In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1

Abstract

Under given environmental conditions, the desert locust (Schistocera gregaria) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the "sensory neuron membrane protein 1" (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1. In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.

Keywords: antenna; desert locust; insect; olfaction; pheromone receptor; sensory neuron membrane protein 1.

Fig 1

Evolutionary relationships among SgreORs and LmigOrs. A neighbor-joining tree was calculated using the MEGA program. For rooting the tree, Orco-encoding sequences of different insect species were used. Calculations are based on a Clustal X alignment of the 117 SgreOR and 138 LmigOr amino acid sequences included in the tree. Branch lengths are proportional to the percentage of sequence differences. The scale bar indicates 10% difference. The numbers in the tree indicate bootstrap support values (in %) based on 1000 replicates (only values above 50% are shown). OR-coding sequences from Schistocerca gregaria are denoted by red squares. SgreOR sequences used in two-color FISH experiments together with a SNMP1-specific probe are highlighted with green squares.

Fig 2

Expression of the b-OR types in the antenna of Schistocerca gregaria. (A-H) In situ hybridizations were performed on longitudinal tissue sections of antennae with antisense RNA probes for the b-OR types indicated. Some of the stained cells are exemplarily denoted by arrows. Scale bars: 40 µm. Scale bar in the inset in A: 20 µm.

Fig 3

Co-expression of different b-OR types with SNMP1. (A-E) Two-color FISH approaches were performed on sections through antennae with antisense RNA probes for SNMP1 (A-E, green) and SgreOR2 (A, red), SgreOR3 (B, red), SgreOR5 (C, red), SgreOR6 (D, red) and SgreOR8 (E, red). The micrographs shown represent single optical planes taken from stacks of confocal images. Cells positive for SgreOR2, SgreOR3, SgreOR5, SgreOR6 or SgreOR8 co-express SNMP1. (A`/A``-E`/E``) Higher magnifications of the boxed areas in A, B, C, D and E are depicted in either the red or the green color channel. Cells co-expressing the relevant SgreOR types and SNMP1 are marked with dashed circles. Scale bars: 10 µm.

Fig 4

Expression of distinct b-OR types in Orco-positive OSNs of sensilla basiconica or sensilla trichodea. (A-B) Two-color FISH experiments on sections through antennae were conducted with labeled antisense RNA probes for Orco (A-B, green) and SgreOR2 (A, red) or SgreOR8 (B, red). Higher magnifications of the regions indicated by dashed boxes are shown for the green and the red color channel in A`-A`` and B`-B``. Cells expressing SgreOR2 or SgreOR8 are located in the Orco-positive neuron clusters characteristic of sensilla basiconica. (C) FISH on antennal tissue employing an antisense RNA probe for SgreOR3 (red). A SgreOR3-positive labeled cell (dashed circle) is located directly below a sensillum trichodeum (arrow). (D) Two-color FISH with antisense probes for SgreOR3 (red) and Orco (green). As exemplarily depicted, SgreOR3-positive neurons were only found within small groups of Orco-expressing cells that are typical for sensilla trichodea. The area circumscribed by the dotted box in D is given in the green and the red color channel in D`-D``. Scale bars: 10 µm.

Fig 5

Expression of SgreOR types outside the b-OR group in antennae of Schistocerca gregaria. (A-D) Non-fluorescent in situ hybridizations were conducted with probes for SgreOR17, SgreOR35, SgreOR31 and SgreOR76 on longitudinal antennal sections. Some of the stained cells are exemplarily denoted by arrows. (E-H) Two-color FISH experiments were performed with probes for the above-mentioned SgreOR types and SNMP1. Cells expressing these SgreORs (denoted by dashed circles) do not co-express SNMP1. Scale bars: A-C 40 μm; E-F 10 μm.