Ewald Grosse-Wilde

Antennal transcriptome of Manduca sexta

In recent years, considerable progress has been made in understanding the molecular mechanisms underlying olfaction in insects. Because of the diverse nature of the gene families involved, this process has largely relied on genomic data. As a consequence, studies have focused on a small subset of species with extensive genomic information. For Lepidoptera, a large order historically crucial to olfactory research, this circumstance has mostly limited advances to the domesticated species Bombyx mori, with some progress in the noctuid Heliothis virescens based on a nonpublic partial genome database. Because of the limited behavioral repertoire and nonexistent ecological importance of Bombyx, molecular data on the tobacco hornworm Manduca sexta are of utmost importance, especially with regards to its position as a classical olfactory model and its complex natural behavior. Here we present the use of transcriptomic and microarray data to identify members of the main olfactory gene families of Manduca. To assess the quality of our data, we correlate information on expressed receptor genes with detailed morphological data on the antennal lobe. Finally, we compare the expression of the near-complete transcript sets in male and female antennae.

Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds

Males of the moth species Heliothis virescens are able to detect the female‐released pheromone with remarkable sensitivity and specificity, distinguishing between highly related pheromonal compounds. In the past, electrophysiological studies succeeded in assigning sensory hairs to identified compounds revealing three functional types of long sensilla trichodea housing neurons specifically responding to distinct semiochemicals. The specific responsiveness implies that the sensory neurons express different receptor types tuned to pheromone components. In this study we demonstrate that heterologously expressed candidate pheromone receptors from Heliothis responded to several pheromonal compounds, including the major sex‐pheromone component Z‐11‐hexadecenal indicating a limited specificity of each receptor type. Nonetheless, based on functional analysis and in situ hybridization studies the analysed receptor types could tentatively be assigned to types of long sensilla trichodea, containing the pheromone‐binding proteins (PBPs) HvirPBP1 and HvirPBP2 in the sensillum lymph. Substituting organic solvent with PBPs to solubilize the hydrophobic pheromone compounds in functional assays revealed an increase in sensitivity and especially specificity. It was found that in the presence of HvirPBP2, cells expressing the receptor type HR13 specifically responded to the main component of the sex pheromone blend only. The data provide further evidence that a combination of a distinct receptor type and binding protein underlie the specific response observed in the detection of a pheromone component in vivo.

Evolution of insect olfactory receptors

The olfactory sense detects a plethora of behaviorally relevant odor molecules; gene families involved in olfaction exhibit high diversity in different animal phyla. Insects detect volatile molecules using olfactory (OR) or ionotropic receptors (IR) and in some cases gustatory receptors (GRs). While IRs are expressed in olfactory organs across Protostomia, ORs have been hypothesized to be an adaptation to a terrestrial insect lifestyle. We investigated the olfactory system of the primary wingless bristletail Lepismachilis y-signata (Archaeognatha), the firebrat Thermobia domestica (Zygentoma) and the neopteran leaf insect Phyllium siccifolium (Phasmatodea). ORs and the olfactory coreceptor (Orco) are with very high probability lacking in Lepismachilis; in Thermobia we have identified three Orco candidates, and in Phyllium a fully developed OR/Orco-based system. We suggest that ORs did not arise as an adaptation to a terrestrial lifestyle, but evolved later in insect evolution, with Orco being present before the appearance of ORs. DOI: http://dx.doi.org/10.7554/eLife.02115.001

Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae)

BackgroundThe European spruce bark beetle, Ips typographus, and the North American mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae), are severe pests of coniferous forests. Both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host trees modulate the pheromone response. Thus, the bark beetle olfactory sense is of utmost importance for fitness. However, information on the genes underlying olfactory detection has been lacking in bark beetles and is limited in Coleoptera. We assembled antennal transcriptomes from next-generation sequencing of I. typographus and D. ponderosae to identify members of the major chemosensory multi-gene families.ResultsGene ontology (GO) annotation indicated that the relative abundance of transcripts associated with specific GO terms was highly similar in the two species. Transcripts with terms related to olfactory function were found in both species. Focusing on the chemosensory gene families, we identified 15 putative odorant binding proteins (OBP), 6 chemosensory proteins (CSP), 3 sensory neuron membrane proteins (SNMP), 43 odorant receptors (OR), 6 gustatory receptors (GR), and 7 ionotropic receptors (IR) in I. typographus; and 31 putative OBPs, 11 CSPs, 3 SNMPs, 49 ORs, 2 GRs, and 15 IRs in D. ponderosae. Predicted protein sequences were compared with counterparts in the flour beetle, Tribolium castaneum, the cerambycid beetle, Megacyllene caryae, and the fruit fly, Drosophila melanogaster. The most notable result was found among the ORs, for which large bark beetle-specific expansions were found. However, some clades contained receptors from all four beetle species, indicating a degree of conservation among some coleopteran OR lineages. Putative GRs for carbon dioxide and orthologues for the conserved antennal IRs were included in the identified receptor sets.ConclusionsThe protein families important for chemoreception have now been identified in three coleopteran species (four species for the ORs). Thus, this study allows for improved evolutionary analyses of coleopteran olfaction. Identification of these proteins in two of the most destructive forest pests, sharing many semiochemicals, is especially important as they might represent novel targets for population control.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.

Sex-Specific Odorant Receptors of the Tobacco Hornworm Manduca Sexta

As odor information plays a vital role in the life of moths, their olfactory sense has evolved into a highly specific and sensitive apparatus relevant to reproduction and survival. The key players in the detection of odorants are olfactory receptor (OR) proteins. Here we identify four OR-encoding genes differentially expressed in the antennae of males and females of the sphingid moth Manduca sexta. Two male-specific receptors (the previously reported MsexOR-1 and the newly identified MsexOR-4) show great resemblance to other male moth pheromone ORs. The putative pheromone receptors are co-expressed with the co-receptor involved in general odorant signal transduction, the DmelOr83b homolog MsexOR-2. One female-specific receptor (MsexOR-5) displays similarities to BmorOR-19, a receptor in Bombyx mori tuned to the detection of the plant odor linalool.

The plastic response of Manduca sexta to host and non-host plants

Specialist insect herbivores have evolved efficient ways to adapt to the major defenses of their host plants. Although Manduca sexta, specialized on Solanaceous plants, has become a model organism for insect molecular biology, little is known about its adaptive responses to the chemical defenses of its hosts. To study larval performance and transcriptomic responses to host and non-host plants, we conducted developmental assays and replicated RNAseq experiments with Manduca larvae fed on different Solanaceous plants as well as on a Brassicaceous non-host plant, Brassica napus. Manduca larvae developed fastest on Nicotiana attenuata, but no significant differences in performance were found on larvae fed on other Solanaceae or the non-host B. napus. The RNAseq experiments revealed that Manduca larvae display plastic responses at the gene expression level, and transcriptional signatures specific to the challenges of each host- and non-host plant. Our observations are not consistent with expectations that specialist herbivores would perform poorly on non-host plants. Instead, our findings demonstrate the ability of this specialized insect herbivore to efficiently use a larger repertoire of host plants than it utilizes in the field.

Expression of ionotropic receptors in terrestrial hermit crab's olfactory sensory neurons

Coenobitidae are one out of at least five crustacean lineages which independently succeeded in the transition from water to land. This change in lifestyle required adaptation of the peripheral olfactory organs, the antennules, in order to sense chemical cues in the new terrestrial habitat. Hermit crab olfactory aesthetascs are arranged in a field on the distal segment of the antennular flagellum. Aesthetascs house approximately 300 dendrites with their cell bodies arranged in spindle-like complexes of ca. 150 cell bodies each. While the aesthetascs of aquatic crustaceans have been shown to be the place of odor uptake and previous studies identified ionotropic receptors (IRs) as the putative chemosensory receptors expressed in decapod antennules, the expression of IRs besides the IR co-receptors IR25a and IR93a in olfactory sensory neurons (OSNs) has not been documented yet. Our goal was to reveal the expression and distribution pattern of non-co-receptor IRs in OSNs of Coenobita clypeatus, a terrestrial hermit crab, with RNA in situ hybridization. We expanded our previously published RNAseq dataset, and revealed 22 novel IR candidates in the Coenobita antennules. We then used RNA probes directed against three different IRs to visualize their expression within the OSN cell body complexes. Furthermore we aimed to characterize ligand spectra of single aesthetascs by recording local field potentials and responses from individual dendrites. This also allowed comparison to functional data from insect OSNs expressing antennal IRs. We show that this orphan receptor subgroup with presumably non-olfactory function in insects is likely the basis of olfaction in terrestrial hermit crabs.

The hermit crab’s nose—antennal transcriptomics

In the course of evolution, crustaceans adapted to a large variety of habitats. Probably the most extreme habitat shift was the transition from water to land, which occurred independently in at least five crustacean lineages. This substantial change in life style required adaptations in sensory organs, as the medium conveying stimuli changed in both chemical and physical properties. One important sensory organ in crustaceans is the first pair of antennae, housing their sense of smell. Previous studies on the crustacean transition from water to land focused on morphological, behavioral, and physiological aspects but did not analyze gene expression. Our goal was to scrutinize the molecular makeup of the crustacean antennulae, comparing the terrestrial Coenobita clypeatus and the marine Pagurus bernhardus. We sequenced and analyzed the antennal transcriptomes of two hermit crab species. Comparison to previously published datasets of similar tissues revealed a comparable quality and GO annotation confirmed a highly similar set of expressed genes in both datasets. The chemosensory gene repertoire of both species displayed a similar set of ionotropic receptors (IRs), most of them belonging to the divergent IR subtype. No binding proteins, gustatory receptors (GRs) or insect-like olfactory receptors (ORs) were present. Additionally to their olfactory function, the antennules were equipped with a variety of pathogen defense mechanisms, producing relevant substances on site. The overall similarity of both transcriptomes is high and does not indicate a general shift in genetic makeup connected to the change in habitat. IRs seem to perform the task of olfactory detection in both hermit crab species studied.

Morphology and Histochemistry of the Aesthetasc-Associated Epidermal Glands in Terrestrial Hermit Crabs of the Genus Coenobita (Decapoda: Paguroidea)

Crustaceans have successfully adapted to a variety of environments including fresh- and saltwater as well as land. Transition from an aquatic to a terrestrial lifestyle required adaptations of the sensory equipment of an animal, particularly in olfaction, where the stimulus itself changes from hydrophilic to mainly hydrophobic, air-borne molecules. Hermit crabs Coenobita spp. (Anomura, Coenobitidae) have adapted to a fully terrestrial lifestyle as adults and have been shown to rely on olfaction in order to detect distant food items. We observed that the specialized olfactory sensilla in Coenobita, named aesthetascs, are immersed in a layer of mucous-like substance. We hypothesized that the mucous is produced by antennal glands and affects functioning of the aesthetascs. Using various microscopic and histochemical techniques we proved that the mucous is produced by aesthetasc-associated epidermal glands, which we consider to be modified rosette-type aesthetasc tegumental glands known from aquatic decapods. These epidermal glands in Coenobita are multicellular exocrine organs of the recto-canal type with tubulo-acinar arrangement of the secretory cells. Two distinct populations of secretory cells were clearly distinguishable with light and electron microscopy. At least part of the secretory cells contains specific enzymes, CUB-serine proteases, which are likely to be secreted on the surface of the aesthetasc pad and take part in antimicrobial defense. Proteomic analysis of the glandular tissue corroborates the idea that the secretions of the aesthetasc-associated epidermal glands are involved in immune responses. We propose that the mucous covering the aesthetascs in Coenobita takes part in antimicrobial defense and at the same time provides the moisture essential for odor perception in terrestrial hermit crabs. We conclude that the morphological modifications of the aesthetasc-associated epidermal glands as well as the functional characteristics of their secretions are important adaptations to a terrestrial lifestyle.