A validated HPLC-MS/MS method for the simultaneous determination of ecdysteroid hormones in subminimal amounts of biological material.

Ecdysteroids represent a large class of polyhydroxylated steroids which, due to their anabolic properties, are marketed as dietary supplements. Some ecdysteroids also act as important hormones in arthropods, where they regulate molting, development, and reproduction and many of these insects are miniature organisms that contain submicroliter levels of circulating biofluids. Analysis of ecdysteroids is further complicated by their very low abundance, large fluctuations during development, and difficult access to a pooled sample, which is important for quantitative measurements. In this work, we propose a new method that overcomes the described difficulties and allows validated quantification of four ecdysteroids in minimal amounts of biological material. After methanolic extraction, detectability of the ecdysteroids is increased 16- to 20-fold by conversion to their 14,15-anhydrooximes. These are further purified by pipette tip solid-phase extraction on a three-layer sorbent and subjected to HPLC-MS/MS analysis. Full validation was achieved using hemolymph from larvae of the firebug Pyrrhocoris apterus as a blank matrix and by the determination of ecdysteroids in a single Drosophila larva. The lower limit of quantifications for the four target ecdysteroids (20-hydroxyecdysone, ecdysone, makisterone A, and 2-deoxyecdysone) were 0.01; 0.1; 0.05; and 0.025 pg·ml-1 (20; 200; 100; 50 fmol ml-1), respectively, with very good accuracy, precision (expressed as relative standard deviation <15%) and recoveries (96%-119.9%). The application potential of the new method was demonstrated by quantification of ecdysteroids in various biological materials including human serum.

Elucidating the ecophysiology of soybean pod-sucking stinkbug Riptortus pedestris (Hemiptera: Alydidae) based on de novo genome assembly and transcriptome analysis.

Food security is important for the ever-growing global population. Soybean, Glycine max (L.) Merr., is cultivated worldwide providing a key source of food, protein and oil. Hence, it is imperative to maintain or to increase its yield under different conditions including challenges caused by abiotic and biotic stresses. In recent years, the soybean pod-sucking stinkbug Riptortus pedestris has emerged as an important agricultural insect pest in East, South and Southeast Asia. Here, we present a genomics resource for R. pedestris including its genome assembly, messenger RNA (mRNA) and microRNA (miRNA) transcriptomes at different developmental stages and from different organs. As insect hormone biosynthesis genes (genes involved in metamorphosis) and their regulators such as miRNAs are potential targets for pest control, we analyzed the sesquiterpenoid (juvenile) and ecdysteroid (molting) hormone biosynthesis pathway genes including their miRNAs and relevant neuropeptides. Temporal gene expression changes of these insect hormone biosynthesis pathways were observed at different developmental stages. Similarly, a diet-specific response in gene expression was also observed in both head and salivary glands. Furthermore, we observed that microRNAs (bantam, miR-14, miR-316, and miR-263) of R. pedestris fed with different types of soybeans were differentially expressed in the salivary glands indicating a diet-specific response. Interestingly, the opposite arms of miR-281 (-5p and -3p), a miRNA involved in regulating development, were predicted to target Hmgs genes of R. pedestris and soybean, respectively. These observations among others highlight stinkbug's responses as a function of its interaction with soybean. In brief, the results of this study not only present salient findings that could be of potential use in pest management and mitigation but also provide an invaluable resource for R. pedestris as an insect model to facilitate studies on plant-pest interactions.

Endocrine Regulation of Aging in the Fruit Fly Drosophila melanogaster.

The past few decades have witnessed increasing research clarifying the role of endocrine signaling in the regulation of aging in both vertebrates and invertebrates. Studies using the model organism fruit fly Drosophila melanogaster have largely advanced our understanding of evolutionarily conserved mechanisms in the endocrinology of aging and anti-aging. Mutations in single genes involved in endocrine signaling modify lifespan, as do alterations of endocrine signaling in a tissue- or cell-specific manner, highlighting a central role of endocrine signaling in coordinating the crosstalk between tissues and cells to determine the pace of aging. Here, we review the current landscape of research in D. melanogaster that offers valuable insights into the endocrine-governed mechanisms which influence lifespan and age-related physiology.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ).

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

Insect Sterols and Steroids.

Insects are incapable of biosynthesising sterols de novo so they need to obtain them from their diets or, in certain cases, from symbiotic microorganisms. Sterols serve a structural role in cellular membranes and act as precursors for signalling molecules and defence compounds. Many phytophagous insects dealkylate phytosterols to yield primarily cholesterol, which is also the main sterol that carnivorous and omnivorous insects obtain in their diets. Some phytophagous species have secondarily lost the capacity to dealkylate and consequently use phytosterols for structural and functional roles. The polyhydroxylated steroid hormones of insects, the ecdysteroids, are derived from cholesterol (or phytosterols in non-dealkylating phytophagous species) and regulate many crucial aspects of insect development and reproduction by means of precisely regulated titres resulting from controlled synthesis, storage and further metabolism/excretion. Ecdysteroids differ significantly from vertebrate steroid hormones in their chemical, biochemical and biological properties. Defensive steroids (cardenolides, bufadienolides, cucurbitacins and ecdysteroids) can be accumulated from host plants or biosynthesised within the insect, depending on species, stored in significant amounts in the insect and released when it is attacked. Other allelochemical steroids serve as pheromones. Vertebrate-type steroids have also been conclusively identified from insect sources, but debate continues about their significance. Side chain dealkylation of phytosterols, ecdysteroid metabolism and ecdysteroid mode of action are targets of potential insect control strategies.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Low concentrations of cyantraniliprole negatively affects the development of Spodoptera frugiperda by disruption of ecdysteroid biosynthesis and carbohydrate and lipid metabolism.

In addition to the acute lethal toxicity, insecticides might affect population dynamics of insect pests by inducing life history trait changes under low concentrations, however, the underlying mechanisms remain not well understood. Here we examined systemic impacts on development and reproduction caused by low concentration exposures to cyantraniliprole in the fall armyworm (FAW), Spodoptera frugiperda, and the putative underlying mechanisms were investigated. The results showed that exposure of third-instar larvae to LC10 and LC30 of cyantraniliprole significantly extended larvae duration by 1.46 and 5.41 days, respectively. Treatment with LC30 of cyantraniliprole significantly decreased the pupae weight and pupation rate as well as the longevity, fecundity and egg hatchability of female adults. Consistently, we found that exposure of FAW to LC30 cyantraniliprole downregulated the mRNA expression of four ecdysteroid biosynthesis genes including SfNobo, SfShd, SfSpo and SfDib and one ecdysone response gene SfE75 in the larvae as well as the gene encoding vitellogenin (SfVg) in the female adults. We also found that treatment with LC30 of cyantraniliprole significantly decreased the whole body levels of glucose, trehalose, glycogen and triglyceride in the larvae. Our results indicate that low concentration of cyantraniliprole inhibited FAW development by disruption of ecdysteroid biosynthesis as well as carbohydrate and lipid metabolism, which have applied implications for the control of FAW.

Piperonyl butoxide elicits a robust transcriptional response in adult Drosophila melanogaster.

While much attention has been devoted to understanding the transcriptional changes underlying resistance to insecticides, comparatively little is known about the transcriptional response of naive insects to agrochemicals. In this study, we analyze the transcriptomic response of an insecticide susceptible strain of Drosophila melanogaster to nine agrochemicals using a robust method that goes beyond classical replication standards. Our findings demonstrate that exposure to piperonyl butoxide (PBO), but not to eight other compounds, elicits a robust transcriptional response in a wild-type strain of Drosophila melanogaster. PBO exposure leads to the upregulation of a subset of Cyps, GSTs, UGTs and EcKls. This response is both time and concentration-dependent, suggesting that the degree of inhibition of P450 activity correlates with the magnitude of the transcriptional response. Furthermore, the upregulation of these enzymes is excluded from reproductive organs. Additionally, different sets of genes are regulated in the digestive/secretory tract and the carcass. Our results suggest that P450s play a role in metabolizing yet unidentified endogenous compounds and are involved in an as-yet-unknown physiological regulatory feedback loop.

The nuclear receptor gene E75 plays a key role in regulating the molting process of the spider mite, Tetranychus urticae.

The nuclear receptor gene Ecdysone-induced protein 75 (E75), as the component of ecdysone response genes in the ecdysone signaling pathway, has important regulatory function for insect molting. However, the regulatory function of E75 during the molting process of spider mites is not yet clear. In this study, the expression pattern of E75 in the molting process of the spider mite Tetranychus urticae was analyzed. The results showed that there was a peak at 8 h post-molting, followed by a decline 8 h after entering each respective quiescent stage across various developmental stages. During the deutonymph stage, the expression dynamics of E75, observed at 4-h intervals, indicated that the transcript levels of TuE75 peaked at 24 h, coinciding with the onset of molting in the mites. To investigate the function of TuE75 during the molting process, silencing TuE75 through dsRNA injection into deutonymph mites at the age of 8 h yielded a notable outcome: 78% of the deutonymph mites were unable to progress to the adult stage. Among these phenotypic mites, 37% were incapable of transitioning into the quiescent state and eventually succumbed after a certain period. An additional 41% of the mites successfully entered the quiescent state but encountered difficulties in shedding the old epidermis, leading to eventual mortality. In summary, these results suggested that TuE75 plays a key role in the molting process of T. urticae.

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

A novel GSTe2 involved in metamorphosis by regulating 20E signal pathway in Tribolium castaneum.

Insect-specific epsilon glutathion S-transferases (GSTs) are a class of multifunctional GST superfamily, which play important roles in detoxification of xenobiotic substances. Most research on GSTs has focused on insecticide detoxification and resistance, with little research on other physiological functions. Here, we identified and cloned the novel GSTe2 from Tribolium castaneum (TcGSTe2). Recombinant TcGSTe2 protein was successfully overexpressed in Escherichia coli and purified with affinity purification, which had high ability to catalyze the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene (CDNB). The expression level of TcGSTe2 was significantly decreased after exposure with four insecticides, phoxim, λ-cyhalothrin, dichlorvos, and carbofuran, in larval stage. Interestingly, RNA interference knockdown of TcGSTe2 caused metamorphosis deficiency in larval and pupal stages by inhibiting the 20E signal pathway. Furthermore, exogenous 20E injection partially rescued this metamorphosis deficiency and also increased the expression levels of 20E downstream response genes. This study illustrated TcGSTe2 plays an important role at metamorphosis beside the insecticide detoxification and resistance in T. castaneum.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis.

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

Ecdysteroid Biosynthesis Halloween Gene Spook Plays an Important Role in the Oviposition Process of Spider Mite, Tetranychus urticae.

In insects, the ecdysteroid hormone regulates development and reproduction. However, its function in the reproduction process of spider mites is still unclear. In this study, we investigated the effect of the Halloween gene Spook on the oviposition of the reproduction process in a spider mite, Tetranychus urticae. The expression patterns of the ecdysteroid biosynthesis and signaling pathway genes, as analyzed by RT-qPCR, showed that the expression pattern of the Halloween genes was similar to the oviposition pattern of the female mite and the expression patterns of the vitellogenesis-related genes TuVg and TuVgR, suggesting that the Halloween genes are involved in the oviposition of spider mites. To investigate the function of the ecdysteroid hormone on the oviposition of the reproduction process, we carried out an RNAi assay against the Halloween gene Spook by injection in female mites. Effective silencing of TuSpo led to a significant reduction of oviposition. In summary, these results provide an initial study on the effect of Halloween genes on the reproduction in T. urticae and may be a foundation for a new strategy to control spider mites.

Control of the ecdysteroid level plays a crucial role in density-dependent metamorphosis in the giant mealworm beetle Zophobas atratus.

Metamorphic transition in some tenebrionid beetles is dependent on population density. This phenomenon is useful for pupae that are vulnerable to cannibalism. The physiological mechanism of this adaptive developmental phenomenon remains unclear. In Zophobas atratus, which show density-dependent metamorphosis, larval isolation can induce metamorphosis. We herein demonstrated that the return of isolated larvae to a crowded condition (re-crowding) inhibited their metamorphosis. The timing of metamorphic initiation was slightly extended according to the duration of re-crowding experienced by the isolated larvae. Therefore, the re-crowding induced physiological changes needed for metamorphic inhibition. We investigated whether hormone-related genes involved in signaling of metamorphic inhibitor (juvenile hormone, JH) and molting hormone (ecdysteroid) responded to the re-crowding. An expression analysis showed that gene expression of ecdysteroid signaling was maintained at low levels under the re-crowded condition. Actually, ecdysteroid levels decreased responding to re-crowding. Ecdysteroid injections induced metamorphosis in re-crowded larvae. In contrast, the JH signaling gene showed little fluctuation in both isolated and re-crowded conditions, and knockdown of JH signaling factors did not affect inhibition of metamorphosis under the re-crowded condition. The present study suggests that regulation of ecdysteroid level rather than JH is more crucial in the density dependent metamorphosis in Z. atratus.

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

Regulation of juvenile hormone and ecdysteroid analogues on the development of the predatory spider, Pardosa pseudoannulata, and its regulatory mechanisms.

Insecticides harm the beneficial organisms, such as predatory spiders, through direct killing or regulation of the development and reproduction. In this study, the bioassay showed that the treatment of juvenile hormone (JH) analogue fenoxycarb delayed the moulting of Pardosa pseudoannulata, a dominant predatory spider in paddy fields. In order to figure out the regulatory mechanism of fenoxycarb on the spider development, we systematically analyzed JH biosynthesis in P. pseudoannulata. All genes involved in JH biosynthesis pathway were retrieved from the genome of P. pseudoannulata, except for CYP15A1. The absence of CYP15A1 was in agreement with the identification of methyl farnesoate (MF) rather than JH III in the spider. The delayed moulting and decreased expression of JH biosynthesis-related genes in the MF-applied spiderlings supported that MF was an active JH. Fenoxycarb treatment significantly upregulated the transcriptional level of JH biosynthesis-related genes and consequently delayed the spiderling moulting. In the spider development, ecdysteroid played the opposite role, in contrast to MF, to accelerate the development, as our previous study. Here we found that the treatment of ecdysteroid analogue tebufenozide accelerated P. pseudoannulata spiderling moulting, which resulted from the expressional suppression of ecdysteroid biosynthesis-related genes. In total, the JH and ecdysteroid analogues affected the development of P. pseudoannulata by the expressional regulation of biosynthesis-related genes, which would be helpful for the evaluation of hormone analogue insecticides in environmental safety, and useful for the protection and application of P. pseudoannulate and related spider species.

Diversity-Oriented Synthesis Catalyzed by Diethylaminosulfur-Trifluoride-Preparation of New Antitumor Ecdysteroid Derivatives.

Fluorine represents a privileged building block in pharmaceutical chemistry. Diethylaminosulfur-trifluoride (DAST) is a reagent commonly used for replacement of alcoholic hydroxyl groups with fluorine and is also known to catalyze water elimination and cyclic Beckmann-rearrangement type reactions. In this work we aimed to use DAST for diversity-oriented semisynthetic transformation of natural products bearing multiple hydroxyl groups to prepare new bioactive compounds. Four ecdysteroids, including a new constituent of Cyanotis arachnoidea, were selected as starting materials for DAST-catalyzed transformations. The newly prepared compounds represented combinations of various structural changes DAST was known to catalyze, and a unique cyclopropane ring closure that was found for the first time. Several compounds demonstrated in vitro antitumor properties. A new 17-N-acetylecdysteroid (13) exerted potent antiproliferative activity and no cytotoxicity on drug susceptible and multi-drug resistant mouse T-cell lymphoma cells. Further, compound 13 acted in significant synergism with doxorubicin without detectable direct ABCB1 inhibition. Our results demonstrate that DAST is a versatile tool for diversity-oriented synthesis to expand chemical space towards new bioactive compounds.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

Characterization of ecdysteroid biosynthesis in the pond wolf spider, Pardosa pseudoannulata.

Ecdysteroids, as the key growth hormones, regulate moulting, metamorphosis and reproduction in arthropods. Ecdysteroid biosynthesis is catalysed by a series of cytochrome P450 monooxygenases (CYP450s) encoded by Halloween genes, including spook (spo), phantom (phm), disembodied (dib), shadow (sad) and shade (shd). The ecdysteroid biosynthesis in insects is clear with 20-hydroxyecdysone (20E) as the main ecdysteroid. However, the information on the major ecdysteroids in arachnids is limited. In this study, Halloween genes spo, dib, sad and shd, but not phm, were identified in the pond wolf spider, Pardosa pseudoannulata. Phylogenetic analysis grouped arachnid and insect Halloween gene products into two CYP450 clades, the CYP2 clan (spo and phm) and the mitochondrial clan (dib, sad, and shd). In P. pseudoannulata, the temporal expression profile of the four Halloween genes in concurrence with spiderling moulting with steady increase in the course of the 2nd instar followed by a rapid dropdown once moulting was completed. Spatially, the four Halloween genes were highly expressed in spiderling abdomen and in the ovaries of female adults. In parallel, ponasterone A (PA), but not 20E, was detected by LC-MS/MS analysis in P. pseudoannulata, and it was demonstrated as a functional ecdysteroid in the spider by accelerating of moulting with PA addition. The present study revealed the different ecdysteroid biosynthesis pathways in spiders and insects.