Across the globe, anthropogenic environmental changes are threatening animal biodiversity and contributing to the emergence of vector-borne and zoonotic pathogens through host range shifts. To combat these challenges, accurate and timely biodiversity assessments and molecular species monitoring efforts are critical. Here, we document how implementation of a portable laboratory in combination with targeted long-read nanopore sequencing can facilitate in situ genomic and systematic analyses across several animal taxa. Working at two ecologically divergent field sites in Guyana, South America, we collected small mammals and blood-feeding insects, including bats, rodents, a marsupial, mosquitoes, and a phlebotomine sand fly. For each specimen sampled, genomic DNA was extracted in the field and used for preparation of nanopore sequencing libraries. For field sequencing, we utilized a novel software-based targeted sequencing approach—nanopore adaptive sampling (NAS)—that enabled selective sequencing of mitochondrial reads using mitogenome assemblies of related taxa as enrichment targets. Basecalled reads from our field sequencing experiments were used to assemble complete mitogenomes and to generate mitochondrial biomarker consensus gene sequences for all nine small mammals and four blood-feeding insects sequenced. Confirmatory molecular identifications were made with a combination of local nucleotide BLAST queries and maximum likelihood analyses using biomarker consensus sequences. Importantly, the mitogenome-based targeted sequencing strategies outlined here are amplification-free and allowed us to bypass time-consuming and potentially troublesome PCR-based methods in the field, streamlining library preparation, sequencing experiments, and on-site analyses. Our findings describe targeted sequencing with NAS as an effective tool for implementation into portable laboratories to widely enhance field-based biodiversity monitoring and rapid molecular species assessments across vertebrate and invertebrate hosts of consequential emerging pathogens.

Rodents are key reservoirs of zoonotic pathogens and play an important role in disease transmission to humans. Importantly, anthropogenic land-use change has been found to increase the abundance of synanthropic rodents, particularly rodent reservoirs of zoonotic disease. Anthropogenic environments also affect the microbiome of synanthropic wildlife, influencing wildlife health and potentially introducing novel pathogens. Our objective was to characterize the microbiome and investigate the prevalence of zoonotic bacterial pathogens in synanthropic rodents in native and anthropogenic environments to better understand their role in pathogen maintenance and transmission. We sampled wild Peromyscus mice in agricultural and undeveloped landscapes and forest and synanthropic habitat in Minnesota, USA and conducted 16S amplicon sequencing using long-read Nanopore sequencing technology on fecal samples to characterize the rodent microbiome. We compared community composition and diversity between habitats and screened for the presence of putative pathogenic bacteria species. Microbiome community composition differed significantly between agricultural and undeveloped landscapes and forest and synanthropic habitat while microbiome richness, diversity, and evenness were lower in undeveloped-forest habitat compared to all other habitats. We detected overall low abundance and diversity of putative pathogenic bacteria, though the greatest number of pathogenic bacteria were detected in the agricultural-forest habitat. Our findings show that rodent microbiome community composition differs across landscapes and habitat types but suggest that landscape-level anthropogenic factors may be most important to predict zoonotic pathogen abundance. Ultimately, understanding how anthropogenic land-use change and synanthropy affect rodent microbiomes and pathogen prevalence is important to managing transmission of rodent-borne zoonotic diseases to humans.