Functional Analysis Of The Genes Involved In Molting And Metamorphosis Signal Pathways In Nilaparvata Lugens

The brown planthopper, Nilaparvata lugens(St?l)(Hemiptera: Delphacidae), is one of the most destructive insect pests of rice in Eastern and Southeastern Asia. The molting and metamorphosis processes are very important for the development and reproduction of N. lugens. And 20-hydroxyecdysone(20E) and juvenile hormone(JH) signaling pathway coordinately orchestrate insect molting and metamorphosis. Based on the transcriptome and genome data, we characterized the nuclear receptor family genes and crucial genes involved in the 20 E and JH signaling pathway in N. lugens. Analysis of conserved domain structures, phylogenetic trees, gene expression patterns was performed and the function characterization of these genes involved in molting and metamorphosis signal pathways in Nilaparvata lugens was studied by RNA interference experiments. The main result are as follows:1. Bioinformatics investigations with genomic and transcriptomic databases identified the relative genes involved in molting and metamorphosis signal pathway. Including ecdysone biosynthesis Halloween genes, ecdysone receptor gene, ecdysone receptor nuclear transportation gene, juvenile hormone biosynthesis genes, JH signal transduction pathway genes, specifies adult metamorphosis gene and 20 nuclear receptor genes(including NlUSP, NlE75, NlHR3 and NlFTZ-F1).2. Based on N. lugens transcriptome data, twenty-two putative cDNAs were cloned. Among them were 5 ecdysone biosynthesis Halloween genes(named NlCYP307, NlCyp306a1, NlCyp302a1, NlCyp315a1, NlCyp314a1), 2 transcripts of ecdysone receptor, 1 ecdysone receptor nuclear transduction gene NlRan, 5 transcripts of ecdysone early response gene NlE75, 2 transcripts of ecdysone response gene NlHR3, 2 transcripts of ecdysone response gene NlFTZ-F1, 2 juvenile hormone biosynthesis genes(NlJHAMT and NlFAMeT), 2 genes involved in juvenile hormone signal transduction pathway(NlBroad-C and NlKr-h1) and a specifies adult metamorphosis gene NlE93. The open reading frames(ORFs), putative amino acids sequences, physico-chemical properties, transmembrane region, conserved domains and gene structure were predicted and annotated. And the function characterization of these genes were preliminary analyzed.3. The expression profile of these genes were estimated by qRT-PCR. The results showed that, two expression peaks of the 5 Halloween genes were found at 24 h and 60 h after ecdysis, indicated the two timepoints are important to ecdysone biosynthesis and regulation of N. lugens molting and metamorphosis. During the nymphal stage, NlUSP, NlRan, NlE75, NlHR3, NlFTZ-F1 shared the same expression patterns, with the highest expression levels occurred in the later stage of each instar or early stage of next instar. Beside, NlFTZ-F1 specific highly expressed in the last day of each instar. The results showed that the 20 E signaling pathway genes highly expressed during molting and have associated with N. lugens ecdysis and metamorphosis. The expression patterns of NlE93 and NlKr-h1 suggested that decreasing NlKr-h1 mRNA levels correlate with increasing NlE93 mRNA levels and increasing NlE93 mRNA levels correlate with decreasing NlKr-h1 mRNA, the balance between the expression patterns of NlE93 and NlKr-h1 may have an association with N. lugens metamorphosis and decide the proper timing for activing metamorphosis action during the nymphal stage of N. lugens.4. The function of these genes were characterized by RNA interference(RNAi). The results showed that NlCyp314a1、NlUSP、NlRan、NlE75、NlHR3 and NlFTZ-F1 are indispensable for N. lugens to complete the molting process and RNAi-mediated knockdown of these genes negatively affect molting and lead to abnormality of phenotype and high lethality. Several phenotypic defects were observed: the cuticle at the notum was split open but cannot completely shed off; duplication of cuticular structures, old cuticle cannot completely shed off and form cuticle tail; extended body form, and failed to molt, and so on. In addition, knockdown of NlCyp314a1 and NlRan caused the ovary development was abnormal and the the oocytes remained at primitive stage with no eggshells, inhibit vitellogenin production and transportation; knockdown of NlHR3 caused small and elliptical oocytes, all of their phenotypic together with no offspring. NlE93 and NlKr-h1 played a decisive function in N. lugens metamorphosis. Knockdown of NlE93 caused supernumerary N6 instar of N. lugens and knockdown of NlKr-h1 caused precocious nymph-adult intermediates(adultoid). The expression pattern and RNAi results showed that they are mutual inhibition relationship, NlKr-h1 can inhibit the expression of NlE93 in the juvenile instar nymphs of N. lugens, and NlE93 not only can promote metamorphosis but also inhibit the expression of NlKr-h1 to ensure the proper metamorphosis action in the later instar nymphs of N. lugens. The balance between the two genes plays a critical role in the metamorphosis of N. lugens deciding the proper timing for activing metamorphosis action during the nymphal stage of N. lugens. At the same time, the effect of these genes knockdown on the transcript levels of the target and related genes was examined to analysis the interaction relation between the genes involved in 20 E and JH signaling pathway.Taken together, we constructed the model for 20 E and JH signal transduction pathway involving in N. lugens molting and metamorphosis, including ecdysone biosynthesis pathway, ecdysone receptor, ecdysone signal transduction pathway and the metamorphosis signal transduction pathway regulated by the interaction between NlE93 and NlKr-h1. This study laid the groundwork for father understand the function of molting and metamorphosis signal transduction pathway in the development of N. lugens, and provided more important reference information for screening the useful RNAi based pest management in N. lugens.