Jürgen Krieger

A candidate olfactory receptor subtype highly conserved across different insect orders

Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. Here we describe the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86–88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors revealed a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects.

A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens

The antennae of moths have been an invaluable model for studying the principles of odour perception. In spite of the enormous progress in understanding olfaction on the molecular level, for the moth one of the key elements in olfactory signalling, the odourant receptors, are still elusive. We have assessed a genome database of a heliothine moth (Heliothis virescens, Noctuidae) and employed exon‐specific probes to screen an antennal cDNA library of this species. Analysis of isolated cDNA‐clones led to the discovery of a divergent gene family encoding putative seven‐transmembrane domain proteins. The notion that they may encode candidate olfactory receptors of the moth, was supported by a tissue‐specific expression; several of the subtypes were exclusively expressed in antennae. By means of double‐labelling in situ hybridization studies it was demonstrated that the receptors are indeed expressed in antennal sensory neurons; moreover, each receptor subtype appears to be expressed in a distinct population of sensory cells. The results strongly suggest that the newly discovered gene family indeed encodes olfactory receptors of moth.

Expression of odorant receptors in spatially restricted subsets of chemosensory neurones

From a rat olfactory library a cDNA clone (OR37) which is supposed to encode an odorant receptor protein has been isolated and characterized. Specific antisense RNA and in situ hybridization techniques have been employed to monitor the olfactory epithelium for the distribution of olfactory neurones expressing the OR37-gene. The OR37-transcripts were detected only in a subset of receptor cells segregated in two restricted areas of the olfactory epithelium. The clusters of reactive cells appear symmetrically in both nasal cavities. Within a reactive region only a subset of the cells expressed the receptor. The segregation of neurones expressing a distinct receptor supports the notion that a spatial component may be involved in coding odour quality.

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.

Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay.

Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs fromMamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds.

Candidate pheromone receptors of the silkmoth Bombyx mori

Communication via specific chemical signals is vitally important for the reproductive behaviour of many species. The first identified sex‐attractant pheromone was bombykol from the silkmoth Bombyx mori. This female‐released signalling compound is perceived by the male moth with extreme sensitivity and specificity. Antennal sensory cells supposedly respond to individual bombykol molecules and can efficiently distinguish bombykol from highly related structural analogues like bombykal, a second female‐released pheromone component. In the four decades since the discovery of bombykol, the Bombyx mori system has continued to serve as an invaluable model system for unraveling the intricacies of chemical communication. The molecular basis for this extraordinary specific recognition of an extraneous compound is still elusive but probably based on specific receptors of the pheromone‐responsive cells. In this study, molecular and bioinformatic approaches were employed to search for candidate pheromone receptors of Bombyx mori. A few receptor types were identified that are related to Heliothis candidate pheromone receptors. They were found to be almost exclusively expressed in male antennae, and double in situ hybridization experiments disclosed a characteristic topographic expression pattern that was reminiscent of pheromone‐responsive cells. Furthermore, the receptor‐expressing cells were closely associated with cells expressing the pheromone‐binding protein. Together, the data support the view that the identified receptor types of Bombyx mori are candidate receptors for pheromone components.

Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae).

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30-76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.

Purification and molecular cloning of chemosensory proteins from Bombyx mori

Soluble low molecular weight acidic proteins are suspected to transport stimulus molecules to the sensory neurons within insect sensilla. From the antennae of Bombyx mori, we have purified and sequenced a protein (BmorCSP1) bearing sequence similarity to a class of soluble chemosensory proteins recently discovered in several orders of insects. Based on its N-terminal sequence, the cDNA encoding this protein has been amplified and cloned. Differential screening of a B. mori antennal cDNA library led to the identification of a second gene encoding a related protein (BmorCSP2), sharing 35-40% identity to BmorCSP1 and chemosensory proteins from other species. The predicted secondary structures of moths, chemosensory proteins comprise alpha-helical foldings at conserved positions and a reduced hydrophobicity with respect to this novel family of chemosensory proteins.

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.