昆虫保幼激素受体的研究与应用进展

保幼激素(juvenile hormone, JH)是昆虫内分泌系统中的关键激素之一,对昆虫生长发育、变态、繁殖起着重要的调控作用。近年来有关JH的分子作用机制取得了极大的进展,主要得益于JH受体的鉴定,大量研究表明JH可通过胞内受体和膜受体两个途径来发挥生理调控功能。本文将从JH胞内受体Met的发现及鉴定、Met转录活性的调控因素、Met功能研究进展,以及Met作为JH受体在JH激动剂及拮抗剂筛选中的应用等方面对JH胞内受体的研究进展进行重点阐述;同时综述了有关JH膜受体的信号通路以及膜受体与核受体的互作等方面的研究进展。     

Photoperiod regulates growth of male accessory glands through juvenile hormone signaling in the linden bug,Pyrrhocoris apterus

Adult reproductive diapause is characterized by lower behavioral activity, ceased reproduction and absence of juvenile hormone (JH). The role of JH receptor Methoprene-tolerant (Met) in female reproduction is well established; however, its function in male reproductive development and behavior is unclear. In the bean bug, Riptortus pedestris, circadian genes are essential for mediating photoperiodically-dependent growth of the male accessory glands (MAGs). The present study explores the role of circadian genes and JH receptor in male diapause in the linden bug, Pyrrhocoris apterus. These data indicate that circadian factors Clock, Cycle and Cry2 are responsible for photoperiod measurement, whereas Met and its partner protein Taiman participate in JH reception. Surprisingly, knockdown of the JH receptor neither lowered locomotor activity nor reduced mating behavior of males. These data suggest existence of a parallel, JH-independent or JH-upstream photoperiodic regulation of reproductive behavior.     

Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.     

Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant

Metamorphosis in insects is regulated by juvenile hormone (JH) and ecdysteroids. The mechanism of 20-hydroxyecdysone (20E), but not of JH action, is well understood. A basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family member, methoprene tolerant (Met), plays an important role in JH action. Microarray analysis and RNA interference (RNAi) were used to identify 69 genes that require Met for their hydroprene-regulated expression in the red flour beetle, Tribolium castaneum. Quantitative real time PCR analysis confirmed microarray data for 13 of the 16 hydroprene-response genes tested. The members of the bHLH-PAS family often function as heterodimers to regulate gene expression and Met is a member of this family. To determine whether other members of the bHLH-PAS family are required for the expression of JH-response genes, we employed RNAi to knockdown the expression of all 11 members of the bHLH-PAS family and studied the expression of JH-response genes in RNAi insects. These studies showed that besides Met, another member of this family, steroid receptor co-activator (SRC) is required for the expression of 15 JH-response genes tested. Moreover, studies in JH responsive Aag-2 cells revealed that Aedes aegypti homologues of both Met and SRC are required for the expression of the JH-response gene, kr-h1, and SRC is required for expression of ecdysone-response genes. These data suggest the steroid receptor co-activator plays key roles in both JH and 20E action suggesting that this may be an important molecule that mediates cross-talk between JH and 20E to prevent metamorphosis.     

Stress-reactivity of a Drosophila melanogaster strain with impaired juvenile hormone action

Met(27) is a null allele of the Methoprene-tolerant gene of D. melanogaster that shows resistance to the toxic effects of both juvenile hormone (JH) and a JH analog, methoprene. The mechanism of resistance appears to be altered JH reception. We measured fertility, JH-hydrolyzing activity, and dopamine (DA) levels in Met(27) and Met(+) flies under normal (25 degrees C) and heat-stress (38 degrees C) conditions. We show that under normal conditions Met(27) females have JH-hydrolyzing activity and fertility lower than Met(+), but DA content did not differ between the two strains. At 38 degrees C Met(27) flies show no impairment in JH-hydrolyzing activity in response to stress, but they do show lower DA levels and impaired reproduction. The results with Met(27) are consistent with the previous hypothesis that the alteration in fertility that follows heat stress in D. melanogaster could result from alteration in the JH endocrine system.     

Genetic Evidence for Function of the bHLH-PAS Protein Gce/Met As a Juvenile Hormone Receptor

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. Here, we show that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone''s vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology.     

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.     

Early manipulation of juvenile hormone has sexually dimorphic effects on mature adult behavior in Drosophila melanogaster

Hormones are critical for the development, maturation, and maintenance of physiological systems; therefore, understanding their involvement during maturation of the brain is important for the elucidation of mechanisms by which adults become behaviorally competent. Changes in exogenous and endogenous factors encountered during sexual maturation can have long lasting effects in mature adults. In this study, we investigated the role of the gonadotropic hormone, juvenile hormone (JH), in the modulation of adult behaviors in Drosophila. Here we utilized methoprene (a synthetic JH analog) and precocene (a JH synthesis inhibitor) to manipulate levels of JH in sexually immature male and female Drosophila with or without decreased synthesis of neuronal dopamine (DA). Locomotion and courtship behavior were assayed once the animals had grown to sexual maturity. The results demonstrate a sexually dimorphic role for JH in the modulation of these centrally controlled behaviors in mature animals that is dependent on the age of the animals assayed, and present DA as a candidate neuronal factor that differentially interacts with JH depending on the sex of the animal. The data also suggest that JH modulates these behaviors through an indirect mechanism. Since gonadotropic hormones and DA interact in mammals to affect brain development and later function, our results suggest that this mechanism for the development of adult behavioral competence may be evolutionarily conserved.     

Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster

The larval fat body of newly eclosed adults of Drosophila melanogaster was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein we have identified mediates this JH effect in male accessory glands and thus is acting as a JH receptor.     

吸血昆虫生殖调控的分子机制

吸血昆虫是可以传播病原微生物的一类节肢动物,包括蚊虫、白蛉、蠓、猎蝽、跳蚤等。由于其特殊的吸血习性,它们成为了疟疾、登革热、丝虫病、锥虫病等急性传染性疾病的媒介载体。虫媒疾病具有传播速度快、扩散面积广和危害重等特点,不仅严重危害人类健康,还容易造成巨大的经济损失。由于针对虫媒传染病的药物匮乏以及虫媒病原对化学药物抗性的不断增加,阻断吸血昆虫的生殖成为控制虫媒疾病传播的有效措施。保幼激素(juvenile hormone, JH)和20-羟基蜕皮激素(20-hydroxyecdysone, 20E)在昆虫生殖过程中扮演着重要的角色。JH与胞内受体复合物Met/Tai结合后调控JH/Met靶基因表达,进而促进卵黄发生过程,为昆虫之后的吸血及产卵提供了必要条件;20E胞内受体为EcR/USP组成的异源二聚体,两者结合后激活下游基因表达,诱导卵黄原蛋白(vitellogenin, Vg)合成,为发育的卵巢提供营养。营养信号通路(胰岛素信号通路以及氨基酸介导的雷帕霉素靶蛋白信号通路)同样可以激活Vg合成,促进昆虫生殖;此外,营养信号通路与JH和20E之间可以相互作用共同调控吸血昆虫发育和繁殖。碳水化合物代谢以及脂代谢等能量代谢过程是昆虫生殖过程中主要能量来源,可以满足吸血昆虫生殖发育不同阶段极高的能量需求。研究表明,JH和20E信号通路在能量代谢过程中起着重要的调控作用;微小RNA在蚊虫这一类吸血昆虫中被证明与肠道微生物稳态、血液消化以及脂代谢等生理学过程密切相关,进一步影响了蚊虫卵巢发育。近年来,随着分子生物学及测序技术的革新,吸血昆虫生殖调控机制的研究不断取得新的进展。本文主要阐述了吸血昆虫生殖调控的分子机制研究进展,以期为通过调控吸血昆虫生殖的方法以阻断病原传播提供重要线索。     

Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila

The Methoprene-tolerant (Met) bHLH-PAS gene is involved in juvenile hormone (JH) action in Drosophila melanogaster as a likely component of a JH receptor. We expressed Met in Drosophila S2 cells and explored for MET partners using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another D. melanogaster bHLH-PAS gene with high homology to Met, and GCE formed heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation was drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain failed to occur. However, JH-dependent interaction occurred between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns.     

Hormonal control of reproduction and reproductive behavior in crickets

In Orthoptera, the endocrine control of reproduction has been investigated mainly in Acrididae. Gryllidae are also good models for the study of hormonal control of reproduction and reproductive behavior. In this review, special attention will be focused on the house cricket, Acheta domesticus. In the house cricket, vitellogenesis is controlled according to the classical model described for most insect orders. However, whereas allatectomy completely abolished oocyte growth in A. domesticus, it did not totally suppress ovarian development and egg-laying in Teleogryllus commodus, Gryllus bimaculatus, and G. campestris. In the Gryllidae studied thus far, juvenile hormone (JH) is not needed for mating behavior. In adult A. domesticus, the expression of oviposition movement, although independent of the presence of the ovaries, is strictly controlled by JH III. Recent findings suggest hormonal actions on the central nervous system of the house cricket, and provide a stimulating basis for further research on the respective involvement of hormones and nervous system in the control of reproductive behavior in adult crickets. Arch. Insect Biochem. Physiol. 35:393–404, 1997. © 1997 Wiley-Liss, Inc.