Expression of odorant-binding proteins in mouthpart palps of the desert locust Schistocerca gregaria

Abstract

Odorant-binding proteins (OBPs) are essential molecular elements of the insect chemosensory system, which is composed of the antennae and the mouthpart palps (maxillary and labial). In this study, we have analysed the expression and the sensilla specificity of 14 OBP subtypes in the palps of the desert locust Schistocerca gregaria. The locust palps comprise only a low number of sensilla basiconica but a high number of sensilla chaetica. Employing a variety of approaches, we found that only a subset of the antennal OBP repertoire was expressed in both palp types. These OBPs were previously shown to be expressed either in sensilla basiconica or sensilla chaetica of the antennae. Comparing the expression pattern in the two chemosensory organs revealed similarities and differences; most remarkably, two OBP subtypes, OBP6 and OBP8, were found in both sensilla types on palps, whereas on the antennae they were solely expressed in one sensillum type. Together, the data indicate a differential, but partly overlapping, expression of OBPs in the two sensilla types of the palps. The differences in the expression pattern of OBP subtypes between antennae and palps might be indicative for distinct functions of the OBPs in the two chemosensory organs.

Keywords: desert locust; expression; insect; odorant-binding protein; palps.

Figure 1

(A, B) Light microscopic images of Schistocerca gregaria maxillary and labial palps. The beginning of the dome region is indicated by a white dashed line. The enriched sensilla density on the dome regions is shown. Below the white dashed line different sensilla in lower density are visible. (C) Immunohistochemistry utilizing a horseradish peroxidase (HRP) Alexa‐488 conjugate utilizing a labial palp cryosection combined with propidium iodide nuclei staining. Neuronal α‐HRP‐labelled cells are visualized by green fluorescence; stained nuclei are shown in red fluorescence. Green‐labelled neuronal structures are visible in a palisade fashion. The white dashed line (1) indicates the beginning of the cuticle, whereas below dashed line (2) the HRP‐labelled neurons can be found. (D, E) Magnification of the indicated regions (white dashed line boxes) in (C), showing the ‘longitude’‐type (D) and the ‘round’‐type (E) of labelled cell clusters in single fluorescence channel images (green). (F, G) Single fluorescence channel images of the merged image in (C). Scale bars: A, B, 200 µm;, C, F, G, 50 µm; D, E, 20 µm.

Figure 2

Analysis of whole‐mount fluorescence in situ hybridization (WM‐FISH)‐treated palps. (A) As a first step in performing WM‐FISH on palps, a slice closely behind the dome region was performed and images were taken using the confocal laser scanning microscope from the angle indicated by the white arrow. (B) Same as in (A), with the difference that after the first slice at the start (1), at the end of the WM‐FISH procedure a second slice (2) was performed and the images were taken as indicated by the white arrows. (C, D) WM‐FISH utilizing digoxigenin and biotin‐labelled riboprobes of SgreOrco. The image in (C), labial palp, was taken with the angle indicated in (A), whereas the image in (D), maxillary palp, was taken as indicated in (B). The white dash line in (D) indicates the boundary to the cuticle. Images represent projections of optical planes from confocal image stacks. Scale bars: C, D, 50 µm.

Figure 3

Reverse transcription PCR conducted with cDNA prepared from maxillary palps and labial palps. The respective odorant‐binding protein (OBP) tested is indicated below the lane; actin (AEV89776) was used for control of the cDNA integrity. The numbers right or left of the image indicate marker bands in base pairs. Expression of the OBPs 1, 2, 4, 5, 6, 7 and 8 in both palp types is shown. The white boxes indicate a weak, but visible PCR band for OBP7.

Figure 4

Expression of subfamily I‐A odorant‐binding proteins (OBPs). Whole‐mount fluorescence in situ hybridization (WM‐FISH) with labial palps (A–C and A) and maxillary (B and C) utilizing digoxigenin‐labelled riboprobes of subfamily I‐A OBPs: OBP1, OBP5 and OBP6. (A–C) Cells labelled by the probes for OBP1, OBP5 or OBP6 are shown by red fluorescence. The images were taken as indicated in Fig. 2A. (A, B) Expression of OBP1 and OBP5 in a lower number of cell clusters is visible. In contrast, OBP6 is expressed in a higher number of cells as shown in (C). (A–C) WM‐FISH utilizing probes for OBP1, OBP5 and OBP6; images were taken as indicated in Fig. 2B. Images represent projections of different optical layers from confocal image stacks. Scale bars: A–C, 50 µm; A–C, 20 µm.

Figure 5

Topography of cells expressing subfamily I‐A OBPs and olfactory receptor co‐receptor (Orco). (A–C) Merged images of fluorescence in situ hybridization on sections through the labial (A) and maxillary (B, C) palps of Schistocerca gregaria using digoxigenin and biotin‐labelled riboprobes of subfamily I‐A odorant‐binding proteins (OBPs) and Orco. Stained cells are either shown in red (Orco) or green fluorescence (OBPs). A close‐up of labelled OBP‐positive cells (green) and Orco‐expressing cells (red) is shown. The white dashed line indicates the boundary to the cuticle. (A–C, A–C) Single fluorescence channel images; either the green or the red fluorescence are shown. Scale bars: 20 µm.

Figure 6

Co‐expression of subfamily I‐A odorant‐binding proteins (OBPs). (A–C) Merged images of fluorescence in situ hybridization on sections of maxillary palps of Schistocerca gregaria using digoxigenin and biotin‐labelled riboprobes of subfamily I‐A. OBP‐expressing cells are depicted in red and green fluorescence. (A) A partial co‐expression of OBP1 and OBP5 is visible (yellow). (B, C) OBP1 and OBP5‐positive cells co‐express OBP6, indicated by yellow fluorescence. The white dashed line indicates the beginning of the cuticle. (A–C, A–C') Single fluorescence channel images; either the green or the red fluorescence are shown. Scale bars: A–C, A, 50 µm; B, C, 20 µm.

Figure 7

Expression of odorant‐binding proteins (OBPs) from different subfamilies. (A–D) Whole‐mount fluorescence in situ hybridization utilizing maxillary (A, B, D) and labial (C) palps of the desert locust using digoxigenin‐labelled riboprobes of subfamily I‐B and subfamily III OBPs (2, 4, 7 and 8). OBPs are visualized by red fluorescence. All four OBPs are expressed in a higher number of cells, similar to OBP6 in Fig. 4. Images represent projections of confocal image stacks representing different optical layers. Scale bars: A–D, 50 µm.

Figure 8

Topography of cells expressing subfamilies I‐B and IV odorant‐binding proteins (OBPs) and olfactory receptor co‐receptor (Orco). (A–D) Merged images from fluorescence in situ hybridization experiments on sections of the maxillary (A–C) and labial (D) palps of Schistocerca gregaria using digoxigenin and biotin‐labelled riboprobes of subfamily I‐B and subfamily IV OBPs and Orco. OBP‐positive cells are visualized by green fluorescence and Orco‐expressing olfactory sensory neurons (OSNs) by red fluorescence. The OBPs 2, 4 and 7 are not co‐localized with Orco‐expressing OSNs. In contrast, in the case of OBP8, a close‐up of OBP8‐positive cells and Orco‐expressing OSNs is shown. The white dashed line indicates the boundary to the cuticle. (A–D, A–D) Single fluorescence channel images; either the green or the red fluorescence are shown. Scale bars: A–D, 50 µm; A–D, A–D, 20 µm.

Figure 9

Co‐expression of subfamily I‐A odorant‐binding proteins (OBPs) and OBP8. (A–C) Fluorescence in situ hybridization on sections of maxillary (A–C) of Schistocerca gregaria with digoxigenin and biotin‐labelled riboprobes of OBP1, OBP5 or OBP6 and OBP8. Family I‐A OBP‐positive cells are depicted by red fluorescence and OBP8‐expressing cells by green fluorescence. Cells expressing OBP1 or OBP5 do co‐express OBP8, indicated by yellow fluorescence. Approximately half of the OBP6‐positive cells co‐express OBP8. The white dashed line indicates the boundary to the cuticle. Images represent projections of different optical layers of confocal image stacks. Scale bars: A–C, A, A, 50 µm; B, C, B, C, 50 µm.