Heterologous expression and functional characterization of Drosophila suzukii OR69a transcript variants unveiled response to kairomones and to a candidate pheromone

Abstract

Drosophila suzukii is a main pest of berries on all continents. Population control relies on insecticides and a current research challenge is to develop alternative strategies. Methods based on behavior-modifying semiochemicals are widely used against other horticultural insects, and the functional characterization of chemoreceptors facilitates the identification of active compounds. Following heterologous expression of single olfactory receptors (ORs) in ab3A olfactory sensory neurons of D. melanogaster, we screened three transcript variants from the OR69a-locus (DsuzOR69aA, DsuzOR69aB, DsuzOR69aC), demonstrating binding to two possible kairomones (3-octanol and R-carvone) and to a possible fly-emitted volatile compound [(Z)-4-nonenal], although with different pharmacological qualities. By coupling Gas Chromatography to SSR (GC-SSR), these ligands enhanced ab3A-spiking at nanogram-aliquots in a complementary fashion among the different OR69a-variants, and we identified another possible kairomone, methyl salicylate, as the most active and specific ligand for the sole DsuzOR69aB. In testing headspaces collected from D. suzukii females and from the fly-associated yeast Hanseniaspora uvarum we did not observe activation from female headspaces but activation from yeast headspaces. In situ hybridization analysis on D. suzukii antennae suggests unique expression of OR69a-subunits in specific neurons, and points toward co-expression within the same neurons. The OR69a-subunits of D. suzukii constitute cation channels, which binding suggests kairomone specificity, even if effects coexist for a complementary binding of (Z)-4-nonenal. Methyl salicylate is the most active ligand and is specific to the sole DsuzOR69aB, inspiring future investigation to validate potentials of this compound for D. suzukii control strategies.

Heterologous expression of insect IRs in transgenicDrosophila melanogaster

“…Since DsuzIR64a was not activated by the carboxylic acids known to activate the orthologous DmelIR64a, we further explored the possible molecular basis of this evolutionary switching. First of all, we checked if DsuzIR64a was expressed at the same location of DmelIR64a, testing a parallel set of antennae for positive (Orco) and negative (IR62a) controls, as done in previous projects (Cattaneo et al, 2022). DmelIR64a is one of the D. melanogaster antennal IRs expressed in the OSNs surrounding the third saccular chamber (Benton et al, 2009; Ai et al, 2010).…”

“…The final strains tested by SSR had the following genotypes: CpomIR41a1 line: w;pIR76a-Gal4;pUAS-CpomIR41a1 ; DsuzIR75d line: w;pUAS-DsuzIR75dHEK/pIR75d-Gal4;IR75d KO ; DsuzIR64a line: w;pUAS-DsuzIR64a;pIR84a-Gal4 ; CpomIR64a line: w;pUAS-CpomIR64a;pIR84a-Gal4 . Insects were reared in our facilities at room temperature (19-22 °C) under a 16:8 hours light:dark photoperiod as described in Cattaneo et al, (2022).…”

“…To screen the panel, we adjusted a similar protocol recently adopted in our labs (Cattaneo et al, 2022). In brief, stimuli were diluted either in water or ethanol (Sigma Aldrich, St. Louis, MO-USA) depending on their solubility (Table 1).…”

“…Stimuli were prepared as done by Silbering et al, (2011), using 20 μL of 1.0% dilutions and 30 μL of 10 μg/μL for phenylacetic acid. As done in our previous experiments (Cattaneo et al, 2022) stimuli aliquots were spread on grade 1 – 20 mm circles filter paper (GE Healthcare Life Science, Little Chalfont, United Kingdom), previously inserted into glass Pasteur pipettes (VWR, Milan, Italy). To minimize possible effects from the solvent, pipettes were let at least 10 minutes after preparation under the fume hood for solvent evaporation.…”

Evidence of a pheromone involved in the behaviour of Drosophila suzukii Matsumura (Diptera: Drosophilidae)

“…In D. melanogaster , a double olfactory channel has been discovered that has a double affinity for sex and for food odours (Lebreton et al., 2017). In D. suzukii , the existence of transcription sensors for kairomones is suggested with possible binding of pheromones in the sensory receptors of the OR69a subunits of the olfactory receptor (Cattaneo et al., 2022; Keesey et al., 2022; Walker et al., 2023). The hypothesis of sensorial drive reflects this interconnection and predicts that adaptation to nature, habitats, and food resources creates a sensorial bias (Boughman, 2002; Endler, 1992; Ramasamy et al., 2016).…”