Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite

A radiochemical assay measuring juvenile hormone synthesis by corpora allata incubated in vitro was adapted for use with the termite Zootermopsis angusticollis. Corpora allata from 3–4-day old virgin female neotenic reproductives were used in these studies because this caste showed the highest rates of juvenile hormone synthesis (0.6 pmol h^?1 per pair corpora allata). Juvenile hormone-III synthesis was linear for up to 6 h over the range of concentrations of labelled l-methionine from 27–280 μM. Rates of juvenile hormone synthesis were stimulated up to 10-fold in a dose-dependent manner by the addition of farnesoic acid to the incubation medium. However, the relatively high concentration of 120 μM farnesoic acid reduced the rates of juvenile hormone synthesis. The radiochemical assay was used to determine rates of juvenile hormone synthesis in vitro by corpora allata from larvae with a queen and king vs orphaned larvae. The presence of reproductives resulted in a suppression of larval corpus allatum activity relative to orphaned controls.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.     

Comparative metabolism of isoleucine by corpora allata of nonlepidopteran insects versus lepidopteran insects,in relation to juvenile hormone biosynthesis

We studied the metabolism of [U-¹⁴C]isoleucine by intact and homogenized corpora allata (CA) from various insect species to determine how this substrate is converted to precursors of juvenile hormone (JH). CA homogenates of the lepidopterans Manduca sexta, Hyalophora cecropia, and Samia cynthia metabolize [U-¹⁴C]isoleucine to several products including 2-keto-3-methyl-valerate, 2-methylbutyrate, CO₂, propionate, and acetate. Intact CA of male H. cecropia produce particularly high levels of 2-keto-3-methylvalerate, indicating a highly active branched-chain-amino acid transaminase. In contrast, CA homogenates from the nonlepidopterans Periplaneta americana, Schistocerca nitens, Tenebrio molitor, and Diploptera punctata barely metabolize [U-¹⁴C]isoleucine. However, P. americana CA homogenate metabolizes [U-¹⁴C]2-keto-3-methylvalerate, the transamination product of [U-¹⁴C]isoleucine, more rapidly than does a homogenate of M. sexta CA. Furthermore, intact CA from P. americana incubated with [U-¹⁴C]2-keto-3-methylvalerate incorporate low levels of ¹⁴C into JH III, but do not metabolize this substrate to JH II or JH I. Intact CA from female Diploptera punctata produce very high levels of JH III, but are also unable to incorporate radiolabel from [U-¹⁴C]isoleucine into JH III, which substantiates our findings with other nonlepidopteran CA. The results suggest that CA of nonlepidopteran insects lack an active branched-chain amino acid transaminase and, consequently, are unable to utilize these substrates for JH biosynthesis.     

Brain-controlled synthesis of juvenile hormone in adult insects

Conclusions and future research There is now quite substantial evidence that the MNSC and the LNSC produce CA regulatory substances which are transported along axons and released in the immediate vicinity of the gland cells and that this system forms the most important means of CA control from the brain. The putative allatostatins and allatotropins are most probably neuropeptides. Isolation and identification of these substances are priorities in the coming years. The neuroendocrinological aspects, such as the precise rôle of the MNSC and the LNSC in different species; whether allatotropins and allatostatins arise from a single precursor molecule, and the dynamics of release of the substances from the nerve terminals, provide highly interesting topics. Furthermore, questions concerning control mechanisms of JH-biosynthesis at the cellular and molecular level, including the role of second messengers, the rate limiting steps in the biosynthetic pathway, and the regulation of gland growth still need to be clarified.     

保幼激素生物合成研究进展

保幼激素（juvenile hormone,JH）是存在于昆虫、甲壳动物和部分植物体内的倍半萜类衍生物。在昆虫和甲壳动物体内,保幼激素主要调节变态和生殖活动。在植物体内,则可能作为异株克生物质发挥作用。保幼激素主要通过细胞质内的甲羟戊酸途径（MVA）合成,植物质体内存在萜类合成的1-去氧木糖-5-磷酸途径（DXP）。MVA和DXP途径通过单向质子协同运输系统进行协调,使DXP途径中形成的前体化合物参与MVA途径的倍半萜合成。JH生物合成的主要步骤己基本查明,但与合成相关的酶学研究还较薄弱。生物合成酶的分子生物学是近来研究的热点,相关酶的cDNA克隆已有报道。JH生物合成酶的进一步研究有助于查明JH生物合成调控机制,深化对节肢动物生殖的理解,还可为新型杀虫剂开发提供可能的靶标。     

昆虫保幼激素的生殖调控机制研究进展

保幼激素（Juvenile hormone,JH）是昆虫特有的一类倍半萜烯类激素。JH在幼/若虫中主要通过拮抗蜕皮激素20E信号影响变态发育,从而维持昆虫幼虫性状。JH在成虫中作为促性腺激素,通过调控雌雄一系列生理和行为促进生殖。JH信号调控雌性生殖主要包括促进卵巢卵子发生、卵黄发生、排卵、滞育、雌性性信息素合成、肠道和卵巢等器官的大小适应性调整等。JH信号促进雄性生殖,主要包括促进附性腺精液蛋白的合成、羽化后调节雄性性行为,进而促进生殖。本文从雌雄性昆虫角度分别阐述JH信号调控生殖的机制,以期为深入探明JH调控昆虫生殖的作用机制提供参考。     

Farnesol oxidation in insects: evidence that the biosynthesis of insect juvenile hormone is mediated by a specific alcohol oxidase

The oxidation of farnesol to farnesoic acid is a key step in insect juvenile hormone biosynthesis. We herein present preliminary characterization of the enzyme-catalyzed oxidation of farnesol to farnesal in larval corpora allata homogenates of the tobacco hornworm, Manduca sexta. This conversion, which is highly substrate specific, has a K(m) apparent of 1 microM and a pH optimum between 6 and 7. Results from chemical modification experiments indicate that the enzyme possesses an active site tyrosine residue. Although farnesol oxidation in adult M. sexta corpora allata homogenates was previously identified as being catalyzed by a dehydrogenase, the corresponding conversion in larvae is not effected by the addition of nicotinamide cofactors. Instead, enzymatic activity is slightly enhanced by the addition of FAD, decreases when incubations are performed anaerobically, and is completely inhibited when either sodium dithionite or glucose oxidase is added. Although the effect of various additives suggests that the oxidation of farnesol to farnesal does not require a metal redox center, 1,10-phenanthroline (but not 4,7-phenanthroline) is a weak irreversible inhibitor of farnesol oxidation (IC(50)=11 mM). The addition of exogenous metals (Fe2+, Cu2+, Ni2+, and Co2+) caused differential effects on farnesol metabolism, with Cu2+ being highly inhibitory. Taken together, this data suggests that the oxidation of farnesol to farnesal in larval corpora allata is mediated by a specific oxygen-dependent enzyme, perhaps a flavin and/or iron-dependent oxidase.     

Comparative genomics of insect juvenile hormone biosynthesis

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

Precursor supply for insect juvenile hormone III biosynthesis in a cockroach

The biosynthesis of the sesquiterpenoid juvenile hormone III (JH III) was studied using corpora allata of the cockroach Diploptera punctata incubated in vitro and a radiochemical assay for the hormone produced. The influence of several exogenous precursors such as glucose, trehalose, acetate, amino acids, and mevalonate on JH synthetic rates was studied. Glucose or trehalose were needed for an optimal rate of JH synthesis. Highest rates were achieved at trehalose concentrations below the normal hemolymph levels (35-40 mM). About one-third of the glucose utilized for the biosynthesis of JH III was metabolized through a pentose pathway, but acetyl-CoA derived from glucose was significantly diluted by acetyl-CoA from other sources. Amino acids provided both a source of carbon for JH III synthesis and a source of energy that allowed JH III synthesis from acetate and stimulated JH III synthesis from glucose. Acetate was a poor substrate, because it could not support JH III synthesis in long term incubations. The incorporation of exogenous mevalonate into JH III was dependent on the physiological state of the glands, but there was a significant dilution with endogenous mevalonate. This dilution reflected in part the poor penetration of mevalonate into the corpora allata cells, because JH synthesis in mevinolin-treated cells was not fully rescued by mevalonate.     

Genome-wide comparison of genes involved in the biosynthesis,metabolism, and signaling of juvenile hormone between silkworm and other insects

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.     

Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis

The activity of 3-hydroxy-3-methylglutaryl CoA reductase in homogenates of the corpora allata of the tobacco hornworm, $\text{Manduca sexta}$, was competitively inhibited by compactin. The K_I for the sodium salt form of compactin was 0.9 nM for the reductase from both male and female corpora allata. In intact female corpora allata juvenile hormone biosynthesis was also inhibited by approximately 50 percent at 10 nM compactin. Following injection with compactin, darkening of the cuticle, an indication of juvenile hormone deficiency, was observed in larvae after ecdysis from third to fourth instar. Hence, compactin shows potential as an inhibitor of insect growth and development.     

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.     

Juvenile hormone biosynthesis in larval and adult stick insects,Carausius morosus

Corpora allata (CA) from adult egg-carrying Indian stick insects, Carausius morosus, synthesise and release juvenile hormone (JH) III in vitro. No JH biosynthesis was observed in larvae, young adults, and old adult females that do not carry sclerotised eggs. In females, which bear sclerotised eggs, a consistent JH biosynthesis was observed. Supplementation of precursors of JH biosynthesis (farnesol, mevalonic acid lactone) greatly enhanced JH biosynthesis in a stage-, age-, and dose-dependent manner, but CA from the last larval instar retained the biosynthesised JH within the gland. Elevated calcium concentration in the incubation medium stimulated JH biosynthesis by CA from older adults but had either no or a poor effect on CA from young adults and larvae. The results obtained with farnesol, mevalonic acid lactone, and calcium indicate that the rate-limiting steps of JH biosynthesis very likely occur before the formation of mevalonic acid and that these early steps cannot be stimulated by elevated calcium concentrations in larvae and young adults. In older adults, in which spontaneous JH biosynthesis occurs, elevated calcium concentration can markedly stimulate JH biosynthesis. A pre-purified extract from brains of adult females had a stimulating effect on JH biosynthesis by CA from adult females. The results indicate that JH biosynthesis in C. morosus may require food-derived farnesol and may be regulated by allatotropic signals from the brain, possibly triggered by sclerotised oocytes in the ovary.     

Resource defense and juvenile hormone: the "challenge hypothesis" extended to insects

As predicted for vertebrates by the challenge hypothesis, the endocrine system of invertebrates can respond to social stimuli to modulate aggression. Testosterone (T) is generally considered to moderate aggression associated with reproduction, i.e. the establishment of breeding territories, mate guarding and offspring defense; juvenile hormone (JH) serves an analogous function in burying beetles. Hemolymph titers of JH increase significantly in Nicrophorus orbicollis, a species with facultative biparental care, when challenged by an intruder to defend their resource. During the first 12 h after the discovery of a carcass, the necessary breeding resource, competition is intrasexual, and JH of males responds only to a challenge by males, and JH of females responds only to a challenge by female intruders. After this period, competition is intersexual, and JH increases significantly in both males and females challenged by an intruder of either sex. In contrast, JH titers in a nonparental species are much higher throughout the breeding season, and neither males nor females respond hormonally to an intruder. These findings support the challenge hypothesis and suggest that mating systems and breeding strategies can promote plastic responses in insect, as well as vertebrate, endocrine systems.