Depletion of juvenile hormone esterase extends larval growth in Bombyx mori

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

Juvenile hormone diol kinase. I. Purification, characterization, and substrate specificity of juvenile hormone-selective diol kinase from Manduca sexta

Manduca sexta juvenile hormone diol kinase (JHDK) catalyzes the conversion of juvenile hormone (JH) diol to JH diol phosphate. JHDK may be the first example of a phosphotransferase directly involved in the catabolism and inactivation of a lipid-soluble hormone. JHDK is an enzyme crucial for secondary metabolism of JH and possesses high specificity and catalytic efficiency for JH diol. In this study, the purification and characterization of native JHDK are described; its enzymatic properties are examined; and its role in cellular JH metabolism is explored. Using a variety of potential substrates, we show that JHDK has a preference for ATP, but will catalyze the formation of JH diol phosphate with GTP as the phosphate donor. JHDK has a nanomolar K(m) for JH I diol and a low micromolar value for MgATP. JH II and III diols also serve as phosphate acceptors with low micromolar K(m), whereas other diol derivatives of terpenoid esters structurally similar to JH metabolites are not phosphorylated. The reaction proceeds via a sequential Bi Bi mechanism. JHDK is active as a homodimer with a subunit molecular mass of 20 kDa. JHDK binds 5'-p-fluorosulfonylbenzoyladenosine and is inhibited by micromolar levels of Ca2+.     

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate‐binding motifs were predicted in the protein. Modeling of the 3‐D structure showed that the protein consisted of eight α‐helixes linked with loops, with no β‐sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

小麦吸浆虫保幼激素酯酶和保幼激素环氧水解酶基因的克隆及在滞育和变态发育过程中的表达动态

【目的】保幼激素(juvenile hormone, JH)在小麦吸浆虫Sitodiplosis mosellana滞育诱导及滞育后静息状态的维持中发挥着重要作用。保幼激素酯酶(hormone esterase, JHE)和保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)是调控JH滴度的重要降解酶。本研究旨在探讨JHE和JHEH在小麦吸浆虫滞育和变态发育中潜在功能。【方法】通过RT-PCR和RACE技术从小麦吸浆虫滞育前幼虫克隆JHE和JHEH全长cDNA序列;利用生物信息学软件分析其核苷酸及编码蛋白特性;采用qPCR技术分析其在小麦吸浆虫滞育不同时期(滞育前、滞育期、滞育后静息期和滞育后发育)3龄幼虫及1龄幼虫到成虫不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹、后蛹、雌成虫和雄成虫)中的表达水平。【结果】克隆获得了cDNA全长分别为3 102和1 980 bp的小麦吸浆虫SmJHE和SmJHEH基因(GenBank登录号分别为MG876768和MG876769),其开放阅读框分别长1 740和1 371 bp,分别编码579和456个氨基酸,预测蛋白分子量分别为65.67和51.65 kD。SmJHE蛋白含有5个JHE家族特有的保守模块,SmJHEH含有催化三联体Asp228, Asp404和His431及组成阴氧离子洞的两个Tyr(Tyr299和Tyr374)和HGWP花样结构。序列比对和进化分析表明,SmJHE和SmJHEH均与双翅目(Diptera)长角亚目(Nematocera)昆虫同源蛋白氨基酸序列一致性较高,亲缘关系最近。不同滞育时期的表达模式表明,SmJHE和SmJHEH在滞育前期(1龄到滞育前的3龄幼虫早期)表达量变化不明显,进入滞育后表达量基本维持恒定,但均在滞育后静息阶段的当年12月至翌年1月最低。发育表达模式表明,幼虫恢复发育后SmJHE表达量逐渐升高,预蛹期达到最高,在雌成虫中的表达量显著低于雄成虫中的;SmJHEH表达量则在预蛹期最低,在雌成虫中最高。【结论】SmJHE和SmJHEH参与小麦吸浆虫滞育调控,其表达量的降低与滞育后静息阶段JH的累积有关;SmJHE在发育过程中表达量的升高可能参与幼虫到蛹的变态,表达量的降低可能与生殖发育有关。     

Characterization and functional study of juvenile hormone diol kinase gene in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (Stål) is an important pest in rice. It has been widely recognized that the juvenile hormone (JH) is regulated by its hydrolase, which includes juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK). In this paper, we cloned the gene of Jhdk and the gene expression at different stages of N. lugens was analysed, and the relationship with Jhe and Jheh was studied after silencing the jhdk gene of N. lugens (Nljhdk) through double-stranded RNA (dsRNA) feeding. We also explored the expression of the three JH hydrolase after indoxacarb treatments. RT-PCR was used to amplify the full length Jhdk cDNA, and the Nljhdk gene was expressed throughout all the development periods tested and showed the lowest level at the 4th instar and the highest in the 5th instar. The expression level of Nljhdk in male adults was higher than that of female adults. Through feeding, dsRNA against Nljhdk successfully knocked down the target gene, which had no significant effect on the expression of the jhe gene of N. lugens (Nljhe), while the expression of Nljheh was upregulated. Indoxacarb could inhibit N. lugens reproduction, and the expression level of Nljhe and Nljhdk increased with the increasing of indoxacarb concentration, but the expression of the jheh gene of N. lugens (Nljheh) was reduced. These studies provide a line of experimental evidence in N. lugens to support that Nljhdk encodes the functional protein involved in JH degradation and further showed the relationship of the three hydrolases and the mechanism of indoxacarb inhibition of the fecundity of N. lugens.     

Fungal Metabolism of trans-2-Octenoic Acid

Juvenile hormone esterases (JHEs) function in juvenile hormone (JH) degradation. In the silkworm, Bombyx mori, we have characterized authentic JHE (Bmjhe) and five other carboxyl/cholinesterase (CCE) genes (Bmcce-1 to -5) with GQSAG, a motif sequence of JHE. But none of the genes appeared to function in vivo as a JHE, except for Bmjhe. Recently it was reported that the GQSAG motif might be dispensable, and that the Thr-316 residue has functional significance for JHE activity. On the basis of these findings, we identified two novel JHE candidates, Bmcce-6 and Bmcce-7, that lack GQSAG but possess Thr-316. In the CCE phylogenetic tree, BmCCE-6 was close to the lepidopteran JHE cluster, while BmCCE-7 constituted the same cluster as pheromone-degrading esterases. The developmental expression profiles were different among Bmjhe, Bmcce-6, and Bmcce-7. None of the proteins hydrolyzed JH in vitro. Our results suggest that only one CCE (BmJHE) functions as JHE in the silkworm.     

Characterization and analysis of novel carboxyl/cholinesterase genes possessing the Thr-316 motif in the silkworm, Bombyx mori

Juvenile hormone esterases (JHEs) function in juvenile hormone (JH) degradation. In the silkworm, Bombyx mori, we have characterized authentic JHE (Bmjhe) and five other carboxyl/cholinesterase (CCE) genes (Bmcce-1 to -5) with GQSAG, a motif sequence of JHE. But none of the genes appeared to function in vivo as a JHE, except for Bmjhe. Recently it was reported that the GQSAG motif might be dispensable, and that the Thr-316 residue has functional significance for JHE activity. On the basis of these findings, we identified two novel JHE candidates, Bmcce-6 and Bmcce-7, that lack GQSAG but possess Thr-316. In the CCE phylogenetic tree, BmCCE-6 was close to the lepidopteran JHE cluster, while BmCCE-7 constituted the same cluster as pheromone-degrading esterases. The developmental expression profiles were different among Bmjhe, Bmcce-6, and Bmcce-7. None of the proteins hydrolyzed JH in vitro. Our results suggest that only one CCE (BmJHE) functions as JHE in the silkworm.     

Identification and partial characterization of juvenile hormone esterase from cotton pest Dysdercus cingulatus

Juvenile hormone esterase (JHE), a selective enzyme that hydrolyzes the methyl ester of insect juvenile hormone plays an important role in regulating metamorphosis in nymphs as well as reproduction in adults. Studies on JH degradation provide insight into the possibilities of physiological disruption in the insects. In the present study, the JH degrading enzyme, JHE from the cotton pest Dysdercus cingulatus (Heteroptera) is characterized. Electrophoretic analysis of haemolymph during various developmental stages showed the JHE bands prominent only on the final day of 5th instar nymph, and the esterase substrate specificity confirmed the presence of JHE isoforms. In an attempt to clone cDNA of JHE gene from the final instar nymphs, mRNA isolated from fat bodies was coupled with JHE gene-specific primers and the cDNA was synthesized using RT-PCR. The PCR amplified cDNA showed the presence of JHE isoforms in D. cingulatus.     

Juvenile hormone (JH) esterase activity but not JH epoxide hydrolase activity is downregulated in larval Adoxophyes honmai following nucleopolyhedroviruses infection

Juvenile hormones (JHs) and ecdysteroids are critical insect developmental hormones. JH esterase (JHE) and JH epoxide hydrolase (JHEH) are JH-selective enzymes that metabolize JH and thus regulate the titer of JH. Baculoviruses are known to alter host endocrine regulation. The nucleopolyhedroviruses, AdhoNPV and AdorNPV, are known to have slow and fast killing activity against Adoxophyes honmai (Lepidoptera: Tortricidae), respectively. Here we found that when penultimate (4th) instar A. honmai are inoculated with AdhoNPV or AdorNPV, the mean survival time is 9.7 and 8.2 days, respectively. The larvae molted once but did not pupate. The AdhoNPV- or AdorNPV-infected larvae did not show a dramatic increase in JHE activity as was found in mock-infected larvae, instead they showed a marked decrease in JHE activity. In contrast, both viral infections had no effect on JHEH activity. In order to further characterize the JHE activity, the JHE-coding sequence of A. honmai (ahjhe) was cloned and confirmed to encode a biologically active JHE. Quantitative real-time PCR analysis of ahjhe expression in 4th and 5th instar A. honmai revealed that AdhoNPV and AdorNPV are able to reduce ahjhe expression levels.     

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

亚洲玉米螟幼虫体内保幼激素二醇激酶cDNA片段克隆与序列分析

为研究亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫体内保幼激素二醇激酶(JHDK)表达调控的分子机理,根据不同昆虫保幼激素二醇激酶基因序列的保守区域,设计合成简并引物,采用RT-PCR技术从亚洲玉米螟5龄幼虫中扩增出一段cDNA片段,大小为189bp,编码63个氨基酸,预测分子量为6.78ku,理论等电点pI值为4.57。该基因序列中含有保守的GTP结合蛋白特征指纹基序∑3和∑1。BlastP分析结果表明:该片段氨基酸序列与烟草天蛾JHDK氨基酸序列的一致性最高,为69%;与家蚕和小菜蛾JHDK氨基酸序列的一致性分别为55%和52%。构建系统发育树分析了3种鳞翅目昆虫JHDK进化关系,结果显示:亚洲玉米螟cDNA片段氨基酸序列与家蚕JHDK的亲缘关系最近,与小菜蛾JHDK的亲缘关系最远。半定量PCR结果表明:JHDK基因在中肠中表达量最高,随着5龄幼虫的发育,JHDK基因在血细胞、脂肪体和体壁组织中表达量有下降趋势,但在中肠组织中表达量明显增强。