Silencing the Olfactory Co-Receptor RferOrco Reduces the Response to Pheromones in the Red Palm Weevil, Rhynchophorus ferrugineus

Abstract

The red palm weevil (RPW, Rhynchophorus ferrugineus), one of the most widespread of all invasive insect pest species, is a major cause of severe damage to economically important palm trees. RPW exhibits behaviors very similar to those of its sympatric species, the Asian palm weevil (R. vulneratus), which is restricted geographically to the southern part of Southeast Asia. Although efficient and sustainable control of these pests remains challenging, olfactory-system disruption has been proposed as a promising approach for controlling palm weevils. Here, we report the cloning and sequencing of an olfactory co-receptor (Orco) from R. ferrugineus (RferOrco) and R. vulneratus (RvulOrco) and examine the effects of RferOrco silencing (RNAi) on odorant detection. RferOrco and RvulOrco encoding 482 amino acids showing 99.58% identity. The injection of double-stranded RNA (dsRNA) from RferOrco into R. ferrugineus pupae significantly reduced RferOrco gene expression and led to the failure of odor-stimulus detection, as confirmed through olfactometer and electroantennography (EAG) assays. These results suggest that olfactory-system disruption leading to reduced pheromone detection holds great potential for RPW pest-control strategies.

Fig 1. Amino acid sequence alignment of RferOrco and RvulOrco with other coleopteran Orco proteins.

D. ponderosae [AEE62122.1], M. caryae [McOr1], T. castaneum [XP_008194693.1], A. corpulenta [AKC58535.1], A. quadriimpressum [AJF94638.2], H. plumbea [ADM35103.1], H. oblita [AEE69033.1], H. parallela [AEG88961.1], T. molitor [AJO62219.1]. Amino acids that are identical in all sequences are indicated by dark shading. The locations of the predicted seven transmembrane domains in the amino acid alignment are indicated with red lines (I-VII) (for RferOrco).

Fig 2. Maximum likelihood phylogenetic tree of the representative insect Orco sequences (from eight different orders).

The species belonging to each order are indicated with bullets of different colors. RferOrco and RvulOrco are located with other coleopteran species in red bullet (RferOrco and RvulOrco are underlined in black).

Fig 3. Expression of GSPOrco (A) and tubulinRfer (B) determined from cDNAs from different R. ferrugineus tissues (1. male antenna; 2. female antenna; 3. male snout; 4. female snout; 5. male thorax; 6. female thorax; 7. male abdomen; 8. female abdomen; 9. male legs; 10. female legs; 11. male wings; and 12. female wings).

Amplification products were analyzed in 3% agarose gels and visualized under UV illumination after ethidium bromide staining. The amplification size (bp) is indicated on the left side of the amplified band, which measured 204 bp for GSPOrco and 196 bp for tubulin R. ferrugineus.

Fig 4

A. Normalized fold expression of dsRNA RferOrco-injected group (dsRNA) compared with no-injection (NI) and nuclease free water-injected (NFW) group. The asterisk (*) above the dsRNA-RferOrco bar indicates significant differences between selected groups (dsRNA) and other groups (LSD at P<0.05). B. Representative visual band of the 1. NI, 2. NFW, and 3. dsRNA groups. The first row shows the expression of GSPOrco in the different experimental groups, and the second row shows tubulin expression in the different experimental groups.

Fig 5. RferOrco expression normalized with tubulin between dsRNA RferOrco injected (dsRNA) and no-injection (NI) RPW across the different post injection periods (10 d, 21 d, 35 d and 60 d).

Letters indicate significant differences between control and dsRNA groups at different post injection periods (days) (LSD at P < 0.05).

Fig 6. Response of dsRNA RferOrco injected (dsRNA) and no-injection (NI) RPW to odor stimulus (sugarcane, live individual, pheromone and ethylacetate) in Y-tube olfactometer assays.

Asterisks (*) indicate significant differences (LSD at P < 0.05) between NI and dsRNA RPW to stimulus, air or “no response” groups. ns: non-significant.

Fig 7. Electroantennographic (EAG) response of dsRNA RferOrco injected (dsRNA), Nuclease free water-injected (NFW) and no-injection (NI) RPW to (4RS,5RS)-4-methylnonan-5-ol, (Pher1); 4(RS)-methylnonan-5-one (Pher2), and ethyl acetate (EA).

The EAG response to different stimuli was subtracted to negative control (air) before proceeded for statistical analysis. The standard errors of the means of the 13 biological replicates (NI and dsRNA) or six for NFW are represented by the error bars. Different letters within each stimulus groups either Pher1, Pher2, or EA signify that the values were significantly different among NI, NFW and dsRNA treatments (LSD at P < 0.05).