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

Abstract: 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.