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

Evan J Kipp, Laramie Lindsey, Marissa S Milstein, Cristina M. Blanco, Julia P. Baker, Christopher Faulk, Jonathan Oliver, Peter Larsen

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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. Graphical Abstract: [Figure not available: see fulltext.].

Original languageEnglish (US)
Article number68
JournalParasites and Vectors
Volume16
Issue number1
DOIs
StatePublished - Dec 2023

Bibliographical note

Funding Information:
This research presented herein was supported in part by the Office Of The Director of the NIH under Award Number T35OD011118, the Office of Graduate Programs, College of Veterinary Medicine, University of Minnesota and startup funds provided to Peter A. Larsen through the Minnesota Agriculture, Research, Education, Extension, and Technology Transfer (AGREETT) program.

Funding Information:
We thank Suzanne Stone for assistance within the molecular laboratory and related logistics. The Minnesota Supercomputing Institute provided essential computational and data storage resources. The staff of the Wildlife Rehabilitation Center of Minnesota (Roseville, MN) and Luciano Caixeta kindly provided access to forested lands and the University of Minnesota dairy barn, respectively, for insect collecting. Financial support for CMB and JB was kindly provided by the University of Minnesota, College of Veterinary Medicine Summer Scholars program with funds originating from the Office Of The Director of the NIH under Award Number T35OD011118 and the Office of Graduate Programs, College of Veterinary Medicine. We thank Tiffany Wolf and Erin Burton for providing dissecting microscopes for morphological identifications of insects. We used BioRender ( www.biorender.com ) to create figures presented herein.

Funding Information:
We thank Suzanne Stone for assistance within the molecular laboratory and related logistics. The Minnesota Supercomputing Institute provided essential computational and data storage resources. The staff of the Wildlife Rehabilitation Center of Minnesota (Roseville, MN) and Luciano Caixeta kindly provided access to forested lands and the University of Minnesota dairy barn, respectively, for insect collecting. Financial support for CMB and JB was kindly provided by the University of Minnesota, College of Veterinary Medicine Summer Scholars program with funds originating from the Office Of The Director of the NIH under Award Number T35OD011118 and the Office of Graduate Programs, College of Veterinary Medicine. We thank Tiffany Wolf and Erin Burton for providing dissecting microscopes for morphological identifications of insects. We used BioRender (www.biorender.com) to create figures presented herein.

Publisher Copyright:
© 2023, The Author(s).

Keywords

  • Culicidae
  • MinION
  • Mitochondrial genomes
  • Molecular barcoding
  • Phylogenetic capture
  • Tabanidae

PubMed: MeSH publication types

  • Journal Article

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