Odorant receptor phylogeny confirms conserved channels for sex pheromone and host plant signals in tortricid moths

The search for mates and food is mediated by volatile chemicals. Insects sense food odorants and sex pheromones through odorant receptors (ORs) and pheromone receptors (PRs), which are expressed in olfactory sensory neurons. Molecular phylogenetics of ORs, informed by behavioral and functional data, generates sound hypotheses for the identification of semiochemicals driving olfactory behavior. Studying orthologous receptors and their ligands across taxa affords insights into the role of chemical communication in reproductive isolation and phylogenetic divergence. The female sex pheromone of green budworm moth Hedya nubiferana (Lepidoptera, Totricidae) is a blend of two unsaturated acetates, only a blend of both elicits male attraction. Females produce in addition codlemone, which is the sex pheromone of another tortricid, codling moth Cydia pomonella. Codlemone also attracts green budworm moth males. Concomitantly, green budworm and codling moth males are attracted to the host plant volatile pear ester. A congruent behavioral response to the same pheromone and plant volatile in two tortricid species suggests co‐occurrence of dedicated olfactory channels. In codling moth, one PR is tuned to both compounds, the sex pheromone codlemone and the plant volatile pear ester. Our phylogenetic analysis finds that green budworm moth expresses an orthologous PR gene. Shared ancestry, and high levels of amino acid identity and sequence similarity, in codling and green budworm moth PRs offer an explanation for parallel attraction of both species to the same compounds. A conserved olfactory channel for a sex pheromone and a host plant volatile substantiates the alliance of social and habitat signals in insect chemical communication. Field attraction assays confirm that in silico investigations of ORs afford powerful predictions for an efficient identification of behavior‐modifying semiochemicals, for an improved understanding of the mechanisms of host plant attraction in insect herbivores and for the further development of sustainable insect control.     

The serendipitous origin of chordate secretin peptide family members

Background  

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.     

Detection of sex pheromone components in Manduca sexta (L.).

The ability of olfactory receptor neurons to detect female-produced sex pheromone components and a limited sample of potential host plant odours was studied by single-sensillum recordings from olfactory sensilla present on male and female antennae in Manduca sexta. The majority of pheromone-sensitive receptor neurons examined in males was specialized for detection of the two major pheromone components, E10,Z12-hexadecadienal and E10,E12,Z14-hexadecatrienal or E10,E12,E14-hexadecatrienal. New olfactory receptor neurons tuned to the minor components E10,E12-hexadecadienal and Z11-hexadecenal were found. In females, olfactory receptor neurons specific to Z11-hexadecanal were discovered. Pheromone components and host volatiles were detected by separate sets of receptor neurons.     

Sex pheromone of a North American population of the spotted tentiform leafminer, Phyllonorycter blancardella

The major sex pheromone compound of the spotted tentiform leafminer, Phyllonorycter blancardella (F.) (Lepidoptera: Gracillariidae), from Ontario, Canada, was identified as (10E)‐dodecen‐1‐yl acetate (E10‐12:Ac) using chemical analysis and field trapping experiments. The minor compounds (10E)‐dodecen‐1‐ol (E10‐12:OH) (4.6%), dodecan‐1‐ol (12:OH) (2.3%), and (10Z)‐dodecen‐1‐yl acetate (Z10‐12:Ac) 1.6% were also identified. The dienic acetate (4E,10E)‐dodecadien‐1‐yl acetate (E4,E10‐12:Ac), a compound reported to be attractive to P. blancardella, was not found in the glands of this population. A two‐component blend of the major and one of each the three minor compounds, in ratios similar to those found in the sex pheromone gland, did not increase the attractiveness of traps baited with synthetic pheromone. The minor compounds E10‐12:OH and 12:OH were not attractive to P. blancardella when tested individually. Z10‐12:Ac was attractive to P. blancardella, although traps baited with this compound captured only 2% of the moths that were captured in traps baited with the main compound. A four‐component blend of the major and each of the three minor compounds (100 : 1 : 1 : 1) was not more attractive than the major compound alone. The related species Phyllonorycter mespilella was captured in traps baited with E10‐12:Ac.     

Persistence of duplicated PAC<Subscript>1</Subscript> receptors in the teleost, <Emphasis Type="Italic">Sparus auratus</Emphasis>

Background:  

Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC₁ receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown.     

Characterization of olfactory receptor neurons for pheromone candidate and plant volatile compounds in the clover root weevil,Sitona lepidus

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sensillum recording technique with five pheromone-related compounds, and 40 host and non-host plant volatile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs were identified in each sex of S. lepidus. Among them, three different types of sensilla in the males and two types in the females housed ORNs specialized for pheromone-related compounds. The ORNs in males were specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactory sensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types in females. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. The most active plant volatile compounds were the four green leaf volatile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of two monoterpenols, (±)-linalool and (±)-α-terpineol, all eliciting strong responses from relatively large numbers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate the behavioral implications of these findings.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

Functional Evolution of a Bark Beetle Odorant Receptor Clade Detecting Monoterpenoids of Different Ecological Origins

Insects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How and which new functions may evolve among related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which physiological and behavioral responses to pheromones, volatiles from host and nonhost trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(–)-ipsenol (ItypOR46) and (R)-(–)-ipsdienol (ItypOR49). These receptors belong to an Ips-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this clade using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids produced in larger relative amounts by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R, 4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. The characterized ORs are under strong purifying selection and demonstrate a shared functional property in that they all primarily respond to monoterpenoids. The variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has occurred early in the evolution of this OR-lineage following gene duplication.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.