Juvenile Hormone Biosynthesis Research Articles

Juvenile Hormone Studies in Drosophila melanogaster.

In the field of insect endocrinology, juvenile hormone (JH) is one of the most wondrous entomological terms. As a unique sesquiterpenoid hormone produced and released by the endocrine gland, corpus allatum (CA), JH is a critical regulator in multiple developmental and physiological processes, such as metamorphosis, reproduction, and behavior. Benefited from the precise genetic interventions and simplicity, the fruit fly, Drosophila melanogaster, is an indispensable model in JH studies. This review is aimed to present the regulatory factors on JH biosynthesis and an overview of the regulatory roles of JH in Drosophila. The future directions of JH studies are also discussed, and a few hot spots are highlighted.

Identification of an allatostatin C signaling system in mollusc Aplysia

Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

In silico identification of potential phytochemical inhibitors targeting farnesyl diphosphate synthase of cotton bollworm (Helicoverpa armigera)

Helicoverpa armigera (Ha), a polyphagous pest, causes significant damage to several crop plants, including cotton. The control of this cosmopolitan pest is largely challenging due to the development of resistance to existing management practices. The Juvenile Hormone (JH) plays a pivotal role in the life cycle of insects by regulating their morphogenetic and gonadotropic development. Hence, enzymes involved in JH biosynthesis are an attractive target for the development of selective insecticides. Farnesyl diphosphate synthase (FPPS), a member protein of (E)-prenyl-transferases, is one of the most crucial enzymes in the biosynthetic pathway of JHs. It catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), forming farnesyl diphosphate (FPP), a precursor of JH. The study was designed to identify an effective small inhibitory molecule that could inhibit the activity of Helicoverpa armigera – FPPS (HaFPPS) for an effective pest control intervention. Therefore, a 3D model of FPPS protein was generated using homology modeling. The FooDB database library of small molecules was selected for virtual screening, following which binding affinities were evaluated using docking studies. Three top-scored molecules were analyzed for various pharmacophore properties. Further, molecular dynamics (MD) simulation analysis showed that the identified molecules (mitraphylline-ZINC1607834, chlorogenic acid-ZINC2138728 and llagate-ZINC3872446) had a reasonably acceptable binding affinity for HaFPPS and resulted in the formation of a stable HaFPPS-inhibitor(s) complex. The identified phytochemical molecules may be used as potent inhibitors of HaFPPS thus, paving the way for further developing environment-friendly insect growth regulator(s). Communicated by Ramaswamy H. Sarma

Genomic Identification and Functional Analysis of JHAMTs in the Pond Wolf Spider, Pardosa pseudoannulata.

Juvenile hormone (JH) plays a critical role in many physiological activities of Arthropoda. Juvenile hormone acid methyltransferase (JHAMT) is involved in the last steps of JH biosynthesis as an important rate-limiting enzyme. In recent studies, an increasing number of JHAMTs were identified in arthropods, but no JHAMT was reported in spiders. Herein, eight JHAMTs were identified in the pond wolf spider, Pardosa pseudoannulata, all containing the well conserved S-adenosyl-L-methionine binding motif. JHAMT-1 and the other seven JHAMTs were located at chromosome 13 and chromosome 1, respectively. Multiple alignment and phylogenetic analysis showed that JHAMT-1 was grouped together with insect JHAMTs independently and shared high similarities with insect JHAMTs compared to the other seven JHAMTs. In addition, JHAMT-1, JHAMT-2, and JHAMT-3 were highly expressed in the abdomen of spiderlings and could respond to the stimulation of exogenous farnesoic acid. Meanwhile, knockdown of these three JHAMTs caused the overweight and accelerated molting of spiderlings. These results demonstrated the cooperation of multi-JHAMTs in spider development and provided a new evolutionary perspective of the expansion of JHAMT in Arachnida.

Gene cloning, recombinant expression, and bioassay of an allatotropin in Spodoptera litura Fabricius\xa0(Lepidoptera: Noctuidae)

BackgroundAllatotropin, a neuropeptide found in several invertebrates indirectly regulates vitellogenesis by stimulating juvenile hormone biosynthesis by the corpora allata. Here, we cloned and expressed the gene encoding allatotropin of Spodoptera litura (tobacco cutworm), a polyphagous pest in the Asian tropics. This study is aimed at studying the effect of recombinant protein on egg-laying in females of S. litura as it could be used as a method to control the pests from a reproductive perspective.ResultsThe protein encodes a full-length open reading frame consisting of 173 amino acids and was rich in arginine (10%) and glutamic acid (9.3%). The theoretical pI of the protein was 5.47 and a hydrophobic signal peptide of 22 residues was predicted. The recombinant allatotropin was expressed in Escherichia coli BL21 (DE3) and purified by nickel exchange chromatography. The molecular weight of the recombinant protein was about 37 kDa and expression levels up to 5.3 mg/ml were achieved. Injection assay in vitro indicated that allatotropin induces egg-laying during the first scotophase after treatment in females of Spodoptera litura.ConclusionAllatotropin induces egg-laying in female moths and could be a potential molecule for the development of control strategies against Spodoptera litura. In this strategy, the protein if delivered to the females before mating may lead to accelerated egg deposition much before she encounters the male moths, thus the population being checked as the eggs deposited by the females are unfertilized. Thus, the present work could lead to the development of a protein based biopesticide resulting into a species-specific and an eco-friendly way of pest control.

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.

Juvenile hormone biosynthetic genes are critical for regulating reproductive diapause in the cabbage beetle

In insects, the juvenile hormone (JH) biosynthetic pathway regulates the in vivo JH titer. Thus, its downregulation potentially contributes to the lowering of JH titers typically observed in insects undergoing reproductive diapause, a developmental arrest at the adult stage. However, no systematic evidence has yet been presented to demonstrate the physiological and genetic roles of JH biosynthetic genes in reproductive diapause. In this work, we performed RNA interference (RNAi)-based reverse genetic analyses by targeting JH biosynthetic genes, followed by analysis of the reproductive diapause traits in Colaphellus bowringi, an economically important cabbage beetle. We identified a total of 22 genes encoding homologues of enzymes involved in the mevalonate pathway and the JH branch of JH biosynthesis in C. bowringi. Among these, 18 genes showed significant downregulation of their expression in the long day-induced diapausing females, compared to the short day-induced reproductive females. RNAi knockdown of almost any one of the 18 genes in reproductive females reduced the expression of the JH-responsive gene, Krüppel homolog1 (Kr-h1), indicating a lowered circulating JH. Most importantly, depleting transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase 2 (HMGR2), farnesyl-pyrophosphate synthase 1 (FPPS1) and juvenile hormone acid methyltransferase 1 (JHAMT1) induced diapause-associated traits, including immature and inactive ovaries, large accumulations of lipids and adult burrowing behavior. Meanwhile, genes related to ovarian development, lipid accumulation and stress response showed expression patterns like those of diapausing females. RNAi-mediated diapause phenotypes could be reversed to reproductive phenotypes by application of methoprene, a JH receptor agonist. These results suggest that photoperiodic reproductive diapause in C. bowringi is triggered by transcriptional suppression of JH biosynthetic genes, with HMGR2, FPPS1 and JHAMT1 playing a critical role in this process. This work provides sufficient evidence to reveal the physiological roles of JH biosynthetic genes in reproductive diapause.

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.

Key role of juvenile hormone in controlling reproductive diapause in females of the Asian lady beetle Harmonia axyridis.

The Asian lady beetle Harmonia axyridis is an important predator of several agricultural pests, including aphids and whiteflies, and thus can contribute to pest management. Commercial viability as a pest control method requires that the beetle can be mass-reared, and that workable conditions for extended shelf-life can be guaranteed. One of the features of Harmonia's life cycle is that it enters diapause in the adult stage when the length of the photophase starts shortening in late summer. Reduction of juvenile hormone (JH) titer has been demonstrated to be the common endocrine mechanism inducing reproductive diapause in insects. However, whether H. axyridis enters diapause dependent on JH shutdown and how the JH level is regulated before diapause remains unknown. Like in other insects, the absence of JH triggers the induction and maintenance of reproductive diapause in H. axyridis, indicated by JH measurements and the knockdown of an intracellular JH receptor methoprene-tolerant (Met). Methoprene, a JH analog, significantly reversed diapause into reproduction via Met. Combined with RNA-sequencing and RNA interference, we also demonstrated that JH biosynthesis rather than the JH degradation pathway determines the reduction of JH titer in diapausing females. Our results reveal the vital role of JH in regulating reproductive diapause in female H. axyridis. Harmonia axyridis diapause could thus be manipulated by targeting JH production and JH signaling. © 2021 Society of Chemical Industry.

Transcriptome Analysis of Hormone-and Cuticle-Related Genes in the Development Process of Deutonymph in Tetranychus urticae.

Simple SummaryThe two-spotted spider mite, Tetranychus urticae, is an important agricultural pest. Gene expression dynamics during different development time points of deutonymphal development process in T. urticae were investigated. The transcriptome sequencing aims to identify hormone- and cuticle-related genes that regulated the development process of spider mite. Moreover, this comprehensive transcriptome will provide a foundation for further research of development process in T. urticae.Tetranychus urticae is an important agricultural pest that feeds on more than 1100 plant species. To investigate gene expression network in development process of deutonymph, a comprehensive transcriptome analysis of different developmental time points of deutonymph in T. urticae was performed. Comparing with expression profile of 7 h, 309, 876, 2736, and 3432 differential expression genes were detected at time points 14 h, 21 h, 28 h, and 35 h, respectively. The expression dynamic analysis indicated that genes in hormone- (ecdysteroid and juvenile hormone) and cuticle- (chitin and cuticle proteins) related pathways were indispensable for development process in deutonymph. Among hormone related pathway genes, the ecdysteroid biosynthesis pathway genes were highly expressed at the growth period of development process, which is opposite to the expression patterns of juvenile hormone biosynthesis pathway genes. For cuticle related pathway genes, 13 chitinase genes were identified in the genome of T. urticae, and 8 chitinase genes were highly expressed in different time points of developmental process in the deutonymph of T. urticae. Additionally, 59 cuticle protein genes were identified from genome, and most of the cuticle protein genes were expressed in the molting period of developmental process in deutonymph. This study reveals critical genes involved in the development process of deutonymph and also provides comprehensive development transcriptome information for finding more molecular targets to control this pest.

Juvenile hormone: Production, regulation, current application in vector control and its future applications.

Juvenile hormone is an exclusive hormone found in insects which involves regulating various insect physiology. A total of eight juvenile hormones have been identified in insects which include JH 0, JH I, JH II, JH III, 4-methyl JH I (Iso- JH 0), JHB III, JHSB III, and MF. Corpora allata are the glands responsible for the production and synthesis of these hormones. They are involved in moulting, reproduction, polyethism, and behavioural regulations in different orders of insects. Factors such as diet temperatures, photoperiods, and plant compounds affect the biosynthesis and regulation of juvenile hormones. Juvenile hormones analogue is usually used to disrupt normal regulation of JH and this analogue is categorized as insect-growth regulators (IGRs) and is widely used in pest control as an alternative to chemical insecticides. Other applications of biosynthesis activities of this hormone have not been explored in the area of JHs. In this review, current applications of JHs with an addition of their future application will be discussed.

Juvenile hormone affects age polyethism, ovarian status and cuticular hydrocarbon profile in workers of the wasp Polybia occidentalis.

Division of labor is one of the most striking features in the evolution of eusociality. Juvenile hormone (JH) mediates reproductive status and aggression among nestmates in primitively eusocial Hymenoptera (species without morphologically distinct castes). In highly social species it has apparently lost its gonadotropic role and primarily regulates the division of labor in the worker caste. Polybia occidentalis, a Neotropical swarm-founding wasp, is an ideal model to understand how JH levels mirror social context and reproductive opportunities because of the absence of a clear morphological caste dimorphism. In this study, we tested the hypothesis that JH influences division of labor, ovary activation and cuticular hydrocarbon profiles of workers. Our observations confirmed that a JH analog (methoprene) and an inhibitor of JH biosynthesis (precocene) affected the cuticular chemical profile associated with age polyethism. Also, methoprene and precocene-I treatment of females influenced ovarian activation differently (individuals treated with methoprene expressed more activated ovaries while precocene treatment did not have significant effect). These results suggest that different hormonal levels induce a differential expression of cuticular chemicals associated with workers' age polyethism, which may be essential for keeping the social cohesion among workers throughout their lives in the colony. Furthermore, JH is likely to play a gonadotropic role in P. occidentalis. JH has apparently undergone certain modifications in social Hymenoptera, presenting multifaceted functions in different species.

MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster

The Drosophila melanogaster corpus allatum (CA) produces and releases three types of sesquiterpenoid hormones, including juvenile hormone III bisepoxide (JHB3), juvenile hormone III (JH III), and methyl farnesoate (MF). JH biosynthesis involves multiple discrete enzymatic reactions and is subjected to a comprehensive regulatory network including microRNAs (miRNAs). Using a high throughput sequencing approach, we have identified abundant miRNAs in the D. melanogaster ring gland, which consists of the CA, prothoracic gland, and corpus cardiaca. Genetic and qPCR screens were then performed in an attempt to uncover the full repertoire of CA miRNAs that are involved in regulating metamorphosis. miR-8 was identified as a potential candidate and further studied for its role in the CA. Overexpression of miR-8 in the CA increased cell size of the gland and expression of Jhamt (a gene coding for a key regulatory enzyme in JH biosynthesis), resulting in pupal lethality. By contrast, sponge-mediated reduction of miR-8 in the CA decreased cell size and Jhamt expression, but did not cause lethality. Further investigation revealed that miR-8 promotes cell growth independent of insulin/IGF signaling. Taken together, these experiments show that miR-8 is highly expressed in the CA and exerts its positive effects on cell growth and JH biosynthesis. The miRNAs data in the ring gland also provide a useful resource to study how miRNAs collaboratively regulate hormone synthesis in D. melanogaster.

A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster.

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis. Although this is also true for the model organism Drosophila melanogaster, neurotransmitters produced in the CA-projecting neurons are yet to be identified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase. We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggest that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. Unexpectedly, the inhibition of CA-projecting hugin neurons did not significantly alter the expression levels of the JH-inducible gene Krüppel-homolog 1, which implies that the CA-projecting neurons are not involved in JH biosynthesis but rather in other known biological processes. This is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

Photoperiodic Response in the Pars Intercerebralis Neurons, Including Plast-MIP Neurons, in the Brown-Winged Green Bug, Plautia stali.

Many insects in temperate regions avoid environmental adversity for reproduction, and thus enter reproductive diapause according to photoperiod. This reproductive diapause is induced by inhibition of juvenile hormone biosynthesis in the corpus allatum. Some neuropeptides that have an effect on juvenile hormone biosynthesis have been detected in insect brains. Thus, the reproductive diapause may be photoperiodically regulated by these juvenile hormones-controlling neuropeptides. However, there is limited understanding of how the neurons expressing these neuropeptides respond to the photoperiod and control the peptide release accordingly. Here, we performed electrophysiological analyses in the pars intercerebralis (PI) of Plautia stali, where juvenile hormone inhibitory neuropeptides, Plautia stali myoinhibitory peptides (Plast-MIPs) are expressed. We found that the large neurons in the PI showed very high firing activity under diapause-inducing short day conditions. Neurotracer staining revealed that all recorded neurons projected to the nervus corporis cardiaci 1, which is known to be connected to the corpus cardiacum-corpus allatum complex. Finally, we determined how many of the large PI cells expressed Plast-MIP by single cell reverse transcription PCR. About half of large PI neurons coexpressed Plast-Mip and other neuropeptides, Diuretic hormone 44 and insulin-like peptide 1. The remaining cells only expressed Diuretic hormone 44 and insulin-like peptide 1. The present results suggested that large PI neurons, including Plast-MIP neurons, have enhanced activity under short day conditions, which may increase Plast-MIP release to the corpus cardiacum-corpus allatum complex and thus contribute to reproductive diapause.

Juvenile hormone in spiders. Is this the solution to a mystery?

The juvenile hormone (JH) plays a crucial role in arthropod physiological processes, e.g., the regulation of metamorphosis, development, and reproduction (the vitellogenesis, the development of gonads, egg production). Still, data about this sesquiterpenoid hormone in spiders (Araneae) are rudimentary and equivocal. The presence of the JH or its precursors (e.g. methyl farnesoate) is not confirmed in spiders. The site of synthesis of its is still undetermined. No receptors of the JH are identified in spiders and thus, the molecular mechanism of action of this group of hormones is still unknown.Here we show by using the phylogenetic analysis and qPCR method the presence of the transcript of the enzyme catalyzing the last phase of the JH biosynthesis pathway (epox CYP15A1), the JH receptor (Met), and a possible candidate to the methyl farnesoate receptor (USP) in the various tissues and stages of ontogenesis in both sexes of spider Parasteatoda tepidariorum. Our results indicate that the juvenile hormone and/or methyl farnesoate presence is possible in the species of spider P. tepidariorum. The presence of the Ptepox CYP15A1 gene suggests that the main site of the juvenile hormone synthesis can be the integument and not the Schneider organ 2. It also seems that the juvenile hormone and/or methyl farnesoate can be hormones with biological activity due to the presence of the transcript of insect and crustacean JH/MG receptor – Met. The Ptepox CYP15A1, PtMet, and Ptusp expression are sex-, tissue-and time-specific.This study is the first report about the presence of the Ptepox CYP15A1 and PtMet transcripts in the Arachnida, which may indicate the presence of the juvenile hormone and/or methyl farnesoate in spiders.

Transcriptional identification of differentially expressed genes during the prepupal-pupal transition in the oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae).

The oriental armyworm, Mythimna separata (Walker) is a serious pest of agriculture that does particular damage to Gramineae crops in Asia, Europe, and Oceania. Metamorphosis is a key developmental stage in insects, although the genes underlying the metamorphic transition in M. separata remain largely unknown. Here, we sequenced the transcriptomes of five stages; mature larvae (ML), wandering (W), and pupation (1, 5, and 10 days after pupation, designated P1, P5, and P10) to identify transition-associated genes. Four libraries were generated, with 22,884, 23,534, 26,643, and 33,238 differentially expressed genes (DEGs) for the ML-vs-W, W-vs-P1, P1-vs-P5, and P5-vs-P10, respectively. Gene ontology enrichment analysis of DEGs showed that genes regulating the biosynthesis of the membrane and integral components of the membrane, which includes the cuticular protein (CP), 20-hydroxyecdysone (20E), and juvenile hormone (JH) biosynthesis, were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs were enriched in the metabolic pathways. Of these DEGs, thirty CP, seventeen 20E, and seven JH genes were differentially expressed across the developmental stages. For transcriptome validation, ten CP, 20E, and JH-related genes were selected and verified by real-time PCR quantitative. Collectively, our results provided a basis for further studies of the molecular mechanism of metamorphosis in M. separata.

Crucial Role of Juvenile Hormone Receptor Components Methoprene-Tolerant and Taiman in Sexual Maturation of Adult Male Desert Locusts.

Currently (2020), Africa and Asia are experiencing the worst desert locust (Schistocerca gregaria) plague in decades. Exceptionally high rainfall in different regions caused favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we investigated the role of methoprene-tolerant (Scg-Met) and Taiman (Scg-Tai), responsible for transducing the juvenile hormone (JH) signal, in adult male locusts. We demonstrated that knockdown of these components by RNA interference strongly inhibits male sexual maturation, severely disrupting reproduction. This was evidenced by the inability to show mating behavior, the absence of a yellow-colored cuticle, the reduction of relative testes weight, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. We also observed a reduced relative weight, as well as relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, as well as the transcript level of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways appeared to be affected by the knockdown, as evidenced by changes in the expression levels of the insulin-related peptide and two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology, illustrating their potential as molecular targets for pest control.

Purification, biochemical characterisation and bioinformatic analysis of recombinant farnesol dehydrogenase from Theobroma cacao

The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.

Steroid hormone ecdysone deficiency stimulates preparation for photoperiodic reproductive diapause.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.