Vomeronasal Research Articles

Structural and ultrastructural studies on the developing vomeronasal sensory epithelium in the grass snake Natrix natrix (Squamata: Colubroidea).

The sensory olfactory epithelium and the vomeronasal sensory epithelium (VSE) are characterized by continuous turnover of the receptor cells during postnatal life and are capable of regeneration after injury. The VSE, like the entire vomeronasal organ, is generally well developed in squamates and is crucial for detection of pheromones and prey odors. Despite the numerous studies on embryonic development of the VSE in squamates, especially in snakes, an ultrastructural analysis, as far as we know, has never been performed. Therefore, we investigated the embryology of the VSE of the grass snake (Natrix natrix) using electron microscopy (SEM and TEM) and light microscopy. As was shown for adult snakes, the hypertrophied ophidian VSE may provide great resolution of changes in neuron morphology located at various epithelial levels. The results of this study suggest that different populations of stem/progenitor cells occur at the base of the ophidian VSE during embryonic development. One of them may be radial glia-like cells, described previously in mouse. The various structure and ultrastructure of neurons located at different parts of the VSE provide evidence for neuronal maturation and aging. Based on these results, a few nonmutually exclusive hypotheses explaining the formation of the peculiar columnar organization of the VSE in snakes were proposed.

Pregnancy Changes the Response of the Vomeronasal and Olfactory Systems to Pups in Mice.

Motherhood entails changes in behavior with increased motivation for pups, induced in part by pregnancy hormones acting upon the brain. This work explores whether this alters sensory processing of pup-derived chemosignals. To do so, we analyse the expression of immediate early genes (IEGs) in the vomeronasal organ (VNO; Egr1) and centers of the olfactory and vomeronasal brain pathways (cFos) in virgin and late-pregnant females exposed to pups, as compared to buttons (socially neutral control). In pup-exposed females, we quantified diverse behaviors including pup retrieval, sniffing, pup-directed attack, nest building and time in nest or on nest, as well as time off nest. Pups induce Egr1 expression in the VNO of females, irrespective of their physiological condition, thus suggesting the existence of VNO-detected pup chemosignals. A similar situation is found in the accessory olfactory bulb (AOB) and posteromedial part of the medial bed nucleus of the stria terminalis (BSTMPM). By contrast, in the medial amygdala and posteromedial cortical amygdala (PMCo), responses to pups-vs-buttons are different in virgin and late-pregnant females, thus suggesting altered sensory processing during late pregnancy. The olfactory system also shows changes in sensory processing with pregnancy. In the main olfactory bulbs, as well as the anterior and posterior piriform cortex, buttons activate cFos expression in virgins more than in pregnant females. By contrast, in the anterior and especially posterior piriform cortex, pregnant females show more activation by pups than buttons. Correlation between IEGs expression and behavior suggests the existence of two vomeronasal subsystems: one associated to pup care (with PMCo as its main center) and another related to pup-directed aggression observed in some pregnant females (with the BSTMPM as the main nucleus). Our data also suggest a coactivation of the olfactory and vomeronasal systems during interaction with pups in pregnant females.

Olfactory subsystems in the peripheral olfactory organ of anuran amphibians.

Anuran amphibians (frogs and toads) typically have a complex life cycle, involving aquatic larvae that metamorphose to semi-terrestrial juveniles and adults. However, the anuran olfactory system is best known in Xenopus laevis, an animal with secondarily aquatic adults. The larval olfactory organ contains two distinct sensory epithelia: the olfactory epithelium (OE) and vomeronasal organ (VNO). The adult organ contains three: the OE, the VNO, and a "middle cavity" epithelium (MCE), each in its own chamber. The sensory epithelia of Xenopus larvae have overlapping sensory neuron morphology (ciliated or microvillus) and olfactory receptor gene expression. The MCE of adults closely resembles the OE of larvae, and senses waterborne odorants; the adult OE is distinct and senses airborne odorants. Olfactory subsystems in other (non-pipid) anurans are diverse. Many anuran larvae show a patch of olfactory epithelium exposed in the buccal cavity (bOE), associated with a grazing feeding mode. And other anuran adults do not have a sensory MCE, but many have a distinct patch of epithelium adjacent to the OE, the recessus olfactorius (RO), which senses waterborne odorants. Olfaction plays a wide variety of roles in the life of larval and adult anurans, and some progress has been made in identifying relevant odorants, including pheromones and feeding cues. Increased knowledge of the diversity of olfactory structure, of odorant receptor expression patterns, and of factors that affect the access of odorants to sensory epithelia will enable us to better understand the adaptation of the anuran olfactory system to aquatic and terrestrial environments.

Coadaptation of the chemosensory system with voluntary exercise behavior in mice.

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process. Although the role of the vomeronasal chemosensory receptors in VWR activity remains to be determined, the current results suggest that these vomeronasal chemosensory receptors are important quantitative trait loci for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals.

Diverse sensory cues for individual recognition.

To be social, the ability to recognize and discriminate conspecific individuals is indispensable in social animals, including primates, rodents, birds, fish, and social insects which live in societies or groups. Recent studies using molecular biology, genetics, in vivo and in vitro physiology, and behavioral neuroscientific approaches have provided detailed insights into how animals process and recognize the information of individuals. Here, we review the most distinct sensory modalities for individual recognition in animals, namely, olfaction and vision. In the case of rodents, two polymorphic gene complexes have been identified in their urine as the key and essential pheromonal components for individual recognition: the major histocompatibility complex (MHC) and the major urinary protein (MUP). Animals flexibly utilize MHC and/or MUP, which are detected by the main olfactory epithelium (MOE) and/or the vomeronasal organ (VNO) for various types of social recognition, such as strain recognition, kin recognition, and individual recognition. In contrast, primates, including humans, primarily use facial appearance to identify others. Face recognition in humans and other animals is naturally unique from genetic, cognitive, developmental, and functional points of view. Importantly note that nurture effects during growth phase such as social experience and environment can also shape and tune this special cognitive ability, in order to distinguish subtle differences between individuals. In this review, we address such unique nature and nurture mechanisms for individual recognition.

The Nasopalatine Ducts Are Required for Proper Pheromone Signaling in Mice.

The vomeronasal organ (VNO) specializes in detection of chemosignals, mainly pheromones, which control social communication and reproduction in many mammals. These pheromones must solubilize with nasal fluids before entering the VNO, and it was suggested that they are delivered to and cleared from the VNO by active pumping. Yet, the details of this pheromone delivery process are unclear. In this study, we first constructed a high-resolution 3D morphological image of the whole adult mouse snout, by using ultra-high-resolution micro-CT. We identified a net of micro tunnels starting from the nostrils and extending around and through the VNO. These micro tunnels connect the nasal cavity with the VNO and the oral cavity via the nasopalatine ducts (NPD). Other micro tunnels connect the nasal cavity to the main olfactory epithelium. We next demonstrated that physical obstruction of the NPD severely impairs the clearance of dissolved compounds from the VNO lumen. Moreover, we found that mice with blocked NPD display alterations in chemosignaling-evoked neuronal activation in brain regions associated with the vomeronasal system. Finally, NPD-blocked male mice exhibit reduced preference for female chemosignals, and impaired social interaction behavior. Taken together, our findings indicate that the NPD in mice are connected to both the nasal and oral cavity, serving an essential role in regulating the flow of soluble chemosignals through the VNO, and are required for proper pheromone-mediated social communication.

Peripheral oxytocin injection modulates vomeronasal sensory activity and reduces pup-directed aggression in male mice

Behaviors are shaped by hormones, which may act either by changing brain circuits or by modifying sensory detection of relevant cues. Pup-directed behaviors have been previously shown to change via action of hormones at the brain level. Here, we investigated hormonal control of pup-induced activity in the vomeronasal organ, an olfactory sensory structure involved in the detection of non-volatile chemosignals. Vomeronasal activity decreases as males switch from a pup-aggressive state to a non-aggressive parenting state, after they socially contact a female. RNA sequencing, qPCR, and in situ hybridization were used to identify expression, in the vomeronasal sensory epithelium, of candidate GPCR hormone receptors chosen by in silico analyses and educated guesses. After identifying that oxytocin and vasopressin receptors are expressed in the vomeronasal organ, we injected the corresponding hormones in mice and showed that oxytocin administration reduced both pup-induced vomeronasal activity and aggressive behavior. Conversely, injection of an oxytocin receptor antagonist in female-primed male animals, which normally exhibit reduced vomeronasal activity, significantly increased the number of active vomeronasal neurons. These data link oxytocin to the modulation of olfactory sensory activity, providing a possible mechanism for changes in male behavior after social experience with females.

Maternally inherited peptides as strain-specific chemosignals

Most mammals rely on chemosensory cues for individual recognition, which is essential to many aspects of social behavior, such as maternal bonding, mate recognition, and inbreeding avoidance. Both volatile molecules and nonvolatile peptides secreted by individual conspecifics are detected by olfactory sensory neurons in the olfactory epithelium and the vomeronasal organ. The pertinent cues used for individual recognition remain largely unidentified. Here we show that nonformylated, but not N-formylated, mitochondrially encoded peptides-that is, the nine N-terminal amino acids of NADH dehydrogenases 1 and 2-can be used to convey strain-specific information among individual mice. We demonstrate that these nonformylated peptides are sufficient to induce a strain-selective pregnancy block. We also observed that the pregnancy block by an unfamiliar peptide derived from a male of a different strain was prevented by a memory formed at the time of mating with that male. Our findings also demonstrate that pregnancy-blocking chemosignals in the urine are maternally inherited, as evidenced by the production of reciprocal sons from two inbred strains and our test of their urine's ability to block pregnancy. We propose that this link between polymorphic mitochondrial peptides and individual recognition provides the molecular means to communicate an individual's maternal lineage and strain.

Embryology of the naso-palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius.

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso-palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho-molecular discordance of squamate phylogeny, since a 'molecular scenario' implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X-ray microtomography and light microscopy to describe morphogenesis of the naso-palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

Unique blood vasculature and innervation in the cavernous tissue of murine vomeronasal organs.

The vomeronasal organ (VNO) is an accessory olfactory device related to reproductive behavior. The soft tissue of the tubular organ is composed of sensory/non-sensory epithelia and a highly developed vasculature, which in the latter the dilation and contraction of blood vessels are thought to contribute to pumping in and out luminal fluid or air, like penile erectile tissue. The present histological observation of the murine VNO revealed a more complicated vasculature than previously evaluated ones with large differences along the rostro-caudal axis. An immunohistochemical study for vasoactive substances displayed extremely dense innervation by cholinergic nerves containing nitric oxide synthase and VIP/PHI in the thick smooth muscle layer surrounding venous sinuses at light and electron microscopic levels. Furthermore, the differential distribution of cholinergic nerves and adrenergic nerves may provide a novel insight into the pumping mechanism of VNO.

Development of the squamate naso-palatal complex: detailed 3D analysis of the vomeronasal organ and nasal cavity in the brown anole Anolis sagrei (Squamata: Iguania)

BackgroundDespite the diverse morphology of the adult squamate naso-palatal complex – consisting of the nasal cavity, vomeronasal organ (VNO), choanal groove, lacrimal duct and superficial palate – little is known about the embryology of these structures. Moreover, there are no comprehensive studies concerning development of the nasal cavity and VNO in relation to the superficial palate. In this investigation, we used X-ray microtomography and histological sections to describe embryonic development of the naso-palatal complex of iguanian lizard, the brown anole (Anolis sagrei). The purpose of the study was to describe the mechanism of formation of adult morphology in this species, which combines the peculiar anole features with typical iguanian conditions. Considering the uncertain phylogenetic position of the Iguania within Squamata, embryological data and future comparative studies may shed new light on the evolution of this large squamate clade.ResultsDevelopment of the naso-palatal complex was divided into three phases: early, middle and late. In the early developmental phase, the vomeronasal pit originates from medial outpocketing of the nasal pit, when the facial prominences are weakly developed. In the middle developmental phase, the following events can be noted: the formation of the frontonasal mass, separation of the vestibulum, appearance of the lacrimal duct, and formation of the choanal groove, which leads to separation of the VNO from the nasal cavity. In late development, the nasal cavity and the VNO attain their adult morphology. The lacrimal duct establishes an extensive connection with the choanal groove, which eventually becomes largely separated from the oral cavity.ConclusionsUnlike in other tetrapods, the primordium of the lacrimal duct in the brown anole develops largely beyond the nasolacrimal groove. In contrast to previous studies on squamates, the maxillary prominence is found to participate in the initial fusion with the frontonasal mass. Moreover, formation of the choanal groove occurs due to the fusion of the vomerine cushion to the subconchal fold, rather than to the choanal fold. The loss or significant reduction of the lateral nasal concha is secondary. Some features of anole adult morphology, such as the closure of the choanal groove, may constitute adaptations to vomeronasal chemoreception.

Origin and Evolution of the Gene Family of Proteinaceous Pheromones, the Exocrine Gland-Secreting Peptides, in Rodents.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Specific Hypothalamic Neurons Required for Sensing Conspecific Male Cues Relevant to Inter-male Aggression

The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By invivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT+) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMvDAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMvDAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors.

Ultrastructural and immunocytochemical studies on the olfactory receptor neurons in the Ichthyosaura alpestris

AbstractMorphological and immunohistochemical studies were conducted on the alpine newt Ichthyosaura alpestris, which is representative of the family Salamandridae. The aim of the study was to determine the differences in olfactory organs structural properties, especially in the olfactory epithelium (OE) of the main olfactory chamber (MOC) and the vomeronasal organ (VNO) between breeding and non‐breeding seasons in this species. A change in environments occurs for alpine newts in the course of the annual cycle because of its aquatic adaptations for reproduction. The results confirmed the variability in the shape and size of olfactory organs between breeding and non‐breeding seasons. The presence of the ovoid cells with microvilli at its apical part was determined in the OE of males and females during breeding season. Giant ORNs, a subtype of ciliated ORNs, were observed only during the newt's non‐breeding season. The presence of the ovoid cells during the terrestrial phase was not determined. Thus, its role might be associated with reproduction.

Macro- and micromorphological remodeling of olfactory organs throughout the ontogeny of the fire salamander Salamandra salamandra (Linnaeus, 1758).

This article studies the morphological remodeling of olfactory organs in the fire salamander (Salamandridae, Caudata), from the larval stages of ontogeny to the adult and throughout the course of the annual cycle. The fire salamander exhibits adaptations to the aquatic environment during premetamorphic life and terrestrial adaptations after metamorphosis. During adulthood, the annual activity of this species is divided into three seasonal periods: a breeding period, a nonbreeding period, and hibernation. We observed significant differences in morphology of olfactory organs between developmental stages as well as between each period within the annual cycle. For the first time in caudates, we examined the morphology of olfactory organs during the winter period (wintering larvae, hibernating adults). The results show that the remodeling of olfactory organs during the life of the fire salamander occurs both on macro- and micromorphological levels. Macromorphological ontogenetic variability includes the shape of the main olfactory chamber (MOC) and the distribution of olfactory epithelium (OE) in the MOC and in the vomeronasal organ (VNO). In larvae, the vomeronasal epithelium (VNE) is in a separate cavity, while in the post-metamorphic stages of ontogeny, the VNE occurs in the diverticulum of the MOC. In adult fire salamanders, both olfactory organs are most developed during the breeding season and reduced during hibernation. The VNE and OE in the MOC are also reduced during hibernation. Micro-morphological changes included different types/subtypes of olfactory receptor neurons (ORNs) in the OE in particular stages of ontogeny and periods within the annual cycle, for example, ciliate ORNs are present in the VNE only in the larval stages and giant ORNs occur only in nonbreeding adults. Also, there was a variable set of types of olfactory supporting cells in the VNO of the fire salamander during pre- and postmetamorphic life stages.

Distinct evolutionary trajectories of V1R clades across mouse species

BackgroundMany 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.ResultsWe 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.ConclusionAnalysis 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.

The expression of androstenone receptor (OR7D4) in vomeronasal organ and olfactory epithelium of horses

Androstenone is the first mammalian steroidal pheromone to be identified. Pheromones are chemicals that animals use to communicate within a species. Pheromone detections are related to vomeronasal organ (VNO) and olfactory epithelium (OE) in mammals. Olfactory Receptor Family 7 Subfamily D Member 4 (OR7D4) is an odorant receptor that responds to androstenone. Several studies indicated that spray with androstenone changes behaviors of the boar and dogs. However, the expression of OR7D4 in VNO and OE was not reported in mammals except human. Thus, the main objectives of this study were to investigate the expression of OR7D4 in VNO and OE of horses. Tissue samples were collected from the VNO and nasal cavity of 6 thoroughbred horses. The presence of OR7D4 gene was investigated with reverse transcription–polymerase chain reaction. The expression of OR7D4 was determined using Western blot and immunofluorescence. As a result, the bands for OR7D4 were observed at approximately 462 bp. The protein band of OR7D4 of VNO and OE was detected at 38 kDa. Immunofluorescence result showed that the cilia and cytoplasm of olfactory receptor cells of VNO and nasal cavity tissues were immunolabeled with OR7D4 antibody. The intensity of OR7D4 protein bands in the ventral region of the ethmoidal concha tissues was not significantly different between mares and geldings. In conclusion, thoroughbred horses are capable of androstenone perception through OR7D4 expressed in the VNO and OE.

The vomeronasal system of the newborn capybara: a morphological and immunohistochemical study

The vomeronasal system (VNS) is responsible for the perception mainly of pheromones and kairomones. Primarily studied in laboratory rodents, it plays a crucial role in their socio-sexual behaviour. As a wild rodent, the capybara offers a more objective and representative perspective to understand the significance of the system in the Rodentia, avoiding the risk of extrapolating from laboratory rodent strains, exposed to high levels of artificial selection pressure. We have studied the main morphological and immunohistochemical features of the capybara vomeronasal organ (VNO) and accessory olfactory bulb (AOB). The study was done in newborn individuals to investigate the maturity of the system at this early stage. We used techniques such as histological stains, lectins-labelling and immunohistochemical characterization of a range of proteins, including G proteins (Gαi2, Gαo) and olfactory marking protein. As a result, we conclude that the VNS of the capybara at birth is capable of establishing the same function as that of the adult, and that it presents unique features as the high degree of differentiation of the AOB and the active cellular migration in the vomeronasal epithelium. All together makes the capybara a promising model for the study of chemical communication in the first days of life.

Representation of Olfactory Information in Organized Active Neural Ensembles in the Hypothalamus

The internal representation of sensory information via coherent activation of specific pathways in the nervous system is key to appropriate behavioral responses. Little is known about how chemical stimuli that elicit instinctive behaviors lead to organized patterns of activity in the hypothalamus. Here, we study how a wide range of chemosignals form a discernible map of olfactory information in the ventromedial nucleus of the hypothalamus (VMH) and show that different stimuli entail distinct active neural ensembles. Importantly, we demonstrate that this map depends on functional inputs from the vomeronasal organ. We present evidence that the spatial locations of active VMH ensembles are correlated with activation of distinct vomeronasal receptors and that disjunct VMH ensembles exhibit differential projection patterns. Moreover, active ensembles with distinct spatial locations are not necessarily associated with different behavior categories, such as defensive or social, calling for a revision of the currently accepted model of VMH organization.

The role of copy-number variation in the reinforcement of sexual isolation between the two European subspecies of the house mouse.

Reinforcement has the potential to generate strong reproductive isolation through the evolution of barrier traits as a response to selection against maladaptive hybridization, but the genetic changes associated with this process remain largely unexplored. Building upon the increasing evidence for a role of structural variants in adaptation and speciation, we addressed the role of copy-number variation in the reinforcement of sexual isolation evidenced between the two European subspecies of the house mouse. We characterized copy-number divergence between populations of Mus musculus musculus that display assortative mate choice, and those that do not, using whole-genome resequencing data. Updating methods to detect deletions and tandem duplications (collectively: copy-number variants, CNVs) in Pool-Seq data, we developed an analytical pipeline dedicated to identifying genomic regions showing the expected pattern of copy-number displacement under a reinforcement scenario. This strategy allowed us to detect 1824 deletions and seven tandem duplications that showed extreme differences in frequency between behavioural classes across replicate comparisons. A subset of 480 deletions and four tandem duplications were specifically associated with the derived trait of assortative mate choice. These ‘Choosiness-associated’ CNVs occur in hundreds of genes. Consistent with our hypothesis, such genes included olfactory receptors potentially involved in the olfactory-based assortative mate choice in this system as well as one gene, Sp110, that is known to show patterns of differential expression between behavioural classes in an organ used in mate choice—the vomeronasal organ. These results demonstrate that fine-scale structural changes are common and highly variable within species, despite being under-studied, and may be important targets of reinforcing selection in this system and others.This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers’.