Chemosensory proteins of Locusta migratoria

Two different classes of chemosensory proteins (CSPs) in Locusta migratoria have been identified on the basis of the molecular cloning of a series of different cDNAs from the antennae of this insect. Several CSP isoforms have been purified and biochemically characterized from antennal and wing extracts, some of them corresponding to expression products predicted for the identified cDNAs. In wings, the nature of the main endogenous ligand binding to these proteins was determined as oleoamide by a gas chromatography-mass spectrometric approach. One of these isoforms has been expressed in a bacterial system with high yield and used in a fluorescent binding assay. Competitive binding experiments have indicated the presence of long-chain compounds among the best ligands.     

dsRNA uptake and persistence account for tissue‐dependent susceptibility to RNA interference in the migratory locust,Locusta migratoria

RNA interference (RNAi) by introducing double‐stranded RNA (dsRNA) is a powerful approach to the analysis of gene function in insects; however, RNAi responses vary dramatically in different insect species and tissues, and the underlying mechanisms remain poorly understood. The migratory locust, a destructive insect pest and a hemimetabolic insect with panoistic ovaries, is considered to be a highly susceptible species to RNAi via dsRNA injection, but its ovary appears to be completely insensitive. In the present study, we showed that dsRNA persisted only briefly in locust haemolymph. The ovariole sheath was permeable to dsRNA, but injected dsRNA was not present in the follicle cells and oocytes. The lack of dsRNA uptake into the follicle cells and oocytes is likely to be the primary factor that contributes to the ineffective RNAi response in locust ovaries. These observations provide insights into tissue‐dependent variability of RNAi and help in achieving successful gene silencing in insensitive tissues.     

Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue

The complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH₂-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corresponding mRNA is present only in epidermal cells secreting the adult fibrous cuticle destined to become heavily sclerotized. In supernumerary pupae obtained after the application of the juvenile hormone analogue (JHA) to newly ecdysed pupae, the mRNA was undetectable, indicating that JHA can prevent the switch to the adult programme. However, in pupal-adult intermediates, obtained when JHA is applied later, the mRNA is detected.     

Evidence for two juvenile hormone esterase-related genes in the Colorado potato beetle

Juvenile hormone esterase (JHE) activity in the haemolymph of the Colorado potato beetle is necessary to initiate pupation in larvae as well as diapause in adults. The enzyme appears in the haemolymph as a dimer consisting of two 57 kDa subunits. The sequence of an encoding cDNA, JHE.A, is distinct from lepidopteran JHEs. In this study, RT-PCR using primers designed on the basis of the 5′- and 3′-ends of the coding region revealed the existence of a related gene, JHE.B. The presence of two JHE-related genes was also shown by PCR amplification on genomic DNA from different individual beetles followed by restriction enzyme analysis. Both forms, probably paralogues, were transcribed since they could be amplified on messenger RNA from fat bodies. The size of the PCR products generated with mRNA and genomic DNA were both 1.6 kb, suggesting the absence of introns in the genomic JHE coding sequence. The sequence of a genomic clone, which encoded JHE.B, was 77% identical and 82% similar in amino acids compared to JHE.A. No introns were found in the coding sequence of these coleopteran JHE-related genes, in contrast to lepidopteran JHE genes. Southern blot analysis of digested genomic DNA confirmed the presence of two JHE-related genes.     

High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics

Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene‐tolerant [Met], Krüppel‐homolog1, Broad‐Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body‐part‐specific activation of JH signalling pathways.     

Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.     

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT‐PCR using degenerate primers based on the N‐terminal amino acid sequence of purified hJHBP and a conserved region near the C‐terminus of other lepidopteran hJHBPs. 5′‐ and 3′‐ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP‐mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS‐PAGE with a M_r of 32 kDa and showed a K_d of 4.5 × 10^?7 M with JH III, both similar to those of native hJHBP.     

Molecular characterization of a gene encoding juvenile hormone esterase in the red flour beetle,Tribolium castaneum

Juvenile hormone esterases (JHEs) are required for the degradation of juvenile hormones (JHs) in insects. Here, we report the cloning and analysis of the jhe gene in the red flour beetle, Tribolium castaneum, a model insect of Coleoptera. The Tcjhe gene was strongly expressed at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. A recombinant TcJHE protein efficiently degraded JH III, suggesting that the enzyme functions in vivo as a JH‐specific degradation enzyme. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was shown in Coleoptera, and the new data reported here will aid elucidation of the mechanism of JH titer regulation in insects.     

Regulation of the brain dopaminergic system by juvenile hormone in honey bee males (Apis mellifera L.)

Dopamine (DA) and juvenile hormone (JH) are multifunctional regulators of behaviour in social insects, with distinct effects across species and even between different dominance positions within the same species. We examined the effects of JH on the brain dopaminergic system in honey bee males to investigate the potential relationship between JH and DA within Apis mellifera. Both DA content and the expression of three DA receptor genes (Amdop1, Amdop2 and Amdop3) increased in the male honey bee brain from day 4 to day 8 after emergence. Treatment of 4-day-old males with a JH analogue (methoprene, JHA) enhanced brain DA levels. Brain expression of Amdop1 was also enhanced by JHA but not by a DA receptor agonist 2-amino 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN), indicating that Amdop1 up-regulation was not mediated by increased DA receptor stimulation. Furthermore, Amdop1 expression was still enhanced when JHA was co-applied with the DA receptor antagonist cis-(Z)-flupenthixol. Expression levels of Amdop2 and Amdop3 were not altered by JHA, 6,7-ADTN or by JHA plus the DA receptor antagonist. Regulation of the brain dopaminergic system by JH, as observed in solitary species, is conserved in male honey bees but not in female honey bees and other advanced eusocial insects.     

Heat stress affects oogenesis differently in wild-type Drosophila virilis and a mutant with altered juvenile hormone and 20-hydroxyecdysone levels

The link between reproduction and environmental signals is poorly understood at the physiological, genetic and molecular levels. We describe a mutant strain of Drosophila virilis that has altered responses to heat stress. Heat stress in wild-type females results in oocyte maturation delays, degradation of early vitellogenic egg chambers, inhibition of yolk protein gene expression in follicle cells and accumulation of mature oocytes. The mutant females have increased levels of ecdysteroids and decreased juvenile hormone degradation, and show all of the heat-stress-induced reproductive effects observed in wild-type flies, without exposure to heat stress. During oogenesis in mutant females following heat stress there is an increase in early vitellogenic oocyte degradation and some degradation of late egg chambers. 20-Hydroxyecdysone levels, but not juvenile hormone degradation, change following heat stress in mutant females.     

Tissue localization and regulation by juvenile hormone of human allergen Bla g 4 from the German cockroach, Blattella germanica (L.)

The German cockroach, Blattella germanica (L.), produces several potent protein aeroallergens, including Bla g 4, a approximately 20 kDa lipocalin. RT-PCR, Northern analyses and in situ hybridization showed that Bla g 4 is expressed only in the adult male reproductive system. Western blotting and ELISA with rBla g 4 antiserum detected immunoreactivity in the utricles and the conglobate gland, but not in other tissues of the male reproductive system. The Bla g 4 protein content of males increased from adult emergence to day 14, but during copulation Bla g 4 was depleted in the male and transferred to the female within the spermatophore. Topical application of juvenile hormone III stimulated Bla g 4 production by both conglobate gland and utricles.     

Absence of juvenile hormone signalling regulates the dynamic expression profiles of nutritional metabolism genes during diapause preparation in the cabbage beetle Colaphellus bowringi

Temperate insects have evolved diapause, a period of programmed developmental arrest during specific life stages, to survive unfavourable conditions. During the diapause preparation phase (DPP), diapause‐destined individuals generally store large amounts of fat by regulating nutrition distribution for the energy requirement during diapause maintenance and postdiapause development. Although nutritional patterns during the DPP have been investigated at physiological and biochemical levels in many insects, it remains largely unknown how nutritional metabolism is regulated during the DPP at molecular levels. We used RNA sequencing to compare gene expression profiles of adult female cabbage beetles Colaphellus bowringi during the preoviposition phase (POP) and the DPP. Most differentially expressed genes were involved in specific metabolic pathways during the DPP. Genes related to lipid and carbohydrate metabolic pathways were clearly highly expressed during the DPP, whereas genes related to protein metabolic pathways were highly expressed during the POP. Hormone challenge and RNA interference experiments revealed that juvenile hormone via its nuclear receptor methoprene‐tolerant mediated the expression of genes associated with nutritional metabolism during the DPP. This work not only sheds light on the mechanisms of diapause preparation, but also provides new insights into the molecular basis of environmental plasticity in insects.     

Genome‐wide analysis of genes related to ovary activation in worker honey bees

A defining characteristic of eusocial animals is their division of labour into reproductive and nonreproductive specialists. Here, we used a microarray study to identify genes associated with functional sterility in the worker honey bee Apis mellifera. We contrasted gene expression in workers from a functionally sterile wild‐type strain with that in a mutant (anarchist) strain selected for high rates of ovary activation. We identified a small set of genes from the brain (n = 7) and from the abdomen (n = 5) that are correlated in their expression with early stages of ovary activation. Sterile wild‐type workers up‐regulated two unknown genes and a homologue of Drosophila CG6004. By contrast, reproductive anarchist workers up‐regulated genes for the yolk protein vitellogenin, venom peptides and a member of the AdoHycase superfamily, among others. The differentially expressed genes identified are likely to be involved in early differentiation into sterile and reproductive worker phenotypes and may therefore form part of the gene networks associated with the regulation of honey bee worker sterility. Our study may have lacked sufficient power to detect all but a minority of biologically relevant changes taking place; however, the differential expression of vitellogenin and a putative AdoHycase suggests that our screen has captured core reproductive genes and that ovary activation may involve an epigenetic mechanism.     

Assay of DNA-binding proteins with a dsDNA-coupled plate

OBJECTIVES: This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP). DESIGN AND METHODS: The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development. RESULTS: The detections of three purified DBPs including NF-kappaB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappaB p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format. CONCLUSIONS: The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.     

Venous thromboembolism risk in relation to use of different types of postmenopausal hormone therapy in a large prospective study

Summary. Background: Current use of menopausal hormone therapy (HT) increases the risk of venous thromboembolism (VTE) and the formulations used may affect risk. Methods: A total of 1 058 259 postmenopausal UK women were followed by record linkage to routinely collected National Health Service hospital admission and death records. HT use and risk of VTE was examined using Cox regression to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: During 3.3 million years of follow‐up, 2200 women had an incident VTE, diagnosed, on average, 1.5 years after last reporting HT use. RRs in current vs. never users at last reporting varied by HT formulation: the risk was significantly greater for oral estrogen‐progestin than oral estrogen‐only therapy (RR = 2.07 [95%CI, 1.86–2.31] vs. 1.42 [1.21–1.66]), with no increased risk with transdermal estrogen‐only therapy (0.82 [0.64–1.06]). Among users of oral estrogen‐progestin, the risk from HT varied by progestin type, with significantly greater risks for preparations containing medroxyprogesterone acetate than other progestins (2.67 [2.25–3.17] vs. 1.91 [1.69–2.17]; P_{heterogeneity} = 0.0007). Current users of oral HT at last reporting had twice the risk of VTE in the first 2 years after starting HT than later (P_{heterogeneity} = 0.0006). Associations were similar for deep vein thrombosis with and without pulmonary embolism. Over 5 years, 1 in 660 who had never used HT were admitted to hospital for (or died from) pulmonary embolism, compared with 1 in 475 current users of oral estrogen‐only HT,1 in 390 users of estrogen‐progestin HT containing norethisterone/norgestrel, and 1 in 250 users of estrogen‐progestin HT containing medroxyprogesterone acetate. Conclusions: The risk of VTE varied considerably by HT formulation, being greatest in users of oral estrogen‐progestin HT, especially formulations containing medroxyprogesterone acetate.