Knockdown of a metathoracic scent gland desaturase enhances the production of (E)-4-oxo-2-hexenal and suppresses female sexual attractiveness in the plant bug Adelphocoris suturalis.

Insect sex pheromones (SPs) are central to mate-finding behaviour, and play an essential role in the survival and reproduction of organisms. Understanding the roles, biosynthetic pathways and evolution of insect chemical communication systems has been an exciting challenge for biologists. Compared with Lepidoptera, little is known about the mechanisms underlying pheromone biosynthesis in Hemiptera. In this study, we isolated and characterized two new desaturase-like genes, termed Asutdes1 and Asutdes2, from Adelphocoris suturalis, an important agricultural pest in China. Although the two genes encode an identical protein, Southern blot analysis revealed that they are duplicated genes. The Asutdes2 transcript is more abundant than Asutdes1 in the tissues tested, in particular the metathoracic scent gland and fat body. Silencing Asutdes expression in females by injecting double-stranded RNA (dsAsutdes) against a portion of the coding sequence shared by the two genes enhanced the production of (E)-4-oxo-2-hexenal, a component of the A. suturalis SP blend, and dramatically suppressed the sexual attractiveness of A. suturalis females. We conclude that dsAsutdes is associated with the SP biosynthetic pathway in A. suturalis.

RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.

Identification and functional characterization of four transient receptor potential ankyrin 1 variants in Apolygus lucorum (Meyer-Dür).

As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from the green plant bug, Apolygus lucorum. Spatiotemporal expression profiling across development and in different adult tissues revealed that the highest relative-transcript levels occurred in first-instar nymphs and antennae, respectively. In Xenopus laevis-based functional assays, Apo. lucorum TRPA1-A (AlucTRPA1-A), AlucTRPA1-B and AlucTRPA1-C were activated by increasing the temperature from 20 to 40 °C with no significant desensitization observed after repeated temperature stimuli. The activation temperature of AlucTRPA1-A and AlucTRPA1-B was < 25 °C, whereas the activation temperature of AlucTRPA1-C was between 25 and 30 °C. Amongst the variants, only AlucTRPA1-A and AlucTRPA1-C were directly activated by high concentrations of allyl isothiocyanate, cinnamaldehyde and citronellal. Taken together, these results suggest that AlucTRPA1 variants may function in vivo as both thermal and chemical sensors, with the four variants potentially mediating different physiological functions. This study not only enriches our understanding of TRPA1 function in Hemiptera (Miridae), but also offers a foundation for developing new pest control strategies.

Identification and characterization of a sex peptide receptor-like transcript from the western tarnished plant bug Lygus hesperus.

Lygus hesperus females exhibit a post-mating behavioural switch that triggers increased egg laying and decreased sexual interest. In Drosophila melanogaster, these changes are controlled by sex peptide (SP) and the sex peptide receptor (DmSPR). In Helicoverpa armigera, SPR (HaSPR) also regulates some post-mating behaviour; however, myoinhibiting peptides (MIPs) have been identified as the SPR ancestral ligand, indicating that SPR is a pleiotropic receptor. In the present study, we identified a transcript, designated L. hesperus SPR (LhSPR), that is homologous to known SPRs and which is expressed throughout development and in most adult tissues. LhSPR was most abundant in female seminal depositories and heads as well as the hindgut/midgut of both sexes. In vitro analyses revealed that fluorescent chimeras of LhSPR, DmSPR and HaSPR localized to the cell surface of cultured insect cells, but only DmSPR and HaSPR bound carboxytetramethylrhodamine-labelled analogues of DmSP21-36 and DmMIP4. Injected DmSP21-36 also failed to have an effect on L. hesperus mating receptivity. Potential divergence in the LhSPR binding pocket may be linked to receptor-ligand co-evolution as 9 of 13 MIPs encoded by a putative L. hesperus MIP precursor exhibit an atypical W-X7 -Wamide motif vs the W-X6 -Wamide and W-X8 -Wamide motifs of Drosophila MIPs and SP.

Cloning and expression profiling of odorant-binding proteins in the tarnished plant bug, Lygus lineolaris.

In insects, the perception and discrimination of odorants requires the involvement of odorant-binding proteins (OBPs). To gain a better molecular understanding of olfaction in the agronomic pest Lygus lineolaris (the tarnished plant bug), we used a transcriptomics-based approach to identify potential OBPs. In total, 33 putative OBP transcripts, including the previously reported Lygus antennal protein (LAP), were identified based on the characteristic OBP Cys signature and/or sequence similarity with annotated orthologous sequences. The L. lineolaris OBP (LylinOBP) repertoire consists of 20 'classic' OBPs, defined by the spacing of six conserved Cys residues, and 12 'Plus-C' OBPs, defined by the spacing of eight conserved Cys and one conserved Pro residue. Alternative splicing of OBP genes appears to contribute significantly to the multiplicity of LylinOBP sequences. Microarray-based analysis of chemosensory tissues (antennae, legs and proboscis) revealed enrichment of 21 LylinOBP transcripts in antennae, 12 in legs, and 15 in proboscis, suggesting potential roles in olfaction and gustation respectively. PCR-based determination of transcript abundance for a subset of the LylinOBP genes across multiple adult tissues yielded results consistent with the hybridization data.

Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor crucial for ligand-induced internalization.

Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Using fluorescent Bombyx mori PBANR (BmPBANR) chimeras to study PBANR regulation, we previously showed that BmPBANR undergoes rapid ligand-induced internalization, that the endocytotic motif resides between residues 358-367 of the BmPBANR C terminus, and that the internalization pathway is clathrin-dependent. Here, we sought to expand our understanding of the molecular mechanisms underlying BmPBANR function and regulation by transiently expressing a series of fluorescent BmPBANR chimeric constructs in cultured Spodoptera frugiperda (Sf9) cells and assaying for internalization of a fluorescently labelled ligand. Pharmacological inhibition of phospholipase C significantly reduced internalization, suggesting that BmPBANR regulation proceeds via a conventional G-protein-dependent pathway. This was further supported by impaired internalization following site-directed mutagenesis of R263 and R264, two basic residues at the transmembrane 6 intracellular junction that are thought to stabilize G-protein coupling via electrostatic interactions. Ala substitution of S333 and S366, two consensus protein kinase C sites in the C terminus, likewise impaired internalization, as did RNA interference-mediated knockdown of Sf9 protein kinase C. N-terminal truncations of BmPBANR indicate that the first 27 residues are not necessary for cell surface trafficking or receptor functionality.

Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States.

The segmented genome of rotaviruses provides an opportunity for rotavirus strains to generate a large genetic diversity through reassortment; however, this mechanism is considered to play little role in the generation of mosaic gene constellations between Wa-like and DS-1-like strains in genes other than the neutralization antigens. A pilot study was undertaken to analyze these two epidemiologically important strains at the genomic level in order to (i) identify intergenogroup reassortment and (ii) to make available additional reference genome sequences of G1P[8] and G2P[4] for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758, and LB2771) and 3 G2P[4] (LB2744, LB2764, and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains. While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post-rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children.

Gqalpha-linked phospholipase Cbeta1 and phospholipase Cgamma are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascade.

Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store-operated Ca(2+) channels, suggesting the involvement of a receptor-activated phospholipase C (PLC). In this study, we found that PLC inhibitors U73122 and compound 48/80 reduced sex pheromone production and that intracellular levels of (3)H-inositol phosphate species increased following PBAN stimulation. In addition, we amplified cDNAs from pheromone glands corresponding to PLCbeta1, PLCbeta4, PLCgamma and two G protein alpha subunits, Go and Gq. In vivo RNA interference-mediated knockdown analyses revealed that BmPLCbeta1, BmGq1, and unexpectedly, BmPLCgamma, are part of the PBAN signal transduction cascade.

Response of prolonged flaccid paralysis to FNS rehabilitation techniques.

PURPOSE: The purpose of this study was to investigate the response of muscles with prolonged flaccid paralysis (a year after stroke) to two types of treatment: (1) functional neuromuscular stimulation (FNS) with surface electrodes; and (2) FNS with intramuscular (IM) electrodes (FNS-IM). A second purpose was to compare FNS-gait versus volitional gait (no FNS activation). METHOD: We used a single case study design; our patient was age 72, with flaccid paralysis of knee flexors and ankle dorsiflexors. RESULTS: Following four months of treatment with surface-stimulation, there was no change in muscle function or gait. Following treatment with FNS-IM, the patient regained partial volitional control of knee flexors and dorsiflexors; untreated muscles did not change. CONCLUSION: FNS-gait provided more normal knee and ankle dorsiflexion during swing phase versus volitional gait swing phase (no FNS activation).

Inner ear barotrauma from scuba diving.

Inner ear barotrauma among scuba divers is believed to be caused by any of three conditions: a hemorrhage in the inner ear, a tear of the labyrinthine membrane, or a perilymphatic fistula. These injuries may occur concurrently or separately. Hemorrhage and membrane rupture are managed conservatively, while fistula requires surgical repair. In this report, we describe three cases of inner ear barotrauma in scuba divers. We also discuss the proposed etiologies of this injury and the controversy over whether or not divers who have suffered an inner ear trauma can safely resume scuba diving. Although the older literature clearly suggests otherwise, we believe that scuba divers who completely recover from inner (or middle) ear barotrauma may return to diving as long as they exercise caution and care.

An integrated algorithm for text recognition: comparison with a cascaded algorithm.

The use of diverse knowledge sources in text recognition and in correction of letter substitution errors in words of text is considered. Three knowledge sources are defined: channel characteristics as probabilities that observed letters are corruptions of other letters, bottom-up context as letter conditional probabilities (when the previous letters of the word are known), and top-down context as a lexicon. Two algorithms, one based on integrating the knowledge sources in a single step and the other based on sequentially cascading bottom-up and top-down processes, are compared in terms of computational/storage requirements and results of experimentation.

Experiments in Text Recognition with Binary n-Gram and Viterbi Algorithms.

The binary n-gram and Viterbi algorithms have been suggested as alternative approaches to contextual postprocessing for text produced by a noisy channel such as an optical character recognizer. This correspondence describes the underlying theory of each approach in unified terminology, and presents new implementation algorithms for each approach. In particular, a storage efficient data structure is proposed for the binary n-gram algorithm and a recursive formulation is given for the Viterbi algorithm. Results of extensive experiments with each algorithm are described.