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.

Ajwa, a variety of date palm Phoenix dactylifera L., produces the most expensive date fruits. Percentages of seed, moisture, fructose, glucose, soluble protein, and fiber in Ajwa dates were 13.24, 6.21, 39.06, 26.35, 1.33, and 11.01, respectively. The ethyl acetate, methanolic, and water extracts of Ajwa dates, active at 250 μg/mL in the MTT assay, inhibited lipid peroxidation (LPO) by 88, 70, and 91% at 250 μg/mL and cyclooxygenase enzymes COX-1 by 30, 31, and 32% and COX-2 by 59, 48, and 45% at 100 μg/mL, respectively. Bioactivity-guided purifications afforded compounds 1-7, in addition to phthalates and fatty acids. Compounds 1-3 showed activity at 100 μg/mL in the MTT assay; inhibited COX-1 enzyme by 59, 48, amd 50% and COX-2 enzyme by 60, 40, amd 39% at 50 μg/mL; and inhibited LPO by 95, 58, amd 66% at 100 μg/mL, respectively. The soluble protein fraction was also very active in both antioxidant and anti-inflammatory assays.

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.

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.

Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective 'single pgFARs' produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a 'single reductase' can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C 14 -and C 16 -specific FARs in moth pheromone biosynthesis.Insects use sophisticated chemical communication systems, including behavior-modifying pheromones, and these systems reinforce intra-and interspecific reproductive isolation and thus play pivotal roles in the evolutionary process. The insect order Lepidoptera (moth and butterflies) contains the second largest number of species (approximately 180,000 species described to date), and their richness and diversity are linked to pheromone-based communication and sophisticated chemosensory systems [1][2][3] . In moths, chemical communication is the major factor in the premating isolation mechanism, and sex pheromone differences inhibit successful mating and disfavor reproductive success 4 . The female signal and male response are highly species specific, but the mechanism regulating concomitant changes in signals and responses has not been established. These changes must occur at the gene level, and it is important to identify and characterize the genes responsible for pheromone production and to thus provide conclusive proof regarding pheromone-driven evolution in Lepidoptera 5,6 . The pheromone race is generally described in moths, and the variability in chemical mating signals and responses is genetically determined and under stabilizing selection [7][8][9] . Studies over the last two decades have pinpointed that the epigenetic effect of pheromone-driven adaptive evolution is one of the major factors driving the successful diversification of Lepidopteran insects 10 . In moths, a few substitutions in critical amino acids in the key pheromone biosynthetic enzymes are sufficient to create a novel pheromone component 11,12 . Few studies have reported the genetic basis of pheromone divergence in insects due to a lack ...

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BackgroundInsects use pheromones, chemical signals that underlie all animal behaviors, for communication and for attracting mates. Synthetic pheromones are widely used in pest control strategies because they are environmentally safe. The production of insect pheromones in transgenic plants, which could be more economical and effective in producing isomerically pure compounds, has recently been successfully demonstrated. This research requires information regarding the pheromone biosynthetic pathways and the characterization of pheromone biosynthetic enzymes (PBEs). We used Illumina sequencing to characterize the pheromone gland (PG) transcriptome of the Pyralid moth, Ephestia cautella, a destructive storage pest, to reveal putative candidate genes involved in pheromone biosynthesis, release, transport and degradation.ResultsWe isolated the E. cautella pheromone compound as (Z,E)-9,12-tetradecadienyl acetate, and the major pheromone precursors 16:acyl, 14:acyl, E14-16:acyl, E12-14:acyl and Z9,E12-14:acyl. Based on the abundance of precursors, two possible pheromone biosynthetic pathways are proposed. Both pathways initiate from C16:acyl-CoA, with one involving ∆14 and ∆9 desaturation to generate Z9,E12-14:acyl, and the other involving the chain shortening of C16:acyl-CoA to C14:acyl-CoA, followed by ∆12 and ∆9 desaturation to generate Z9,E12-14:acyl-CoA. Then, a final reduction and acetylation generates Z9,E12-14:OAc. Illumina sequencing yielded 83,792 transcripts, and we obtained a PG transcriptome of ~49.5 Mb. A total of 191 PBE transcripts, which included pheromone biosynthesis activating neuropeptides, fatty acid transport proteins, acetyl-CoA carboxylases, fatty acid synthases, desaturases, β-oxidation enzymes, fatty acyl-CoA reductases (FARs) and fatty acetyltransferases (FATs), were selected from the dataset. A comparison of the E. cautella transcriptome data with three other Lepidoptera PG datasets revealed that 45 % of the sequences were shared. Phylogenetic trees were constructed for desaturases, FARs and FATs, and transcripts that clustered with the ∆14, ∆12 and ∆9 desaturases, PG-specific FARs and potential candidate FATs, respectively, were identified. Transcripts encoding putative pheromone degrading enzymes, and candidate pheromone carrier and receptor proteins expressed in the E. cautella PG, were also identified.ConclusionsOur study provides important background information on the enzymes involved in pheromone biosynthesis. This information will be useful for the in vitro production of E. cautella sex pheromones and may provide potential targets for disrupting the pheromone-based communication system of E. cautella to prevent infestations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1710-2) contains supplementary material, which is available to authorized users.

Plant cell wall degrading enzymes (PCWDEs) from insects were recently identified as a multigene family of proteins that consist primarily of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) and play essential roles in the degradation of the cellulose/hemicellulose/pectin network in the invaded host plant. Here we applied transcriptomic and degenerate PCR approaches to identify the PCWDEs from a destructive pest of palm trees, Rhynchophorus ferrugineus, followed by a gut-specific and stage-specific differential expression analysis. We identified a total of 27 transcripts encoding GH family members and three transcripts of the CE family with cellulase, hemicellulase and pectinase activities. We also identified two GH9 candidates, which have not previously been reported from Curculionidae. The gut-specific quantitative expression analysis identified key cellulases, hemicellulases and pectinases from R. ferrugineus. The expression analysis revealed a pectin methylesterase, RferCE8u02, and a cellulase, GH45c34485, which showed the highest gut enriched expression. Comparison of PCWDE expression patterns revealed that cellulases and pectinases are significantly upregulated in the adult stages, and we observed specific high expression of the hemicellulase RferGH16c4170. Overall, our study revealed the potential of PCWDEs from R. ferrugineus, which may be useful in biotechnological applications and may represent new tools in R. ferrugineus pest management strategies.