Juvenile Hormone Acid Methyltransferase Research Articles

Transcriptomics Provide Insights into the Photoperiodic Regulation of Reproductive Diapause in the Green Lacewing, Chrysoperla nipponensis (Okamoto) (Neuroptera: Chrysopidae).

Chrysoperla nipponensis (Okamoto) displays typical adult reproductive diapause under short photoperiods; however, our understanding of the molecular mechanism underlying photoperiod-sensitive reproduction remains limited. In this study, we performed transcriptome profiling of four treatments (the diapause-sensitive stage and pre-diapause phase under long and short photoperiods) of C. nipponensis using RNA sequencing (RNA-seq). A total of 71,654 unigenes were obtained from the samples. Enrichment analysis showed that fatty acid metabolism-related pathways were altered under a short photoperiod. Moreover, β-oxidation-related gene expression was active during the diapause-sensitive period under a short photoperiod. The knockdown of juvenile hormone acid methyltransferase 1 (Jhamt1) prolonged the pre-oviposition period but did not affect the reproductive ability of female individuals in C. nipponensis. These findings provided us with a more comprehensive understanding of the molecular mechanisms of photoperiod-sensitive diapause and show that groundwork is crucial for bolstering the long-term storage and biocontrol potential of C. nipponensis.

Carbohydrate nutrition associated with health of overwintering honey bees.

In temperate climates, honey bees rely on stored carbohydrates to sustain them throughout the winter. In nature, honey serves as the bees' source of carbohydrates, but when managed, beekeepers often harvest honey and replace it with cheaper, artificial feed. The effects of alternative carbohydrate sources on colony survival, strength, and individual bee metabolic health are poorly understood. We assessed the impacts of carbohydrate diets (honey, sucrose syrup, high-fructose corn syrup, and invert syrup) on colony winter survival, population size, and worker bee nutritional state (i.e., fat content and gene expression of overwintered bees and emerging callow bees). We observed a nonsignificant trend for greater survival and larger adult population size among colonies overwintered on honey compared to the artificial feeds, with colonies fed high-fructose corn syrup performing particularly poorly. These trends were mirrored in individual bee physiology, with bees from colonies fed honey having significantly larger fat bodies than those from colonies fed high-fructose corn syrup. For bees fed honey or sucrose, we also observed gene expression profiles consistent with a higher nutritional state, associated with physiologically younger individuals. That is, there was significantly higher expression of vitellogenin and insulin-like peptide 2 and lower expression of insulin-like peptide 1 and juvenile hormone acid methyltransferase in the brains of bees that consumed honey or sucrose syrup relative to those that consumed invert syrup or high-fructose corn syrup. These findings further our understanding of the physiological implications of carbohydrate nutrition in honey bees and have applied implications for colony management.

Evolution of proteins involved in the final steps of juvenile hormone synthesis

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), is a key regulator of insect metamorphosis, reproduction, caste differentiation, and polyphenism. The first part of JH biosynthesis occurs via the universal eukaryotic mevalonate pathway. The final steps involve epoxidation and methylation. However, the sequence of these steps might not be conserved among all insects and Crustacea. Therefore, we used available genomic and transcriptomic data and identified JH acid methyltransferase (JHAMT), analyzed their genomic duplications in selected model organisms, and reconstructed their phylogeny. We have further reconstructed phylogeny of FAMeT proteins and show that evolution of this protein group is more complicated than originally appreciated. The analysis delineates important milestones in the evolution of several JH biosynthetic enzymes in arthropods, reviews major literature data on the last steps of JH synthesis, and defines questions and some hypotheses worth pursuing experimentally.

Egfr signaling promotes juvenile hormone biosynthesis in the German cockroach

BackgroundIn insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females.ResultsWe found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the − 941~ − 886 nt region of the Jhamt promoter.ConclusionsThis study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development.

Genetics tools for corpora allata specific gene expression in Aedes aegypti mosquitoes

Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.

Genome-wide screening of genes involved in programming diapause in the next generation in silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Maternal silkworms (Bombyx mori) of bivoltine strains are destined to produce either diapause or non-diapause eggs depending on environmental factors, such as, temperature and photoperiod experienced during the egg and larval stages. However, the molecular mechanisms that program diapause, which depend on information about the environment, remain unclear. We aimed to identify genes that are involved in programming diapause in the next generation in bivoltine silkworms. We therefore screened differentially expressed genes (DEGs) in the larval brains of diapause- and non-diapause-egg producers kept under three different diapause-inducing conditions using cap analysis of gene expression. Under each condition, only temperature, illumination or photoperiod was changed during the egg or larval stage as a diapause-controlling stimulus to induce the production of diapause or non-diapause eggs. We then verified the expression of DEGs that were common to all the three conditions using real-time quantitative PCR. We investigated the functional involvement of candidate genes in programming diapause using double-stranded RNA interference (RNAi) for gene knockdown. The results showed more abundant juvenile hormone acid methyltransferase (Jhamt) and proton-coupled folate transporter (Pcft) gene expression in the brains of fifth instar larvae of producers of diapause eggs than those of non-diapause eggs under the three conditions. Furthermore, RNAi against either of these genes significantly decreased the incidence of diapause in the next generation. These findings indicate that both Jhamt and Pcft are involved in the programming of diapause in the silkworm brain. These genes could function in retaining information that leads to diapause in the next generation.

Structural characterization and functional analysis of juvenile hormone acid methyltransferase JHAMT3 from the silkworm, Bombyx mori

Juvenile hormone acid methyltransferase (JHAMT) is a rate-limiting enzyme of juvenile hormone (JH) biosynthesis in insects. It transfers the methyl group of S-adenosyl methionine to either the carboxyl group of JH acids or farnesoic acid to produce JH. Six JHAMT paralogues have been identified in the silkworm (Bombyx mori); among them, JHAMT1 and JHAMT2 display a methyltransferase activity. Here, the three-dimensional crystal structure of inactive JHAMT3 and the binary complex of JHAMT3 with its cofactor S-adenosyl-l-homocysteine were determined through X-ray crystallization. Comparative structural analysis revealed that JHAMT3 adopted a similar structural pattern to that of functional JHAMT2, which comprised one core Rossmann fold domain and one substrate-binding domain. Similar to JHAMT2, JHAMT3 underwent a conformational change at the Rossmann fold domain because of cofactor binding, which promoted ligand accommodation. However, it exhibited a relatively rigid substrate-binding pocket compared with that of JHAMT2. JHAMT3 was also highly expressed in the silk gland of fourth- and fifth-instar B. mori larvae. The results of expression profiling combined with activity analysis suggested that JHAMT3 might function as a binding protein of JH acids for the regulation of JH acid titers. These findings provide a structural basis for enhancing the understanding of the physiological function of JHAMT3 and a rational framework for the development of potent and specific inhibitors of JHAMT family members.

Identification and Evolution Analysis of the Complete Methyl Farnesoate Biosynthesis and Related Pathway Genes in the Mud Crab, Scylla paramamosain.

The sesquiterpenoid hormone methyl farnesoate (MF) plays a vital role during crustacean development, which is mainly evidenced by its varied titers during different developmental stages. However, the biosynthesis pathways of MF remain obscure to some extent. In this study, we identified the complete MF biosynthesis and related pathway genes in Scylla paramamosain, including three involved in acetyl-CoA metabolism, eight in the mevalonate pathway, five in the sesquiterpenoids synthesis pathway, and five in the methionine cycle pathway. Bioinformatics, genomic structure, and phylogenetic analysis indicated that the JH biosynthesis genes might have experienced evolution after species differentiation. The mRNA tissue distribution analysis revealed that almost all genes involving in or relating to MF syntheses were highly expressed in the mandibular organ (MO), among which juvenile hormone acid methyltransferase was exclusively expressed in the MO, suggesting that most of these genes might mainly function in MF biosynthesis and that the methionine cycle pathway genes might play a crucial regulatory role during MF synthesis. In addition, the phylogenetic and tissue distribution analysis of the cytochrome P450 CYP15-like gene suggested that the epoxidized JHs might exist in crustaceans, but are mainly synthesized in hepatopancreas rather than the MO. Finally, we also found that betaine-homocysteine S-methyltransferase genes were lost in insects while methionine synthase was probably lost in most insects except Folsomia candida, indicating a regulatory discrepancy in the methionine cycle between crustaceans and insects. This study might increase our understanding of synthetic metabolism tailored for sesquiterpenoid hormones in S. paramamosain and other closely related species.

Toxicity of Ribavirin to Spodoptera litura by Inhibiting the Juvenile Hormone.

Ribavirin is an antiviral drug showing high and delayed toxicity to the destructive agricultural pest Spodoptera litura. Larvae fed with artificial diets containing ribavirin could not molt successfully and showed abnormal phenotypes, including cuticle melanization and heavy wrinkle of the newly formed procuticle. RNA-Seq analysis suggested that ribavirin has great negative influence on cuticle. Quantitative real-time-polymerase chain reaction results indicated that ribavirin treatment decreased the expression of key genes in juvenile hormone (JH) biosynthesis (CYP15C1 and JH acid methyltransferase) and most cuticle protein genes, whereas the genes in melanin biosynthesis and bursicon genes were upregulated by ribavirin treatment. These results coincided with the decreased titer of JH I, JH II, and JH III determined by enzyme-linked immunosorbent assay, the much thinner procuticle layer exhibited by histopathological examination, and the cuticle melanization after ribavirin treatment. These results provided a valuable theoretical basis for the creation of green insecticides targeting JH and the development of new insecticide derivatives from 1,2,4-triazole.

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.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

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.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Regulation of Juvenile Hormone on Summer Diapause of Geleruca daurica and Its Pathway Analysis.

Simple SummaryDiapause is an arrestment state in development, and plays an important role in life history in insects. It has been thought that a lack in juvenile hormone (JH) results in reproductive diapause occurring at the adult stage. However, we do not fully know about the underlying molecular mechanism. In this study, we proved that the topical application of a JH analog methoprene caused the changes at the transcriptional levels of a great number of genes, inhibited lipid accumulation, and finally delayed the adults entering diapause. Therefore, JH signaling plays an important role in regulating reproductive diapause of G. daurica, a new pest with great outbreaks in Inner Mongolia.Juvenile hormone (JH) signaling plays an important role in regulation of reproductive diapause in insects. However, we have little understanding of the effect of JH on gene expression at the transcriptome level in diapause. Galeruca daurica is a new pest in the Inner Mongolia grasslands with obligatory summer diapause in the adult stage. Topical application of a JH analog methoprene at the pre-diapause stage delayed the adults entering diapause and inhibited lipid accumulation whereas it did not during diapause. Using Illumina sequencing technology and bioinformatics tools, 54 and 138 differentially expressed genes (DEGs) were detected at 1 and 2 d after treatment, respectively. The KEGG analysis showed that the DEGs were mainly enriched in the metabolism pathways. qRT-PCR analysis indicated that methoprene promoted the expression of genes encoding vitellogenin, fork head transcription factor and Krüppel homolog 1, whereas suppressed the expression of genes encoding juvenile hormone-binding protein, juvenile hormone esterase, juvenile hormone acid methyltransferase, juvenile hormone epoxide hydrolase and fatty acid synthase 2. These results indicate that JH signaling plays an important role in regulating reproductive diapause of G. daurica.

Seed dressing with triflumezopyrim controls brown planthopper populations by inhibiting feeding behavior, fecundity and enhancing rice plant resistance.

Triflumezopyrim (TFM), a novel mesoionic insecticide, has high efficiency at a low dosage, and is mainly used to control hopper species. A previous study demonstrated that seed dressing with TFM effectively controlled rice planthopper populations in mechanically transplanted rice fields; however, mode of action for control was unclear. The study shows that seed dressing with TFM resulted in elevated levels of oxalic acid, flavonoids, phenolic substances, callose and other compounds associated with Nilaparvata lugens resistance in rice plants, and low TFM residue content in rice plant stem and grain. Host choice behavioral experiments showed that N. lugens females prefer feeding on untreated rice plants. Electrical penetration graph (EPG) data showed that seed dressing with TFM at medium and high doses significantly prolonged the non-probing period and inhibited phloem ingestion in N. lugens females. These changes led to a significant decrease in female secretion of honeydew, expression of genes encoding vitellogenin and juvenile hormone acid methyltransferase, body weight and longevity, and significantly influenced several physiological parameters resulting in impaired oocyte growth, fecundity and population. Field survey data showed that seed dressing with TFM was efficacious and relatively durable in protecting rice plants from infestation by planthoppers. This study revealed that seed dressing with TFM enhances rice plant resistance to N. lugens by limiting phloem ingestion and increasing the N. lugens non-probing period; this leads to reduced fecundity of females and lowers N. lugens numbers in the field. © 2021 Society of Chemical Industry.

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.

RNA silencing of hormonal biosynthetic genes impairs larval growth and development in cotton bollworm, Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera, is a highly polyphagous pest, causing enormous losses to various economically important crops. The identification and in vitro functional validation of target genes of a pest is a prerequisite to combat pest via host-mediated RNA interference (RNAi). In the present study, six hormonal biosynthesis genes of H. armigera were chosen and evaluated by feeding insect larvae with dsRNAs corresponding to each target gene, viz., juvenile hormone acid methyltransferase (HaJHAMT), prothoracicotropic hormone (HaPTTH), pheromone biosynthesis-activating peptide (HaPBAP), molt regulating transcription factor (HaHR3), activated protein 4 (HaAP-4) and eclosion hormone precursor (HaEHP). The loss of function phenotypes for these hormonal genes were observed by releasing second instar larvae on to artificial diet containing target gene-specific dsRNAs. Ingestion of dsRNAs resulted in mortality ranging from 60% to 90%, reduced larval weight, phenotypic deformities and delayed pupation. The quantitative real-time PCR (qRT-PCR) analysis showed that the target gene transcript levels were decreased drastically (31% to 77%) as compared to control or unrelated control (GFP-dsRNA), and correlated well with the mortality and developmental defects of larvae. Also, a comparison of the silencing efficacy of un-diced long HaPTTH -dsRNAwith RNase III diced HaPTTH-dsRNA (siRNAs) revealed that long dsRNAs were more efficient in silencing the target gene. These results indicated that the hormonal biosynthesis genes have varied sensitivity towards RNAi and could be the vital targets for insect resistance in crop plants like cotton which are infested by H. armigera.

Quantitative proteomic analysis of ovaries from Nosema bombycis-infected silkworm (Bombyx mori)

The microsporidium Nosema bombycis is an obligate intracellular parasite of Bombyx mori and causes serious losses in the sericulture industry. The isobaric tags for relative and absolute quantitation (iTRAQ) methods have been used to study numerous pathogen-host interactions. Here, using iTRAQ technology, we explored the quantitative proteomics by gene ontology and KEGG. The proteins in the ovaries of B. mori infected with N. bombycis were identified and compared to those in uninfected ovaries by iTRAQ. A total of 5401 proteins were identified, and 70 of them were differentially expressed. The differentially quantified proteins were involved in a variety of important processes and pathways, such as host development, host metabolism or host defense system. Most proteins involved in basic metabolism were up-regulated following infection, and the expression levels of some proteins related to the host immunity, such as the lipid droplet protein prilipin, 30 K proteins, HDD13, and beta-1,3-glucan recognition protein, were altered after infection with N. bombycis. Juvenile hormone acid methyltransferase, which regulates insect development, and ATG8, which is a key factor in autophagy, were also induced by N. bombycis infection. Our comparative and quantitative proteomic data will provide new insights into the interaction between N. bombycis and B. mori, especially in the host ovary.

Juvenile hormone-mediated reproduction induced by a sublethal concentration of imidacloprid in Cyrtorhinus lividipennis (Hemiptera: Miridae)

Abstract The Hemipteran predator, Cyrtorhinus lividipennis, feeds on the eggs and nymphs of rice planthoppers and leafhoppers. We previously demonstrated that sublethal concentrations of imidacloprid stimulated the reproduction of C. lividipennis. Considering the essential roles of juvenile hormone (JH) in insect reproduction, we speculated that sublethal concentrations of imidacloprid may stimulate the reproduction of C. lividipennis by regulating JH level. To test this, we cloned C. lividipennis JH acid methyl transferase (ClJHAMT) and JH esterase (ClJHE), which are responsible for JH biosynthesis and degradation genes, respectively. We then knocked down ClJHAMT by injecting dsRNA into C. lividipennis nymphs and found that emerging female adults exhibited 88.8% lower expression of the vitellogenin gene (ClVg) and the number of eggs was reduced by 41.5% as compared with controls. Silencing ClJHE increased ClVg mRNA expression by 275.0% but did not affect fecundity. A sublethal concentration of imidacloprid (LC20) increased the JH titer in females by 35.3% and 60.6% at 24 and 48 h post-emergence, respectively. In treatments containing both imidacloprid and dsJHAMT, the silencing of CLJHAMT reduced the number of eggs produced by adult females by 21.4% as compared to the control (imidacloprid + dsGFP). Our results indicated that sublethal concentration of imidacloprid may induce C. lividipennis reproduction by upregulating JH level via JHAMT. The finding could provide valuable information for improved integration of C. lividipennis and insecticides in pest management.