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Predatory syrphid larvae are an important natural enemy of aphids in cotton agro-ecosystems in China. Their behaviors in prey foraging, localization and oviposition greatly rely on the perception of chemical cues. As a first step to better understand syrphid olfaction at the molecular level, we have performed a systematic identification of their major chemosensory genes. Male and female antennal transcriptomes of Episyrphus balteatus and Eupeodes corollae were sequenced and assembled using Illumina HiSeq2000 technology. A total of 154 chemosensory genes in E. balteatus transcriptome, including candidate 51 odorant receptors (ORs), 32 ionotropic receptors (IRs), 14 gustatory receptors (GRs), 49 odorant-binding proteins (OBPs), 6 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs) were identified. In E. corollae transcriptome, we identified 134 genes including 42 ORs, 23 IRs, 16 GRs, 44 OBPs, 7 CSPs and 2 SNMPs. We have provided full-length sequences of the highly conserved co-receptor Orco, IR8a/25a family and carbon dioxide gustatory receptor in both syrphid species. The expression of candidate OR genes in the two syrphid species was evaluated by semi-quantitative reverse transcription PCR. There were no significant differences of transcript abundances in the respective male and female antenna, which is consistent with differentially expressed genes (DEGs) analysis using the FPKM value. The sequences of candidate chemosensory genes were confirmed and phylogenetic analysis was performed. This research comprehensively analyzed and identified many novel candidate chemosensory genes regarding syrphid olfaction. It provides an opportunity for understanding how syrphid insects use chemical cues to conduct their behaviors among tritrophic interactions of plants, herbivorous insects, and natural enemies in agricultural ecosystems.

The cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation. M. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in mitochondrial respiratory chain. This is the first study to perform a comparative transcriptome analysis of S. zeamais in response to M. alternifolia essential oil fumigation. Our results provide new insights into the insecticidal mechanism of M. alternifolia essential oil fumigation against S. zeamais and eventually contribute to the management of this important agricultural pest.

Olfaction is important for mediating aphid behaviors and is involved in host location and mating. Antennal primary rhinaria play a key role in the chemoreception of aphids. The function of the peripheral olfactory system in the subfamily Aphidinae has been intensively studied, but little is known about other subfamilies of Aphididae. Therefore, three aphid species were selected to study the olfactory reception of plant volatiles: Cinara cedri (Lachninae), Eriosoma lanigerum (Eriosomatinae), and Therioaphis trifolii (Calaphidinae). In this study, the morphology and distribution of the antennal sensilla of apterous adults were observed by scanning electron microscopy. Three morphological types were identified (placoid sensilla, coeloconic sensilla, and trichoid sensilla); the first two were distributed on the antennal primary rhinaria. A pattern of primary rhinaria in C. cedri was found that differed from that of E. lanigerum and T. trifolii and consists of 1 large placoid sensillum (LP) on the 4th segment, 2 LPs on the 5th segment, and a group of sensilla on the 6th antennal segments. Later, we recorded and compared neuronal responses of the distinct placoid sensilla in the primary rhinaria of the three aphid species to 18 plant volatiles using a single sensillum recording (SSR) technique. The results indicated that the functional profiles based on the tested odorants of the primary rhinaria of the three investigated aphid species were clustered into three classes, and exhibited excitatory responses to certain types of odorants, especially terpenes. In C. cedri , the ORNs in LP6 exhibited the highest responses to (±)-citronellal across all tested chemicals, and showed greater sensitivity to (±)-citronellal than to (+)-limonene. ORNs in LP5 were partially responsive to α-pinene and (–)-β-pinene in a dose-dependent manner. Across different species, E. lanigerum showed significantly stronger neuronal responses of LP5 to several terpenes, such as (–)-linalool and α-terpineol, compared to other species. In T. trifolii , the neuronal activities in LP6 showed a greater response to methyl salicylate as compared to LP5. Overall, our results preliminarily illustrate the functional divergence of ORNs in the primary rhinaria of aphids from three subfamilies of Aphididae and provide a basis for better understanding the mechanism of olfactory recognition in aphids.

Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.

The olfactory sensing system of the syrphid fly Eupeodes corollae is essential in pollination and prey localization, but little is known about the ultrastructural organization of their olfactory organs. In this study, the morphology, distribution, and ultrastructural organization of antennal sensilla of E. corollae in both sexes were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Neuronal responses of a subtype of sensilla basiconica to floral scent compounds were recorded by single sensillum recording (SSR). Ten morphological types, including Böhm bristles, sensilla chaetica, microtrichiae, sensilla trichodea, sensilla basiconica, sensilla clavate, sensilla coeloconica, sensilla styloconica, sensilla placodea, and sensory pit, were identified. Except for Böhm bristles and sensilla chaetica, which were distributed on the scape and pedicel of E. corollae antennae, innervated sensilla were densely distributed on the flagellum, a vital sensory organ. Further, observing ultrastructural organization showed that the sensilla trichodea, basiconica, and clavate are single-walled with multiple nanoscale pores perforating the cuticle. Sensilla coeloconica are double-walled and have no wall pores, but instead, have longitudinal grooves along with the pegs. Sensilla chaetica, Böhm bristles, and microtrichiae did not have wall pores on the cuticle or sensory cells at the base. The SSR results indicated that neuron B housed in the subtype of sensilla basiconica I (SBI) mainly responded to methyl eugenol and other aromatic compounds. Overall, our results provide valuable information to understand the morphology and ultrastructure of antennal sensilla from E. corollae . These findings are beneficial for the studies of the neuronal function map of olfactory sensilla and for determining evolutionary relationships in Diptera.

Heortia vitessoides Moore, 1885 (Lepidoptera: Crambidae) is an economically important lepidopteran pest that caused severe damage to the plantation area of Aquilaria sinensis (Lour.) Gilg, 1825 (Thymelaeaceae), resulting in extensive defoliation of the trees during an epidemic. In this study, we used scanning electron microscopy (SEM) to analyze the external morphology and ultrastructure of sensilla on various body parts of H. vitessoides. Specifically, seven, four, four, and five types of sensilla were found, respectively, on the antennae, proboscis, labial palps, and legs. We described the types, distributions, and sexual dimorphism of these sensilla on antennae, and found that the number and size of sensilla differed significantly between males and females. This study provides crucial information for future investigations into the function of these sensilla in H. vitessoides.

Synchronous gastric tumors that consist of both gastrointestinal stromal tumor (GIST) and adenocarcinoma are rare. We studied the clinicopathological and molecular characteristics of six cases containing both gastric adenocarcinoma and GIST. By means of immunohistochemical analysis, all GIST cells expressed CD117, CD34 and Dog1 in all six synchronous gastric adenocarcinomas with GIST, and in GIST alone. Sequencing analysis demonstrated that exon 11 c-kit mutations were present in two of six synchronous tumors and four of five GISTs. One of the two exon 11 c-kit mutations in synchronous adenocarcinomas with GISTs was an uncommon mutation of CTT > CCA at amino acid 576, and the other was a GTT deletion at amino acid 560. The mutation was a homozygous A > G mutation in exon 12 (amino acid 567) of PDGFR-α . We concluded that the exon 11 mutations were the most important in both cases of synchronous gastric adenocarcinoma with GIST and GIST alone. The mutation rate was higher in GIST alone than in synchronous adenocarcinoma with GIST.

Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C)) to the pathogenic one (PrP(Sc)). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.

Background: Interferon-gamma release assay (IGRA) has been used in latent tuberculosis (TB) infection and TB diagnosis, but the results from different high TB-endemic countries are different. The aim of this study was to investigate the value of IGRA in the diagnosis of active pulmonary TB (PTB) in China. Methods: We conducted a large-scale retrospective multicenter investigation to further evaluate the role of IGRA in the diagnosis of active PTB in high TB-epidemic populations and the factors affecting the performance of the assay. All patients who underwent valid T-SPOT.TB assays from December 2012 to November 2015 in six large-scale specialized TB hospitals in China and met the study criteria were retrospectively evaluated. Patients were divided into three groups: Group 1, sputum culture-positive PTB patients, confirmed by positive Mycobacterium tuberculosis sputum culture; Group 2, sputum culture-negative PTB patients; and Group 3, non-TB respiratory diseases. The medical records of all patients were collected. Chi-square tests and Fisher's exact test were used to compare categorical data. Multivariable logistic analyses were performed to evaluate the relationship between the results of T-SPOT in TB patients and other factors. Results: A total of 3082 patients for whom complete information was available were included in the investigation, including 905 sputum culture-positive PTB cases, 914 sputum culture-negative PTB cases, and 1263 non-TB respiratory disease cases. The positive rate of T-SPOT.TB was 93.3% in the culture-positive PTB group and 86.1% in the culture-negative PTB group. In the non-PTB group, the positive rate of T-SPOT.TB was 43.6%. The positive rate of T-SPOT.TB in the culture-positive PTB group was significantly higher than that in the culture-negative PTB group (χ2 = 25.118, P < 0.01), which in turn was significantly higher than that in the non-TB group (χ2 = 566.116, P < 0.01). The overall results were as follows: sensitivity, 89.7%; specificity, 56.37%; positive predictive value, 74.75%; negative predictive value, 79.11%; and accuracy, 76.02%. Conclusions: High false-positive rates of T-SPOT.TB assays in the non-TB group limit the usefulness as a single test to diagnose active TB in China. We highly recommend that IGRAs not be used for the diagnosis of active TB in high-burden TB settings.

The oriental armyworm, (Walker), is a polyphagous, migratory pest relying on olfactory cues to find mates, locate nectar, and guide long-distance flight behavior. In the present study, a combination of neuroanatomical techniques were utilized on this species, including backfills, confocal microscopy, and three-dimensional reconstructions, to trace the central projections of sensory neurons from the antenna and the labial pit organ, respectively. As previously shown, the axons of the labial sensory neurons project via the ipsilateral labial nerve and terminate in three main areas of the central nervous system: (1) the labial-palp pit organ glomerulus of each antennal lobe, (2) the gnathal ganglion, and (3) the prothoracic ganglion of the ventral nerve cord. Similarly, the antennal sensory axons project to multiple areas of the central nervous system. The ipsilateral antennal nerve targets mainly the antennal lobe, the antennal mechanosensory and motor center, and the prothoracic and mesothoracic ganglia. Specific staining experiments including dye application to each of the three antennal segments indicate that the antennal lobe receives input from flagellar olfactory neurons exclusively, while the antennal mechanosensory and motor center is innervated by mechanosensory neurons from the whole antenna, comprising the flagellum, pedicle, and scape. The terminals in the mechanosensory and motor center are organized in segregated zones relating to the origin of neurons. The flagellar mechanosensory axons target anterior zones, while the pedicular and scapal axons terminate in posterior zones. In the ventral nerve cord, the processes from the antennal sensory neurons terminate in the motor area of the thoracic ganglia, suggesting a close connection with motor neurons. Taken together, the numerous neuropils innervated by axons both from the antenna and labial palp indicate the multiple roles these sensory organs serve in insect behavior.