Juvenile Hormone Acid O-methyltransferase Research Articles

Saccharomyces cerevisiae cellular engineering for the production of FAME biodiesel

The unsustainable and widespread utilization of fossil fuels continues to drive the rapid depletion of global supplies. Biodiesel has emerged as one of the most promising alternatives to conventional diesel, leading to growing research interest in its production. Microbes can facilitate the de novo synthesis of a type of biodiesel in the form of fatty acid methyl esters (FAMEs). In this study, Saccharomyces cerevisiae metabolic activity was engineered to facilitate enhanced FAME production. Initially, free fatty acid concentrations were increased by deleting two acetyl-CoA synthetase genes (FAA1, FAA4) and an acyl-CoA oxidase gene (POX1). Intracellular S-adenosylmethionine (SAM) levels were then enhanced via the deletion of an adenosine kinase gene (ADO1) and the overexpression of a SAM synthetase gene (SAM2). Lastly, the S. cerevisiae strain overproducing free fatty acids and SAM were manipulated to express a plasmid encoding the Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). Using this combination of engineering approaches, a FAME concentration of 5.79 ± 0.56 mg/L was achieved using these cells in the context of shaking flask fermentation. To the best of our knowledge, this is the first detailed study of FAME production in S. cerevisiae. These results will provide a valuable basis for future efforts to engineer S. cerevisiae strains for highly efficient production of biodiesel.

Fertility differences between two wild-type Drosophila melanogaster lines correlate with differences in the expression of the Jheh1 gene, which codes for an enzyme degrading juvenile hormone.

Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes, ensuring the ovaries' preparedness for vitellogenesis. The decreased level of juvenile hormone results in reproductive disorders in D. melanogaster females including a delay in the oviposition onset and a fertility decrease. Another factor that can affect the insect reproduction is an infection with the maternally inherited symbiotic α-proteobacterium Wolbachia. The present study is devoted to the analysis of the expression of two juvenile hormone metabolism genes encoding enzymes of its synthesis and degradation, juvenile hormone acid O-methyltransferase ( jhamt) and juvenile hormone epoxide hydrase (Jheh1), respectively, in four wild-type D. melanogaster lines, two of them being infected with Wolbachia. Lines w153 and Bi90 were both derived from an individual wild-caught females infected with Wolbachia, while lines w153T and Bi90T were derived from them by tetracycline treatment and are free of infection. Line Bi90 is known to be infected with the Wolbachia strain wMel, and line w153, with the Wolbachia strain wMelPlus belonging to the wMelCS genotype. It was found that infection with either Wolbachia strain does not affect the expression of the studied genes. At the same time, it was shown that the w153 and w153T lines differ from the Bi90 and Bi90T lines by an increased level of the Jheh1 gene expression and do not differ in the jhamt gene expression level. Analysis of the fertility of these four lines showed that it does not depend on Wolbachia infection either, but differs between lines with different nuclear genotypes: in w153 and w153T, it is significantly lower than in lines Bi90 and Bi90T. The data obtained allow us to reasonably propose that the inter-line D. melanogaster polymorphism in the metabolism of the juvenile hormone is determined by its degradation (not by its synthesis) and correlates with the fertility level.

Aphid gene expression following polerovirus acquisition is host species dependent.

Upon acquisition of persistent circulative viruses such as poleroviruses, the virus particles transcytose through membrane barriers of aphids at the midgut and salivary glands via hemolymph. Such intricate interactions can influence aphid behavior and fitness and induce associated gene expression in viruliferous aphids. Differential gene expression can be evaluated by omics approaches such as transcriptomics. Previously conducted aphid transcriptome studies used only one host species as the source of virus inoculum. Viruses typically have alternate hosts. Hence, it is not clear how alternate hosts infected with the same virus isolate alter gene expression in viruliferous vectors. To address the question, this study conducted a transcriptome analysis of viruliferous aphids that acquired the virus from different host species. A polerovirus, cotton leafroll dwarf virus (CLRDV), which induced gene expression in the cotton aphid, Aphis gossypii Glover, was assessed using four alternate hosts, viz., cotton, hibiscus, okra, and prickly sida. Among a total of 2,942 differentially expressed genes (DEGs), 750, 310, 1,193, and 689 genes were identified in A. gossypii that acquired CLRDV from infected cotton, hibiscus, okra, and prickly sida, respectively, compared with non-viruliferous aphids that developed on non-infected hosts. A higher proportion of aphid genes were overexpressed than underexpressed following CLRDV acquisition from cotton, hibiscus, and prickly sida. In contrast, more aphid genes were underexpressed than overexpressed following CLRDV acquisition from okra plants. Only four common DEGs (heat shock protein, juvenile hormone acid O-methyltransferase, and two unannotated genes) were identified among viruliferous aphids from four alternate hosts. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated that the acquisition of CLRDV induced DEGs in aphids associated with virus infection, signal transduction, immune systems, and fitness. However, these induced changes were not consistent across four alternate hosts. These data indicate that alternate hosts could differentially influence gene expression in aphids and presumably aphid behavior and fitness despite being infected with the same virus isolate.

Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT–PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 μL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase disrupts larval development in the oriental fruit fly, Bactrocera dorsalis (Hendel)

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a notoriously agricultural pest that causes serious economic losses to fruits and vegetables. Widespread insecticide resistance in B. dorsalis is a major obstacle in successful control. Therefore, new pest control strategies, such as those targeting specific genes that can block pest development, are urgently needed. In the current study, the function of JHAMT in B. dorsalis was systematically investigated. A methyltransferase gene in B. dorsalis (BdJHAMT) that is homologous to JHAMT of Drosophila melanogaster was cloned firstly. The subsequently spatiotemporal expression analysis indicated that BdJHAMT mRNA was continuously present in the larval stage, declined sharply immediately before pupation, and then increased in the adult. Subcellular localization showed that BdJHAMT was localized in the adult corpora allata and larval intestinal wall cells. The JH III titer in B. dorsalis was closely related to the transcription level of BdJHAMT in different developmental stages. The dsBdJHAMT feeding-based RNAi resulted in a greatly decreased JH III titer that disrupted fly development. The slow growth caused by BdJHAMT silencing was partially rescued by application of the JH mimic, methoprene. These results demonstrated that BdJHAMT was crucial for JH biosynthesis and thus regulated larval development in B. dorsalis, indicating it may serve as a prospective target for the development of novel control strategies against this pest.

Impact of Imidacloprid Resistance on the Demographic Traits and Expressions of Associated Genes in Aphis gossypii Glover.

Imidacloprid is one of the most widely used neonicotinoid insecticides to control sap-sucking insect pests, including Aphis gossypii. The intensive application of chemical insecticides to A. gossypii led to the development of resistance against several insecticides, including imidacloprid. Therefore, it is crucial to understand the association between imidacloprid resistance and the fitness of A. gossypii to limit the spread of the resistant population under field contexts. In this study, we used the age-stage, two-sex life table method to comprehensively investigate the fitness of imidacloprid resistant (ImR) and susceptible strains (SS) of melon aphids. Results showed that ImR aphids have prolonged developmental stages and decreased longevity, fecundity, and reproductive days. The key demographic parameters (r, λ, and R0) were significantly reduced in ImR strain compared to SS aphids. Additionally, the molecular mechanism for fitness costs was investigated by comparing the expression profile of juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), juvenile hormone acid O-methyltransferase (JHAMT), Vitellogenin (Vg), ecdysone receptor (EcR), and ultraspiracle protein (USP) supposed to be associated with development and reproduction in insects. The results of RT-qPCR showed that EcR, JHBP, JHAMT, JHEH, and Vg genes were downregulated, while USP was statistically the same in ImR A. gossypii compared to the SS strain. Together, these results provide in-depth information about the occurrence and magnitude of fitness costs against imidacloprid resistance that could help manage the evolution and spread of A. gossypii resistance in field populations.

Ginsenosides improve reproductive capability of aged female Drosophila through mechanism dependent on ecdysteroid receptor (ECR) and steroid signaling pathway.

Aging ovaries caused diminished fertility and depleted steroid hormone level. Ginsenosides, the active ingredient in ginseng, had estrogen-like hormonal effects. Although ginsenosides were well known for their ability to alleviate many age-related degenerative diseases, the effect of ginsenosides on the decline in reproductive capability caused by aging, as well as the mechanism, are unknown. We found that ginsenosides improved the quantity and quality of the offspring, prolonged life and restored muscle ability in aged female Drosophila. In addition, ginsenosides inhibited ovarian atrophy and maintained steroid hormone 20-Hydroxyecdysone (20E) and juvenile-preserving hormone (JH)) levels. Ginsenosides activated ecdysteroid receptor (ECR) and increased the expression of the early transcription genes E74 and Broad (Br), which triggered steroid signaling pathway. Meanwhile, ginsenosides promoted JH biosynthesis by increasing the expression of Hydroxyl-methylglutaryl-CoA reductase (HMGR) and juvenile hormone acid O-methyltransferase (JHAMT). Subsequently, JH was bound to Methoprene Tolerant (Met) and activated the transcription of the responsive gene Kruppel Homolog 1 (Kr-h1), which coordinated with 20E signaling to promote the reproduction of aged female Drosophila. The reproductive capacity and steroid hormone levels were not improved and the steroid signaling pathway was not activated in ginsenoside-treated ECR knockout Drosophila. This suggested that ginsenosides played a role dependent on targeted ECR. Furthermore, 17 kinds of ginsenoside monomers were identified from the total ginsenosides. Among them, Rg1, Re and Rb1 improved the reproductive capacity and steroid hormone levels of aged female Drosophila, which has similar effects to the total ginsenoside. These results indicated that ginsenosides could enhance the reproductive capacity of aged female Drosophila by activating steroid signals dependent on nuclear receptor ECR. In addition, ginsenoside monomers Rg1, Rb1 and Re are the main active components of total ginsenosides to improve reproductive ability. This will provide strong evidence that ginsenosides had the potential to alleviate age-induced reproductive degradation.

Comparative Transcriptome Analysis Reveals the Process of Ovarian Development and Nutrition Metabolism in Chinese Mitten Crab, Eriocheir Sinensis.

Ovarian development is a key physiological process that holds great significance in the reproduction of the Chinese mitten crab (Eriocheir sinensis), which is an economically important crab species for aquaculture. However, there is limited knowledge for the regulatory mechanisms of ovarian development. To study the molecular mechanisms of its ovarian development, transcriptome analysis was performed in the ovary and hepatopancreas of E. sinensis during ovarian stages I (oogonium proliferation), II (endogenous vitellogenesis), and III (exogenous vitellogenesis). The results showed that 5,520 and 226 genes were differentially expressed in the ovary and hepatopancreas, respectively. For KEGG enrichment analysis, the differentially expressed genes in the ovary were significantly clustered in phototransduction-fly, phagosome, and ECM-receptor interaction. Significantly enriched pathways in the hepatopancreas included fatty acid biosynthesis, fatty acid metabolism, and riboflavin metabolism. Further analysis showed that 25 genes and several pathways were mainly involved in oogenesis, including the ubiquitin-proteasome pathway, cyclic AMP-protein kinase A signaling pathway, and mitogen-activated protein kinase signaling pathway. Twenty-five candidate genes involved in vitellogenesis and endocrine regulation were identified, such as vitellogenin, vitellogenin receptor, estrogen sulfotransferase, ecdysone receptor, prostaglandin reductase 1, hematopoietic prostaglandin D synthase and juvenile hormone acid O-methyltransferase. Fifty-six genes related to nutritional metabolism were identified, such as fatty acid synthase, long-chain-fatty-acid-CoA ligase 4, 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, fatty acid-binding protein, and glycerol-3-phosphate acyltransferase 1. These results highlight the genes involved in ovarian development and nutrition deposition, which enhance our understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

Juvenile Hormone Is an Important Factor in Regulating Aspongopus chinensis Dallas Diapause.

Aspongopus chinensis is a Chinese traditional edible and medicinal insect, which is in great demand in the society. This insect reproduces once a year which is caused by reproductive diapause resulting in insufficient production in wild resources. However, the mechanism of diapause in A. chinensis is still unclear. In this study, we focus on the relationship between juvenile hormones (JHs) and A. chinensis diapause. The results showed that JHIII concentration in diapause adult individuals was significantly lower than that in diapause termination adult individuals. When exogenous JHⅢ was injected into diapause adults, the rate of mating was increased significantly, development of the reproductive systems was accelerated, consumption of fat intensified, the expression of juvenile hormone acid o-methyl-transferase (JHAMT) was upregulated, and juvenile hormone epoxide hydrolase (JHEH) and fatty acid synthase (FAS) gene expressions were downregulated. In addition, RNAi of JHAMT decreased JH concentration, delayed the development of reproductive systems, slowed down fat consumption, and delayed the mean mating occurrence time significantly. Conversely, RNAi of JHEH resulted in an increased concentration of JH, development of reproductive systems was accelerated, consumption of fat was intensified, and mean mating occurrence time advanced significantly. Taken together, these findings uncovered that JH plays an important role in regulating reproductive diapause in A. chinensis and, thus, could provide a theoretical basis for further research on the diapause of A. chinensis.

POU-M2 promotes juvenile hormone biosynthesis by directly activating the transcription of juvenile hormone synthetic enzyme genes in Bombyx mori.

Juvenile hormone (JH) plays a key role in preventing larval precocious metamorphosis, maintaining larval state, controlling adult sexual development and promoting insect egg maturation. Genetic studies have shown that POU factor ventral veins lacking regulates JH synthesis to control the timing of insect metamorphosis. However, how POU factor regulates JH synthesis is largely unknown. Here, we found POU-M2 was highly expressed in corpora allata (CA) and specifically localized in the nucleus of CA. The overexpression of POU-M2 promoted the expression of JH synthase genes and kr-h1 and enhanced the activity of JH synthase genes promoter. Further, POU-M2 promoted the transcription of JH acid O-methyltransferase (JHAMT) by directly binding to the key cis-regulatory elements -207, -249 and -453 within the proximal regions of JHAMT promoter. Both the POU domain and homeodomain were vital for the activation of POU-M2 on JHAMT transcription. Our study reveals the mechanism by which POU-M2 regulates JHAMT transcription.

Juvenile hormone as a causal factor for maternal regulation of diapause in a wasp

Most temperate multivoltine insects enter diapause, a hormonally controlled developmental suspension, in response to seasonal photoperiodic and/or thermal cues. Some insect species exhibit maternal regulation of diapause in which developmental trajectories of the offspring are determined by mothers in response to environmental cues that the mother received. Although maternally regulated diapause is common among insects, the maternal endocrinological mechanisms are largely veiled. To approach this issue, we used the jewel wasp Nasonia vitripennis, which produces non-diapause-destined offspring under long days and diapause-destined offspring under short days or low temperatures. Comparative transcriptomics of these wasps revealed possible involvement of the juvenile hormone (JH) biosynthetic cascade in maternal diapause regulation. The expression of juvenile hormone acid O-methyltransferase (jhamt) was typically downregulated in short-day wasps, and this was reflected by a reduction in haemolymph JH concentrations. RNAi targeted at jhamt reduced haemolymph JH concentration and induced wasps to produce diapause-destined offspring even under long days. In addition, topical application of JH suppressed the production of diapause-destined offspring under short days or low temperatures. These results indicate that diapause in N. vitripennis is determined by maternal jhamt expression and haemolymph JH concentration in response to day length. We therefore report a novel role for JH in insect seasonality.

Functional Characterization of Allatostatin C (PISCF/AST) and Juvenile Hormone Acid O-Methyltransferase in Dendroctonus armandi

Allatostatin C (PISCF/AST) is a neuropeptide gene that affects juvenile hormone (JH) synthesis in the corpora allata. Juvenile hormone acid O-methyltransferase (JHAMT) is a key gene in the JH biosynthetic pathway. In this study, two genes encoding DaAST and DaJHAMT were cloned. Both DaAST and DaJHAMT were expressed in the larvae, pupae and adults of Chinese white pine beetle (Dendroctonus armandi), and highly expressed in the head and the gut. The expression of the two genes was induced by JH analog (JHA) methoprene and the functions of the two genes were then investigated by RNAi. Considering the role of hormones in metamorphosis, JHA significantly induced DaAST and DaJHAMT in the larval stage. DaAST knockdown in larvae, pupae and adults significantly increased the DaJHAMT mRNA levels. Moreover, knockdown of DaAST instead of DaJHAMT increased pupae mortality and the abnormal rate of emergence morphology and reduced emergence rates. However, knockdown of DaJHAMT instead of DaAST significantly reduced frontalin biosynthesis in adult males. The results showed that DaAST acts as an allatostatin and inhibits JH biosynthesis, and that JHAMT is a key regulatory enzyme for JH synthesis in the D. armandi.

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.

Role of SfJHAMT and SfFAMeT in the reproductive regulation of Sogatella furcifera and its expression under insecticide stress

The isoprene branching pathway is a unique downstream synthesis pathway of juvenile hormone (JH) in arthropods, which plays an important role in the growth, development, and reproduction of insects. Juvenile hormone acid O-methyltransferase (JHAMT) and farnesoic acid O-methyltransferase (FAMeT) are two key proteins that are regulated in the isoprene branching pathway. Based on the available transcriptomic and genomic data of Sogatella furcifera, full-length cDNAs of SfJHAMT and SfFAMeT were identified. In vitro injection of dsRNA targeted to silence SfJHAMT and SfFAMeT inhibited the fecundity, ovarian development, and transcription levels of SfKr-h1 and SfVg significantly. Of note, The transcription levels of SfJHAMT and SfFAMeT are regulated mutually; i.e., silencing of SfJHAMT causes an increase in the SfFAMeT transcription level and vice versa, and the negative effect of simultaneous silencing on reproduction is greater. The results revealed a coordinated effect of SfJHAMT and SfFAMeT on the reproductive capabilities of S. furcifera. Furthermore, a JH analog (methoprene) partially rescued the negative effect of simultaneous silencing by SfJHAMT and SfFAMeT on reproduction. In addition, the expression profile analysis after insecticide stress showed that triazophos (LC25) can induce the transcription of SfMet and SfKr-h1 to promote JH signal transduction, which affects the transcription of SfVg and ultimately promotes the reproduction of S. furcifera. The results of the present study lay a foundation to further explain the isoprene branch pathway function in insect reproduction and can open up new avenues for sustainable pest control while expanding the current understanding of molecular mechanisms through which insecticides stimulate reproduction and lead to pest resurgence.

Roles of and cross-talk between ecdysteroid and sesquiterpenoid pathways in embryogenesis of branchiopod crustacean Daphnia magna.

The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.

Cross-resistance and Fitness Cost Analysis of Resistance to Thiamethoxam in Melon and Cotton Aphid (Hemiptera: Aphididae).

The melon/cotton aphid, Aphis gossypii Glover, is a notorious pest in many crops. The neonicotinoid insecticide thiamethoxam is widely used for A. gossypii control. To evaluate thiamethoxam resistance risk, a melon/cotton aphid strain with an extremely high level of resistance to thiamethoxam (>2,325.6-fold) was established after selection with thiamethoxam for 24 generations. Additionally, the cross-resistance pattern to other neonicotinoids and fitness were analyzed. The cross-resistance results showed the thiamethoxam-resistant strain had extremely high levels of cross-resistance against clothianidin (>311.7-fold) and nitenpyram (299.9-fold), high levels of cross-resistance against dinotefuran (142.3-fold) and acetamiprid (76.6-fold), and low cross-resistance against imidacloprid (9.3-fold). Compared with the life table of susceptible strain, the thiamethoxam-resistant strain had a relative fitness of 0.950, with significant decreases in oviposition days and fecundity and prolonged developmental duration. The molecular mechanism for fitness costs was studied by comparing the mRNA expression levels of juvenile hormone acid O-methyltransferase (JHAMT), juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), ecdysone receptor (EcR), ultraspiracle protein (USP), and Vitellogenin (Vg) in the susceptible and thiamethoxam-resistant strains. Significant overexpression of JHEH and JHBP and downregulation of EcR and Vg expression were found in the thiamethoxam-resistant strain. These results indicate that A. gossypii has the potential to develop extremely high resistance to thiamethoxam after continuous exposure, with a considerable fitness cost and cross-resistance to other neonicotinoids.

Juvenile hormone suppresses aggregation behavior through influencing antennal gene expression in locusts.

Animals often exhibit dramatically behavioral plasticity depending on their internal physiological state, yet little is known about the underlying molecular mechanisms. The migratory locust, Locusta migratoria, provides an excellent model for addressing these questions because of their famous phase polyphenism involving remarkably behavioral plasticity between gregarious and solitarious phases. Here, we report that a major insect hormone, juvenile hormone, is involved in the regulation of this behavioral plasticity related to phase change by influencing the expression levels of olfactory-related genes in the migratory locust. We found that the treatment of juvenile hormone analog, methoprene, can significantly shift the olfactory responses of gregarious nymphs from attraction to repulsion to the volatiles released by gregarious nymphs. In contrast, the repulsion behavior of solitarious nymphs significantly decreased when they were treated with precocene or injected with double-stranded RNA of JHAMT, a juvenile hormone acid O-methyltransferase. Further, JH receptor Met or JH-response gene Kr-h1 knockdown phenocopied the JH-deprivation effects on olfactory behavior. RNA-seq analysis identified 122 differentially expressed genes in antennae after methoprene application on gregarious nymphs. Interestingly, several olfactory-related genes were especially enriched, including takeout (TO) and chemosensory protein (CSP) which have key roles in behavioral phase change of locusts. Furthermore, methoprene application and Met or Kr-h1 knockdown resulted in simultaneous changes of both TO1 and CSP3 expression to reverse pattern, which mediated the transition between repulsion and attraction responses to gregarious volatiles. Our results suggest the regulatory roles of a pleiotropic hormone in locust behavioral plasticity through modulating gene expression in the peripheral olfactory system.

Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely ’UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that ’UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO2 into excreted FAME.

Enzymes in the juvenile hormone biosynthetic pathway can be potential targets for pest control.

Discovery of novel insecticides and targets has received global attention in recent years. Ten genes coding for enzymes involved in the juvenile hormone biosynthetic pathway of Manduca sexta were studied as potential insecticide targets. We determined the expression of genes encoding some critical enzymes in the JH biosynthetic pathway. Farnesol dehydrogenase (FOLD), Juvenile hormone acid O-methyltransferase (JHAMT) and Juvenile hormone epoxidase (CYP15C1) were selected as the candidate targets based on gene expression results. RNAi silencing and enzyme inhibitor tests were performed to validate whether these candidate genes could be the potential insecticide targets. The down-regulation of FOLD, JHAMT and CYP15C1 resulted in a 68%, 82% and 79% reduction in the rates of JH biosynthesis in vitro, respectively. In addition, RNA interference and inhibitor studies of these enzymes following oral administration demonstrated the potential application in pest management, with respect to high mortality and effects on growth. Based on our study, FOLD, JHAMT and CYP15C1 could be potential targets for pest control as a consequence of their important roles in insect development. © 2019 Society of Chemical Industry.