The genome sequence of Molossusnigricans (Chiroptera, Molossidae; Miller, 1902).

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.

Metagenomic surveillance for bacterial tick-borne pathogens using nanopore adaptive sampling.

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) strategy facilitates immediate mapping of individual nucleotide molecules to a given reference as each molecule is being sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence DNA of multiple bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis.

Nanopore adaptive sampling for targeted mitochondrial genome sequencing and bloodmeal identification in hematophagous insects.

BACKGROUND: Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. METHODS: We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. RESULTS: Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. CONCLUSIONS: Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.

Reevaluation of the phylogenetic relationships among Neotomini rodents (Hodomys, Neotoma, and Xenomys) and comments on the woodrat classification.

The woodrats or packrats of the genus Neotoma have been the subject of a wide array of research including paleoecology, physiology, morphological evolution, systematics, speciation, and hybridization. In recent years, much work has been done to elucidate evolutionary relationships within and between closely related species of the genus; in particular the addition of newly collected specimens from critical geographic regions has provided new opportunities for taxonomic assessment. Given these new data and their potential, parsimony (PARS), maximum likelihood (ML), and Bayesian inference (BI) analyses were conducted on DNA sequences obtained from nine individual genes (four mitochondrial loci: 12S, 16S, CoII, and Cytb; five nuclear loci: AdhI2, BfibI7, En2, Mlr, and Myh6) to estimate the phylogenetic relationships among 23 species of Neotoma. Results of these analyses depicted a wide array of phylogenetic relationships among taxa; with substantial nodal support recovered in both the ML and PARS analyses at some mid-level and terminal positions. Several individual genes, particularly 12S, AdhI2, BfibI7, CoII, and Cytb, provided support at several basal positions; however, phylogenetic resolution was limited in the other genes. A final BI analysis where the nine genes were concatenated into a single data set produced several supported clades that corresponded to previously recognized species groups (floridana, micropus, mexicana, and lepida) and the subgenus Homodontomys. Levels of genetic divergence for within-species comparisons (estimated from the Cytb data set) ranged from 0.88% (N. magister) to 6.82% (N. fuscipes); for between sister species comparisons ranged from 4.68% (N. devia and N. lepida) to 12.70% (N. angustapalata and N. nelsoni); and for members within closely related clades ranged from 8.70% (N. bryanti and N. lepida) to 12.57% (N. goldmani and N. magister). Evaluations of generic, subgeneric, and species group boundaries were explored using phylogenetic principles on the DNA sequence data presented herein, as well as morphological findings from previous studies. Results obtained suggest that the most conservative taxonomic interpretation involves the abandonment of subgeneric delineations and relies on the recognition of eight species groups (cinerea, floridana, fuscipes, lepida, mexicana, micropus, phenax, and stephensi) as the backbone of the woodrat classification.

Las ratas cambalacheras del género Neotoma han sido estudiadas en varios tipos de investigaciones incluyendo paleoecología, fisiología, evolución morfológica, sistemática, especiación e hibridación. Recientemente, se han realizado numerosos estudios para elucidar las relaciones evolutivas dentro del género y entre especies cercanamente relacionadas al mismo; en particular la inclusión de nuevos especímenes provenientes de regiones geográficas críticas han brindado nuevas oportunidades para evaluaciones taxonómicas. A partir de estos nuevos datos se realizaron análisis de parsimonia (PARS), Máxima Verosimilitud (MV), e Inferencia Bayesiana (IB) en secuencias de ADN provenientes de nueve genes individuales (cuatro loci mitocondriales: 12S, 16S, CoII, y Cytb; cinco loci nucleares: Adh-I2, Bfib-I7, En2, Mlr, and Myh6) para determinar la relación filogenética de 23 especies de Neotoma. Los resultados de estos análisis presentan una amplia gama de relaciones filogenéticas entre taxa con un soporte nodal importante en los análisis de MV y PARS en algunas posiciones terminales de nivel medio. Varios genes individuales, en particular 12S, Adh-I2, Bfib-I7, CoII, and Cytb, ofrecieron soporte en varias posiciones basales; sin embargo, la resolución filogenética fue reducida en los demás genes. El último análisis de IB, en donde nueve genes se concatenaron en un solo conjunto de datos, produjo soporte en varios clados que correspondieron a especies de grupos previamente reconocidos (floridana, micropus, mexicana, y lepida) y el sub-género Homodontomys. Los niveles de divergencia genética para comparaciones intraespecíficas fluctuaron entre 0.88% (N. magister) y 6.82% (N. fuscipes); para especies hermanas (4.68%­N. devia y N. lepida hasta 12.70%­N. angustapalata y N. nelsoni); y para los miembros de clados cercanos (8.70%­N. bryanti y N. lepida hasta 12.57%­N. goldmani y N. magister). Las evaluaciones de los limites genéricos, subgenéricos y de grupos de especies fueron explorados usando principios filogenéticos en las secuencias de ADN de este trabajo, y también se basaron en las conclusiones morfológicas de estudios previos. Los resultados obtenidos sugieren que la interpretación taxonómica más conservadora incluye el abandono de las delineaciones subgenéricas y se depende en el reconocimiento de ocho grupos de especies (cinerea, floridana, fuscipes, lepida, mexicana, micropus, phenax, y stephensi) como el pilar central de la clasificación de las ratas cambalacheras.

Nanopore-Based Surveillance of Zoonotic Bacterial Pathogens in Farm-Dwelling Peridomestic Rodents.

The effective control of rodent populations on farms is crucial for food safety, as rodents are reservoirs and vectors for several zoonotic pathogens. Clear links have been identified between rodents and farm-level outbreaks of pathogens throughout Europe and Asia; however, comparatively little research has been devoted to studying the rodent-agricultural interface in the USA. Here, we address this knowledge gap by metabarcoding bacterial communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of putative zoonotic foodborne pathogens, among others. Rodents were live trapped (n = 90) from three dairy and mixed animal farms. DNA extraction was performed on 63 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed. Our farm-level rodent-metabarcoding data indicate the presence of multiple foodborne pathogens, including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many mastitis pathogens circulating within five rodent species (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and a shrew (Blarina brevicauda). Interestingly, we observed a higher abundance of enteric pathogens (e.g., Salmonella) in shrew feces compared to the rodents analyzed in our study. Knowledge gained from our research efforts will directly inform and improve farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

The Role of Peridomestic Rodents as Reservoirs for Zoonotic Foodborne Pathogens.

Although rodents are well-known reservoirs and vectors for a number of zoonoses, the functional role that peridomestic rodents serve in the amplification and transmission of foodborne pathogens is likely underappreciated. Clear links have been identified between commensal rodents and outbreaks of foodborne pathogens throughout Europe and Asia; however, comparatively little research has been devoted to studying this relationship in the United States. In particular, regional studies focused on specific rodent species and their foodborne pathogen reservoir status across the diverse agricultural landscapes of the United States are lacking. We posit that both native and invasive species of rodents associated with food-production pipelines are likely sources of seasonal outbreaks of foodborne pathogens throughout the United States. In this study, we review the evidence that identifies peridomestic rodents as reservoirs for foodborne pathogens, and we call for novel research focused on the metagenomic communities residing at the rodent-agriculture interface. Such data will likely result in the identification of new reservoirs for foodborne pathogens and species-specific demographic traits that might underlie seasonal enteric disease outbreaks. Moreover, we anticipate that a One Health metagenomic research approach will result in the discovery of new strains of zoonotic pathogens circulating in peridomestic rodents. Data resulting from such research efforts would directly inform and improve upon biosecurity efforts, ultimately serving to protect our food supply.

Differential Expression in Testis and Liver Transcriptomes from Four Species of Peromyscus (Rodentia: Cricetidae).

The genus Peromyscus represents a rapidly diverged clade of Cricetid rodents that contains multiple cryptic species and has a propensity for morphologic conservation across its members. The unresolved relationships in previously proposed phylogenies reflect a suspected rapid adaptive radiation. To identify functional groups of genes that may be important in reproductive isolation in a reoccurring fashion across the Peromyscus phylogeny, liver and testis transcriptomes from four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) were generated and differential expression (DE) tests were conducted. Taxa were selected to represent members diverged from a common ancestor: P. attwateri + P. boylii (clade A), and P. leucopus + P. maniculatus (clade B). Comparison of clades (A vs. B) suggested that 252 transcripts had significant DE in the liver data set, whereas significant DE was identified for 657 transcripts in the testis data set. Further, 45 genes had DE isoforms in the 657 testis transcripts and most of these functioned in major reproductive roles such as acrosome assembly, spermatogenesis, and cell cycle processes (meiosis). DE transcripts in the liver mapped to more broad gene ontology terms (metabolic processes, catabolic processes, response to chemical, and regulatory processes), and DE transcripts in the testis mapped to gene ontology terms associated with reproductive processes, such as meiosis, sperm motility, acrosome assembly, and sperm-egg fusion. These results suggest that a suite of genes that conduct similar functions in the testes may be responsible for the adaptive radiation events and potential reoccurring speciation of Peromyscus in terms of reproduction through varying expression levels.

A new species of big-eared climbing rat, genus Ototylomys (Cricetidae: Tylomyinae), from Chiapas, Mexico.

An allopatric population of big-eared climbing rats (Ototylomys) from the Northern Highlands of Chiapas, Mexico, is described as a new species. The new taxon is part of a unique montane rainforest community that includes several other endemic species in the limited geographic range between the Río Grijalva and the Central Depression of Chiapas. Several cranial, external, and molecular characters distinguish this new species of big-eared climbing rat from its more widely distributed congener, Ototylomys phyllotis. We performed principal component and discriminate function analyses of cranial measurements, and found that specimens of the new species consistently could be distinguished from other Ototylomys with strong statistical support. Compared with exemplars of Ototylomys from elsewhere in their range, the new species possesses a karyotype that differs by 3 additional biarmed chromosome pairs, is fixed or nearly fixed for distinct electromorphs at 12 allozyme loci, and the mean genetic distance exceeds 14%, based on comparisons of the mitochondrial cytochrome b gene between the new species of Ototylomys and representatives of O. phyllotis. The restricted distribution in montane karst rainforest suggests that the species and its habitat may be a matter of conservation concern.

Una población alopátrica de rata orejuda trepadora (Ototylomys) de las Tierras Altas del Norte de Chiapas, México se describe como una nueva especie. El nuevo taxón es parte de una comunidad única de bosque lluvioso montano que incluye varias especies endémicas en el área de distribución geográfica limitada entre el Río Grijalva y la Depresión Central de Chiapas. Varios caracteres craneales, externos, y moleculares distinguen la nueva rata orejuda trepadora de su congénere más ampliamente distribuido, Ototylomys phyllotis. Se realizaron análisis de componentes principales y de función discriminante de los caracteres craneales, y se encontró que los especímenes de La Pera fueron consistentemente distinguidos de otros Ototylomys con un fuerte soporte estadístico. En comparación con ejemplares de Ototylomys del rango, la nueva especie posee un cariotipo que difiere por 3 pares adicionales de cromosomas biarmados, está fijo o casi fijo por distintos electromorfos en 12 loci alozímicos. Adicionalmente, la media de la distancia genética comparada del gen mitochondrial citocromo b entre la nueva especie de Ototylomys y representantes de O. phyllotis, excede el 14%. La distribución restringida en el bosque lluvioso montano kárstico sugiere que la especie y su hábitat pueden ser de importancia para la conservación.