Isolation and characterization of the insect growth regulatory substances from actinomycetes.

Insect growth regulators (IGRs) are attractive alternatives to chemical insecticides. Since it has been reported that secondary metabolites from actinomycetes show insecticidal activities against various insect pests, actinomycetes could be a potential source of novel IGR compounds. In the present study, insect juvenile hormone antagonists (JHANs) were identified from actinomycetes and their insect growth regulatory and insecticidal activities were investigated. A total of 363 actinomycetes were screened for their insect growth regulatory and insecticidal activities against Aedes albopictus and Plutella xylostella. Among them, Streptomyces sp. AN120537 showed the highest JHAN and insecticidal activities. Five antimycins were isolated as active compounds by assay-guided fractionation and showed high JHAN activities. These antimycins also exhibited significant insecticidal activities against A. albopictus, P. xylostella, F. occidentalis, and T. urticae. Moreover, dead larvae treated with these antimycins displayed morphological deformities that are similar to those of JH-based IGR-treated insects. This is the first report demonstrating that the insecticidal activities of antimycins resulted from their possible JHAN activity. Based on our results, it is expected that novel JHAN compounds potentially derived from actinomycetes could be efficiently applied as IGR insecticides with a broad insecticidal spectrum.

Plant-derived compounds regulate formation of the insect juvenile hormone receptor complex.

Insect growth regulators (IGRs) are attractive pest control agents due to their high target specificity and relative safety to the environment. Recently, plants have been shown to synthesize IGRs that affect the insect juvenile hormone (JH) as a part of their defense mechanisms. Using a yeast two-hybrid system transformed with the Aedes aegypti JH receptor as a reporter system, we identified several JH agonists (JHAs) and antagonists (JHANs) causing retardation in the ovarian development of female Asian tiger mosquito, Aedes albopictus, from plant essential oil compounds. While the JHAs increased the expression of a JH-induced gene, the JHANs caused a reduction in the expression of the same gene. The compounds identified in this study could provide insights into plant-insect interactions and may be useful for the development of novel IGR insecticides.

Simultaneous determination of neonicotinoid insecticides and insect growth regulators residues in honey using LC-MS/MS with anion exchanger-disposable pipette extraction.

In this study, we developed an anion exchanger-disposable pipette extraction (DPX) method to detect the residual concentrations of eight neonicotinoid insecticides (dinotefuran, acetamiprid, clothianidin, thiacloprid, imidachloprid, imidaclothiz, nitenpyram, and thiamethoxam) and eight insect growth regulators (IGRs; triflumuron, cyromazine, buprofezin, methoxyfenozide, tebufenozide, chromafenozide, fenoxycarb, and RH 5849) in Chinese honey samples collected from different floral sources and different geographical regions using liquid chromatography tandem mass spectrometry (LC-MS/MS). QAE Sephadex A-25 was used as the anion exchanger in the DPX column for the purification and cleanup of honey samples. Analytes were eluted with a mixture of acetonitrile and 0.1 M HCl, and the elution was subjected to LC analysis. This method was thoroughly validated for its reproducibility, linearity, trueness, and recovery. Satisfactory recovery of pesticides was obtained ranging from 72% to 111% with intraday RSDs (n = 5) of 1%-10%. High linearity (R2 ≥ 0.9987) was observed for all 16 pesticides. Limits of detection and quantification for all 16 compounds ranged from 0.3 to 3 µg/kg and from 1 to 10 µg/kg, respectively. Pesticide residues (9-113 µg/kg) were found in Chinese honey samples. The anion exchanger-DPX method was effective for removing sugars and retaining target analytes. Moreover, this method was highly reliable and sensitive for detecting neonicotinoids and IGRs in different floral sources of honey and will be applicable to matrixes with high sugar content.

S-(-)-10,11-dihydroxyfarnesoic acid methyl ester inhibits melanin synthesis in murine melanocyte cells.

The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasmas, freckles, age spots, and chloasmas. In the course of screening for melanin synthesis inhibitors, we found that the culture broth from an insect morphopathogenic fungus, Beauveria bassiana CS1029, exhibits potent antimelanogenic activity. We isolated and purified an active metabolite and identified it as S-(-)-10,11-dihydroxyfarnesoic acid methyl ester (dhFAME), an insect juvenile hormone. To address whether dhFAME inhibits melanin synthesis, we first measured the size of the melanin biosynthesis inhibition zone caused by dhFAME. dhFAME also showed inhibitory activity against mushroom tyrosinase in Melan-a cells. Intracellular, dose-dependent tyrosinase inhibition activity was also confirmed by zymography. In addition, we showed that dhFAME strongly inhibits melanin synthesis in Melan-a cells. Furthermore, we compared levels of TYR, TRP-1, TRP-2, MITF, and MC1R mRNA expression by reverse-transcription polymerase chain reaction and showed that treatment of Melan-a cells with 35 µM dhFAME led to an 11-fold decrease in TYR expression, a 6-fold decrease in TRP-2 expression, and a 5-fold decrease in MITF expression. Together, these results indicate that dhFAME is a potent inhibitor of melanin synthesis that can potentially be used for cosmetic biomaterial(s).

Development of a novel screening system for allatostatin receptor agonists in search of new candidate insect growth regulators.

Allatostatins (ASTs) are insect neuropeptide hormones that regulate diverse physiological functions, including feeding, growth and development, and reproduction. Therefore, regulation of allatostatin receptor (AstR) activity can be an effective tool for controlling insect growth and proliferation. Here, we describe a novel screening system using a mammalian cell line in which AstR is ectopically expressed, combined with fluorescence-based measurements of the membrane potential. HEK293T cells that do not express cognate receptors for AST became responsive to AST upon transfection with AstR. The response of the membrane potential to AST could be reliably detected by measuring the fluorescence of DiBAC4(3), a voltage-sensitive dye. We also discovered that overexpressing GIRK1/2 in this cell line could augment the magnitude of hyperpolarization by AST. Our screening system produces a fast and reliable readout for the efficient screening of AstR agonists.

Plant-derived juvenile hormone III analogues and other sesquiterpenes from the stem bark of Cananga latifolia.

Juvenile hormone III (JH III) is a larval metamorphosis-regulating hormone present in most insect species. JH III was first isolated from the plant, Cyperus iria L., but the presence of JH III has not been reported in other plant species. In the present study, proof of the existence of JH III and its analogues from Cananga latifolia was established. From an aqueous MeOH extract of C. latifolia stem bark, six compounds were isolated along with nine known compounds. These were identified by using spectroscopic analyses as: (2E,6E,10R)-11-butoxy-10-hydroxy-3,7,11-trimethyldodeca-2,6-dienoic acid methyl ester, (2E,6E)-3,7,11-trimethyl-10-oxododeca-2,6-dienoic acid methyl ester, (2E)-3-methyl-5-[(1S,2R,6R)-1,2,6-trimethyl-3-oxocyclohexyl]-pent-2-enoic acid methyl ester, 1ß-hydroxy-3-oxo-4ß, 5α,7α-H-eudesmane 11-O-α-l-rhamnopyranoside, 4-epi-aubergenone 11-O-2',3'-di-O-acetyl-α-l-rhamnopyranoside and 4-epi-aubergenone 11-O-2',4'-di-O-acetyl-α-l-rhamnopyranoside. Three of the previously known compounds, (2E,6E,10R)-10-hydroxy-3,7,11-trimethyldodeca-2,6,11-trienoaic acid methyl ester, (2E,6E,10R)-10,11-dihydroxy-3,7,11-trimethyldodeca-2,6-dienoic acid and (2E,6S)-3-methyl-6-hydroxy-6-[(2R,5R)-5-(2-hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl]-hex-2-enoaic acid methyl ester have now been found in a plant species. Ultra performance liquid chromatography-quadruple time-of-flight mass spectroscopy (UPLC-QTOF/MS) analysis of the chemical constituents of C. latifolia showed that several were predominant in the sub-fractions of a C. latifolia stem bark extract.

Juvenile hormone extraction, purification, and quantification in ants.

Juvenile hormone (JH) is an important insect hormone known to have many effects on development, reproduction, and behavior in both solitary and social insects. A number of questions using ants as a model involve JH. This procedure allows for quantification of circulating levels of JH III, which can be an important factor in many questions relating to insect research. The JH III is extracted from a subject, purified, and converted to a d(3)-methoxyhydrin derivative that can be quantified by gas chromatography-mass spectrometry (GC-MS). The major advantages of this protocol are its high resolution, and its ability to quantify significant differences between relatively small quantities of the hormone. Its major limitations are the time necessary to process samples, its relatively high cost, and maintaining the sensitivity of the equipment.

Phytochemical and biological studies of Abies species.

In the present review, the literature data on the phytochemical and biological investigations on the genus of Abies are summarized with 110 references. Up to now, 277 compounds were isolated from 19 plants of Abies species. The chemical constituents are mostly terpenoids, flavonoids, and lignans, together with minor constituents of phenols, steroids, and others. The crude extracts and metabolites have been found to possess various bioactivities including insect juvenile hormone, antitumor, antimicrobial, anti-ulcerogenic, anti-inflammatory, antihypertensive, antitussive, and CNS (central nervous system) activities.

A mathematical model for the regulation of juvenile hormone titers.

The titer of juvenile hormone (JH) is determined by three factors: its rate of synthesis, its rate of degradation, and the degree to which JH is protected from degradation by binding to a diversity of JH-binding proteins. All three of these factors vary throughout the life history of an insect and contribute to variation in the JH titer. The relative importance of each of these factors in determining variation in the JH titer is not known and can, presumably, differ in different life stages and different species. Here we develop a mathematical model for JH synthesis, degradation, and sequestration that allows us to describe quantitatively how each of these contribute to the titer of total JH and free JH in the hemolymph. Our model allows for a diversity of JH-binding proteins with different dissociation constants, and also for a number of different modes of degradation and inactivation. The model can be used to analyze whether data on synthesis and degradation are compatible with the observed titer data. We use the model to analyze two data sets, from Manduca and Gryllus, and show that in both cases, the known data on synthesis and degradation cannot account for the observed JH titers because the role of JH sequestration by binding proteins is greatly underestimated, and/or the in vivo rate of JH degradation is greatly overestimated. These analyses suggest that there is a critical need to develop a better understanding of the in vivo role of synthesis, sequestration and degradation in JH titer regulation.

A nutrient sensor mechanism controls Drosophila growth.

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. Here, we provide evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. In a genetic screen for growth modifiers, we identified slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. Our results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism.

Growth inhibitory effects on fall armyworm Spodoptera frugiperda of some limonoids isolated from Cedrela spp. (Meliaceae).

Dichloromethane extracts of Cedrela salvadorensis and Cedrela dugessi afforded a photogedunin epimeric mixture, gedunin and cedrelanolide. These compounds and the photogedunin epimeric acetates 3 and 4 at the 23-OH position were evaluated against Spodoptera frugiperda. Toosendanin, isolated from Melia azedarach, was used as a positive control. When tested for activity on neonate larvae into the no-choice bioassays, gedunin, photogedunin epimeric mixture, and photogedunin acetates mixture caused significant larval mortality with LC(50) of 39.0, 10.0, and 8.0 ppm at 7 days, respectively, as well as growth reduction. All the compounds tested inhibited larval growth, compared to the control, in a concentration-dependent manner. In addition, it was possible to observe significant reduced pupal weights and adult emergence. All the tested compounds except cedrelanolide showed comparable activity to that of toosendanin.

Characterization of antibody 444 using chromatographically purified enantiomers of juvenile hormones I, II, and III: implications for radioimmunoassays.

Optically pure (> 99.5%) enantiomers of insect juvenile hormones (JH) I, II, and III were obtained by injection of racemic mixtures onto a chiral HPLC column using hexane:2-propanol (99.5:0.5) as the mobile phase. The enantiomers of JH III were the best resolved (R = 4.26), followed by those of JH II (R = 2.29) and JH I (R = 1.47). These purified natural and unnatural enantiomers were used to further characterize an antiserum (444) developed for JH radioimmunoassays (RIAs). Based on ED50 values generated using optically pure [methyl-3H]-10R,11S-JH II as a tracer, the natural isomers of JH I, JH II, and JH III were 30, 87, and 36 times more immunoreactive, respectively, than the unnatural isomers. When compared with the racemates, the natural isomers were approximately twice as immunoreactive. In competitive displacement studies where the natural enantiomers of the three JHs were compared, immunoreactivities were in the order JH II > JH I > JH III (ED50 = 109, 198, and 300, respectively). Availability of pure natural enantiomers of JH, both as tracers and competitors, should improve the sensitivity and accuracy of JH titer determinations made by RIA and facilitate various enzyme, binding protein, and receptor studies.

Separation of juvenile hormone metabolites with a silica-based gel permeation column.

Metabolites of juvenile hormone (JH) III, JH acid, JH diol and JH acid diol in an aqueous solution were separated by gel permeation chromatography using an UltraSpherogel SEC 2000 column. Each metabolite was eluted in an inclusion volume in the order JH acid diol, JH acid and JH diol. Although JH was the last compound eluted, it was co-eluted with a JH-binding protein (JHBP) when JHBP was present in the solution. Using this method, in vivo and in vitro JH catabolism studies were performed in the fifth stadium larvae of Bombyx mori.

Synergy between the hunchback and bicoid morphogens is required for anterior patterning in Drosophila.

Anterior patterning of the Drosophila embryo is specified by the localized expression of the gap genes, which is controlled by the gradient of the maternal morphogen bicoid (bcd). Another maternal component, hunchback (hb), can substitute for bcd in the thorax and abdomen. Here we show that hb is required for bcd to execute all of its functions. Removal of both maternal and zygotic hb produces embryos with disrupted polarity that fail to express all known bcd target genes correctly. Proper expression of hb and the head gap genes requires synergistic activation by hb and bcd. We propose that it is the combined activity of bcd and hb, and not bcd alone, that forms the morphogenetic gradient that specifies polarity along the embryonic axis and patterns the embryo. bcd may be a newly acquired Drosophila gene, which is gradually replacing some of the functions performed by maternal hb in other species.

De novo biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito.

The role of the male accessory glands (MAG) in reproduction was investigated in the mosquito Aedes aegypti. MAG incubated with [14C]acetate synthesized radioactively labeled JH III, JH III bisepoxide and methyl farnesoate. MAG incubated with L-[methyl-3H]methionine synthesized [3H]JH III and a molecule that chromatographed on HPLC with JH I. Analysis of MAG and whole males extract by glass capillary combined gas-chromatography-selected ion monitoring mass spectrometry identified JH III and I as the main analogs that were synthesized by male mosquitoes. MAG of Culex nigripalpus, Anopheles rangeli and Anopheles trinkae also synthesized JH III from L-[methyl-3H]methionine, which indicates that the male mosquito has a complete JH III biosynthetic pathway. Unfed and unmated Culex quinquefasciatus do not develop their ovaries to the resting stage. Females injected with one MAG extract equivalent or implanted with A. aegypti MAG developed their ovaries to the resting previtellogenic stage, whereas females that were injected with saline did not. These results indicate that MAG synthesize and secrete JH III. The corpora allata (CA) of the male Aedes aegypti also synthesize JH III from L-[methyl-3H]methionine. This observation may suggest that JH synthesized by the male's CA is used for internal regulation, whereas JH synthesized by the MAG is transferred with the sperm into the female.

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.

Reversed-phase liquid chromatographic separation of juvenile hormone and its metabolites, and its application for an in vivo juvenile hormone catabolism study in Manduca sexta.

A convenient reversed-phase liquid chromatographic method was developed to separate juvenile hormone (JH) and its metabolites. The known metabolites including JH acid, JH diol, and JH acid-diol, as well as an unknown metabolite, were efficiently separated within 25 min on a 50 X 4.6 mm polymer column using a linear gradient of acetonitrile:5 mM Hepes (pH 7.4) buffer. Use of the polymer column diminished tailing observed for the diol metabolite on a C18 silica column, and allowed use of slightly basic buffers without concern of column instability. Use of buffer was essential to give good peak shape and reproducible retention behavior for the acidic metabolites. Using this method, an in vivo JH catabolism study was performed in fifth stadium larvae of Manduca sexta. Injected (10R)-[3H]JH III was rapidly converted to JH acid-diol and to an unknown compound(s) indicating that, in addition to JH esterase, epoxide hydrolase and other reactions play an important role in the catabolism of JH.

Molecular cloning, regulation, and complete sequence of a hemocyanin-related, juvenile hormone-suppressible protein from insect hemolymph.

A cDNA library was prepared from mRNA isolated from the lepidopteran Trichoplusia ni during larval-pupal metamorphosis. Differential probing was used to identify clones for mRNAs which are suppressible by exogenous juvenile hormone treatment. In vitro transcribed cRNAs from these clones were translated in vitro and challenged with antiserum specific for a known acidic, juvenile hormone-suppressible hemolymph protein (AJSP-1) that is associated with larval metamorphosis. Three clones were found which encoded immunoreactive translation products; their identity was confirmed by comparison of the N-terminal sequence of the mature AJSP-1 protein with the cDNA sequence. As inferred from the cDNA sequence, the protein encompasses 704 amino acid residues, including a N-terminal signal peptide; widely distributed as well as more localized stronger sequence similarities indicate that the protein is distantly related to hemocyanins and hemocyanin-like insect proteins. However, on the basis of amino acid sequence and composition, immunological reactivity, and hormonal sensitivity, the protein is distinct from previously described insect proteins. Its juvenile hormone suppressibility can be ascribed to suppression of the mRNA. RNA blot analysis using the cloned cDNA as a probe demonstrated that the transcript (approximately 2.8 kilobases) is of very low abundance during the penultimate stadium but becomes very abundant during the last larval stadium, when juvenile hormone rapidly declines. Furthermore, treatment of larvae with a juvenile hormone analog strongly suppresses the abundance of the message.