5 ApoD genes,respectively;Hemipteran B.tabaci,N.lugens and A.pisum have 5,7 and 1 ApoD genes,respectively;Coleopteran L.decemlineata has 7 ApoD genes.Whereas there are 62,21 and 16 ApoD genes in T.urticae,P.citri and P.ulmi,respectively,which are far more numerous than other species,suggesting the intensive expansion of ApoD in spider mites.2.Induction of ApoD expression upon mite feedingBased on the transcriptome data of different developmental stages of T.urticae and P.citri,as well as the transcriptome data of host alternatives of these two species,it was found that ApoD is closely related to the feeding stage of mites as well host plant alternatives.Using RT-q PCR technology,the expression level of ApoD in two spider mites gradually increased with the prolonged feeding time,and then tend to stabilize or decrease.ApoD is thereby related to the duration that spider mites feed on plants.Immunohistochemistry analysis showed that immunofluorescence signals were concentrated near the intestines of the two species of spider mites,and they were distributed as dots.It was speculated that the fluorescence signals were localized in gaint digestive cells in the intestine.3.Involvement of ApoD in the adaptation to oxidative stress in spider mitesBy detecting the levels of LPO content in spider mites,the relationship between oxidative stress and cumulative feeding time was analyzed.The results showed that the content of LPO in the spider mites gradually increased with feeding in a short period of time,indicating that the feeding of spider mites would increase the lipid peroxide content in the body.Using RNAi technology,the expression levels of ApoD genes of T.urticae(tetur01g5730,tetur01g5740)and P.citri(EVM0011211,EVM0006972,EVM0004493 and Pc-newgene11497)were decreased,and the feeding performance of spider mites was observed.The results showed that after silencing ApoD in P.citri,the reproduciton of mites significantly decreased after feeding on leaves treated by jasmonic acid and salicylic acid,suggesting that silencing of the ApoD reduced the host adaptability of the spider mite.While silencing ApoD in T.urticae,the survival rate and reproduction did not change.However,LPO levels in both mites were significantly upregulated after RNAi,suggesting that ApoD in spider mites plays an important role in stabilizing homeostasis.When expressing ApoD(tetur01g5730)in vitro,it was found that ApoD(tetur01g05730)of T.urticae improves cell survival under oxidative stress and have antioxidant function.