The Function of Nilaparvata lugens (Hemiptera: Delphacidae) E74 and Its Interaction With ßFtz-F1.

Drosophila E74 is an early gene located in the polytene chromosome 74EF puff position. E74 controls the production of late genes, indicating that it plays a crucial role in this cascade model. Nilaparvata lugens E74 is closely related to Diaphorina citri, Bemisia tabaci, and Laodelphax striatellus. After downregulating E74, molting, and nymphal mortality were increased, and ovarian development was delayed. Moreover, the expression of Vg was reduced at the transcriptional level, as measured by qRT-PCR, and the content of Vg protein was reduced, as detected by Western blotting. After downregulating E74, the expression of hormone-related genes, including Tai, ßFtz-F1, Met, Kr-h1, UspA, UspB, E93, and Br, was changed. The expression of E74 was significantly decreased after downregulating hormone-related genes. When the expression of E74 and ßFtz-F1 was downregulated together, nymph mortality and molting mortality were higher than those when E74 or ßFtz-F1 was downregulated alone. Thus, E74 probably interacts with ßFtz-F1 at the genetic level. In summary, this study showed that E74 plays a crucial role in the development, metamorphosis and reproduction of N. lugens, possibly via the interaction with ßFtz-F1 at the genetic level. This study provides a basis for the development of new target-based pesticides and new methods for the effective control of N. lugens.

Cuticular protein and transcription factor genes expressed during prepupal-pupal transition and by ecdysone pulse treatment in wing discs of Bombyx mori.

We aimed to understand the underlying mechanism that regulates successively expressed cuticular protein (CP) genes around pupation in Bombyx mori. Quantitative PCR was conducted to clarify the expression profile of CP genes and ecdysone-responsive transcription factor (ERTF) genes around pupation. Ecdysone pulse treatment was also conducted to compare the developmental profiles and the ecdysone induction of the CP and ERTF genes. Fifty-two CP genes (RR-1 13, RR-2 18, CPG 8, CPT 3, CPFL 2, CPH 8) in wing discs of B. mori were examined. Different expression profiles were found, which suggests the existence of a mechanism that regulates CP genes. We divided the genes into five groups according to their peak stages of expression. RR-2 genes were expressed until the day of pupation and RR-1 genes were expressed before and after pupation and for longer than RR-2 genes; this suggests different construction of exo- and endocuticular layers. CPG, CPT, CPFL and CPH genes were expressed before and after pupation, which implies their involvement in both cuticular layers. Expression profiles of ERTFs corresponded with previous reports. Ecdysone pulse treatment showed that the induction of CP and ERTF genes in vitro reflected developmental expression, from which we speculated that ERTFs regulate CP gene expression around pupation.

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ßFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ßFTZ-F1. Selective RNA silencing of ßftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ßftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ßFTZ-F1 expression. Moreover, premature ßftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ßFTZ-F1 expression late in the molt as steroids decline.

Function of the nuclear receptor FTZ-F1 during the pupal stage in Drosophila melanogaster.

The nuclear receptor ßFTZ-F1 is expressed in most cells in a temporally specific manner, and its expression is induced immediately after decline in ecdysteroid levels. This factor plays important roles during embryogenesis, larval ecdysis, and early metamorphic stages. However, little is known about the expression pattern, regulation and function of this receptor during the pupal stage. We analyzed the expression pattern and regulation of ftz-f1 during the pupal period, as well as the phenotypes of RNAi knockdown or mutant animals, to elucidate its function during this stage. Western blotting revealed that ßFTZ-F1 is expressed at a high level during the late pupal stage, and this expression is dependent on decreasing ecdysteroid levels. By immunohistological analysis of the late pupal stage, FTZ-F1 was detected in the nuclei of most cells, but cytoplasmic localization was observed only in the oogonia and follicle cells of the ovary. Both the ftz-f1 genetic mutant and temporally specific ftz-f1 knockdown using RNAi during the pupal stage showed defects in eclosion and in the eye, the antennal segment, the wing and the leg, including bristle color and sclerosis. These results suggest that ßFTZ-F1 is expressed in most cells at the late pupal stage, under the control of ecdysteroids and plays important roles during pupal development.

Cuticular protein genes showing peaks at different stages are probably regulated by different ecdysone responsive transcription factors during larval-pupal transformation.

We aimed to explain the reason and function of the successive expression of ecdysone-responsive transcription factors (ERTFs) and related cuticular protein (CP) genes during transformation from larva to pupa. The regulation of the expression of CP genes by ERTFs was examined by in vitro wing disc culture and reporter assay using a gene gun transduction system. Two CP genes that showed expression peaks at different stages-BmorCPG12 at W3L and BmorCPH2 at P0 stage-were selected and examined. Reporter constructs conveying putative BHR3, ßFTZ-F1, BHR39, and E74A binding sites of BmorCPG12 and BmorCPH2 showed promoter activity when introduced into wing discs. In the present study, we showed the functioning of the putative BHR3 and E74A binding sites, together with putative ßFTZ-F1 binding sites, on the activation of CP genes, and different ERTF binding sites functioned in one CP gene. From these, we conclude that BHR3, ßFTZ-F1, and E74A that are successively expressed bring about the successive expression of CP genes, resulting in insect metamorphosis. In addition to this, reporter constructs conveying putative BHR39 binding sites of BmorCPG12 and BmorCPH2 showed negative regulation.

Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori.

We aimed to clarify the regulation of cuticular protein (CP) gene expression and the resulting insect cuticular layers by comparing the expression pattern of CP genes and related ecdysone-responsive transcription factor (ERTF) genes, the coding amino acid sequences of CP genes, and histological observation. The expression of CP and ERTF genes during pupal and adult stages was examined via qPCR. The number of CP genes expressed during pupal and adult stages decreased as compared to that during prepupal to pupation stages, particularly in CPRs. The peaks of transcripts were observed at P5, P6, P9, A0, and A1. The order of the ERTF and CP genes expression resembled that at prepupal and pupation stages, suggesting the relatedness of ERTFs with the same CP genes at both stages. Moreover, the order of expression of CP genes resembled that in prepupal to pupation stages, by which we presumed the spaces of CPs in the epicuticle, outer-exocuticle, inner-exocuticle, endocuticle layer.