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Abstract Background Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood. Results We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2+ vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks. Conclusions Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice.

Background: Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood. Results: We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2+ vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks. Conclusions: Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice. [ABSTRACT FROM AUTHOR]

[Display omitted] • Six predominantly expressed vomeronasal receptor genes (OzVRs) were identified in the female muskrat. • OzVR genes were clustered into two clades: OzV1Rs and OzV2Rs. • OzV1R and OzV2R were predicted associate with testosterone and MUPs, respectively. • The expression levels of OzVRs were highest in the vomeronasal organ. • OzV1Rs were highly expressed in the breeding season, while OzV2Rs showed the opposite. Vomeronasal receptors (VRs) play a crucial role in recognizing pheromones, which are essential for social chemical communication. The male muskrat (Ondatra zibethicus) secretes musk, which contains pheromones as a reproductive signal, and the female can recognize it through the VNO to mediate social communication behavior. This study aimed to identify the genomic information of VRs (OzVRs) in the female muskrat and elucidate their physicochemical properties and evolutionary relationship. Six predominantly expressed OzVR genes were identified using the RACE technique, and a comprehensive analysis was conducted on their gene structure, subcellular distribution, functional predictions, and mRNA levels, revealed that all OzVRs were transmembrane proteins. Phylogenetic analysis clustered OzVR genes into two clades (V1Rs: OzV1R21 , OzV1R81 , OzV1R105 ; V2Rs: OzV2R33 , OzV2R44 , OzV2R60). Physiochemically, OzV1Rs were basic proteins, while OzV2Rs exhibited weakly acidic character. Among them, OzV1R81 and OzV2R44 were identified as hydrophobicitystable proteins, with the remainder categorized as hydrophobicity-unstable proteins. Promoters analysis revealed the involvement of transcription factors and complexes, including Ahr::Arnt , Runx1 , Arnt , and TFAP2A , in regulating the expression of the OzVR genes. Conserved domain and motif analyses demonstrated a high conservation of the VRs superfamily in rodents, with many conserved domains linked to pheromone binding. Functional predictions confirmed that OzVRs were associated with pheromones detection. Finally, the expression patterns of OzVR genes in different tissues and seasons indicated that OzVRs have the highest level of expression in the vomeronasal organ, and OzV1Rs notably higher in the breeding season than that in the non-breeding season, however the expression levels of OzV2Rs were higher in the non-breeding season. This study provided insights into the phylogenetic relationships, gene structure, physicochemical properties, promoter binding sites, functions and gene expression patterns of OzVRs , offering a theoretical reference for further examination of VR gene functions and a foundation for understanding chemical signaling mechanisms in the muskrat. [ABSTRACT FROM AUTHOR]

Ancient vomeronasal receptor type-1 (ancV1R), a putative vomeronasal receptor, is highly conserved across a wide range of vertebrates and is expressed in the majority of vomeronasal sensory neurons, co-expressing with canonical vomeronasal receptors, V1Rs and V2Rs. The pseudogenization of ancV1R is closely associated with vomeronasal organ (VNO) degeneration, indicating its critical role in pheromone sensing. However, the specific role of ancV1R remains unknown. In this study, to elucidate the function of ancV1R, we conducted phenotypic analyses of ancV1R-deficient female mice. Behavioral analyses showed that ancV1R-deficient females exhibited rejective responses toward male sexual behavior and displayed no preference for male urine. Physiological analyses demonstrate that the loss-of-function mutation of ancV1R reduced VNO response to various pheromone cues, including male urine, the sexual enhancing pheromone exocrine gland-secreting peptide 1 (ESP1), and β-estradiol 3-sulfate. Pre-exposure to ESP1 did not overcome the rejection behavior caused by ancV1R deficiency. Analysis of neural activity in the vomeronasal system revealed increased responses in the medial amygdala and posteromedial cortical amygdala of mutant females upon contact with males but not in response to male urine alone. Additionally, upon male contacts, ancV1R-deficient females exhibited increased neural activity in the lateral septum, a stress-associated brain region, along with elevated stress hormone levels. Such effects were not observed in females exposed solely to male urine. These findings suggest that, in females, ancV1R facilitates VNO responses to pheromone stimuli and plays a crucial role in perceiving males as mating partners. The absence of ancV1R results in failure of male perception, leading to abnormal sexual behaviors and stress responses upon male contact., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract Background Many animals rely heavily on olfaction to navigate their environment. Among rodents, olfaction is crucial for a wide range of social behaviors. The vomeronasal olfactory system in particular plays an important role in mediating social communication, including the detection of pheromones and recognition signals. In this study we examine patterns of vomeronasal type-1 receptor (V1R) evolution in the house mouse and related species within the genus Mus. We report the extent of gene repertoire turnover and conservation among species and clades, as well as the prevalence of positive selection on gene sequences across the V1R tree. By exploring the evolution of these receptors, we provide insight into the functional roles of receptor subtypes as well as the dynamics of gene family evolution. Results We generated transcriptomes from the vomeronasal organs of 5 Mus species, and produced high quality V1R repertoires for each species. We find that V1R clades in the house mouse and relatives exhibit distinct evolutionary trajectories. We identify putative species-specific gene expansions, including a large clade D expansion in the house mouse. While gene gains are abundant, we detect very few gene losses. We describe a novel V1R clade and highlight candidate receptors for future study. We find evidence for distinct evolutionary processes across different clades, from largescale turnover to highly conserved repertoires. Patterns of positive selection are similarly variable, as some clades exhibit abundant positive selection while others display high gene sequence conservation. Based on clade-level evolutionary patterns, we identify receptor families that are strong candidates for detecting social signals and predator cues. Our results reveal clades with receptors detecting female reproductive status are among the most conserved across species, suggesting an important role in V1R chemosensation. Conclusion Analysis of clade-level evolution is critical for understanding species’ chemosensory adaptations. This study provides clear evidence that V1R clades are characterized by distinct evolutionary trajectories. As receptor evolution is shaped by ligand identity, these results provide a framework for examining the functional roles of receptors.

Abstract Background Vomeronasal type 1 receptor genes (V1Rs) are expected to detect intraspecific pheromones. It is believed that rodents rely heavily on pheromonal communication mediated by V1Rs, but pheromonal signals are thought to be confined in subterranean rodents that live in underground burrows. Thus, subterranean rodents may show a contrasting mode of V1R evolution compared with their superterranean relatives. Results We examined the V1R evolution in subterranean rodents by analyzing currently available genomes of 24 rodents, including 19 superterranean and 5 subterranean species from three independent lineages. We identified a lower number of putatively functional V1R genes in each subterranean rodent (a range of 22–40) compared with superterranean species (a range of 63–221). After correcting phylogenetic inertia, the positive correlation remains significant between the small V1R repertoire size and the subterranean lifestyle. To test whether V1Rs have been relaxed from functional constraints in subterranean rodents, we sequenced 22 intact V1Rs in 29 individuals of one subterranean rodent (Spalax galili) from two soil populations, which have been proposed to undergo incipient speciation. We found 12 of the 22 V1Rs to show significant genetic differentiations between the two natural populations, indicative of diversifying selection. Conclusion Our study demonstrates convergent reduction of V1Rs in subterranean rodents from three independent lineages. Meanwhile, it is noteworthy that most V1Rs in the two Spalax populations are under diversifying selection rather than relaxed selection, suggesting that functional constraints on these genes may have retained in some subterranean species.

Background: Many animals rely heavily on olfaction to navigate their environment. Among rodents, olfaction is crucial for a wide range of social behaviors. The vomeronasal olfactory system in particular plays an important role in mediating social communication, including the detection of pheromones and recognition signals. In this study we examine patterns of vomeronasal type-1 receptor (V1R) evolution in the house mouse and related species within the genus Mus. We report the extent of gene repertoire turnover and conservation among species and clades, as well as the prevalence of positive selection on gene sequences across the V1R tree. By exploring the evolution of these receptors, we provide insight into the functional roles of receptor subtypes as well as the dynamics of gene family evolution. Results: We generated transcriptomes from the vomeronasal organs of 5 Mus species, and produced high quality V1R repertoires for each species. We find that V1R clades in the house mouse and relatives exhibit distinct evolutionary trajectories. We identify putative species-specific gene expansions, including a large clade D expansion in the house mouse. While gene gains are abundant, we detect very few gene losses. We describe a novel V1R clade and highlight candidate receptors for future study. We find evidence for distinct evolutionary processes across different clades, from largescale turnover to highly conserved repertoires. Patterns of positive selection are similarly variable, as some clades exhibit abundant positive selection while others display high gene sequence conservation. Based on clade-level evolutionary patterns, we identify receptor families that are strong candidates for detecting social signals and predator cues. Our results reveal clades with receptors detecting female reproductive status are among the most conserved across species, suggesting an important role in V1R chemosensation. Conclusion: Analysis of clade-level evolution is critical for understanding species' chemosensory adaptations. This study provides clear evidence that V1R clades are characterized by distinct evolutionary trajectories. As receptor evolution is shaped by ligand identity, these results provide a framework for examining the functional roles of receptors. [ABSTRACT FROM AUTHOR]

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus , respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R "hotspot." [ABSTRACT FROM AUTHOR]

In mammals, social and reproductive behaviors are mediated by chemical cues encoded by hyperdiverse families of receptors expressed in the vomeronasal organ. Between species, the number of intact receptors can vary by orders of magnitude. However, the evolutionary processes behind variation in receptor number, and its link to fitness-related behaviors are not well understood. From vomeronasal transcriptomes, we discovered the first evidence of intact vomeronasal type-1 receptor (V1r) genes in bats, and we tested whether putatively functional bat receptors were orthologous to those of related taxa, or whether bats have evolved novel receptors. Instead of lineage-specific duplications, we found that bat V1r s show high levels of orthology to those of their relatives, and receptors are under comparative levels of purifying selection as non-bats. Despite widespread vomeronasal organ loss in bats, V1r copies have been retained for >65 million years. The highly conserved nature of bat V1r s challenges our current understanding of mammalian V1r function and suggests roles other than conspecific recognition or mating initiation in social behavior. [ABSTRACT FROM AUTHOR]

Marine amniotes, a polyphyletic group, provide an excellent opportunity for studying convergent evolution. Their sense of smell tends to degenerate, but this process has not been explored by comparing fully aquatic species with their amphibious relatives in an evolutionary context. Here, we sequenced the genomes of fully aquatic and amphibious sea snakes and identified repertoires of chemosensory receptor genes involved in olfaction. Snakes possess large numbers of the olfactory receptor (OR) genes and the type-2 vomeronasal receptor (V2R) genes, and expression profiling in the olfactory tissues suggests that snakes use the ORs in the main olfactory system (MOS) and the V2Rs in the vomeronasal system (VNS). The number of OR genes has decreased in sea snakes, and fully aquatic species lost MOS which is responsible for detecting airborne odours. By contrast, sea snakes including fully aquatic species retain a number of V2R genes and a well-developed VNS for smelling underwater. This study suggests that the sense of smell also degenerated in sea snakes, particularly in fully aquatic species, but their residual olfactory capability is distinct from that of other fully aquatic amniotes. Amphibious species show an intermediate status between terrestrial and fully aquatic snakes, implying their importance in understanding the process of aquatic adaptation. [ABSTRACT FROM AUTHOR]

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicit innate responses and induce social and sexual behaviors. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the VNO. Previous studies have revealed that the V1R family shows extraordinary variety among mammalian species owing to successive gene gains and losses. Because species-specific pheromonal interaction may facilitate species-specific reproductive behaviors, understanding the evolution of V1Rs in terms of their origin, repertoire and phylogeny should provide insight into the mechanisms of animal diversification. Here I summarize recent studies about the V1R family from its initial discovery in the rat genome to extensive comparative analyses among vertebrates. I further introduce our recent findings for V1Rs in a broad range of vertebrates, which reveal unexpected diversity as well as shared features common among lineages. [ABSTRACT FROM AUTHOR]

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1R s, which are sparsely expressed in a manner that is consistent with the "one neuron–one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ. [ABSTRACT FROM AUTHOR]

In contrast to other olfactory receptor families that exhibit frequent lineage-specific expansions, the vomeronasal type 1 receptor (V1R) family exhibits a canonical six-member repertoire in teleosts. V1r1 and V1r2 are present in no more than one copy in all examined teleosts, including salmons, which are ancient polyploids, implying strict evolutionary constraints. However, recent polyploids have not been examined. Here, we identified a young allotetraploid lineage of weatherfishes and investigated their V1r1-V1r2 cluster. We found a novel pattern that the parental V1r1-V1r2 clusters had recombined in the tetraploid genome and that the recombinant was nearly fixed in the tetraploid population. Subsequent analyses suggested strong selective pressure, for both a new combination of paralogs and homogeneity among gene duplicates, acting on the V1r1-V1r2 pair. [ABSTRACT FROM AUTHOR]

Background: A major effort is underway to use population genetic approaches to identify loci involved in adaptation. One issue that has so far received limited attention is whether loci that show a phylogenetic signal of positive selection in the past also show evidence of ongoing positive selection at the population level. We address this issue using vomeronasal receptors (VRs), a diverse gene family in mammals involved in intraspecific communication and predator detection. In mouse lemurs, we previously demonstrated that both subfamilies of VRs (V1Rs and V2Rs) show a strong signal of directional selection in interspecific analyses. We predicted that ongoing sexual selection and/or co-evolution with predators may lead to current directional or balancing selection on VRs. Here, we re-sequence 17 VRs and perform a suite of selection and demographic analyses in sympatric populations of two species of mouse lemurs (Microcebus murinus and M. ravelobensis) in northwestern Madagascar. Results: M. ravelobensis had consistently higher genetic diversity at VRs than M. murinus. In general, we find little evidence for positive selection, with most loci evolving under purifying selection and one locus even showing evidence of functional loss in M. ravelobensis. However, a few loci in M. ravelobensis show potential evidence of positive selection. Using mismatch distributions and expansion models, we infer a more recent colonisation of the habitat by M. murinus than by M. ravelobensis, which most likely speciated in this region earlier on. Conclusions: These findings suggest that the analysis of VR variation is useful in inferring demographic and phylogeographic history of mouse lemurs. In conclusion, this study reveals a substantial heterogeneity over time in selection on VR loci, suggesting that VR evolution is episodic. [ABSTRACT FROM AUTHOR]

In most mammals, the vomeronasal system detects a variety of (semio)chemicals that mediate olfactory-driven social and sexual behaviors. Vomeronasal chemosensation depends on G proteincoupled receptors (V1R, V2R, and FPR-rs) that operate at remarkably low stimulus concentrations, thus, indicating a highly sensitive and efficient signaling pathway. We identified the PDZ domaincontaining protein, Na+/H+ exchanger regulatory factor-1 (NHERF1), as putative molecular organizer of signal transduction in vomeronasal neurons. NHERF1 is a protein that contains 2 PDZ domains and a carboxy-terminal ezrin-binding domain. It localizes to microvilli of vomeronasal sensory neurons and interacts with V1Rs. Furthermore, NHERF1 and Gαi2 are closely colocalized. These findings open up new aspects of the functional organization and regulation of vomeronasal signal transduction by PDZ scaffolding proteins. [ABSTRACT FROM AUTHOR]

The vomeronasal organ (VNO) is functional in most terrestrial mammals, though progressively reduced in the primate lineage, and is used for intraspecific communication and predator recognition. Vomeronasal receptor (VR) genes comprise two families of chemosensory genes (V1R and V2R) that have been considered to be specific for the VNO. However, recently a large number of VRs were reported to be expressed in the main olfactory epithelium (MOE) of mice, but there is little knowledge of the expression of these genes outside of rodents. To explore the function of VR genes in mammalian evolution, we analyzed and compared the expression of 64 V1R and 2 V2R genes in the VNO and the MOE of the grey mouse lemur (Microcebus murinus), the primate with the largest known VR repertoire. We furthermore compared expression patterns in adults of both sexes and seasons, and in an infant. A large proportion (83% – 97%) of the VR loci was expressed in the VNO of all individuals. The repertoire in the infant was as rich as in adults, indicating reliance on olfactory communication from early postnatal development onwards. In concordance with mice, we also detected extensive expression of VRs in the MOE, with proportions of expressed loci in individuals ranging from 29% to 45%. TRPC2, which encodes a channel protein crucial for signal transduction via VRs, was co-expressed in the MOE in all individuals indicating likely functionality of expressed VR genes in the MOE. In summary, the large VR repertoire in mouse lemurs seems to be highly functional. Given the differences in the neural pathways of MOE and VNO signals, which project to higher cortical brain centers or the limbic system, respectively, this raises the intriguing possibility that the evolution of MOE-expression of VRs enabled mouse lemurs to adaptively diversify the processing of VR-encoded olfactory information.

The vomeronasal organ (VNO) is functional in most terrestrial mammals, though progressively reduced in the primate lineage, and is used for intraspecific communication and predator recognition. Vomeronasal receptor (VR) genes comprise two families of chemosensory genes (V1R and V2R) that have been considered to be specific for the VNO. However, recently a large number of VRs were reported to be expressed in the main olfactory epithelium (MOE) of mice, but there is little knowledge of the expression of these genes outside of rodents. To explore the function of VR genes in mammalian evolution, we analyzed and compared the expression of 64 V1R and 2 V2R genes in the VNO and the MOE of the gray mouse lemur (Microcebus murinus), the primate with the largest known VR repertoire. We furthermore compared expression patterns in adults of both sexes and seasons, and in an infant. A large proportion (83-97%) of the VR loci was expressed in the VNO of all individuals. The repertoire in the infant was as rich as in adults, indicating reliance on olfactory communication from early postnatal development onwards. In concordance with mice, we also detected extensive expression of VRs in the MOE, with proportions of expressed loci in individuals ranging from 29 to 45%. TRPC2, which encodes a channel protein crucial for signal transduction via VRs, was co-expressed in the MOE in all individuals indicating likely functionality of expressed VR genes in the MOE. In summary, the large VR repertoire in mouse lemurs seems to be highly functional. Given the differences in the neural pathways of MOE and VNO signals, which project to higher cortical brain centers or the limbic system, respectively, this raises the intriguing possibility that the evolution of MOE-expression of VRs enabled mouse lemurs to adaptively diversify he processing of VR-encoded olfactory information. [ABSTRACT FROM AUTHOR]

We performed comprehensive data mining to explore the vomeronasal receptor (V1R & V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. 168 V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO), and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.

Fishes use olfaction to gain varied information vital for survival and communication. To understand biodiversity in fishes, it is important to identify what receptors individual fish use to detect specific chemical compounds. However, studies of fish olfactory receptors and their ligands are still limited to a few model organisms represented primarily by zebrafish. Here, we tested the c-fos expression of olfactory sensory neurons (OSNs) in an East African cichlid, the most diversified teleost lineage, by in situ hybridization with a c-fos riboprobe. We confirmed that microvillous neurons contributed the most to the detection of amino acids, as in other fishes. Conversely, we found that ciliated neurons contributed the most to the detection of conjugated steroids, known as pheromone candidates. We also found that V2Rs, the major receptor type in microvillous neurons, exhibited differential responsiveness to amino acids, and further suggest that the cichlid-specific duplication of V2R led to ligand differentiation by demonstrating a differential response to arginine. Finally, we established a non-lethal method to collect cichlid urine and showed how various OSNs, including V1R+ neurons, respond to male urine. This study provides an experimental basis for understanding how cichlids encode natural odours, which ultimately provides insight into how olfaction has contributed to the diversification of cichlids., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Chemosensory genes are frequently the target of positive selection and are often present in large gene families, but little is known about heterogeneity of selection in these cases and its relation to function. Here, we use the vomeronasal-1 receptor (V1R) repertoire of mouse lemurs (Microcebus spp.) as a model system to study patterns of selection of chemosensory genes at several different levels. Mouse lemurs are small nocturnal strepsirrhine primates and have a large (∼200 loci) repertoire of V1R loci that are likely important for intraspecific pheromonal communication and interspecific interactions, for example, recognition of predator cues. We investigated signals and patterns of positive selection among the 105 identified full length V1R loci in the gray mouse lemur and within 7 V1R loci amplified across multiple mouse lemur species. Phylogenetic reconstructions of published sequences revealed at least nine monophyletic clusters of V1Rs in gray mouse lemurs that have diversified since the split between lemurs and lorisoid primates. A large majority of clusters evolved under significant positive selection. Similar results were found in V1Rs of closely related greater galagos. Comparison with function of related V1R clusters in mice suggested a potential relationship between receptor function and strength of selection. Interestingly, most codons identified as being under positive selection are located in the extracellular domains of the receptors and hence likely indicate the position of residues involved in ligand binding. Positive selection was also detected within five V1R loci (=71% of analyzed loci) sequenced from 6 to 10 mouse lemur species, indicating ongoing selection within the genus, which may be related to sexual selection and, potentially, speciation processes. Variation in strength of positive selection on V1Rs showed no simple relationship to cluster size. The diversity of V1R loci in mouse lemurs reflects their adaptive evolution and is most likely related to the fundamental relevance of olfactory communication and predator recognition in these primates. Overall, adaptive evolution is the predominant mode of evolution of V1R loci at all levels, and the substantial heterogeneity in the strength of selection may be related to receptor function. [ABSTRACT FROM PUBLISHER]

Vomeronasal sensitivity is important for detecting intraspecific pheromonal cues as well as environmental odorants and is involved in mating, social interaction, and other daily activities of many vertebrates. Two large families of seven-transmembrane G-protein–coupled receptors, V1rs and V2rs, bind to various ligands to initiate vomeronasal signal transduction. Although the macroevolution of V1r and V2r genes has been well characterized throughout vertebrates, especially mammals, little is known about their microevolutionary patterns, which hampers a clear understanding of the evolutionary forces behind the rapid evolutionary turnover of V1r and V2r genes and the great diversity in receptor repertoire across species. Furthermore, the role of divergent vomeronasal perception in enhancing premating isolation and maintaining species identity has not been evaluated. Here we sequenced 44 V1r genes and 25 presumably neutral noncoding regions in 14 wild-caught mice belonging to Mus musculus and M. domesticus, two closely related species with strong yet incomplete reproductive isolation. We found that nucleotide changes in V1rs are generally under weak purifying selection and that only ∼5% of V1rs may have been subject to positive selection that promotes nonsynonymous substitutions. Consistent with the low functional constraints on V1rs, 18 of the 44 V1rs have null alleles segregating in one or both species. Together, our results demonstrate that, despite occasional actions of positive selection, the evolution of V1rs is in a large part shaped by purifying selection and random drift. These findings have broad implications for understanding the driving forces of rapid gene turnovers that are often observed in the evolution of large gene families. [ABSTRACT FROM AUTHOR]

V1r-like Ora genes express putative chemoreceptors that may function as pheromone receptors in fishes. We used a candidate gene approach to test whether V1r-like Ora2 genes show evidence of positive selection that could suggest a role in mate recognition and the avoidance of hybridization between closely related rockfishes. We amplified a 492-bp fragment of a single V1r-like Ora2 gene from each of 5 species of rockfish. Despite separation of up to 7.8 My, the sequence of V1r-like Ora2 is highly conserved. Genetic distances are small, and all our study species shared at least one sequence with another species. Sequence comparisons suggested that, although most amino acids were subject to purifying selection, 9 amino acids showed evidence of positive selection. Because many of these amino acids were not associated with the areas of the protein suggested to be involved in ligand binding based on structural similarity to other olfactory receptors, this signal may reflect an echo of the relaxation of selection associated with the speciation events that separate these species. Strong sequence conservation suggests that this gene is of functional significance. However, because of shared alleles among species, the V1r-like Ora2 gene, in isolation, would be unlikely to differentiate species during mating season. [ABSTRACT FROM PUBLISHER]

Abstract: We applied a comprehensive data-mining strategy to examine the repertoires of rat and mouse odorant receptors (ORs) and type 1 pheromone receptors (V1Rs) using the mm5 (mouse) and rn3 (rat) genomes. We identified 1576 rat OR genes, including 292 pseudogenes. The rat V1R repertoire is composed of 115 intact genes and 72 pseudogenes. The mouse OR and V1R databases were updated using the new assembly mm5, from which 1375 mouse ORs and 308 V1Rs were identified, with more than 100 putative pseudogenes from mm2 now identified as intact because of the higher sequence quality. With these new data we have conducted a series of genomic analyses of the OR and V1R genes from mouse and rat. Orthologous OR clusters were identified in mouse and rat and comparison analysis was performed at three incremental levels: families, coding sequences, and motifs. At the family level, we found that V1R genes have more species-specific families than OR genes. About 20% of intact V1R genes have no orthologous counterpart in the same family, whereas less than 1% of intact ORs are similarly isolated. At the coding sequence level, OR genes are more conserved between mouse and rat than V1R genes. OR genes share greater similarity with their orthologous counterparts than with their closest neighbor, whereas V1R genes show the opposite tendency. Motifs were identified to obtain biological insights. Motifs specific for species or families were found in OR and V1R genes, which may result in the differential pheromone-dependent behaviors and perception of odors between mouse and rat. [Copyright &y& Elsevier]

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicit innate responses and induce social and sexual behaviors. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the VNO. Previous studies have revealed that the V1R family shows extraordinary variety among mammalian species owing to successive gene gains and losses. Because species-specific pheromonal interaction may facilitate species-specific reproductive behaviors, understanding the evolution of V1Rs in terms of their origin, repertoire and phylogeny should provide insight into the mechanisms of animal diversification. Here I summarize recent studies about the V1R family from its initial discovery in the rat genome to extensive comparative analyses among vertebrates. I further introduce our recent findings for V1Rs in a broad range of vertebrates, which reveal unexpected diversity as well as shared features common among lineages.

In contrast to other olfactory receptor families that exhibit frequent lineage-specific expansions, the vomeronasal type 1 receptor (V1R) family exhibits a canonical six-member repertoire in teleosts. V1r1 and V1r2 are present in no more than one copy in all examined teleosts, including salmons, which are ancient polyploids, implying strict evolutionary constraints. However, recent polyploids have not been examined. Here, we identified a young allotetraploid lineage of weatherfishes and investigated their V1r1 - V1r2 cluster. We found a novel pattern that the parental V1r1 - V1r2 clusters had recombined in the tetraploid genome and that the recombinant was nearly fixed in the tetraploid population. Subsequent analyses suggested strong selective pressure, for both a new combination of paralogs and homogeneity among gene duplicates, acting on the V1r1 - V1r2 pair., Competing Interests: The authors declare no conflict of interest.

Chemical communication is widely used among various organisms to obtain essential information from their environment required for life. Although a large variety of molecules have been shown to act as chemical cues, the molecular and neural basis underlying the behaviors elicited by these molecules has been revealed for only a limited number of molecules. Here, we review the current knowledge regarding the signaling molecules whose flow from receptor to specific behavior has been characterized. Discussing the molecules utilized by mice, insects, and the worm, we focus on how each organism has optimized its reception system to suit its living style. We also highlight how the production of these signaling molecules is regulated, an area in which considerable progress has been recently made., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

We performed comprehensive data mining to explore the vomeronasal receptor (V1R and V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. One hundred sixty-eight V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO), and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.