Identification of odorant binding proteins and chemosensory proteins in <i>Microplitis mediator</i> as well as functional characterization of chemosensory protein 3

<div><p>Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play important roles in transporting semiochemicals through the sensillar lymph to olfactory receptors in insect antennae. In the present study, twenty OBPs and three CSPs were identified from the antennal transcriptome of <i>Microplitis mediator</i>. Ten OBPs (<i>MmedOBP11–20</i>) and two CSPs (<i>MmedCSP2–3</i>) were newly identified. The expression patterns of these new genes in olfactory and non-olfactory tissues were investigated by real-time quantitative PCR (qPCR) measurement. The results indicated that <i>MmedOBP14</i>, <i>MmedOBP18</i>, <i>MmedCSP2</i> and <i>MmedCSP3</i> were primarily expressed in antennae suggesting potential olfactory roles in <i>M</i>. <i>mediator</i>. However, other genes including <i>MmedOBP11</i>–<i>13</i>, <i>15–17</i>, <i>19</i>–<i>20</i> appeared to be expressed at higher levels in body parts than in antennae. Focusing on the functional characterization of MmedCSP3, immunocytochemistry and fluorescent competitive binding assays were conducted indoors. It was found that MmedCSP3 was specifically located in the sensillum lymph of olfactory sensilla basiconca type 2. The recombinant MmedCSP3 could bind several types of host insects odors and plant volatiles. Interestingly, three sex pheromone components of Noctuidae insects, <i>cis</i>-11-hexadecenyl aldehyde (<i>Z</i>11-16: Ald), <i>cis</i>-11-hexadecanol (<i>Z</i>11-16: OH), and <i>trans</i>-11-tetradecenyl acetate (<i>E</i>11-14: Ac), showed high binding affinities (Ki = 17.24–18.77 μM). The MmedCSP3 may be involved in locating host insects. Our data provide a base for further investigating the physiological roles of OBPs and CSPs in <i>M</i>. <i>mediator</i>, and extend the function of MmedCSP3 in chemoreception of <i>M</i>. <i>mediator</i>.</p></div

Immunocytochemical localization of MmedCSP3 in sensilla on female antennae.

<p>(A): The sensilla basiconica type 2 was labeled strongly, whereas the sensilla placodea were not labeled. (B): The sensillum lymph in the sensilla basiconica type 2 was strong labeled. sp, s. placodea; st, s. trichodea; c, cavity; d, dendrites; p, pore; w, sensillum wall.</p

Primers used in this study.

<p>Primers used in this study.</p

Sequence alignment of all identified OBPs and CSPs in <i>M</i>. <i>mediator</i>.

<p>A: Sequence alignment of OBPs, six conserved cysteine residues are marked in black; B: Sequence alignment of CSPs, four conserved cysteine residues are marked in black. Predicted signal peptides are boxed in the figure.</p

Phylogenetic tree of OBPs and CSPs from Hymenoptera species.

<p>Mmed (red): <i>M</i>. <i>mediator</i>; Ccun (purple): <i>Chouioia cunea</i>; Ssp (green): <i>Sclerodermus sp</i>.; Amel (blue): <i>Apis mellifera</i>.</p

SDS-PAGE and western blot analysis of recombinant MmedCSP3.

<p>M: Molecular weight marker; 1: Non-induced pET-30a (+) / MmedCSP3; 2: Induced pET-30a (+) / MmedCSP3; 3: pET-30a (+) / MmedCSP3 supernatant; 4: pET-30a (+) / MmedCSP3 pellet; 5: Purified MmedCSP3 with His-tag; 6: Purified MmedCSP3 without His-tag; 7: Western blot analysis of MmedCSP3.</p

qPCR analysis of <i>MmedOBPs</i> and <i>MmedCSPs</i> expression in different tissues.

<p>The error bars represent standard error and the different small letters above each bar indicate significant differences in transcript abundances (<i>P</i> < 0.05).</p

Binding affinities of selected ligands with recombinant MmedCSP3.

<p>Binding affinities of selected ligands with recombinant MmedCSP3.</p

Binding characteristic of selected ligands to MmedCSP3.

<p>(A) Binding curve and relative Scatchard plot of bis-ANS to MmedCSP3. (B) Competitive binding curves of four selected plant volatiles to MmedCSP3 (C) Competitive binding curves of three sex pheromone components to MmedCSP3. (D) Competitive binding curved of two fatty acids to MmedCSP3.</p