Candidate chemosensory ionotropic receptors in a Lepidoptera.

A new family of candidate chemosensory ionotropic receptors (IRs) related to ionotropic glutamate receptors (iGluRs) was recently discovered in Drosophila melanogaster. Through Blast analyses of an expressed sequenced tag library prepared from male antennae of the noctuid moth Spodoptera littoralis, we identified 12 unigenes encoding proteins related to D. melanogaster and Bombyx mori IRs. Their full length sequences were obtained and the analyses of their expression patterns suggest that they were exclusively expressed or clearly enriched in chemosensory organs. The deduced protein sequences were more similar to B. mori and D. melanogaster IRs than to iGluRs and showed considerable variations in the predicted ligand-binding domains; none have the three glutamate-interacting residues found in iGluRs, suggesting different binding specificities. Our data suggest that we identified members of the insect IR chemosensory receptor family in S. littoralis and we report here the first demonstration of IR expression in Lepidoptera.

Molecular identification and characterization of two new Lepidoptera chemoreceptors belonging to the Drosophila melanogaster OR83b family.

In insect antennae, olfaction depends on olfactory receptors (ORs) that function through heterodimerization with an unusually highly conserved partner orthologue to the Drosophila melanogaster DOR83b. Here, we report the identification of two cDNAs encoding new DOR83b orthologues that represent the first members, although nonconventional, of the OR families of two noctuid crop pests, the cotton leafworm Spodoptera littoralis and the cabbage armyworm Mamestra brassicae. They both displayed high protein sequence conservation with previously identified DOR83b orthologues. Transcripts were abundantly detected in adult chemosensory organs as well as in fifth instar larvae heads. In adult antennae, the expression patterns of both genes revealed common features with other members of the OR83b subfamily: they appeared to be expressed at the bases of numerous olfactory sensilla belonging to different functional categories, suggesting that both receptors may be co-expressed with yet unidentified conventional ORs. Bioinformatic analyses predicted the occurrence of seven transmembrane domains and an unusual topology with intracellular N-termini and extracellular C-termini, extending to Lepidoptera the hypothesis of an inverted topology for DOR83b orthologues, demonstrated to date only in D. melanogaster.

Antennal esterase cDNAs from two pest moths, Spodoptera littoralis and Sesamia nonagrioides, potentially involved in odourant degradation.

Rapid degradation of odours after interaction with olfactory receptors is a critical step of the signal reception process. However, the implied mechanisms are still largely unknown in vertebrates as well as in insects. Involvement of odourant-degrading enzymes in odourant degradation within the antennae has been shown in some insect species and, in particular, esterases could play a key role in degradation of sex pheromones from Lepidoptera. Using a PCR-based strategy, we isolated cDNAs encoding two new esterases from two moths which used acetates as pheromone compounds: the Egyptian armyworm Spodoptera littoralis and the Mediterranean corn borer Sesamia nonagrioides. In antennae, both transcripts were clearly restricted to olfactory sensilla, suggesting their involvement in the degradation of odourant acetate components.

Evidence for a putative antennal clock in Mamestra brassicae: molecular cloning and characterization of two clock genes--period and cryptochrome-- in antennae.

Circadian rhythms are generated by endogenous circadian clocks, organized in central and peripheral clocks. An antennal peripheral clock has been demonstrated to be necessary and sufficient to generate Drosophila olfactory rhythms in response to food odours. As moth pheromonal communication has been demonstrated to follow daily rhythms, we thus investigated the occurence of a putative antennal clock in the noctuid Mamestra brassicae. From moth antennae, we isolated two full-length cDNAs encoding clock genes, period and cryptochrome, which appeared to be expressed throughout the body. In the antennae, expression of both transcripts was restricted to cells that likely represent olfactory sensory neurones. Our results suggest the occurence of a putative antennal clock that could participate in the pheromonal communication rhythms observed in vivo.

A new aldehyde oxidase selectively expressed in chemosensory organs of insects.

Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant's volatiles.

cDNA cloning of biotransformation enzymes belonging to the cytochrome P450 family in the antennae of the noctuid moth Mamestra brassicae.

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae - CYP4L4 and CYP4S4 - which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae - the sensilla trichodea - which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.

Functional and expression pattern analysis of chemosensory proteins expressed in antennae and pheromonal gland of Mamestra brassicae.

Sequences coding for chemosensory proteins (CSP) CSPMbraA and CSPMbraB, soluble proteins of low mol. wt, have been amplified using polymerase chain reaction on antennal and pheromonal gland complementary DNAs. On the basis of their sequences, these proteins could be classed in the 'OS-D like' protein family whose first member was described in Drosophila, and that includes proteins characterized in chemosensory organs of many insect phylla, including our recent identification in Mamestra brassicae proboscis. Binding assays have shown that these proteins bind the pheromonal component (Z)-11-hexadecenyl-1-acetate (Z11-16:Ac) as well as (Z)-11-octadecenyl-1-acetate (Z11-18:Ac), an other putative component of the M. brassicae pheromonal blend. Furthermore, binding with fatty acids, but not with progesterone that is a structurally unrelated compound, leads to the hypothesis that the odorant-binding capability of the MbraCSPs may be restricted to fatty acids and/or to 16-18 carbon backbone skeletons. Thus, these proteins do not show the same highly binding specificity as the pheromone-binding proteins do. The CSP-related proteins appear homologous based on sequence identity, conserved cysteine residues and general patterns of expression. However, phylogenetic analyses suggest the presence of multiple classes of CSP within a given species and possible diversification of CSPs within different orders. This diversity perhaps contributes to the many CSP functions proposed in the literature. In M. brassicae, we localized the CSPMbraA expression to the sensilla trichodea, devoted to pheromone reception, suggesting a role in the chemosensory pathway. However, we also localized such proteins in the pheromonal gland, devoid of any chemosensory structure. This suggests that the M. brassicae CSP could be involved in transport of hydrophobic molecules through different aqueous media, such as the sensillar lymph, as well as the pheromonal gland cytosol.

Chemosensory proteins from the proboscis of mamestra brassicae.

Soluble, low molecular weight proteins were immunodetected in proboscis extracts of Mamestra brassicae males by Western blot, using antibodies raised against the general odorant-binding protein of the moth Antheraea polyphemus. The same antibodies weakly labelled the sensillum lymph and subcuticular space of sensilla styloconica on ultrathin sections of the proboscis. The morphology of sensilla styloconica is described. The immunodetected proteins yielded several N-terminal sequences, three of which showed strong affinity for tritiated analogues of pheromonal compounds of M. brassicae in binding assays. The cDNAs coding for these sequences were cloned and it was shown that the new proteins are related to the OS-D protein of Drosophila. They are named chemosensory proteins (CSP-MBRA:A1-CSP-MBRA:A5 and CSP-MBRA:B1 and CSP-MBRA:B2) and may have an odorant-binding protein-like function. A common localization in both olfaction and taste organs suggests a physiological role depending on the cellular environment.

Characterization of the general odorant-binding protein 2 in the molecular coding of odorants in Mamestra brassicae.

The general odorant-binding protein 2 of Mamestra brassicae males has been purified from antennal extracts and examined in binding assays with pheromone components of this species and a behavioral antagonist, cis-11-hexadecenol. The protein showed high affinity for the latter compound and no affinity for the pheromone components. In addition, expression of the protein, studied by in situ hybridization, was restricted to the long sensilla trichodea, which house the neuron that responds to cis-11-hexadecenol. The expression in a functionally defined population of sensilla, together with binding specificity and previous electrophysiological data, suggest an unsuspected role for the general odorant-binding protein 2 in M. brassicae. It may be involved in the transduction process for the behavioral antagonist to which neurons are specifically tuned and always cocompartmentalized in long trichodeal hairs, with neurons responding to the major pheromonal compound, cis-11-hexadecenyl acetate. These data are consistent with the involvement of odorant-binding proteins in the fine discrimination between pheromone and antagonist, which is related to avoidance of interspecific mating mistakes.

Molecular cloning of two pheromone binding proteins in the cabbage armyworm Mamestra brassicae.

Two cDNA clones encoding pheromone binding proteins (PBPs) were isolated from antennal cDNA of Mamestra brassicae by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR) performed with specific primers deduced from the N-terminal sequences of two PBPs previously reported. The deduced protein sequences of the two PBPs showed a strong relationship between primary structures and functional properties of the corresponding mature proteins.

cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of Mamestra brassicae: a new member of the PBAN family.

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis activating neuropeptide). Here, using immunoblotting and bioassay, we present evidence for the presence of a very similar peptide, called Mab-PBAN, in the brain-subesophageal ganglion complex of Mamestra brassicae females. A partial Mab-PBAN encoding cDNA was isolated using 3'RACE. The deduced amino acid sequence for Mab-PBAN is: LADDMPATPADQEMYRPDPEQIDSRTKYFSPRL with a presumed amidated C-terminus. Mab-PBAN has high homology to the other members of the PBAN peptide family: 94% with Hez-PBAN, 87.9% with Lyd-PBAN and 78.8% with Bom-PBAN. The Mab-PBAN gene encodes, beside Mab-PBAN, at least three putative amidated peptides in the same reading frame, all of them having a common C-terminal pentapeptide motif F(T/S)P(R/K)L-NH2.