| Mitochondria,as the main production site of reactive oxygen species(ROS)in organisms,play an important role in the process of organism resistance to adversity in organisms.The oriental fruit fly(Bactrocera dorsalis),is a serious insect pest of fruits and vegetables with strong environmental adaptability.With the global warming,B.dorsalis has spread to Hebei,Shandong and other places in China,and the insecticide resistance problem is becoming more and more serious.Therefore,it is of great practical significance to study the stress adaptability of B.dorsalis.However,previous studies on the molecular mechanism of stress resistance in B.dorsalis mainly focused on nuclear genes,and fewer studies on genes encoded by organelle.In this study,taking mitochondrial as the breakthrough point,the function of mitochondrial genes in adaptation process of B.dorsalis were analyzed through determining physiological parameters and expression profiles,constructing the mitochondrial transcription maps,and identification of RNA editing sites.The main results are listed as below:1.Mitochondria of B.dorsalis positively respond to stress at physiological levelThe physiological parameters of mitochondria in B.dorsalis responses to short-term extreme temperature,insecticides and mitochondrial inhibitors were determined by measuring the changes of three parameters,i.e.,respiratory rate,complex Ⅳ activity and ATP content.The results showed that respiration rate and ATP content of B.dorsalis decreased significantly after 0°C treatment for 1 h.After 40°C treatment for 1 h,the respiration rate of B.dorsalis increased significantly,and ATP content increased but the difference was not significant.All three insecticides(malathion,abamectin and betacypermethrin)can reduce mitochondrial complex Ⅳ activity and ATP content in B.dorsalis.Malathion treatment increased respiration rate significantly,while abamectin and beta-cypermethrin treatment reduced respiration rate significantly.Meanwhile,the respiratory rate,complex Ⅳ activity and ATP content were significantly decreased by two mitochondrial inhibitors(rotenone and antimycin A)treatment.The above results suggest that stress will affect mitochondrial homeostasis and functions in B.dorsalis,and the response trends of mitochondria were different under various environmental stresses.2.The expression profiles of mitochondrial gene at different development stages,tissues and upon environmental stresses in B.dorsalisThe expression patterns of mitochondrial genes in different developmental stages,tissues and stress were analyzed by RT-q PCR.The results showed that mitochondrial genes were expressed in all stages and tissues of B.dorsalis,and most of them were relatively high expressed in the middle or late periods of each metamorphosis stage and important metabolic tissues(e.g.,midgut of larval,fat body and Malpighian tubules of adult).Our results suggested that the expression of mitochondrial genes was adapted to the energy needs of B.dorsalis.In addition,the expression level of mitochondrial genes varied under different stress conditions.Among them,the expression level of atp6,nad1,nad3 and nad6 were significantly up-regulated in low temperature adapted strain,but did not respond to short-term cold stress at 0°C.The expression level of cox3,cytb and nad2 were significantly down-regulated in response to short-term heat stress at 40°C,but did not change significantly in high temperature adapted strain.Furthermore,the expression level of cox3 was significantly up-regulated in malathion resistant strain,and atp6,cox2,cytb and nad3 were significantly up-regulated after exposing to malathion treatment.In summary,mitochondrial genes are not only closely related to growth and metabolism of B.dorsalis,but also play a crucial role in its adaptation to stress.It is worth mentioning that the expression level of mitochondrial genes in response to different stresses and their time effects(such as short-term stress and long-term acclimation)were different,and the category and expression level of mitochondrial genes varied with the type and intensity of stress.3.Construction of mitochondrial transcription maps and identification of RNA editing sites in B.dorsalisBased on the transcriptomes of different developmental stages,tissues,temperature adapted strains and insecticide resistant strains of B.dorsalis,several mitochondrial transcription maps were constructed,and the differences of relative expression levels of different genes in the same state were also evaluated,which provided a basic data for the study of mitochondrial genes transcription and expression in B.dorsalis.Interestingly,the reads coverage of 16 S r RNA(rrn L)was much higher than that of 12 S r RNA(rrn S)and other protein-coding genes.As two consecutive genes,atp6 and atp8 have obvious continuity in the coverage of reads,indicating that they are bicistronic.Meanwhile,protein-coding genes without complete stop codons such as nad3 and cytb can form complete stop codons by adding poly A tail.In addition,based on the transcriptome and mitochondrial genome and its polymorphism data,2,274 RNA editing sites were identified,and the distribution of RNA editing sites was different in different stages,tissues and strains.A > G,C > T,G > A,T > C are the main RNA editing types.Furthermore,RNA editing sites tend to be distributed in the third base of code.In tissues,the number of RNA editing sites in fat body was far more than that in other tissues,which indicating that the highly metabolic tissues regulated gene expression through RNA editing.Among the temperature adapted strains,the number of RNA editing sites of the high and low temperature adapted strains were much lower than that of the control strains,which indicating that RNA editing played an important role in the long-term temperature acclimation.In addition,four meaningful editing sites were found,i.e.,cox11,462 T > C and cox11,463 C > A in fat body and nad1220 G > A and nad2814 G > A in malathion resistant strains.It suggests that these genes may have functional adaptation changes through posttranscriptional modification to cope with environmental pressure.4.Functional study of mitochondrial genes atp6 and cox2 based on RNAi technologyRNAi was used to silence 11 mitochondrial genes in B.dorsalis.The results showed that nad6,cox1,cox2 and atp6 could be stably silenced,and the silencing efficiency of cox2 was up to 76.3%.It suggesting that the mitochondrial genes of B.dorsalis can be successfully silenced,but the silence efficiency of different genes varied significantly.Meanwhile,mitochondrial genes encoding mitochondrial respiratory chain complex IV subunit are more likely to be stably silenced.Further studies showed that after atp6 silencing,the tolerance of B.dorsalis to 40°C was enhanced,while the tolerance to malathion was reduced.After cox2 silencing,the complex Ⅳ activity of B.dorsalis and its tolerance to malathion decreased.The results showed that atp6 and cox2 were involved in the regulation of malathion resistance,and atp6 was also involved in the resistance to high temperature(40°C). |
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