Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria)

Abstract

The behaviour of the desert locust, Schistocera gregaria, is largely directed by volatile olfactory cues. The relevant odorants are detected by specialized antennal sensory neurons which project their sensory dendrites into hair-like structures, the sensilla. Generally, the responsiveness of the antennal chemosensory cells is determined by specific receptors which may be either odorant receptors (ORs) or variant ionotropic receptors (IRs). Previously, we demonstrated that in locust the co-receptor for ORs (ORco) is only expressed in cells of sensilla basiconica and sensilla trichodea, suggesting that cells in sensilla coeloconica may express different types of chemosensory receptors. In this study, we have identified the genes of S. gregaria which encode homologues of co-receptors for the variant ionotropic receptors, the subtypes IR8a and IR25a. It was found that both subtypes, SgreIR8a and SgreIR25a, are expressed in the antennae of all five nymphal stages and in adults. Attempts to assign the relevant cell types by means of in situ hybridization revealed that SgreIR8a and SgreIR25a are expressed in cells of sensilla coeloconica. Double fluorescence in situ hybridization experiments disclosed that the two IR-subtypes are co-expressed in some cells of this sensillum type. Expression of SgreIR25a was also found in some of the sensilla chaetica, however, neither SgreIR25a nor SgreIR8a was found to be expressed in sensilla basiconica and sensilla trichodea. This observation was substantiated by the results of double FISH experiments demonstrating that cells expressing SgreIR8a or SgreIR25a do not express ORco. These results support the notion that the antenna of the desert locust employs two different populations of OSNs to sense odors; cells which express IRs in sensilla coeloconica and cells which express ORs in sensilla basiconica and sensilla trichodea.

Keywords: in situ hybridization; ionotropic receptors; locust; olfaction.

Figure 1

Alignment of the SgreIR8a amino acid sequence with IR8a sequences from other insects. Positions of the amino terminal domain (ATD), the binding domain lobes (S1 and S2), the pore loop (P) and the transmembrane segments (M1, M2, M3) are marked by bars of different colors referring to their position in DmelIR8a . The positions of key ligand binding residues in iGluRs are marked by asterisks above the sequences. Numbers on the right refer to the number of the last amino acid in the line. Amino acids with at least 50% identity or similarity between sequences are shaded black and grey, respectively. The IR8a amino acid sequences from Agam = Anopheles gambiae, Amel = Apis melifera, Apis = Acyrthosiphon pisum, Bmor = Bombyx mori, Dmel = Drosophila melanogaster, and Tcas = Tribolium castaneum were taken from .

Figure 2

Alignment of the SgreIR25a amino acid sequence with IR25a sequences identified in other insects. Numbers on the right refer to the number of the last amino acid in the line. Black and grey shadings indicate amino acids which show at least 70% identity, respectively similarity, between sequences. Labeling of protein domains, abbreviations and origin of sequences are the same as indicated in figure 1. LmigIR25a = Locusta migratoria IR25a (GenBank: AFP33229.1)

Figure 3

Phylogenetic relationship of IR8a and IR25a sequences from S. gregaria and species belonging to various insect orders. A neighbour-joining tree was constructed using MEGA5 based on a ClustalW alignment of the IRs indicated in figures 1 and 2, as well as sequences from Aaeg = Aedes aegypti, Cpom = Cydia pomonella, Cqui = Culex quinquefasciatus, Dgri = D. grimshawi, Dpse= D. pseudoobscura, Msex = Manduca sexta, Nvit = Nasonia vitripennis and Phum = Pedeculus humanus, , , . Bootstrap support values are based on 1000 replicates, only support values above 80% are shown. Branch lengths are proportional.

Figure 4

Tissue specificity and developmental expression of SgreIR8a and SgreIR25a. RT-PCR experiments were performed using cDNAs prepared from the tissues indicated and primer pairs specific for SgreIR8a and SgreIR25a, respectively. Primers to actin were used as control for the integrity of the cDNA preparations. A, Expression of the IRs in different locust tissues. FA, female antenna; MA, male antenna; M, mouthparts (maxillary and labial palps); B, brain; T, tarsi. B, Comparison of the IR expression in the antennae of different nympal stages (1^st to 5^th instar) and adults. Amplification products were analysed on agarose gels and visualized by UV illumination after ethidium bromide staining.

Figure 5

Topography of SgreIR8a gene expression in the antenna S. gregaria. In situ hybridization using SgreIR8a-specific Dig-labeled sense and anti-sense riboprobes and visualization with color substrates. A, Labeling of cells by the SgreIR8a anti-sense RNA probe in two antennal segments of the desert locust. B, Higher magnification of the area boxed in A. C - F, The SgreIR8a anti-sense RNA probe labeled one to three cells under sensilla coeloconica (s.co), but never cells under sensilla basiconica (s.ba, C) , sensilla chaetica (s.ch, E) or sensilla trichodea (s.tr, F). G, Labeling of cells by a Dig-labeled SgreORco-specific anti-sense RNA probe. H, No hybridization signals were observed with the SgreIR8a sense probe. A, B, D, E: female antennae; C, F, G, H: male antennae. Scale bars: 100 μm in A; 50 μm in B, G, H; 20 μm in C, D, E, F.

Figure 6

Localisation of SgreIR25a gene expression in the antenna of S. gregaria. In situ hybridization using SgreIR25a-specific Dig-labeled sense and anti-sense riboprobes and chromogenic visualization. A - D, The SgreIR25a anti-sense RNA probe labeled one (A), two (B), three (C) or four (D) cells under sensilla coeloconica (s.co). No cells under sensilla trichodea (s.tr, E) and sensilla basiconica (s.ba, F) were labeled. For sensilla chaetica (s.ch), cases of no labeled cells (C) and SgreIR25a-positive cells (G) were found. H, No labeling of cells were obtained with the SgreIR25a sense riboprobe. B, C, E, H: female antennae; A, D, G, F: male antennae. Scale bars: 20 μm in A - G; 50 μm in H.

Figure 7

Partial overlap of SgreIR8a and SgreIR25a expression. Double FISH on antennal sections using Dig-labeled SgreIR25a and biotin-labeled SgreIR8a probes with visualization of FISH signals in red (SgreIR25a) and green (SgreIR8a). A - C, Cluster of three cells labeled by both (C), the SgreIR25 probe (red, A) and the SgreIR8a probe (green, B). D - F, Distinct cells that only express SgreIR25a (red, D) or SgreIR8a (green, E) without overlap (F). G - I, Cluster of three cells, with one cell co-expressing SgreIR8a and SgreIR25a (I), the other two cells express SgreIR25a (red, G) but not SgreIR8a (H, green). A - C, G - I: female antennae; D - F: male antennae. Scale bars: 20 μm.

Figure 8

SgreIR8a is not expressed in cells expressing SgreORco. Two-color FISH on antennal sections of male locust using a biotin-labeled SgreIR8a and a Dig-labeled SgreORco probe and detection by green (SgreIR8a) and red (SgreORco) fluorescence. A - D, Hybridization signals on a sagital section of the antenna. The IR8a anti-sense RNA probe labeled cells under sensilla coeloconica which are not labeled by the ORco probe. Pictures show projections of confocal image stacks showing the separated (A, green; B, red) or overlaid (C) fluorescence channels. To better show the morphology of the section the transmitted light channel has been overlaid with the fluorescence channel in D. E - G, Two-color FISH on a more horizontal section of the antenna section confirming the expression of SgreIR8a (green) and SgreORco (red) in different cells. Clusters of ORco-positive cells are intermingled with SgreIR8a-positive cells. Pictures show the separated green (E) and red (F) fluorescence channels and the overlay at higher magnification in G. Scale bars: 20 μm.

Figure 9

Expression of SgreIR25a and SgreORco locate in different cells. Double FISH on female antennal sections was performed employing Dig-labeled SgreIR25a and biotin-labeled SgreORco probes. Positive cell were visualized by red (SgreIR25a) and green (SgreORco) fluorescence. A - C, Hybridization signals on a longitudinal section of the antenna. The IR25a anti-sense RNA probe labeled cells under sensilla coeloconica which are not labeled by the ORco probe. D - F, The SgreIR25a anti-sense RNA probe labeled cells under sensilla cheatica which do not express SgreORco. Pictures are projections of confocal image stacks showing the separated (A, D, red; B, E, green) or overlaid fluorescence channels (C, F with transmitted light channel). Scale bars: 20 μm.