Ecdysteroid Pathway Research Articles

De novo RNA-Seq and Annotation of Sesquiterpenoid and Ecdysteroid Biosynthesis Genes and MicroRNAs in a Spider Mite Eotetranychus kankitus.

Eotetranychus kankitus is an important mite pest in citrus, but molecular data on the developmental processes of E. kankitus are lacking. The different development stages mix of E. kankitus was used to sequence for transcriptome and small RNAs to identify genes and predict miRNAs associated with sesquiterpenoid and ecdysteroid biosynthesis and signaling pathways. More than 36 million clean reads were assembled and 67,927 unigenes were generated. Of the unigenes, 19,300 were successfully annotated through annotation databases NR, SwissProt, COG, GO, KEGG, PFAM, and KOG. The transcripts were involved in sesquiterpenoid biosynthesis (11 genes) and ecdysteroid biosynthesis and signaling pathway (13 genes). Another, small RNA library was obtained and 31 conserved miRNAs were identified. Five most abundant miRNAs were Ek-miR-5735, Ek-miR-1, Ek-miR-263a, Ek-miR-184, and Ek-miR-8. The target genes related to sesquiterpenoid and ecdysteroid showed that 10 of the conserved miRNAs could potentially target the sesquiterpenoid and ecdysteroid pathway according to four-prediction software, sRNAT, miRanda, RNAhybrid, and Risearch2. Thus, the results of this study will provide bioinformatics information for further molecular studies of E. kankitus which may facilitate improved pest control strategies.

Testes morphology and the identification of transcripts of the hormonal pathways of the velvetbean caterpillar Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Erebidae)

Anticarsia gemmatalis is one of the main defoliating pests of soybeans in Brazil. In the current study, we characterized the histomorphology of the testes and the spermatogenesis process in A. gemmatalis. We also identified transcripts involved in the biosynthesis, metabolism, and signaling of juvenile and ecdysteroid hormones, in order to provide information about potential mechanisms of regulation of hormonal pathways in this species. Our analyses revealed that the A. gemmatalis larvae have a pair of kidney-shaped testicles. These are divided into four testicular follicles, where there are germ cell cysts at different stages of development. In the pupal stage, the testicles are fused, so adults have a single spherical testis, with a variable number of follicles. The A. gemmatalis has centripetal spermatogenesis and exhibits spermatic dimorphism. We identified 31 transcripts that encode proteins involved in juvenile hormone and ecdysteroid pathways, such as mevalonate kinase, CYP14A1, ecdysone receptor, among others. Our results on the morphology of the testes and spermatogenesis process, as well as identification of the genes involved in hormonal pathways in A. gemmatalis, provide important data for understanding the biology of this agricultural pest, which can be used as a basis for further research in other economically important lepidopterans.

Effects of ultraviolet-filters on Daphnia magna development and endocrine-related gene expression.

Ultraviolet (UV) filters are emerging contaminants of concern that are widely spread throughout the aquatic environment. Many organic UV filters are endocrine disrupting compounds (EDCs) in vertebrates. However, few studies have assessed their effects on invertebrates. Molting, or the shedding of the exoskeleton, may be affected by exposure to these compounds in Arthropods (the largest phylum of invertebrates). Molting is necessary for growth and development and is regulated by an arthropod specific endocrine system, the ecdysteroid pathway. Alterations of this process by EDCs can result in improper development, reduced growth, and even death. We investigated the sublethal effects of chronic exposure to three organic UV filters (4-methylbenzylidene camphor (4MBC), octylmethoxycinnamate (OMC), and benzophenone-3 (BP3) in a crustacean, Daphnia magna, with particular emphasis on molting and development. We demonstrate that 4MBC, OMC, and BP3 affect development and long-term health in neonates of exposed parents at concentrations of 130µg/L, 75µg/L, and 166µg/L, respectively. Additionally, the expression of endocrine-related genes (including ultraspiracle protein, usp) are significantly altered by 4MBC and BP3 exposure, which may relate to their developmental toxicity.

Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor.

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20‐hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa's reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa's ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH‐like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa's initiation of egg laying.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

Transcriptional response of springtail (Folsomia candida) exposed to decabromodiphenyl ether-contaminated soil

Decabromodiphenyl ether (BDE209) is a widely used brominated flame retardant that has become a common soil contaminant of concern due to its persistence and toxicity. However, little is known about molecular-level effects of BDE209 on soil invertebrates. Here, we detected changes in gene transcription of the soil springtail, Folsomia candida, exposed to BDE209 (0.81 mg/kg) in soil for 2, 7 and 14 days. We identified 16 and 771 significantly differentially expressed genes after 2 and 7 days of exposure respectively, and no significantly regulated genes were shared among the two time points. No genes were affected after 14 days of exposure. According to the annotation of the significantly differently expressed genes at 2 and 7 day exposure, we found that BDE209 affected the transcription of genes involved in moulting, neural signal transmission and detoxification. Our results suggested that BDE209 could disrupt moulting of F. candida via the ecdysteroid pathway, and cause neurotoxicity through disrupting some neurotransmitter signalling pathways. This study provided insights into the toxic mechanism of BDE209 on F. candida.

Acute toxicity of bisphenol A and its structural analogues and transcriptional modulation of the ecdysone-mediated pathway in the brackish water flea Diaphanosoma celebensis

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.

Relative expression of three key genes involved in the hormonal cycle of the freshwater amphipod, Gammarus pulex.

Ecdysteroids and sesquiterpenoids are the two groups of hormones controlling molt and growth processes in amphipods, and are mostly represented by ecdysone (i.e., 20-hydroxyecdysone) and methyl farnesoate (MF), respectively. The endocrine system responsible for their syntheses is located in the cephalothorax and is composed of three main organs: the X-organ/sinus gland complex (XO), the Y-organ (YO) and the mandibular organ. Ecdysone synthesis is negatively controlled by the molt-inhibiting hormone (MIH) produced by XO, and its signal is mediated through the ecdysteroid receptor (EcR); whereas the MF production is limited by the farnesoic acid O-methyltransferase enzyme (FAMeT). As little is known on MIH, EcR, and FAMeT in amphipods, this study focused on the evaluation of the expression variations of the gene of these three proteins in Gammarus pulex, during the embryonic development and the molt cycle of females. Results highlighted the presence of ecr and famet genes from the first stages of the embryogenesis, suggesting key roles in the embryo development. The mih gene was only measured from Stage 3 of embryogenesis, probably related to the gastrulation and the cephalothorax development. Regardless of the gene, a strong overexpression was also measured at the hatch time. Besides, differential expression levels of mih, ecr, and famet genes through the molt cycle were observed. The highest expressions of the three genes were measured at the premolt stage, confirming key roles of MIH and EcR in the ecdysteroid pathways, and also suggesting the involvement of the FAMeT enzyme during the ecdysis in G. pulex.

Effects of DEHP on post-embryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.

Molecular Mechanisms of Wing Polymorphism in Insects.

Many insects are capable of developing into either long-winged or short-winged (or wingless) morphs, which enables them to rapidly match heterogeneous environments. Thus, the wing polymorphism is an adaptation at the root of their ecological success. Wing polymorphism is orchestrated at various levels, starting with the insect's perception of environmental cues, then signal transduction and signal execution, and ultimately the transmitting of signals into physiological adaption in accordance with the particular morph produced. Juvenile hormone and ecdysteroid pathways have long been proposed to regulate wing polymorphism in insects, but rigorous experimental evidence is lacking. The breakthrough findings of ecdysone receptor regulation on transgenerational wing dimorphism in the aphid Acyrthosiphon pisum and of insulin signaling in the planthopper Nilaparvata lugens greatly broaden our understanding of wing polymorphism at the molecular level. Recently, the advent of high-throughput sequencing coupled with functional genomics provides powerful genetic tools for future insights into the molecular bases underlying wing polymorphism in insects.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

Rice stripe virus counters reduced fecundity in its insect vector by modifying insect physiology, primary endosymbionts and feeding behavior.

Virus-vector relationships can be complex and diverse as a result of long-term coevolution. Understanding these interactions is crucial for disease and vector management. Rice stripe virus (RSV) is known to be transovarially transmitted within its vector, Laodelphax striatellus, and causes serious rice stripe disease. In RSV-infected L. striatellus, we found contrasting changes in vector fecundity, physiology, primary endosymbionts (i.e. yeast-like symbionts, YLS) and feeding behavior that can interact to affect the spread of RSV. RSV-infected L. striatellus exhibited a significant decrease in fecundity that could lead a reduction of viruliferous individuals in populations. As a potential response to this loss, RSV infection also significantly shortened nymphal stage duration, which can strengthen RSV vertical circulation in L. striatellus populations and promote RSV spreading by adult migration and dispersal. Down-regulated JHAMT and up-regulated CYP307A1 in the juvenile hormone and ecdysteroid pathways, respectively, were linked to accelerated development. RSV-infected adults were also found to have higher body weight in conjunction with increased YLS abundance. Furthermore, prolonged host plant phloem exposure to salivation by RSV-infected adults should further enhance RSV horizontal transmission. Our study highlights potential strategies of RSV in enhancing its transmission, and provides new insights into the complexity of virus-vector interactions.

How Did Arthropod Sesquiterpenoids and Ecdysteroids Arise? Comparison of Hormonal Pathway Genes in Noninsect Arthropod Genomes.

The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the “Broad-Complex” was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor (“Methoprene-tolerant”). Furthermore, the gain of “Phantom” differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.

Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata

Although the sesquiterpenoid juvenile hormone (JH) and the steroidal ecdysteroids are of vital importance to the development and reproduction of insects, our understanding of the evolution of these crucial hormonal regulators in other arthropods is limited. To better understand arthropod hormone evolution and regulation, here we describe the hormonal pathway genes (e.g. those involved in hormone biosynthesis, degradation, regulation and signal transduction) of a new decapod model, the shrimp Neocaridina denticulata. The majority of known insect sesquiterpenoid and ecdysteroid pathway genes and their regulators are contained in the N. denticulata genome. In the sesquiterpenoid pathway, these include biosynthetic pathway components: juvenile hormone acid methyltransferase (JHAMT); hormone binding protein: juvenile hormone binding protein (JHBP); and degradation pathway components: juvenile hormone esterase (JHE), juvenile hormone esterase binding protein (JHEBP) and juvenile hormone epoxide hydrolase (JHEH), with the JHBP, JHEBP and JHEH genes being discovered in a crustacean for the first time here. Ecdysteroid biosynthetic pathway genes identified include spook, phantom, disembodied, shadow and CYP18. Potential hormonal regulators and signal transducers such as allatostatins (ASTs), Methoprene-tolerant (Met), Retinoid X receptor (RXR), Ecdysone receptor (EcR), calponin-like protein Chd64, FK509-binding protein (FKBP39), Broad-complex (Br-c), and crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone (CHH/MIH/GIH) genes are all present in the shrimp N. denticulata. To our knowledge, this is the first report of these hormonal pathways and their regulatory genes together in a single decapod, providing a vital resource for further research into development, reproduction, endocrinology and evolution of crustaceans, and arthropods in general.

Cloning of a cDNA Encoding a Novel Cytochrome P450 from the Insect Locusta migratoria: CYP6H1, a Putative Ecdysone 20-Hydroxylase

The biosynthesis of the steroidal molting hormone, 20-hydroxyecdysone, of arthropods involves a series of cytochrome P450-catalyzed hydroxylations. None of the many sequences of insect cytochromes P450, known to date, is related to ecdysteroid pathways. Here, we report the cloning and sequencing of a full-length cDNA of a new cytochrome P450, classified as CYP6H1, from malpighian tubules of the locust, Locusta migratoria. The 1854 bp DNA contained an open reading frame coding for a protein of 542 amino acids, a 5′-leader sequence and a 3′-untranslated region containing a polyadenylation signal and a poly(A) tail. The encoded protein had been isolated as an ecdysone-binding cytochrome P450 from microsomes of the same tissue in previous work. The closest homolog of CYP6H1 was CYP6A2 from Drosophila with 42.1% identity. According to Northern analysis, CYP6H1 is predominantly expressed at larval instars and in malpighian tubules. Evidence is presented for a functional assigment of CYP6H1 to microsomal ecdysone 20-hydroxylase of the locust.

Biosynthesis of ecdysone and ecdysone phosphates in spinach

The polar ecdysteroid conjugate, ecdysone phosphate (2β,3β,14α,22 R,25-pentahydroxy-7-en-6-one-3- phosphate) was identified in excised first leaves of spinach, where it is endogenously produced during 20- hydroxyecdysone biosynthesis. Radiolabelled [ 14C]ecdysone phosphate was isolated from several excised leaf assays and was hydrolysed with wheat germ acid phosphatase to yield [ 14C]ecdysone. Incorporation of ecdysone into excised first leaves followed by 32P exposure produced a compound with 32P activity, with chromatographic properties identical to those of the isolated [ 14C]ecdysone phosphate and upon hydrolysis released ecdysone. In spinach first leaves with active ecdysteroid biosynthesis, ecdysone is present at 0.004% of the total free ecdysteroid and contained 6% of the total radioactivity from [2- 14C]mevalonic acid (MVA). These biosynthetically active tissues also produce radiolabelled lathosterol, ecdysone-3-phosphate and 20-hydroxyecdysone. In biosynthetically inactive tissue (immature apical organs) no radiolabelled lathosterol, ecdysone-3-phosphate, ecdysone or 20-hydroxyecdysone was produced from [2- 14C]MVA despite an active biosynthesis of C 29-sterols. Several intermediate and end product ecdysteroids, when incorporated into excised first leaves of spinach produced conjugates which were readily cleaved by wheat germ acid phosphatase. The ecdysteroid pathway appears to be regulated by the presence of ecdysteroid substrates.

Regulation of ecdysteroid biosynthesis in crayfish Y‐organs: I. Role of cyclic nucleotides

AbstractEcdysteroidogenesis in crustacean Y‐organs is negatively regulated by the so‐called molt‐inhibiting hormone (MIH), which is thought to inhibit the ecdysteroid pathway either directly through phosphorylation of the key enzyme(s) and/or through action on the synthesis of these enzymes. In the present study the role of cyclic nucleotides in the mode of action of MIH in the astacuran Orconectes limosus was investigated. Orconectes Y‐organs were incubated in vitro with sinus gland conditioned medium (SGCM) and cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured. A striking increase of cGMP values (4 to 43 pmoles/mg protein) was observed after 2 hours, whereas cAMP levels were unchanged. Using the established in vitro bioassay for the measurement of inhibitory action on ecdysteroid release from Y‐organs (Soumoff and O'Connor, '82) the potential of dBcAMP, dBcGMP, forskolin, and IBMX to diminish this release was demonstrated, suggesting a possible role of both cyclic nucleotides in the mode of action of MIH. The present findings are discussed in view of the effect of MIH‐material on cyclic nucleotide levels in crab Y‐organs (Mattson, '86). © 1993 Wiley‐Liss, Inc.