Sexual discrimination and attraction through scents in the water vole, Arvicola terrestris.

In mammals, especially rodents, social behaviours, such as parenting, territoriality or mate attraction, are largely based on olfactory communication through chemosignals. These behaviours are mediated by species-specific chemosignals, including small organic molecules and proteins that are secreted in the urine or in various fluids from exocrine glands. Chemosignal detection is mainly ensured by olfactory neurons in two specific sensory organs, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). This study aimed to characterise the olfactory communication in the fossorial ecotype of the water voles, Arvicola terrestris. We first measured the olfactory investigation of urine and lateral scent gland secretions from conspecifics. Our results showed that water voles can discriminate the sex of conspecifics based on the smell of urine, and that urinary male odour is attractive for female voles. Then, we demonstrated the ability of the VNO and MOE to detect volatile organic compounds (VOCs) found in water vole secretions using live-cell calcium imaging in dissociated cells. Finally, we evaluated the attractiveness of two mixtures of VOCs from urine or lateral scent glands in the field during a cyclical outbreak of vole populations.

Pheromones, binding proteins, and olfactory systems in the pig (Sus scrofa): An updated review.

Pigs utilize multimodal communication for reproductive and other behaviors, and chemical communication is one of the key components. The success of reproduction relies on chemical communication favored by the steroid pheromones from boar saliva. These steroids were proven to be involved in advancing puberty in gilts (the boar effect) and in promoting estrus behaviors in gilts/sows, thereby helping to detect estrus and facilitating the timing of artificial insemination. The steroid pheromones bound with carrier proteins are evidenced in the mandibular (submandibular) salivary secretions of the boar. These salivary steroids bind with carrier proteins in the nasal mucus and vomeronasal organ (VNO) of the sows, eventually triggering a cascade of activities at the olfactory and endocrine levels. Besides steroid pheromones, pig appeasing pheromones (from mammary skin secretions of sows) have also been demonstrated to bind with carrier proteins in the nasal mucus and VNO of the piglets. Thus far, four different proteins have been identified and confirmed in the nasal mucus and VNO of pigs, including odorant binding proteins (OBPs), salivary lipocalin (SAL), pheromaxein, and Von Ebner's Gland Protein (VEGP). The critical roles of the chemosensory systems, main olfactory systems and VNO, have been comprehensively reported for pigs. This review summarizes the current knowledge on pheromones, their receptor proteins, and the olfactory systems of porcine species.

Oleic Acid and Palmitic Acid from Bacteroides thetaiotaomicron and Lactobacillus johnsonii Exhibit Anti-Inflammatory and Antifungal Properties.

A decrease in populations of Bacteroides thetaiotaomicron and Lactobacillus johnsonii is observed during the development of colitis and fungal overgrowth, while restoration of these populations reduces inflammatory parameters and fungal overgrowth in mice. This study investigated the effect of two fatty acids from B. thetaiotaomicron and L. johnsonii on macrophages and Caco-2 cells, as well as their impact on the inflammatory immune response and on Candida glabrata overgrowth in a murine model of dextran sulfate sodium (DSS)-induced colitis. Oleic acid (OA) and palmitic acid (PA) from L. johnsonii and B. thetaiotaomicron were detected during their interaction with epithelial cells from colon samples. OA alone or OA combined with PA (FAs) reduced the expression of proinflammatory mediators in intestinal epithelial Caco-2 cells challenged with DSS. OA alone or FAs increased FFAR1, FFAR2, AMPK, and IL-10 expression in macrophages. Additionally, OA alone or FAs decreased COX-2, TNFα, IL-6, and IL-12 expression in LPS-stimulated macrophages. In the DSS murine model, oral administration of FAs reduced inflammatory parameters, decreased Escherichia coli and Enterococcus faecalis populations, and eliminated C. glabrata from the gut. Overall, these findings provide evidence that OA combined with PA exhibits anti-inflammatory and antifungal properties.

Inflammation interferes with chemoreception in pigs by altering the neuronal layout of the vomeronasal sensory epithelium.

Chemical communication is widely used by animals to exchange information in their environment, through the emission and detection of semiochemicals to maintain social organization and hierarchical rules in groups. The vomeronasal organ (VNO) is one of the main detectors of these messages, and its inflammation has been linked to behavioral changes because it potentially prevents molecule detection and, consequently, the translation of the signal into action. Our previous study highlighted the link between the intensity of vomeronasal sensory epithelium (VNSE) inflammation, probably induced by farm contaminant exposure, and intraspecific aggression in pigs. The aim of this study was to evaluate the cellular and molecular changes that occur during vomeronasalitis in 76 vomeronasal sensorial epithelia from 38 intensive-farmed pigs. Histology was used to evaluate the condition of each VNO and classify inflammation as healthy, weak, moderate, or strong. These data were compared to the thickness of the sensorial epithelium and the number of type 1 vomeronasal receptor cells using anti-Gαi2 protein immunohistochemistry (IHC) and analysis. The presence of odorant-binding proteins (OBPs) in the areas surrounding the VNO was also analyzed by IHC and compared to inflammation intensity since its role as a molecule transporter to sensory neurons has been well-established. Of the 76 samples, 13 (17%) were healthy, 31 (41%) presented with weak inflammation, and 32 (42%) presented with moderate inflammation. No severe inflammation was observed. Epithelial thickness and the number of Gαi2+ cells were inversely correlated with inflammation intensity (Kruskal-Wallis and ANOVA tests, p < 0.0001), while OBP expression in areas around the VNO was increased in inflamed VNO (Kruskal-Wallis test, p = 0.0094), regardless of intensity. This study showed that inflammation was associated with a reduction in the thickness of the sensory epithelium and Gαi2+ cell number, suggesting that this condition can induce different degrees of neuronal loss. This finding could explain how vomeronasalitis may prevent the correct functioning of chemical communication, leading to social conflict with a potential negative impact on welfare, which is one of the most important challenges in pig farming.

Variation of ewe olfactory secretome during a ram effect.

Introduction: Under temperate latitudes, reproduction in Ovis aries displays a marked seasonality, governed by the photoperiod. In natural conditions, the transition between sexual rest and sexual activity in both sexes is induced by the decrease of day light. Meanwhile, specific odors emitted by a sexually active male are able to reactivate the gonadotropic axis of anovulatory ewes. This physiological effect is called "male effect", precisely ram effect in the ovine species. We have previously shown that the secreted proteins, namely Olfactory Binding Proteins (OBP), contained in the nasal mucus constitute the olfactory secretome (OS), the composition of which is determined by the status of oestrus cycle of females and differs between sexual rest and sexual activity periods. The objective of this study was to test the hypothesis that exposure to sexually active male can also modify the composition of ewes olfactory secretome during a male effect, as well as hormones produced by the reactivation of the oestrus cycle in sexual activity period under natural conditions. Methods: We have set up a new non-invasive protocol of nasal mucus sampling and collected it from 12 ewes at different times during a ram effect. We analyzed the composition of their olfactory secretome by proteomics, mainly SDS-PAGE and MALDI-TOF mass spectrometry. As post-translational modifications of OBPs were a hallmark of ewes' sexual activity period, we were looking for glycosylation by western-blot and mass spectrometry. Results: The efficiency of male effect was low in stimulated ewes as only 3 females displayed elevated progesterone levels in their blood. Besides, half of control ewes (non-stimulated ones) were cycled. We noticed a common OS profile in ewes in anoestrus, versus OS of cycled ones. A very clear and important result was the apparition of O-GlcNAcylation, previously detected only in sexual activity, after only 30 min of male introduction into the flock. Discussion: This exploratory study paves the way for further experiments with larger flock to confirm and reinforce these results, and for eventually exploiting the nasal mucus as an indicator of females' receptivity to male odors.

Identification of potential chemosignals in the European water vole Arvicola terrestris.

The water vole Arvicola terrestris is endemic to Europe where its outbreak generates severe economic losses for farmers. Our project aimed at characterising putative chemical signals used by this species, to develop new sustainable methods for population control that could also be used for this species protection in Great Britain. The water vole, as well as other rodents, uses specific urination sites as territorial and sex pheromone markers, still unidentified. Lateral scent glands and urine samples were collected from wild males and females caught in the field, at different periods of the year. Their volatile composition was analysed for each individual and not on pooled samples, revealing a specific profile of flank glands in October and a specific profile of urinary volatiles in July. The urinary protein content appeared more contrasted as males secrete higher levels of a lipocalin than females, whenever the trapping period. We named this protein arvicolin. Male and female liver transcript sequencing did not identify any expression of other odorant-binding protein sequence. This work demonstrates that even in absence of genome, identification of chemical signals from wild animals is possible and could be helpful in strategies of species control and protection.

The olfactory secretome varies according to season in female sheep and goat.

BACKGROUND: Small ungulates (sheep and goat) display a seasonal breeding, characterised by two successive periods, sexual activity (SA) and sexual rest (SR). Odours emitted by a sexually active male can reactivate the ovulatory cycle of anoestrus females. The plasticity of the olfactory system under these hormonal changes has never been explored at the peripheral level of odours reception. As it was shown in pig that the olfactory secretome (proteins secreted in the nasal mucus) could be modified under hormonal control, we monitored its composition in females of both species through several reproductive seasons, thanks to a non-invasive sampling of olfactory mucus. For this purpose, two-dimensional gel electrophoresis (2D-E), western-blot with specific antibodies, MALDI-TOF and high-resolution (nano-LC-MS/MS) mass spectrometry, RACE-PCR and molecular modelling were used. RESULTS: In both species the olfactory secretome is composed of isoforms of OBP-like proteins, generated by post-translational modifications, as phosphorylation, N-glycosylation and O-GlcNAcylation. Important changes were observed in the olfactory secretome between the sexual rest and the sexual activity periods, characterised in ewe by the specific expression of SAL-like proteins and the emergence of OBPs O-GlcNAcylation. In goat, the differences between SA and SR did not come from new proteins expression, but from different post-translational modifications, the main difference between the SA and SR secretome being the number of isoforms of each protein. Proteomics data are available via ProteomeXchange with identifier PXD014833. CONCLUSION: Despite common behaviour, seasonal breeding, and genetic resources, the two species seem to adapt their olfactory equipment in SA by different modalities: the variation of olfactory secretome in ewe could correspond to a specialization to detect male odours only in SA, whereas in goat the stability of the olfactory secretome could indicate a constant capacity of odours detection suggesting that the hallmark of SA in goat might be the emission of specific odours by the sexually active male. In both species, the olfactory secretome is a phenotype reflecting the physiological status of females, and could be used by breeders to monitor their receptivity to the male effect.

Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

Enhancing bull sexual behavior using estrus-specific molecules identified in cow urine.

Deficiencies in bull mating behavior have implications for bovine artificial insemination activities. The aim of this study was to identify the compounds present in fluids emitted by cows during estrus, which could enhance bull libido. Chemical analysis of urine samples from cows led to the characterization of molecules varying specifically at the preestrous and estrous stages. The synthetic counterpart molecules (1,2-dichloroethylene, squalene, coumarin, 2-butanone, oleic acid) were used to investigate the biological effects on male sexual behavior and sperm production. When presented to males, 2-butanone and oleic acid synthetic molecules significantly lowered mounting reaction time and ejaculation time (-33% and 21% after 2-butanone inhalation, respectively, P < 0.05). The "squalene +1,2-dichloroethylene" combination induced a 9% increase of sperm quantity (P < 0.05). This study suggests that the identified estrous-specific molecules could be part of the chemical signals involved in male and female mating behavior and may be used for a wide range of applications. The identification of these molecules may have implications for the cattle breeding industry.

Proteomic Analysis of Pig (Sus scrofa) Olfactory Soluble Proteome Reveals O-Linked-N-Acetylglucosaminylation of Secreted Odorant-Binding Proteins.

The diversity of olfactory binding proteins (OBPs) is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry, and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT) was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis.

BACKGROUND: In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. METHODS: Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. RESULTS: rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. CONCLUSIONS: A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.

The evolutionary history of the SAL1 gene family in eutherian mammals.

BACKGROUND: SAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions. RESULTS: Here, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection. CONCLUSIONS: This work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

Binding specificity of recombinant odorant-binding protein isoforms is driven by phosphorylation.

Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.

Phosphorylation of native porcine olfactory binding proteins.

The identification of various isoforms of olfactory binding proteins is of major importance to elucidate their involvement in detection of pheromones and other odors. Here, we report the characterization of the phosphorylation of OBP (odorant binding protein) and Von Ebner's gland protein (VEG) from the pig, Sus scrofa. After labeling with specific antibodies raised against the three types of phosphorylation (Ser, Thr, Tyr), the phosphate-modified residues were mapped by using the beta-elimination followed by Michael addition of dithiothreitol (BEMAD) method. Eleven phosphorylation sites were localized in the pOBP sequence and nine sites in the VEG sequence. OBPs are secreted by Bowman's gland cells in the extracellular mucus lining the nasal cavity. After tracking the secretion pathway in the rough endoplasmic reticulum of these cells, we hypothesize that these proteins may be phosphorylated by ectokinases that remain to be characterized. The existence of such a regulatory mechanism theoretically increases the number of OBP variants, and it suggests a more specific role for OBPs in odorant coding than the one of odorant solubilizer and transporter.

Natural ligands of porcine olfactory binding proteins.

Knowledge of endogenous ligands of olfactory binding proteins is a prerequisite for studying their role in odor and pheromone transduction. Here, we report the extraction, derivatization, and characterization by gas chromatography-mass spectrometry of the natural ligands of pig, Sus scrofa (L.), Von Ebner's Gland protein (VEG) and odorant binding protein (OBP). We identified two isoforms (VEG1 and VEG2), which differed only by the linkage of an O-N-acetylglucosamine (O-GlcNac) group on VEG1. The natural ligands of VEG1 were characterized as two isomers of testosterone, whereas ligands of VEG2 and OBP were fatty acids or their derivatives. Our findings suggest that the binding specificity of VEG1 for steroids is governed by the presence of an O-GlcNac moiety on the protein. This specificity was confirmed by the binding of radiolabeled testosterone only by VEG1 in an in-gel binding assay. This is the first evidence for a post-translational modification in the process of odorant discrimination by olfactory binding proteins.

Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant-binding protein.

Structural and molecular dynamics studies have pointed out the role of aromatic residues in the uptake of ligand by porcine odorant-binding protein (pOBP). The shift of Tyr82 from its position during the opening of the binding cavity has been shown, and was supposed to participate in the entrance of the ligand. Several Phe residues in the vicinity of Tyr82 could also participate in the binding process. To clarify their involvement, we performed molecular dynamics studies to simulate the dissociation of undecanal, a ligand previously co-crystallized with pOBP. The results confirmed the key-role of Tyr82 and pointed out the participation of Phe35 in controlling the reorientation of undecanal towards the exit. To bring experimental support to both published (binding) and present simulations (dissociation), we have mutated these two residues and over expressed the wild type pOBP, the two single mutants and the double mutant in the yeast Pichia pastoris. As fluorescence spectroscopy implies the uptake of the fluorescent probe and release in displacement experiments, we monitored the binding ability of the four proteins for 1-aminoanthracene (1-AMA). The experimental results indicated that both residues are involved in the uptake of ligand as the three mutated proteins were unable to bind 1-AMA, contrary to the wild type recombinant pOBP that bound 1-AMA with the expected affinity.

Heterologous expression of piglet odorant-binding protein in Pichia pastoris: a comparative structural and functional characterization with native forms.

This study targets to express the piglet odorant-binding protein (plOBP) and compare the engineered product to the corresponding native protein forms, i.e. plOBP and adult porcine OBP (pOBP). Using the natural signal peptide from the cDNA sequence, up to 40 mg l(-1) of secreted recombinant piglet OBP (rOBP) has been produced in a minimal culture medium. No significant difference in molecular mass between rOBP and native plOBP could be observed by mass spectrometry following or not trypsin digestion. rOBP and pOBP shared similar immunoreactivity towards polyclonal anti-pOBP antibodies, suggesting a proper processing and folding of the recombinant product. Both plOBP and rOBP displayed comparable binding properties towards fatty acids present in the putative maternal pheromone and a steroid, component of the boar sex pheromone. Furthermore, the rOBP product was found to bind to an olfactory receptor, for which pOBP binding was previously characterized. Taken together, these findings suggest that rOBP, produced in Pichia pastoris, exhibits structural and functional properties comparable to those of the native lipocalins from both young or adult animal.

Biochemical and chemical supports for a transnatal olfactory continuity through sow maternal fluids.

Recognition of the mother is of major importance for the survival of mammalian neonates. This recognition is based, immediately after birth, on the detection of odours that have been learned by the fetus in utero. If the ethological basis of a transnatal olfactory continuity is well established, little is known on the nature of its olfactory cues, and nothing about the presence of potential carrier proteins in the maternal fluids such as amniotic fluid, colostrum and milk. We have identified the components of the pig putative maternal pheromone in these fluids of the sow. We also used a ligand-oriented approach to functionally characterize carrier proteins for these compounds in the maternal fluids. Six proteins were identified, using binding assay, immunodetection and peptide mapping by mass spectrometry. These proteins are known to transport hydrophobic ligands in biological fluids. Among them, alpha-1 acid glycoprotein (AGP) and odorant-binding protein (OBP) have been described in the oral sphere of piglets as being involved in the detection of pig putative maternal pheromone components. These are the first chemical and biochemical data supporting a transnatal olfactory continuity between the fetal and the postnatal environments.

Identification and molecular cloning of putative odorant-binding proteins from the American palm weevil, Rhynchophorus palmarum L.

We have identified and cloned the cDNAs encoding two odorant-binding proteins (OBPs) from the American palm weevil (APW) Rhynchophorus palmarum (Coleoptera, Curculionidae). Degenerate primers were designed from the N-terminal sequences and were used in polymerase chain reaction (PCR) in order to obtain full-length sequences in both males and females. In both sexes, two different cDNAs were obtained, encoding 123 and 115 amino acid-deduced sequences. Each sequence showed few amino acid differences between the sexes. The proteins were named RpalOBP2 and RpalOBP4 for male, RpalOBP2' and RpalOBP4' for female, with the types 2 and 4 presenting only 34% identities. These proteins shared high identity with previously described coleopteran OBPs. In native gels, RpalOBP2 clearly separated into two bands and RpalOBP4 into three bands, suggesting the presence of several conformational isomers. Thus, OBP diversity in this species may rely on both the presence of OBPs from different classes and the occurrence of isoforms for each OBP.

Functional characterization of olfactory binding proteins for appeasing compounds and molecular cloning in the vomeronasal organ of pre-pubertal pigs.

The appeasing behaviour of pre-pubertal pigs appears to result from the perception of maternal odours (fatty acids) and of steroids coming from the male. We have used a ligand-oriented approach to functionally characterize olfactory binding proteins involved in the detection of appeasing compounds in the nasal mucosa (NM) and the vomeronasal organ (VNO) of pre-pubertal pigs. Several proteins were identified, combining binding assay, immunodetection and protein sequencing. Their sites of expression in nasal and vomeronasal tissues were studied by reverse transcription polymerase chain reaction (RT-PCR). The proteins belong to the lipocalin superfamily: Alpha-1-acid glycoprotein (AGP), odorant-binding protein (OBP), salivary lipocalin (SAL) and Von Ebner's gland protein (VEG), and displayed different binding capacities for the appeasing compounds. RT-PCR experiments showed that OBP and VEG are expressed not only in the NM, but also in the VNO and that SAL is only expressed in the VNO. This is the first report of the expression of these lipocalins in the VNO. Different binding affinities between lipocalins and appeasing compounds, together with their different localizations in the olfactory systems, suggest multiple possibilities for the peripheral coding of appeasing signals.