Identification and Expression Analysis of Candidate Odorant-Binding Protein and Chemosensory Protein Genes by Antennal Transcriptome of <i>Sitobion avenae</i>

<div><p>Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) of aphids are thought to be responsible for the initial molecular interactions during olfaction that mediate detection of chemical signals. Analysis of the diversity of proteins involved comprises critical basic research work that will facilitate the development of sustainable pest control strategies. To help us better understand differences in the olfactory system between winged and wingless grain aphids, we constructed an antennal transcriptome from winged and wingless <i>Sitobion avenae</i> (Fabricius), one of the most serious pests of cereal fields worldwide. Among the 133,331 unigenes in the antennal assembly, 13 OBP and 5 CSP putative transcripts were identified with 6 OBP and 3 CSP sequences representing new <i>S</i>. <i>avenae</i> annotations. We used qPCR to examine the expression profile of these genes sets across <i>S</i>. <i>avenae</i> development and in various tissues. We found 7 SaveOBPs and 1 SaveCSP were specifically or significantly elevated in antennae compared with other tissues, and that some transcripts (<i>SaveOBP8</i>, <i>SaveCSP2</i> and <i>SaveCSP</i>5) were abundantly expressed in the legs of winged or wingless aphids. The expression levels of the SaveOBPs and SaveCSPs varied depending on the developmental stage. Possible physiological functions of these genes are discussed. Further molecular and functional studies of these olfactory related genes will explore their potential as novel targets for controlling <i>S</i>. <i>avenae</i>.</p></div

Expression profiles of <i>SaveOBP6 SaveOBP8</i> and <i>SaveOBP10</i> in different ages of <i>S</i>. <i>avenae</i>.

<p>Fold-changes are relative to transcript levels in 1st instar nymphs. Differences in mean transcript levels were compared using one-way ANOVA, followed by the least-significant difference (LSD) method. Bars with different letters indicate significant differences (<i>p</i> < 0.05). 1 In: 1st instar nymph; 2 In: 2nd instar nymph; 3W In: 3rd winged instar nymph; 3WL In: 3rd wingless instar nymph; 4W In: 4th winged instar nymph; 4WL In: 4th wingless instar nymph; W: winged adult; WL: wingless adult.</p

Tissue expression profiles of candidate OBPs in <i>S</i>. <i>avenae</i>.

<p>Fold-changes are relative to transcript levels in abdomens of winged adult aphids. Differences in mean transcript levels were compared using one-way ANOVA, followed by the least-significant difference (LSD) method. Bars with different letters indicate significant differences (<i>p</i> < 0.05). An: antennae, H: heads, T: thoraxes, Ab: abdomens, L: legs.</p

Phylogenetic tree of 167 OBPs from 21 hemipteran species.

<p>The tree was constructed using MEGA 5.0 with bootstrap support based on 1000 iterations. Aphid sequences are in red. Bug sequences are in blue. Planthopper sequences are in gray. Major clades for aphid OBPs are marked in a different color. Abbreviation for these aphid species: Save, <i>S</i>. <i>avenae</i>; Acra, <i>A</i>. <i>craccivora</i>; Psal, <i>P</i>. <i>salicis</i>; Brebr, <i>B</i>. <i>brassicae</i>; Apis, <i>A</i>. <i>pisum</i>; Dpla, <i>D</i>. <i>platanoidis</i>; Mper, <i>M</i>. <i>persicae</i>; Nrib, <i>N</i>. <i>ribisnigri</i>; Rpad, <i>R</i>. <i>padi</i>; Mvic, <i>M</i>. <i>viciae</i>; Tsal, <i>T</i>. <i>salignus</i>; Afab, <i>A</i>. <i>fabae</i>; Agos, <i>A</i>. <i>gossypii</i>; Mdir, <i>M</i>. <i>dirhodum</i>; Lery, <i>L</i>. <i>erysimi</i>; Agly, <i>A</i>. <i>glycines;</i> Aluc, <i>A</i>. <i>lucorum</i>; Alin, <i>A</i>. <i>lineolatus</i>; Llin, <i>L</i>. <i>lineolaris</i>; Nlug, <i>N</i>. <i>lugens</i>; Sfur, <i>S</i>. <i>furcifera</i>.</p

Expression profiles of candidate OBPs in each age of <i>S</i>. <i>avenae</i>.

<p>Fold changes for 1st or 2nd instar nymphs are relative to transcript levels of <i>SaveOBP15</i>. Fold-changes for other stages are relative to transcript levels of wingless <i>SaveOBP15</i> in wingless aphids of the same age. Differences in mean transcript levels were compared using one-way ANOVA, followed by the least-significant difference (LSD) method. Bars with different letters indicate significant differences (<i>p</i> < 0.05).</p

Noise Reduction Method for Quantifying Nanoparticle Light Scattering in Low Magnification Dark-Field Microscope Far-Field Images

Nanoparticles have become a powerful tool for cell imaging and biomolecule, cell and protein interaction studies, but are difficult to rapidly and accurately measure in most assays. Dark-field microscope (DFM) image analysis approaches used to quantify nanoparticles require high-magnification near-field (HN) images that are labor intensive due to a requirement for manual image selection and focal adjustments needed when identifying and capturing new regions of interest. Low-magnification far-field (LF) DFM imagery is technically simpler to perform but cannot be used as an alternate to HN-DFM quantification, since it is highly sensitive to surface artifacts and debris that can easily mask nanoparticle signal. We now describe a new noise reduction approach that markedly reduces LF-DFM image artifacts to allow sensitive and accurate nanoparticle signal quantification from LF-DFM images. We have used this approach to develop a “Dark Scatter Master” (DSM) algorithm for the popular NIH image analysis program ImageJ, which can be readily adapted for use with automated high-throughput assay analyses. This method demonstrated robust performance quantifying nanoparticles in different assay formats, including a novel method that quantified extracellular vesicles in patient blood sample to detect pancreatic cancer cases. Based on these results, we believe our LF-DFM quantification method can markedly decrease the analysis time of most nanoparticle-based assays to impact both basic research and clinical analyses

MOESM1 of Antigen 85B peptidomic analysis allows species-specific mycobacterial identification

Additional file 1: Table S1. Ag85B target peptide identification and quantification by LC-MS/MS PRM mode

Alignment of amino acid sequences of the OBPs and CSPs in <i>S</i>. <i>avenae</i>.

<p>Sequences were aligned by Clustal Omega and edited using BoxShade. Black boxes show conserved cysteines. The conserved Cys residues are indicated. Shading represents sequence identity > 70%.</p

Phylogenetic tree of 51 CSPs from 7 hemipteran species.

<p>The tree was constructed using MEGA 5.0 with bootstrap support based on 1000 iterations. SaveOBP sequences are in bold. Abbreviation of these aphid spaces are as follows: Save, <i>S</i>. <i>avenae</i>; Mper, <i>M</i>. <i>persicae</i>; Agos, <i>A</i>. <i>gossypii</i>; Aluc, <i>A</i>. <i>lucorum</i>; Alin, <i>A</i>. <i>lineolatus</i>; Nlug, <i>N</i>. <i>lugens</i>; Sfur, <i>S</i>. <i>furcifera</i>.</p

Tissue expression profiles of candidate CSPs in <i>S</i>. <i>avenae</i>.

<p>Fold-changes are relative to transcript levels in abdomens of winged adult aphids. Differences in mean transcript levels were compared using one-way ANOVA, followed by the least-significant difference (LSD) method. Bars with different letters indicate significant differences (<i>p</i> < 0.05). An: antennae, H: heads, T: thoraxes, Ab: abdomens, L: legs.</p