Host influence on the eukaryotic virome of sympatric mosquitoes and abundance of diverse viruses with a broad host range.

Mosquitoes harbor a large diversity of eukaryotic viruses. Those viromes probably influence mosquito physiology and the transmission of human pathogens. Nevertheless, their ecology remains largely unstudied. Here, we address two key questions in virome ecology. First, we assessed the influence of mosquito species on virome taxonomic diversity and relative abundance. Contrary to most previous studies, the potential effect of the habitat was explicitly included. Thousands of individuals of Culex poicilipes and Culex tritaeniorhynchus, two vectors of viral diseases, were concomitantly sampled in three habitats over two years. A total of 95 viral taxa from 25 families were identified with meta-transcriptomics, with 75% of taxa shared by both mosquitoes. Viromes significantly differed by mosquito species but not by habitat. Differences were largely due to changes in relative abundance of shared taxa. Then, we studied the diversity of viruses with a broad host range. We searched for viral taxa shared by the two Culex species and Aedes vexans, another disease vector, present in one of the habitats. Twenty-six out of the 163 viral taxa were found in the three mosquitoes. These taxa encompassed 14 families. A database analysis supported broad host ranges for many of those viruses, as well as a widespread geographical distribution. Thus, the viromes of mosquitoes from the same genera mainly differed in the relative abundance of shared taxa, whereas differences in viral diversity dominated between mosquito genera. Whether this new model of virome diversity and structure applies to other mosquito communities remains to be determined.

Deep sequencing and variant frequency analysis for the quality control of a live bacterial vaccine against contagious bovine pleuropneumonia, strain T1.

Vaccination is the most cost-effective tool to control contagious bovine pleuropneumonia. The vaccines currently used in Africa are derived from a live strain called T1, which was attenuated by passage in embryonated eggs and broth culture. The number of passages is directly correlated to the degree of attenuation of the vaccinal strains and inversely correlated to their immunogenicity in cattle. Current quality control protocols applied to vaccine batches allow the assessment of identity, purity, and titers, but cannot assess the level of genetic drift form the parental vaccine strains. Deep sequencing was used to assess the genetic drift generated over controlled in vitro passages of the parental strain, as well as on commercial vaccine batches. Signatures of cloning procedures were detected in some batches, which imply a deviation from the standard production protocol. Deep sequencing is proposed as a new tool for the identity and stability control of T1 vaccines.

Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

One Health compartment analysis of ESBL-producing Escherichia coli reveals multiple transmission events in a rural area of Madagascar.

BACKGROUND: ESBL-producing Escherichia coli (ESBL-Ec) is considered a key indicator for antimicrobial resistance (AMR) epidemiological surveillance in animal, human and environment compartments. There is likelihood of ESBL-Ec animal-human transmission but proof of cross-compartment transmission is still unclear. OBJECTIVES: To characterize ESBL-Ec genetic similarity in various compartments (humans, animals and environment) from a rural area of Madagascar. METHODS: We collected ESBL-Ec isolates prospectively from humans, animals and the environment (water) between April and October 2018. These isolates were subject to WGS and analysed with cutting-edge phylogenomic methods to characterize population genetic structure and infer putative transmission events among compartments. RESULTS: Of the 1454 samples collected, 512 tested positive for ESBL-Ec. We successfully sequenced 510 samples, and a phylogenomic tree based on 179 365 SNPs was produced. Phylogenetic distances between and amongst compartments were indistinguishable, and 104 clusters of recent transmission events between compartments were highlighted. Amongst a large diversity of ESBL-Ec genotypes, no lineage host specificity was observed, indicating the regular occurrence of ESBL-Ec transfer among compartments in rural Madagascar. CONCLUSIONS: Our findings stress the importance of using a phylogenomic approach on ESBL-Ec samples in various putative compartments to obtain a clear baseline of AMR transmissions in rural settings, where one wants to identify risk factors associated with transmission or to measure the effect of 'One Health' interventions in low- and middle-income countries.

SILVI, an open-source pipeline for T-cell epitope selection.

High-throughput screening of available genomic data and identification of potential antigenic candidates have promoted the development of epitope-based vaccines and therapeutics. Several immunoinformatic tools are available to predict potential epitopes and other immunogenicity-related features, yet it is still challenging and time-consuming to compare and integrate results from different algorithms. We developed the R script SILVI (short for: from in silico to in vivo), to assist in the selection of the potentially most immunogenic T-cell epitopes from Human Leukocyte Antigen (HLA)-binding prediction data. SILVI merges and compares data from available HLA-binding prediction servers, and integrates additional relevant information of predicted epitopes, namely BLASTp alignments with host proteins and physical-chemical properties. The two default criteria applied by SILVI and additional filtering allow the fast selection of the most conserved, promiscuous, strong binding T-cell epitopes. Users may adapt the script at their discretion as it is written in open-source R language. To demonstrate the workflow and present selection options, SILVI was used to integrate HLA-binding prediction results of three example proteins, from viral, bacterial and parasitic microorganisms, containing validated epitopes included in the Immune Epitope Database (IEDB), plus the Human Papillomavirus (HPV) proteome. Applying different filters on predicted IC50, hydrophobicity and mismatches with host proteins allows to significantly reduce the epitope lists with favourable sensitivity and specificity to select immunogenic epitopes. We contemplate SILVI will assist T-cell epitope selections and can be continuously refined in a community-driven manner, helping the improvement and design of peptide-based vaccines or immunotherapies. SILVI development version is available at: github.com/JoanaPissarra/SILVI2020 and https://doi.org/10.5281/zenodo.6865909.

Characterisation of chicken farms in Vietnam: A typology of antimicrobial use among different production systems.

The usage of antimicrobials in livestock production is a driver for antimicrobial resistance worldwide. Reducing the use of antibiotics in the animal sector is a priority and requires a change in practices. Vietnam has diverse husbandry and antimicrobial use practices. The objective of this study was to determine the socio-economic and technical factors associated with antibiotic usage patterns on chicken farms in the north and south of Vietnam. Semi-structured interviews (n = 34) and on-farm questionnaires (n = 125) were conducted to collect socio-economic, technical, biosecurity, health management, and antibiotic usage data. Using Multivariate Corresponding Analysis, we identified three production systems (A, B, C) and three patterns of antibiotic usage (1, 2, 3). Group A raised indoor exotic chickens in an intensive setting and was associated with group 1, which used antibiotics according to company recommendations for both treatment and prevention. Group C raised free-range chickens for their own consumption and was associated with group 2, which used antibiotics according to drugstore advice for treatment. Finally, group B was a market-oriented, semi-confined system associated with group 3, which practiced experience-based antibiotic use and overuse. Farms in the south of Vietnam were associated with group 3 and those in the north with group 2. The prediction of antibiotic usage patterns based on farming practices could lead to the identification of a group of farms to be targeted in order to foster the more prudent use of antibiotics in Vietnam.

A participatory epidemiological and One Health approach to explore the community's capacity to detect emerging zoonoses and surveillance network opportunities in the forest region of Guinea.

The Ebola virus disease epidemic that threatened West Africa between 2013 and 2016 was of unprecedented health magnitude. After this health crisis, studies highlighted the need to introduce community-based surveillance systems and to adopt a One Health approach. This study aimed to provide preparatory insights for the definition of a community-based surveillance system for emerging zoonoses such as viral hemorrhagic fevers in Guinea. The objective was to explore the disease detection capacity and the surveillance network opportunities at the community level in two pilot areas in the forest region of Guinea, where the epidemic emerged. Based on a participatory epidemiological and One Health approach, we conducted Focus Group Discussions with human, animal and ecosystem health actors. We used a range of participatory tools, included semi-structured interviews, ranking, scoring and flow diagram, to estimate the local knowledge and perception of diseases and clinical signs and to investigate the existing health information exchange network and its related strengths and weaknesses. The results showed that there is heterogeneity in knowledge of diseases and perception of the clinical signs among actors and that there are preferred and more effective health communication channels opportunities. This preparatory study suggests that it is necessary to adapt the case definitions and the health communication channels to the different actors who can play a role in a future community-based surveillance system and provides recommendations for future surveillance activities to be carried out in West Africa.

Ant phylogenomics reveals a natural selection hotspot preceding the origin of complex eusociality.

The evolution of eusociality has allowed ants to become one of the most conspicuous and ecologically dominant groups of organisms in the world. A large majority of the current ∼14,000 ant species belong to the formicoids,1 a clade of nine subfamilies that exhibit the most extreme forms of reproductive division of labor, large colony size,2 worker polymorphism,3 and extended queen longevity.4 The eight remaining non-formicoid subfamilies are less well studied, with few genomes having been sequenced so far and unclear phylogenetic relationships.5 By sequencing 65 genomes, we provide a robust phylogeny of the 17 ant subfamilies, retrieving high support to the controversial leptanillomorph clade (Leptanillinae and Martialinae) as the sister group to all other extant ants. Moreover, our genomic analyses revealed that the emergence of the formicoids was accompanied by an elevated number of positive selection events. Importantly, the top three gene functions under selection are linked to key features of complex eusociality, with histone acetylation being implicated in caste differentiation, gene silencing by RNA in worker sterility, and autophagy in longevity. These results show that the key pathways associated with eusociality have been under strong selection during the Cretaceous, suggesting that the molecular foundations of complex eusociality may have evolved rapidly in less than 20 Ma.

A library preparation optimized for metagenomics of RNA viruses.

Our understanding of the viral communities associated to animals has not yet reached the level attained on the bacteriome. This situation is due to, among others, technical challenges in adapting metagenomics using high-throughput sequencing to the study of RNA viromes in animals. Although important developments have been achieved in most steps of viral metagenomics, there is yet a key step that has received little attention: the library preparation. This situation differs from bacteriome studies in which developments in library preparation have largely contributed to the democratisation of metagenomics. Here, we present a library preparation optimized for metagenomics of RNA viruses from insect vectors of viral diseases. The library design allows a simple PCR-based preparation, such as those routinely used in bacterial metabarcoding, that is adapted to shotgun sequencing as required in viral metagenomics. We first optimized our library preparation using mock viral communities and then validated a full metagenomic approach incorporating our preparation in two pilot studies with field-caught insect vectors; one including a comparison with a published metagenomic protocol. Our approach provided a fold increase in virus-like sequences compared to other studies, and nearly-full genomes from new virus species. Moreover, our results suggested conserved trends in virome composition within a population of a mosquito species. Finally, the sensitivity of our approach was compared to a commercial diagnostic PCR for the detection of an arbovirus in field-caught insect vectors. Our approach could facilitate studies on viral communities from animals and the democratization of metagenomics in community ecology of viruses.

DILS: Demographic inferences with linked selection by using ABC.

We present DILS, a deployable statistical analysis platform for conducting demographic inferences with linked selection from population genomic data using an Approximate Bayesian Computation framework. DILS takes as input single-population or two-population data sets (multilocus fasta sequences) and performs three types of analyses in a hierarchical manner, identifying: (a) the best demographic model to study the importance of gene flow and population size change on the genetic patterns of polymorphism and divergence, (b) the best genomic model to determine whether the effective size Ne and migration rate N, m are heterogeneously distributed along the genome (implying linked selection) and (c) loci in genomic regions most associated with barriers to gene flow. Also available via a Web interface, an objective of DILS is to facilitate collaborative research in speciation genomics. Here, we show the performance and limitations of DILS by using simulations and finally apply the method to published data on a divergence continuum composed by 28 pairs of Mytilus mussel populations/species.

A whole-genome worldwide molecular epidemiology approach for contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia is an infectious and contagious disease affecting goats and wildlife ruminants, mostly in Africa and Asia. It is caused by a mycoplasma, Mycoplasma capricolum susbp. capripneumoniae, which is very fastidious. This may be the reason why there are few reports of its isolation and characterization. This study describes the development of a whole genome typing strategy based on sequencing reads assemblies on a reference genome (Abomsa, GenBank accession LM995445) and extraction of informative single nucleotide polymorphism. FASTA sequences inferred from the variant calling files were used to establish a comprehensive phylogenetic tree based on 2880 SNPs. This tree included a total of 34 strains originating from all the regions where CCPP has been detected, as well as strains isolated from wildlife. A recent isolate from West-Niger was positioned closely to another 1995 East-Niger isolate, an indication that CCPP may be extending westward in Africa. Six 2013 Tanzanian isolates had identical sequences in spite of diverse geographical origins. This could be explained by the clonal expansion of a virulent strain at that time in East Africa. Although all strains isolated from wildlife in the Middle East were in the same phylogenetic group, this may not sign an adaptation to new hosts. The most probable explanation for wildlife contamination remains the contact with goats. This strategy will easily accommodate new data in the near future and should become a gold-standard high-resolution typing procedure for the surveillance of contagious caprine pleuropneumonia.

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner.

Genome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and Culicoides biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments.IMPORTANCE The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and Culicoides biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses.

In vitro shared transcriptomic responses of Aedes aegypti to arboviral infections: example of dengue and Rift Valley fever viruses.

BACKGROUND: Arthropod borne virus infections are the cause of severe emerging diseases. Among the diseases due to arboviruses, dengue (DEN) and Rift Valley fever (RVF) are in the top ten in the list of diseases responsible of severe human cases worldwide. Understanding the effects of viral infection on gene expression in competent vectors is a challenge for the development of early diagnostic tools and may enable researchers and policy makers to better anticipate outbreaks in the next future. METHODS: In this study, alterations in gene expression across the entire Aedes aegypti genome during infection with DENV and RVFV were investigated in vitro at two time points of infection, the early phase (24 h) and the late phase (6 days) of infection using the RNA sequencing approach RESULTS: A total of 10 upregulated genes that share a similar expression profile during infection with both viruses at early and late phases of infection were identified. Family B and D clip-domain serine proteases (CLIP) were clearly overrepresented as well as C-type lectins and transferrin. CONCLUSIONS: Our data highlight the presence of 10 viral genes upregulated in Ae. aegypti during infection. They may also be targeted in the case of the development of broad-spectrum anti-viral diagnostic tools focusing the mosquito vectors rather than the mammalian hosts as they may predict the emergence of outbreaks.

A novel Borrelia species, intermediate between Lyme disease and relapsing fever groups, in neotropical passerine-associated ticks.

Lyme disease (LD) and relapsing fevers (RF) are vector-borne diseases caused by bacteria of the Borrelia genus. Here, we report on the widespread infection by a non-described Borrelia species in passerine-associated ticks in tropical rainforests of French Guiana, South America. This novel Borrelia species is common in two tick species, Amblyomma longirostre and A. geayi, which feed on a broad variety of neotropical mammal and bird species, including migratory species moving to North America. The novel Borrelia species is divergent from the LD and RF species, and is more closely related to the reptile- and echidna-associated Borrelia group that was recently described. Genome sequencing showed that this novel Borrelia sp. has a relatively small genome consisting of a 0.9-Mb-large chromosome and an additional 0.3 Mb dispersed on plasmids. It harbors an RF-like genomic organization but with a unique mixture of LD- and RF-specific genes, including genes used by RF Borrelia for the multiphasic antigen-switching system and a number of immune-reactive protein genes used for the diagnosis of LD. Overall, our data indicate that this novel Borrelia is an intermediate taxon between the LD and RF species that may impact a large host spectrum, including American mammals. The designation "Candidatus Borrelia mahuryensis" is proposed for this species.

Evolution of Attenuation and Risk of Reversal in Peste des Petits Ruminants Vaccine Strain Nigeria 75/1.

Peste des Petits Ruminants (PPR) is a highly infectious disease caused by a virus of the Morbillivirus genus. The current PPR eradication effort relies mainly on the implementation of massive vaccination campaigns. One of the most widely used PPR vaccines is the Nigeria 75/1 strain obtained after attenuation by 75 serial passages of the wild type isolate in cell cultures. Here we use high throughput deep sequencing of the historical passages that led to the Nigeria 75/1 attenuated strain to understand the evolution of PPRV attenuation and to assess the risk of reversal in different cell types. Comparison of the consensus sequences of the wild type and vaccine strains showed that only 18 fixed mutations separate the two strains. At the earliest attenuation passage at our disposal (passage 47), 12 out of the 18 mutations were already present at a frequency of 100%. Low-frequency variants were identified along the genome in all passages. Sequencing of passages after the vaccine strain showed evidence of genetic drift during cell passages, especially in cells expressing the SLAM receptor targeted by PPRV. However, 15 out of the 18 mutations related to attenuation remained fixed in the population. In vitro experiments suggest that one mutation in the leader region of the PPRV genome affects virus replication. Our results suggest that only a few mutations can have a serious impact on the pathogenicity of PPRV. Risk of reversion to virulence of the attenuated PPRV strain Nigeria 75/1 during serial passages in cell cultures seems low but limiting the number of passages during vaccine production is recommended.

Identification of Eilat virus and prevalence of infection among Culex pipiens L. populations, Morocco, 2016.

Eilat virus (EILV) is described as one of the few alphaviruses restricted to insects. We report the record of a nearly-complete sequence of an alphavirus genome showing 95% identity with EILV during a metagenomic analysis performed on 1488 unblood-fed females and 1076 larvae of the mosquito Culex pipiens captured in Rabat (Morocco). Genetic distance and phylogenetic analyses placed the EILV-Morocco as a variant of EILV. The observed infection rates in both larvae and adults suggested an active circulation of the virus in Rabat and its maintenance in the environment either through vertical transmission or through horizontal infection of larvae in breeding sites. This is the first report of EILV out of Israel and in Culex pipiens populations.

Nanopore-based detection and characterization of yam viruses.

We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.

The Genomic Basis of Color Pattern Polymorphism in the Harlequin Ladybird.

Many animal species comprise discrete phenotypic forms. A common example in natural populations of insects is the occurrence of different color patterns, which has motivated a rich body of ecological and genetic research [1-6]. The occurrence of dark, i.e., melanic, forms displaying discrete color patterns is found across multiple taxa, but the underlying genomic basis remains poorly characterized. In numerous ladybird species (Coccinellidae), the spatial arrangement of black and red patches on adult elytra varies wildly within species, forming strikingly different complex color patterns [7, 8]. In the harlequin ladybird, Harmonia axyridis, more than 200 distinct color forms have been described, which classic genetic studies suggest result from allelic variation at a single, unknown, locus [9, 10]. Here, we combined whole-genome sequencing, population-based genome-wide association studies, gene expression, and functional analyses to establish that the transcription factor Pannier controls melanic pattern polymorphism in H. axyridis. We show that pannier is necessary for the formation of melanic elements on the elytra. Allelic variation in pannier leads to protein expression in distinct domains on the elytra and thus determines the distinct color patterns in H. axyridis. Recombination between pannier alleles may be reduced by a highly divergent sequence of ∼170 kb in the cis-regulatory regions of pannier, with a 50 kb inversion between color forms. This most likely helps maintain the distinct alleles found in natural populations. Thus, we propose that highly variable discrete color forms can arise in natural populations through cis-regulatory allelic variation of a single gene.

Tick-Bacteria Mutualism Depends on B Vitamin Synthesis Pathways.

Mutualistic interactions with microbes have facilitated the radiation of major eukaryotic lineages [1, 2]. Microbes can notably provide biochemical abilities, allowing eukaryotes to adapt to novel habitats or to specialize on particular feeding niches [2-4]. To investigate the importance of mutualisms for the exclusive blood feeding habits of ticks, we focused on a bacterial genus of medical interest, Francisella, which is known to include both virulent intracellular pathogens of vertebrates [5, 6] and maternally inherited symbionts of ticks [7-9]. Through a series of physiological experiments, we identified a Francisella type, F-Om, as an obligate nutritional mutualist in the life cycle of the African soft tick Ornithodoros moubata. Francisella F-Om mutualism synthesizes B vitamins that are deficient in the blood meal of ticks. Indeed, experimental elimination of Francisella F-Om resulted in alteration of tick life history traits and physical abnormalities, deficiencies which were fully restored with an oral supplement of B vitamins. We also show that Francisella F-Om is maternally transmitted to all maturing tick oocytes, suggesting that this heritable symbiont is an essential adaptive element in the life cycle of O. moubata. The Francisella F-Om genome further revealed a recent origin from a Francisella pathogenic life style, as observed in other Francisella symbionts [6, 7, 10]. Though half of its protein-coding sequences are now pseudogenized or lost, Francisella F-Om has kept several B vitamin synthesis pathways intact, confirming the importance of these genes in evolution of its nutritional mutualism with ticks.

Do changes in gene expression contribute to sexual isolation and reinforcement in the house mouse?

Expression divergence, rather than sequence divergence, has been shown to be important in speciation, particularly in the early stages of divergence of traits involved in reproductive isolation. In the two European subspecies of house mice, Mus musculus musculus and Mus musculus domesticus, earlier studies have demonstrated olfactory-based assortative mate preference in populations close to their hybrid zone. It has been suggested that this behaviour evolved following the recent secondary contact between the two taxa (~3,000 years ago) in response to selection against hybridization. To test for a role of changes in gene expression in the observed behavioural shift, we conducted a RNA sequencing experiment on mouse vomeronasal organs. Key candidate genes for pheromone-based subspecies recognition, the vomeronasal receptors, are expressed in these organs. Overall patterns of gene expression varied significantly between samples from the two subspecies, with a large number of differentially expressed genes between the two taxa. In contrast, only ~200 genes were found repeatedly differentially expressed between populations within M. m. musculus that did or did not display assortative mate preferences (close to or more distant from the hybrid zone, respectively), with an overrepresentation of genes belonging to vomeronasal receptor family 2. These receptors are known to play a key role in recognition of chemical cues that handle information about genetic identity. Interestingly, four of five of these differentially expressed receptors belong to the same phylogenetic cluster, suggesting specialization of a group of closely related receptors in the recognition of odorant signals that may allow subspecies recognition and assortative mating.