Olfactory Receptor Neurons for Plant Volatiles and Pheromone Compounds in the Lucerne Weevil, Sitona discoideus

Abstract

Antennal olfactory receptor neurons (ORNs) for pheromone-related and plant volatile compounds were identified and characterized in the lucerne weevil, Sitona discoideus (Gyllenhal), using the single sensillum recording technique. Our study using five pheromone-related compounds and 42 plant volatile compounds indicates that S. discoideus have highly specialized ORNs for pheromone and plant volatile compounds. Different groups of ORNs present in both males and females of S. discoideus were highly sensitive to 4-methylheptane-3,5-dione (diketone) and four isomers (RR, RS, SR and SS) of 5-hydroxy-4-methylheptan-3-one, respectively. Our results also indicate that male S. discoideus, using the sensory input from antennal ORNs, can distinguish both diketone and the RR-isomer from others, and RS- and SS-isomers from others, although it was unclear if they can distinguish between RS-isomer and SS-isomer, or between diketone and the SR-isomer. It also appeared that female S. discoideus could distinguish between RS-isomer and SS-isomers. The antennae of S. discoideus thus contain sex-specific sets of ORNs for host- and non-host plant volatile compounds. Both sexes of S. discoideus have highly sensitive and selective ORNs for some green-leaf volatiles, such as (Z)-3-hexenol and (E)-2-hexenal. In contrast, male antennae of S. discoideus house three distinct groups of ORNs specialized for myrcene and (E)-β-ocimene, 2-phenylethanol, and phenylacetaldehyde, respectively, whereas female antennae contain three groups of ORNs specialized for (±)-linalool and (±)-α-terpineol, myrcene and (E)-β-ocimene, (±)-1-octen-3-ol, and 3-octanone. Our results suggest that S. discoideus use a multi-component pheromone communication system, and a sex-specific set of ORNs with a narrow range of response spectra for host-plant location.

Keywords: 4-methyl-3,5- heptanedione; 5-hydroxy-4-methyl-3-heptanone; Host-plant volatiles; Olfactory sensilla; Pheromone; Single-sensillum recording.

Fig. 1

4-Methyl-3,5-heptanedione (‘diketone’) and its enol form, and the four stereoisomers of sitophilure, 5-hydroxy-4-methylheptan-3-one, pheromone components in the Sithophilus and Sitona genera

Fig. 2

Antennal morphology of Sitona discoideus. a: The seven-segment antennal flagella (F1 - F7) bear sensilla, with the majority of the sensilla being located at the terminal club (b). c: A number of basiconic sensilla (asterisks) are located at the circumferential regions around the distal area of the first three (b: 1 ~ 3), forming a so-called ‘sensory band’, in four subsections of the terminal club (F7). d: The cross-section of a flagellum observed by transmission electron microscopy shows that two axon bundles (ab) run inside the length of the flagella segments. S: scape; P: pedicel

Fig. 3

Responses of olfactory receptor neurons (ORNs) housed in three different types of sensilla in male Sitona discoideus to the pheromone-related compounds, 4-methylheptane-3,5-dione (diketone), and the four stereoisomers (RR, RS, SR and SS) of 5-hydroxy-4-methylheptan-3-one. Red triangles indicate stimulation for 0.1 s. The presence of two different ORNs co-compartmentalized in type M-PM-C sensilla was verified with the presence of two distinct groups of action potentials with different spike sizes, respectively (bottom right: Histogram distributions display two groups of action potentials, each of which, marked with different colors and responsive to the RR isomer and diketone, respectively, could be clearly distinguished in the spike trains)

Fig. 4

Responses of two olfactory receptor neurons (ORNs) co-compartmentalized in type F-PL-F sensilla in female Sitona discoideus to three different doses of 1-octen-3-ol and 3-octanone, demonstrating that these ORNs are highly sensitive to the corresponding stimuli. The ORN generating small spikes (red dots) was exclusively responsive to 1-octen-3-ol, whereas the ORN generating large spikes (blue dots) was exclusively responsive to 3-octanone. Red triangle indicates stimulation for 0.1 s

Fig. 5

The structures of six green leaf volatiles that elicited strong responses from the antennal olfactory receptor neurons (ORNs) in both sexes of Sitona discoideus

Fig. 6

The structures of four plant volatile compounds that elicited stronger responses from the antennal olfactory receptor neurons (ORNs) in male than in female Sitona discoideus

Fig. 7

The structures of five plant volatile compounds that elicited stronger responses from the antennal olfactory receptor neurons (ORNs) in female than in male Sitona discoideus