Pheromone-based behaviours are crucial in animals from insects to mammals, and reproductive isolation is often based on pheromone differences. However, the genetic mechanisms by which pheromone signals change during the evolution of new species are largely unknown. In the sexual communication system of moths (Insecta: Lepidoptera), females emit a species-specific pheromone blend that attracts males over long distances. The European corn borer, Ostrinia nubilalis, consists of two sex pheromone races, Z and E, that use different ratios of the cis and trans isomers of acetate pheromone components. This subtle difference leads to strong reproductive isolation in the field between the two races, which could represent a first step in speciation. Female sex pheromone production and male behavioural response are under the control of different major genes, but the identity of these genes is unknown. Here we show that allelic variation in a fatty-acyl reductase gene essential for pheromone biosynthesis accounts for the phenotypic variation in female pheromone production, leading to race-specific signals. Both the cis and trans isomers of the pheromone precursors are produced by both races, but the precursors are differentially reduced to yield opposite ratios in the final pheromone blend as a result of the substrate specificity of the enzymes encoded by the Z and E alleles. This is the first functional characterization of a gene contributing to intraspecific behavioural reproductive isolation in moths, highlighting the importance of evolutionary diversification in a lepidopteran-specific family of reductases. Accumulation of substitutions in the coding region of a single biosynthetic enzyme can produce pheromone differences resulting in reproductive isolation, with speciation as a potential end result.

BackgroundThe Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Oliver) is one of the most damaging invasive insect species in the world. This weevil is highly specialized to thrive in adverse desert climates, and it causes major economic losses due to its effects on palm trees around the world. RPWs locate palm trees by means of plant volatile cues and use an aggregation pheromone to coordinate a mass-attack. Here we report on the high throughput sequencing of the RPW antennal transcriptome and present a description of the highly expressed chemosensory gene families.ResultsDeep sequencing and assembly of the RPW antennal transcriptome yielded 35,667 transcripts with an average length of 857 bp and identified a large number of highly expressed transcripts of odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors/co-receptors (ORs/Orcos), sensory neuron membrane proteins (SNMPs), gustatory receptors (GRs) and ionotropic receptors (IRs). In total, 38 OBPs, 12 CSPs, 76 ORs, 1 Orco, 6 SNMPs, 15 GRs and 10 IRs were annotated in the R. ferrugineus antennal transcriptome. A comparative transcriptome analysis with the bark beetle showed that 25 % of the blast hits were unique to R. ferrugineus, indicating a higher, more complete transcript coverage for R. ferrugineus. We categorized the RPW ORs into seven subfamilies of coleopteran ORs and predicted two new subfamilies of ORs. The OR protein sequences were compared with those of the flour beetle, the cerambycid beetle and the bark beetle, and we identified coleopteran-specific, highly conserved ORs as well as unique ORs that are putatively involved in RPW aggregation pheromone detection. We identified 26 Minus-C OBPs and 8 Plus-C OBPs and grouped R. ferrugineus OBPs into different OBP-subfamilies according to phylogeny, which indicated significant species-specific expansion and divergence in R. ferrugineus. We also identified a diverse family of CSP proteins, as well as a coleopteran-specific CSP lineage that diverged from Diptera and Lepidoptera. We identified several extremely diverged IR orthologues as well as highly conserved insect IR co-receptor orthologous transcripts in R. ferrugineus. Notably, GR orthologous transcripts for CO2-sensing and sweet tastants were identified in R. ferrugineus, and we found a great diversity of GRs within the coleopteran family. With respect to SNMP-1 and SNMP-2 orthologous transcripts, one SNMP-1 orthologue was found to be strikingly highly expressed in the R. ferrugineus antennal transcriptome.ConclusionOur study presents the first comprehensive catalogue of olfactory gene families involved in pheromone and general odorant detection in R. ferrugineus, which are potential novel targets for pest control strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2362-6) contains supplementary material, which is available to authorized users.

Pheromones are central to the mating systems of a wide range of organisms, and reproductive isolation between closely related species is often achieved by subtle differences in pheromone composition. In insects and moths in particular, the use of structurally similar components in different blend ratios is usually sufficient to impede gene flow between taxa. To date, the genetic changes associated with variation and divergence in pheromone signals remain largely unknown. Using the emerging model system Ostrinia, we show the functional consequences of mutations in the protein-coding region of the pheromone biosynthetic fatty-acyl reductase gene pgFAR. Heterologous expression confirmed that pgFAR orthologs encode enzymes exhibiting different substrate specificities that are the direct consequences of extensive nonsynonymous substitutions. When taking natural ratios of pheromone precursors into account, our data reveal that pgFAR substrate preference provides a good explanation of how species-specific ratios of pheromone components are obtained among Ostrinia species. Moreover, our data indicate that positive selection may have promoted the observed accumulation of nonsynonymous amino acid substitutions. Site-directed mutagenesis experiments substantiate the idea that amino acid polymorphisms underlie subtle or drastic changes in pgFAR substrate preference. Altogether, this study identifies the reduction step as a potential source of variation in pheromone signals in the moth genus Ostrinia and suggests that selection acting on particular mutations provides a mechanism allowing pheromone reductases to evolve new functional properties that may contribute to variation in the composition of pheromone signals.

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In insects, perception of the environment—food, mates, and prey—is mainly guided by chemical signals. The dynamic process of signal perception involves transport to odorant receptors (ORs) by soluble secretory proteins, odorant binding proteins (OBPs), which form the first stage in the process of olfactory recognition and are analogous to lipocalin family proteins in vertebrates. Although OBPs involved in the transport of pheromones to ORs have been functionally identified in insects, there is to date no report for Coleoptera. Furthermore, there is a lack of information on olfactory perception and the molecular mechanism by which OBPs participate in the transport of aggregation pheromones. We focus on the red palm weevil (RPW) Rhynchophorus ferrugineus, the most devastating quarantine pest of palm trees worldwide. In this work, we constructed libraries of all OBPs and selected antenna-specific and highly expressed OBPs for silencing through RNA interference. Aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol) and 4-methyl-5-nonanone (ferruginone), and a kairomone, ethyl acetate, were then sequentially presented to individual RPWs. The results showed that antenna-specific RferOBP1768 aids in the capture and transport of ferrugineol to ORs. Silencing of RferOBP1768, which is responsible for pheromone binding, significantly disrupted pheromone communication. Study of odorant perception in palm weevil is important because the availability of literature regarding the nature and role of olfactory signaling in this insect may reveal likely candidates representative of animal olfaction and, more generally, of molecular recognition. Knowledge of OBPs recognizing the specific pheromone ferrugineol will allow for designing biosensors for the detection of this key compound in weevil monitoring in date palm fields.

For decades, the American palm weevil (APW), Rhynchophorus palmarum, has been a threat to coconut and oil palm production in the Americas. It has recently spread towards North America, endangering ornamental palms, and the expanding date palm production. Its behavior presents several parallelisms with a closely related species, R. ferrugineus, the red palm weevil (RPW), which is the biggest threat to palms in Asia and Europe. For both species, semiochemicals have been used for management. However, their control is far from complete. We generated an adult antennal transcriptome from APW and annotated chemosensory related gene families to obtain a better understanding of these species' olfaction mechanism. We identified unigenes encoding 37 odorant-binding proteins (OBPs), ten chemosensory proteins (CSPs), four sensory neuron membrane proteins (SNMPs), seven gustatory receptors (GRs), 63 odorant receptors (ORs), and 28 ionotropic receptors (IRs). Noticeably, we find out the R. ferrugineus pheromone-binding protein and pheromone receptor orthologs from R. palmarum. Candidate genes identified and annotated in this study allow us to compare these palm weevils' chemosensory gene sets. Most importantly, this study provides the foundation for functional studies that could materialize as novel pest management strategies.

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.

Palm trees are of immense economic, sociocultural, touristic and patrimonial significance all over the world, and date palm-related knowledge, traditions and practices are now included in UNESCO’s list of the Intangible Cultural Heritage of Humanity. Of all the pests that infest these trees, the red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) is its primary enemy. The RPW is a category-1 quarantine insect pest that causes enormous economic losses in the cultivation of palm trees worldwide. The RPW synchronizes mass gathering on the palm tree for feeding and mating, regulated by a male-produced pheromone composed of two methyl-branched compounds, (4RS,5RS)-4-methylnonan-5-ol (ferrugineol) and 4(RS)-methylnonan-5-one (ferrugineone). Despite the importance of odorant detection in long-range orientation towards palm trees, palm colonization and mating, nothing regarding the molecular mechanisms of pheromone detection in this species is known. In this study, we report the identification and characterization of the first RPW pheromone receptor, RferOR1. Using gene silencing and functional expression in Drosophila olfactory receptor neurons, we demonstrate that RferOR1 is tuned to both ferrugineol and ferrugineone and binds five other structurally related molecules. We reveal the lifetime expression of RferOR1, which correlates with adult mating success irrespective of age, a factor that could explain the wide distribution and spread of this pest. As palm weevils are challenging to control based on conventional methods, elucidation of the mechanisms of pheromone detection opens new routes for mating disruption and the early detection of this pest via the development of pheromone receptor-based biosensors.