The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern

Abstract

In locusts, olfaction plays a crucial role for initiating and controlling behaviours, including food seeking and aggregation with conspecifics, which underlie the agricultural pest capacity of the animals. In this context, the molecular basis of olfaction in these insects is of particular interest. Here, we have identified genes of two orthopteran species, Locusta migratoria and Schistocera gregaria, which encode the olfactory receptor co-receptor (Orco). It was found that the sequences of LmigOrco and SgreOrco share a high degree of identity to each other and also to Orco proteins from different insect orders. The Orco-expressing cells in the antenna of S. gregaria and L. migratoria were visualized by in situ hybridization. Orco expression could be assigned to clusters of cells in sensilla basiconica and few cells in sensilla trichodea, most likely representing olfactory sensory neurons. No Orco-positive cells were detected in sensilla coeloconica and sensilla chaetica. Orco expression was found already in all nymphal stages and was verified in some other tissues which are equipped with chemosensory hairs (mouthparts, tarsi, wings). Together, the results support the notion for a decisive role of Orco in locust olfaction.

Keywords: Orco; gene expression; in situ hybridization.; locust; olfaction.

Figure 1

Alignment of the amino acid sequences of LmigOrco and SgreOrco to selected Orco proteins of insects species from different insect orders. Amino acids identical in all sequences are marked with grey shading. Numbers to the right refer to the position of the last residue in a line in each Orco sequence. The abbreviations indicate (accession numbers in brackets): Amel = Apis melifera (XP_001121145); Dmel = Drosophila melanogaster (AAF52031); Bmor = Bombyx mori (AJ555487); Tcas = Tribolium castaneum (AM689911). Dashes indicate gaps introduced into the sequences for optimal alignment or missing amino acids at the C-terminus of the locust Orcos. The positions of putative transmembrane domains (TMD1 -TMD7), which were predicted based on DmelOrco, are indicated. The amino acid stretches corresponding to the cloning primers are boxed: 1 = Orco-deg sense, 2 = Orco-deg antisense, 3 = Sgre5'race, 4 = Sgre5'race nested, 5 = LmigOrco-NT sense, 6 = LmigOrco-NT antisense, 7 = LmigOrco5'race, 8 = LmigOrco5'race nested.

Figure 2

Neighbor joining tree of Orco sequences from insects belonging to different insect orders. The tree was constructed with MEGA5 based on a ClustAl alignment of the amino acid sequences shown in Fig. 1 and selected Orco sequences from other insects deposited in Genbank. Bootstrap support values are based on 1000 replicates; only support values above 80% are shown. Branch lengths are proportional. Abbreviations as in figure 1 and (accession numbers in brackets): Apis = Acyrthosiphon pisum (XP_001951646); Aaeg = Aedes aegypti (AAT01220); Agam = Anopheles gambiae (AY363725); Aper = Anthereae pernyi (AJ555486); Bcuc = Bactrocera curcurbitae (ADK97803); Bdor = Bactrocera dorsalis (ACC86853); Ccap = Ceratitis capitata (AAX14775); Hobl = Holotricha oblita (AEE69033); Hplu = Holotricha plumbea (ADM35103); Hvir = Heliothis virescens (AJ487477); Hzea = Helicoverpa zea (AAX14773); Hass = Helicoverpa assulta (ABU45983); Phum = Pediculus humanus (EEB12924); Pxyl = Plutella xylostella (BAG71421); Save = Sitobion avenae (ACT37280). Hemi. Ass. = Hemipteroid Assemplage.

Figure 3

Expression of LmigOrco in the antenna of the migratory locust L. migratoria. In situ hybridizations were performed on horizontal tissue sections of male antennae with DIG-labeled antisense (A - C) or sense (D) RNA probes for LmigOrco. Signals were visualized using an anti-DIG antibody and color substrates. A, Hybridization signals in a segment of the filamentous locust antenna. B, Higher magnification of the area boxed in A showing a cluster of labeled cells. C, Hybridization signals can be assigned to clusters of cells bordering the antennal surface. D, No hybridization signals were obtained in ISH experiments with an Orco-specific sense RNA probe. Scale bars: 20 μm in A and B, 50 μm in C and D.

Figure 4

LmigOrco gene expression in cells of sensilla basiconica and sensilla trichodea. In situ hybridizations were performed using a DIG-labeled Orco-specific antisense RNA probes on sections of male antenna. A, shows a cluster of LmigOrco-positive cells under a short sensillum basiconicum. B, Three LmigOrco-expressing cells can be assigned to a longer sensillum trichodeum. C and D, No labeling of cells was detected under sensilla chaetica (C) or coeloconica (D). Scale bars: 10 μm.

Figure 5

SgreOrco gene expression in the antenna of the desert locust S. gregaria. In situ hybridization on sections of the antennae with DIG-labeled antisense (A - C and E - H) or sense (D) RNA probes for SgreOrco. A, Hybridization on a horizontal section of an antennal segment. B, Higher magnification of the area boxed in A showing several clusters of labeled cells. C, Clusters of labeled cells bordering the antennal surface. D, No hybridization signals were obtained with a sense RNA probe. E, LmigOrco-positive cells under a short sensillum basiconicum. F, Two SgreOrco-expressing cells can be assigned to a sensillum trichodeum. G and H, No labeling of cells was detected under a sensillum chaeticum (G) or sensilla coeloconica (H and G). A - E and G = female antenna; F and H, male antenna. Scale bars: 50 μm in A and D, 20 μm in B and C, 10 μm in E - H.

Figure 6

Expression of LmigOrco in different tissues and developmental stages of L. migartoria. RT-PCRs were performed using specific primer pairs and cDNAs prepared from the tissues indicated. A, RT-PCRs with cDNA prepared from the antenna of different nymphal stages (1^st to 5^th instar) and adults. B, Tissue specificity of LmigOrco expression. M ant, male antenna; F ant, female antenna; Mouthp, mouthparts. Amplification products were analysed on agarose gels and visualized by UV illumination after ethidium bromide staining. Based on the primer design the expected size of the PCR-products is 342 bp for LmigOrco and 314 bp for the actin control gene. The position of molecular weight markers (in bp) is indicated.