Study On The Function Of Mir-9a?decapentaplegic?spineless On Wing Development In Brown Planthopper

Polymorphism contributes significantly to the ecological success of some insect species in natural and agricultural habitats.The brown planthopper(BPH)Nilaparvata lugens,which can develop as one of two wing-morphs(long-winged or short-winged),is the most destructive pest of rice in Asia.Long-winged BPH morphs can migrate to escape from unsuitable growth environment,whereas short-winged BPH morphs are less mobile but have higher fertility and destructiveness.This polyphenism in BPHs greatly exacerbates the extent of the harm and makes this pest difficult to control.Various environmental cues such as host plant quality,crowding,photoperiod,and temperature are known to influence which type of morph will develop,and wing-morph differentiation is thought to be most strongly influenced by the nutritional status of the host plant rice.In recent years,two insulin receptors of N.lugens,Nl In R1 and Nl In R2,have been shown to function as master regulators of wing morph type which can respond to the nutritional signals of rice plant and play opposite roles in wing-morph determination in the BPH.During the process of wing formation and development,however,many of the downstream events from Nl In R1/2 and the regulation mechanism require more in-depth study.With wild-type BPH population and our lab-maintained stable populations of LW and SW BPH,some research methods,including transcriptome sequencing,software analysis,dual luciferase reporter assay,RNA interference,real-time quantitative PCR,fluorescence in situ hybridization,Western blot,and Northern blot,were used to verify the interaction between Nlu-mir-9a and Ultrabithorax of N.lugens(Nl Ubx),and to clarify the regulation mechanism of wing morph.The functions of decapentaplegic(Nldpp)and spineless(Nlss)of N.lugens in the process of wing development were also studied and elucidated.The main results are as follows:Nlu-mir-9a controls wing size in BPH by inhibiting Nl Ubx:A recent study from our research group reported the highly provocative result that the Hox gene Ubx is expressed in BPH forewings and showed that this wing-development gene is differentially expressed in nymphs that develop into long-winged vs.short-winged morphs through dose-effect.mi RNAs are known to be able to post-transcriptionally fine-tune the levels of transcripts that context-appropriate phenotypes.Working under the basis of previous work,the forewing-and hindwing-pads of early 5^th instar BPH nymph of LW and SW BPH population were sampled for transcriptome sequencing and a total of 35 known mature mi RNAs and 21 novel mature mi RNAs were annotated.We employed five widely-used algorithms for mi RNA target prediction and found that Nl Ubx may be a target of Nlu-mir-9a.We measured the expression profiles of Nlu-mir-9a and Nl Ubx in BPH nymphs,especially in wing-pads,and found that the expression of these two biomolecules was negatively correlated.Then we used dual luciferase reporter assays and injection of mi RNA mimics and inhibitors to confirm the interactions between Nlu-mi R-9a and Nl Ubx in vitro and in vivo.After overexpression of Nlu-mir-9a in BPH nymphs,we found that the phenotypic changes of wings were similar to those of Nl Ubx-RNAi.By rearing BPH nymphs on host rice plans with low or high quality nutrition status,we also studied the nutritional status of the host and the regulation of In R1/2 on Nlu-mir-9a and Nl Ubx.When the host plant nutritional status is not ideal for BPH growth,the expression level of Nl In R1 in the tergum increased,a situation that leads to a higher proportion of LW morphs.In this process,the up-regulation of Nl In R1 induced higher levels of Nlu-mi R-9a,which in turn reduced the amount of Nl Ubx transcripts,ultimately resulting in longer wing lengths.Beyond extending our understanding of the interplay between host plant nutrition status and genetic events that modulate polyphenism,our study demonstrates the both the upstream signal and the mi RNA-based regulatory mechanism that control Ubx in BPH wing morph.decapentaplegic function in wing vein development and wing morph transformation in BPH:Silencing of Nldpp in the SW population led to the significant lengthening of the forewing,while Nldpp-knockdown in the LW population resulted in twisted wings.Moreover,knockdown of Nldpp caused the complete absence of wing veins.During the development of wing-pads,the Nldpp abundance in the terga of the SW population was significantly higher than that of the LW population.Through controlling the direction of wing morph transformation,we found that the expression level of Nldpp increased in the Nl In R1-knockdown BPH(tending to SW)and abundance of Nldpp declined after ds Nl In R2 injection(tending to LW).In addition,Nldpp-RNAi can significantly alter the expression of several wing development genes.Our results showed that Nldpp is mainly responsible for the formation and development of veins in BPH.Also,Nldpp can be regulated by Nl In R1/2 and participate in the wing morph transformation.spineless function in wing and antenna development in BPH:The high level of Nlss expression was detected in the head and thorax of 3^rd-and 4^th-instar nymphs.Nlss-knockdown in 3^rd-instar nymphs resulted in twisted wings in both long-winged and short-winged BPHs.This is the first report that ss could function in wing development of insects.Knockdown of Nlss also led to a decrease in the number and size of plaque organs.Moreover,the flagella of the treated insects were poorly developed,wilted,and even dropped off from the pedicel.Y-type olfactometer analyses indicated that antenna defects originating from Nlss depletion resulted in less sensitivity to host volatiles.Our study represents the first report of the characteristics and functions of Nlss in antenna and wing development and illuminates the function of the plaque organ of BPH in host volatile perception.Our study clarified the regulatory relationship between Nlu-mir-9a and Nl Ubx and their regulatory mechanism affecting wing size,as well as the functions of Nldpp and Nlss in the wing development of brown planthopper.These findings might be helpful to deepen the understanding of the molecular regulation mechanism during wing differentiation and wing development of brown planthopper,and might be of great significance to reveal the intrinsic molecular mechanism of brown planthopper disasters and the control work in the future.