A Sex Pheromone Receptor in the Hessian Fly Mayetiola destructor (Diptera, Cecidomyiidae)

Abstract

The Hessian fly, Mayetiola destructor Say (Diptera, Cecidomyiidae), is a pest of wheat and belongs to a group of gall-inducing herbivores. This species has a unique life history and several ecological features that differentiate it from other Diptera such as Drosophila melanogaster and blood-feeding mosquitoes. These features include a short, non-feeding adult life stage (1-2 days) and the use of a long-range sex pheromone produced and released by adult females. Sex pheromones are detected by members of the odorant receptor (OR) family within the Lepidoptera, but no receptors for similar long-range sex pheromones have been characterized from the Diptera. Previously, 122 OR genes have been annotated from the Hessian fly genome, with many of them showing sex-biased expression in the antennae. Here we have expressed, in HEK293 cells, five MdesORs that display male-biased expression in antennae, and we have identified MdesOR115 as a Hessian fly sex pheromone receptor. MdesOR115 responds primarily to the sex pheromone component (2S,8E,10E)-8,10-tridecadien-2-yl acetate, and secondarily to the corresponding Z,E-isomer. Certain sensory neuron membrane proteins (i.e., SNMP1) are important for responses of pheromone receptors in flies and moths. The Hessian fly genome is unusual in that it encodes six SNMP1 paralogs, of which five are expressed in antennae. We co-expressed each of the five antennal SNMP1 paralogs together with each of the five candidate sex pheromone receptors from the Hessian fly and found that they do not influence the response of MdesOR115, nor do they confer responsiveness in any of the non-responsive ORs to any of the sex pheromone components identified to date in the Hessian fly. Using Western blots, we detected protein expression of MdesOrco, all MdesSNMPs, and all MdesORs except for MdesOR113, potentially explaining the lack of response from this OR. In conclusion, we report the first functional characterization of an OR from the Cecidomyiidae, extending the role of ORs as long-range sex pheromone detectors from the Lepidoptera into the Diptera.

Keywords: HEK293 cells; deorphanization; functional characterization; heterologous expression; odorant receptor; pheromone receptor; sensory neuron membrane protein.

FIGURE 1

Protein expression of myc-tagged Orco (A) and V5-tagged ORs and SNMPs (B) in cell lines with induced (+) gene expression, as indicated by Western blots. No proteins were detected from non-induced cells (-). Numbers (112, 113, 115, 116, and 120) refer to the MdesORs, and letters (A, B, C, E and F) to the SNMP1 paralogs. The OR113 protein could not be detected.

FIGURE 2

Responses of six HEK293/TMO/MdesOR115 cell lines, with or without SNMP1 (S1), to Hessian fly sex pheromone components (10 μM concentration), including both induced cells (red bars) and non-induced cells (black bars). Shown are also responses to vehicle control (0.5% DMSO) and the Orco agonist VUAA1 (50 μM). Asterisks indicate significant differences between non-induced and induced cells (all p < 0.001). Different lower-case letters indicate significant (all p < 0.007) differences between the response to 2S-8Z,10E-13:OAc and 2S-8E,10E-13:OAc. Means (±SEM) are derived from three biological replicates (n = 3), each including three technical replicates (i.e., total n = 9). Cell lines: (A) HEK293/TMO/MdesOR115, (B) HEK293/TMO/MdesOR115/SNMP1A, (C) HEK293/TMO/MdesOR115/SNMP1B, (D) HEK293/TMO/MdesOR115/SNMP1C, (E) HEK293/TMO/MdesOR115/SNMP1E, (F) HEK293/TMO/MdesOR115/SNMP1F.

FIGURE 3

Dose-response curves of six HEK293/TMO/MdesOR115 cell lines, with or without SNMP1 (S1). Means (±SEM) are derived from three to four biological replicates (n = 3–4), each including three technical replicates (i.e., total n = 9–12). (A) Responses to 2S-8E,10E-13:OAc, and (B) 2S-8Z,10E-13:OAc.

FIGURE 4

Temporal response change post treatment of 2S-8E,10E-13:OAc in six HEK293/TMO/MdesOR115 cell lines, with or without SNMP1 (S1). (A–D) show responses to decreasing ligand concentrations. Analyses of covariance show that the slopes do not differ between different cell lines. Each data point (biological replicate) is the average of three technical replicates (n = 3–4 biological replicates, total n = 9–12).