Characterization of olfactory sensory neurons in the striped ambrosia beetle Trypodendron lineatum

Introduction: The striped ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) is a major forest pest in the Holarctic region. It uses an aggregation pheromone and host and non-host volatiles to locate suitable host trees, primarily stressed or dying conifer trees. The beetles bore into the xylem and inoculate spores of their obligate fungal mutualist Phialophoropsis ferruginea inside their excavated egg galleries, with the fungus serving as the main food source for the developing larvae. Olfactory sensory neuron (OSN) responses to pheromones and host volatiles are poorly understood in T. lineatum and other ambrosia beetles, and nothing is known about potential responses to fungal volatiles.Methods: We screened responses of OSNs present in 170 antennal olfactory sensilla using single sensillum recordings (SSR) and 57 odor stimuli, including pheromones, host and non-host compounds, as well as volatiles produced by P. ferruginea and fungal symbionts of other scolytine beetles.Results and Discussion: Thirteen OSN classes were characterized based on their characteristic response profiles. An OSN class responding to the aggregation pheromone lineatin was clearly the most abundant on the antennae. In addition, four OSN classes responded specifically to volatile compounds originating from the obligate fungal mutualist and three responded to non-host plant volatiles. Our data also show that T. lineatum has OSN classes tuned to pheromones of other bark beetles. Several OSN classes showed similar response profiles to those previously described in the sympatric bark beetle Ips typographus, which may reflect their shared ancestry

Evolution of olfaction in Lepidoptera and Trichoptera : Gene families and antennal morphology

In moths, females produce sex pheromone compounds to attract males over a long distance for mating. The antennae of moths and many other insects have specialized odorant receptors (ORs), called pheromone receptors (PRs), to sense the pheromone compounds and they group in a monophyletic clade (PR clade).In this thesis, I investigated and compared various components of the olfactory system in different species of Trichoptera and Lepidoptera (moths and butterflies). I made an effort to particularly understand the origin of thePR clade, the pheromone binding proteins (PBPs) and other chemosensory genes, differences in antennal morphology, presence of Macro glomerular complex (MGC). I used a variety of experimental approaches ranging from microscopy studies, next-generation sequencing and in vitro functional characterization of receptors.Eriocrania semipupurella (Eriocranidae: Lepidoptera) is more basal among the moths than Lampronia capitella (Prodoxidae: Lepidoptera). However, L. capitella is the most basal moth species using Type I pheromone compound. I functionally characterized three receptors from E. semipupurella, two of them responding to primitive pheromone compounds (Type 0 pheromone compounds) and structurally similar plant volatiles, indicating that these receptors likely have evolved from common plant volatile-detecting ORs. One receptor positioned at the base of the conserved pheromone receptor (PR) clade selectively responded to a plant volatile β-caryophyllene, which suggests that PRs of derived moths may also have evolved their function from plant volatile detecting ORs. In addition, a L. capitella specific clade of ORs falls in between the classical PR clade and the β-caryophyllene receptor. The functional activity of three L. capitella ORs, that responded to Type I sex pheromone compounds, suggests that the PR clade can be expanded with these receptors.The antennal transcriptome analysis provided the first set of chemosensory gene families from Trichoptera and basal Lepidoptera. Furthermore, the L. capitella transcriptome comprised chemosensory genes that group within the PR and PBP clades, which contain specialized proteins involved in sex pheromone detection so far only reported in more derived, so-called ditrysian moths. These findings suggest that specialized chemosensory proteins have evolved in parallel with the transition of different sex pheromone types in Lepidoptera.Antennal morphology studies revealed that there was a shift in the major sensilla type, from sensilla auricillica in Trichoptera to sensilla trichoidea in derived Lepidoptera. Preliminary results from immunocytochemistry studies ofantennal lobes show the presence of MGC-like structures in male E. semipupurella and both sexes of R. nubile which possibly are homologous to MGCs of derived moth. On the other hand, the MGC is present only in male ALof L. capitella which may correspond to detection of female-produced pheromone compounds by the male. This is in line with what previously was shown in derived moths that pheromone detecting neurons of sensilla trichoideaproject into MGC and that these enlarged glomeruli are dimorphic and mostly present in males. Interestingly, in the butterfly Bicyclus anynana the MGC-like glomeruli seem to present only in female AL. In addition, the number of ORs found in the antennal transcriptome roughly correspond to the number of glomeruli’s found in the antennal lobes of R. nubila, E. semipurpurella and L. capitella.My work on olfaction in Trichoptera and primitive Lepidoptera has demonstrated that (1) receptors involved in detection of Type 0 and I pheromone compounds have possibly evolved independently from different plant volatile detecting ORs, (2) the functional studies of L. capitella PRs add functional support to the PR clade, and (3) some Lepidoptera specific chemosensory proteins are only present in L. capitella which use Type I pheromone for sex communication. This illustrates that the chemosensory gene families, at least at the level of antennal expression may be associated with different pheromone types. (4) Similarly, antennal morphology studies show a shift in major types of olfactory sensilla, from sensilla placodea in basal moths to sensilla trichoidea in derived moths

Codon optimization of insect odorant receptor genes may increase yheir stable expression for functional characterization in HEK293 cells

Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization (“de-orphanization”) using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus (“Ityp”) and Dendroctonus ponderosae (“Dpon”), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(−)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes

Diversity of olfactory structures: A comparative study of antennal sensilla in Trichoptera and Lepidoptera

The antenna is the main sensory organ of insects, housing different types of sensilla dedicated to detect chemicalcues, motion, humidity and temperature. Sensilla are divided into different types based on their wall structureand morphology. Among the olfactory sensilla, there is an enormous variation in the numbers and morphologicaltypes present in different insect taxa. The reasons for this variation remain obscure, though there may be acorrelation between sensillum morphology and the characteristics of the stimulus that the olfactory sensoryneurons inside the sensillum detect. Here, we report the first comparative analysis of the morphology and ultrastructureof sensilla from Rhyacophila nubila (Rhyacophilidae: Trichoptera) and three species of Lepidoptera,Eriocrania semipurpurella (Eriocraniidae), Lampronia capitella (Prodoxidae), and Bicyclus anynana (Nymphalidae),which use different chemical types of pheromones. Our results, together with a thorough literature review,suggest a shift in major types of olfactory sensilla, from a high proportion of sensilla placodea or auricillica inTrichoptera and the most basal moth lineages (including Eriocraniidae), respectively, to sensilla trichodea in themore derived Lepidoptera (including Prodoxidae and the Ditrysia clade), which parallels the change in the typesof sex pheromones used

Antennal transcriptome analysis of the chemosensory gene families from Trichoptera and basal Lepidoptera

The chemosensory gene families of insects encode proteins that are crucial for hostlocation, mate finding, oviposition, and avoidance behaviors. The insect peripheralchemosensory system comprises odorant receptors (ORs), gustatory receptors (GRs),ionotropic receptors (IRs), odorant binding proteins (OBPs), chemosensory proteins(CSPs), and sensory neuron membrane proteins (SNMPs). These protein families havebeen identified from a large number of insect species, however, they still remainunidentified from several taxa that could provide important clues to their evolution. Thesetaxa include older lepidopteran lineages and the sister order of Lepidoptera, Trichoptera(caddisflies). Studies of these insects should improve evolutionary analyses of insectchemoreception, and in particular shed light on the origin of certain lepidopteran proteinsubfamilies. These include the pheromone receptors (PRs) in the “PR clade”, thepheromone binding proteins (PBPs), general odorant binding proteins (GOBPs), andcertain presumably Lepidoptera-specific IR subfamilies. Hence, we analyzed antennaltranscriptomes from Rhyacophila nubila (Trichoptera), Eriocrania semipurpurella, andLampronia capitella (representing two old lepidopteran lineages). We report 37 ORs,17 IRs, 9 GRs, 30 OBPs, 7 CSPs, and 2 SNMPs in R. nubila; 37 ORs, 17 IRs, 3 GRs,23 OBPs, 14 CSPs, and 2 SNMPs in E. semipurpurella; and 53 ORs, 20 IRs, 5 GRs,29 OBPs, 17 CSPs, and 3 SNMPs in L. capitella. We identified IR members of the“Lepidoptera-specific” subfamilies IR1 and IR87a also in R. nubila, demonstrating thatthese IRs also occur in Trichoptera. Members of the GOBP subfamily were only foundin the two lepidopterans. ORs grouping within the PR clade, as well as PBPs, were onlyfound in L. capitella, a species that in contrast to R. nubila and E. semipurpurella usesa so-called Type I pheromone similar to the pheromones of most species of the derivedLepidoptera (Ditrysia). Thus, in addition to providing increased coverage for evolutionaryanalyses of chemoreception in insects, our findings suggest that certain subfamilies ofchemosensory genes have evolved in parallel with the transition of sex pheromone typesin Lepidoptera. In addition, other chemoreceptor subfamilies show a broader taxonomicoccurrence than hitherto acknowledged

Specificity and sensitivity of plant odor-detecting olfactory sensory neurons in Ctenarytaina eucalypti (Sternorrhyncha: Psyllidae).

The blue gum psyllid, Ctenarytaina eucalypti (Sternorrhyncha: Psyllidae), is an economic threat to Eucalyptus subgenus Symphyomyrtus plantations worldwide. To date, no generally applicable control method is available and the potential for semiochemical-based monitoring or control methods has not yet been investigated. Hence, we conducted the first study on the olfactory sense of C. eucalypti, investigating the specificity and sensitivity of its olfactory sensory neurons (OSNs) to host plant volatiles using single sensillum recordings (SSR). Synthetic compounds were selected from published identifications of Eucalyptus volatiles and after analysis of headspace collections from Eucalyptus cordata. The antenna of C. eucalypti carries four cavities containing olfactory sensilla (S1-S4). Our recordings revealed that each of these sensilla houses three OSNs that could be distinguished electrophysiologically based on spike amplitude differences (A, B, and C neuron with large, intermediate, and small amplitude, respectively). The A neuron in sensillum S1 responded primarily to β-caryophyllene and weaker to β-ocimene, whereas the accompanying B-neuron responded strongly and very specifically to linalool. Furthermore, the B-neuron in both S2 and S3 responded strongly to 1-hexanol, Z3-hexenol, and Z3-hexenyl acetate. OSNs in S4 responded only weakly to a few of the synthetic compounds. Response thresholds in strongly responding OSNs to putative key compounds were close to the 1ng dose on the filter paper and responses exhibited a phasic-tonic profile irrespective of compound dose. C. eucalypti may use the physiologically active compounds for long-range host finding. Future laboratory and field experiments will reveal whether plant volatiles can be used in the management and monitoring of C. eucalypti

Functional characterization of odorant receptors from Lampronia capitella suggests a non-ditrysian origin of the lepidopteran pheromone receptor clade

The odorant receptors (ORs) of insects are crucial for host and mate recognition. In moths (Lepidoptera), specialized ORs are involved in male detection of the sex pheromone produced by females. Most moth sex pheromones are C10-C18 acetates, alcohols, and aldehydes (Type I pheromones), and most pheromone receptors (PRs) characterized to date are from higher Lepidoptera (Ditrysia), responding to these types of compounds. With few exceptions, functionally characterized PRs fall into what has been called the “PR-clade” which also contains receptors that have yet to be characterized. While it has been suggested that moth PRs have evolved from plant odor-detecting ORs, it is not known when receptors for Type I pheromones arose. This is largely due to a lack of functionally characterized PRs from non-ditrysian Lepidoptera. The currant shoot borer moth, Lampronia capitella (Prodoxidae), belongs to a non-ditrysian lineage, and uses Type I pheromone compounds. We identified 53 ORs from antennal transcriptomes of this species, and analyzed their phylogenetic relationships with known lepidopteran ORs. Using a HEK293 cell-based assay, we showed that three of the LcapORs with male-biased expression (based on FPKM values) respond to Type I pheromone compounds. Two of them responded to pheromone components of L. capitella and one to a structurally related compound. These PRs are the first from a non-ditrysian moth species reported to respond to Type I compounds. They belong to two of the more early-diverging subfamilies of the PR-clade for which a role in pheromone detection had not previously been demonstrated. Hence, our definition of the monophyletic lepidopteran PR-clade includes these receptors from a non-ditrysian species, based on functional support

Identification of sesquisabinene B in carrot (Daucus carota L.) leaves as a compound electrophysiologically active to the carrot psyllid (Trioza apicalis Forster)

The Carrot psyllid, Trioza apicalis Forster (Homoptera: Psylloidea: Triozidae) is one of the major insect pests of carrots (Daucus carota L.) in parts of northern and central Europe. Gas chromatography-single-sensillum recording (GC-SSR) previously confirmed several active compounds in a carrot leaf extract, but the most active compound remained unidentified. Mass fragmentation patterns observed from the unidentified active compound when analyzed by gas chromatography and mass spectrometry (GC-MS) was used to propose -sesquiphellandrene and -cis-bergamotene to be candidates as the unidentified compound. The compounds were synthesized and their mass spectra were nearly identical with the unknown active compound. But, the retention times differed from the compound in the carrot leaf extract. Thus, to obtain the unidentified compound pure enough and in adequate amounts for nuclear magnetic resonance (NMR) analysis, preparative gas chromatography was applied to separate and concentrate this biologically active compound. Analysis by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF) confirmed the unidentified compound to be a compound with theformula of C15H24 and together with GC-MS, H-1 and C-13 NMR analysis sesquisabinene B was identified as the unidentified compound in the extract. GC-SSR was then used to finally confirm the biological activity of sesquisabinene B isolated from the carrot leaf extract via preparative GC

Characterization of odorant receptors from a non-ditrysian moth, Eriocrania semipurpurella sheds light on the origin of the sex pheromone receptors in Lepidoptera

Pheromone receptors (PRs) are essential in moths to detect sex pheromones for mate finding. However, it remainsunknown from which ancestral proteins these specialized receptors arose. The oldest lineages of moths, so-callednon-ditrysian moths, use short-chain pheromone components, secondary alcohols, or ketones, so called Type 0 pheromonesthat are similar to many common plant volatiles. It is, therefore, possible that receptors for these ancestralpheromones evolved from receptors detecting plant volatiles. Hence, we identified the odorant receptors (ORs) from anon-ditrysian moth, Eriocrania semipurpurella (Eriocraniidae, Lepidoptera), and performed functional characterizationof ORs using HEK293 cells. We report the first receptors that respond to Type 0 pheromone compounds; EsemOR3displayed highest sensitivity toward (2S, 6Z)-6-nonen-2-ol, whereas EsemOR5 was most sensitive to the behavioralantagonist (Z)-6-nonen-2-one. These receptors also respond to plant volatiles of similar chemical structures, but withlower sensitivity. Phylogenetically, EsemOR3 and EsemOR5 group with a plant volatile-responding receptor from thetortricid moth Epiphyas postvittana (EposOR3), which together reside outside the previously defined lepidopteran PRclade that contains the PRs from more derived lepidopteran families. In addition, one receptor (EsemOR1) that falls atthe base of the lepidopteran PR clade, responded specifically to b-caryophyllene and not to any other additional plant orpheromone compounds. Our results suggest that PRs for Type 0 pheromones have evolved from ORs that detectstructurally-related plant volatiles. They are unrelated to PRs detecting pheromones inmore derived Lepidoptera, which,in turn, also independently may have evolved a novel function from ORs detecting plant volatiles