DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3-5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.     

Final steps in juvenile hormone biosynthesis in the desert locust, Schistocerca gregaria

Two genes coding for enzymes previously reported to be involved in the final steps of juvenile hormone (JH) biosynthesis in different insect species, were characterised in the desert locust, Schistocerca gregaria. Juvenile hormone acid O-methyltransferase (JHAMT) was previously described to catalyse the conversion of farnesoic acid (FA) and JH acid to their methyl esters, methyl farnesoate (MF) and JH respectively. A second gene, CYP15A1 was reported to encode a cytochrome P450 enzyme responsible for the epoxidation of MF to JH. Additionally, a third gene, FAMeT (originally reported to encode a farnesoic acid methyltransferase) was included in this study. Using q-RT-PCR, all three genes (JHAMT, CYP15A1 and FAMeT) were found to be primarily expressed in the CA of the desert locust, the main biosynthetic tissue of JH. An RNA interference approach was used to verify the orthologous function of these genes in S. gregaria. Knockdown of the three genes in adult animals followed by the radiochemical assay (RCA) for JH biosynthesis and release showed that SgJHAMT and SgCYP15A1 are responsible for synthesis of MF and JH respectively. Our experiments did not show any involvement of SgFAMeT in JH biosynthesis in the desert locust. Effective and selective inhibitors of SgJHAMT and SgCYP15A1 would likely represent selective biorational locust control agents.     

暗褐网柄牛肝菌菌核形成相关基因分析

【背景】暗褐网柄牛肝菌(Phlebopus portentosus)是第一个能够人工栽培的食用牛肝菌,人工栽培过程中,不同菌株会形成数量不等的菌核。【目的】探明不同菌株产核差异机制。【方法】采集多菌核(JH1)、寡菌核(JH2)菌株的成熟菌核及无菌核(JH3)菌株培养相同时间的菌丝体进行转录组测序,分析差异表达基因对菌核形成的作用和功能。【结果】KEGG富集分析显示,JH2 vs. JH1互比,苯丙氨酸、酪氨酸和色氨酸的生物合成,精氨酸和脯氨酸代谢,半胱氨酸及蛋氨酸代谢显著富集;JH3 vs. JH1互比,乙醛酸和二羧酸代谢显著富集;JH3 vs. JH2互比,谷胱甘肽、乙醛酸和二羧酸代谢显著富集。菌核形成相关基因分析显示,从JH2 vs. JH1、JH3 vs. JH1和JH3 vs. JH2的差异表达基因中分别筛选到69、118和82条与信号转导、感知刺激、防御、碳水化合物活性酶等有关的基因,其中碳水化合物活性酶基因数量最多。三个比较组共有的碳水化合物活性酶基因在JH1中的表达量高于JH2、JH3,表明JH1更能充分利用底物营养以形成更多菌核。【结论】本研究从转录组水平初步分析了暗...     

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Transcriptomic analysis reveals the significant effects of fertilization on the biosynthesis of sesquiterpenes in Phoebe bournei

Phoebe bournei is a potential medicinal plant. Its essential oils (Eos) are mainly composed of sesquiterpenes that has potential activities of anti-bacteria and anti-tumors. In this study, we evaluated the effects of compost and compound fertilizer on the total amount and main components of Eos in P. bournei, we also studied the molecular mechanism undergoing this process by deep sequencing the genes involved in the biosynthesis of sesquiterpenes. Fertilization enhanced the total amount of main components in Eos from both leaves and twigs. Bicyclogermacrene, the primary sesquiterpene in the leaf EO, was significantly increased under compost treatment, while bicyclogermacrene and δ-cadinene (the second most abundant sesquiterpene) were decreased under compound fertilizer treatment. The two fertilizers had no significant effect on the abundance of the primary (+) - δ-cadinene in the twig EO, but had a positive effect on the second most abundant sesquiterpene copaene. Significant differences were observed in the number of differentially expressed genes (DEGs) with the leaves showing greater number of DEGs as compared to the twigs after compost treatment. Terpenoid backbone biosynthesis (TBB) is a key pathway of sesquiterpenes synthesis. The expression of genes regulating several important enzymes in TBB was altered after fertilization. After the compost treatment, the expression of the leaf DXS gene (ACQ66107.1), being closely related to the sesquiterpene biosynthesis in P. bournei leaves, was decreased. Compost and compound fertilizer altered the expression of the two important branch-point enzymes (FPPS and GGPPS) genes (ART33314.1 and ATT59265.1), which contributed to the changes of the total amount and components of P. bournei sesquiterpenes. This study provides a new insight into the future use of P. bournei for Eos.     

A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg²⁺ ions lead to the production of FPP, while the presence of Co²⁺ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg²⁺ the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways.     

Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation. However, the order of activity was changed (JH III > bisJH III > MF) with respect to preventing prepupae from eclosing as normal adults, whether administered in the larval diet or as topically applied at the white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation. Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval attainment of pupariation. However, in this background dietary MF had increased activity in preventing puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the binding of secreted MF to USP.     

Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce.     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Methyl farnesoate and its role in crustacean reproduction and development

Studies with Libinia emarginata suggest that methyl farnesoate (MF), a product of the mandibular organs (MOs), may be a crustacean juvenile hormone. In order to better understand the significance of this compound in crustacean physiology, we first investigated the presence of MF in other decapods. MF was synthesized and secreted by MOs from all species tested. However, large differences in the level of MF secretion were observed between species and also between individuals of a species. For example, the level of secretion by MOs from L. emarginata was 100-fold greater than that observed in MOs from Homarus americanus. Analysis of hemolymph from these two species by GC-MS indicated comparable differences in the amount of MF present. Differences in the level of MF secretion by MOs from individuals of a species appear to reflect the physiological roles of this compound. For example, a close relationship was seen between MF secretion and gametogenesis in females of L. emarginata. Finally, treatment of lobster larvae with seawater containing MF caused a small but significant delay in their metamorphosis when compared with untreated larvae. These data suggest that MF affects reproduction in a manner similar to the effects of JH on insects, and may also have effects on the development of crustacean larvae. Taken together, these data support the classification of MF as a crustacean JH.