Barcode 100K Specimens: In a Single Nanopore Run.

It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition.

DNA barcodes from over-a-century-old type specimens shed light on the taxonomy of a group of rare butterflies (Lepidoptera: Nymphalidae: Calinaginae).

We analyzed COI barcode sequences from 138 over-a-century old specimens of Calinaga including 36 name-bearing type specimens stored at the Natural History Museum London. These new data, combined with previously available RPS5 sequences, divide the Calinaga samples into four well-supported mitochondrial lineages that together with a novel wing-pattern analysis, support the recognition of six species (lhatso, buddha, brahma, aborica, formosana and davidis), with all other names subsumed either as subspecies or synonyms. One new taxon is described, Calinaga aborica naima Vane-Wright, ssp.

A workflow for expanding DNA barcode reference libraries through 'museum harvesting' of natural history collections.

Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society - through, for example, digitisation or the construction of a validated, global DNA barcode library - is of crucial importance. To this end, we developed and streamlined a workflow for 'museum harvesting' of authoritatively identified Diptera specimens from the Smithsonian Institution's National Museum of Natural History.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases.

Interrogating 1000 insect genomes for NUMTs: A risk assessment for estimates of species richness.

The nuclear genomes of most animal species include NUMTs, segments of the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study of their frequency/attributes in the most diverse group of terrestrial organisms, insects. This study examines NUMTs derived from a 658 bp 5' segment of the cytochrome c oxidase I (COI) gene, the barcode region for the animal kingdom.

Culicoides species community composition and feeding preferences in two aquatic ecosystems in northern Spain.

Background: Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019.

Methods: Culicoides were captured by light suction traps baited with CO and by sweep netting vegetation.

Drought at a coastal wetland affects refuelling and migration strategies of shorebirds.

Droughts can affect invertebrate communities in wetlands, which can have bottom-up effects on the condition and survival of top predators. Shorebirds, key predators at coastal wetlands, have experienced widespread population declines and could be negatively affected by droughts. We explored, in detail, the effects of drought on multiple aspects of shorebird stopover and migration ecology by contrasting a year with average wet/dry conditions (2016) with a year with moderate drought (2017) at a major subarctic stopover site on southbound migration.

An Integrated Molecular Approach to Untangling Host-Vector-Pathogen Interactions in Mosquitoes (Diptera: Culicidae) From Sylvan Communities in Mexico.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector-host-pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract.

A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome oxidase I (COI) gene from 380 butterflies with an average age of 50 years.

Vertebrate-Aedes aegypti and Culex quinquefasciatus (Diptera)-arbovirus transmission networks: Non-human feeding revealed by meta-barcoding and next-generation sequencing.

Background: Aedes aegypti mosquito-borne viruses including Zika (ZIKV), dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) have emerged and re-emerged globally, resulting in an elevated burden of human disease. Aedes aegypti is found worldwide in tropical, sub-tropical, and temperate areas. The characterization of mosquito blood meals is essential to understand the transmission dynamics of mosquito-vectored pathogens.

Next generation sequencing-aided comprehensive geographic coverage sheds light on the status of rare and extinct populations of Aporia butterflies (Lepidoptera: Pieridae).

The Black-veined White Aporia crataegi (Linnaeus, 1758), a common and widespread butterfly ranging from northwestern Africa to Europe and Asia, has been extinct in Britain since the 1920s and is on a steady decline in several other parts of its range. In order to investigate genetic diversity within A. crataegi and its correspondence with current subspecies-level taxonomy, we barcoded 173 specimens from across its range including, for the first time, extinct populations from Britain and Korea.

Dynamics of a host-parasitoid interaction clarified by modelling and DNA sequencing.

It has been hypothesised that the 2-year oscillations in abundance of Xestia moths are mediated by interactions with 1-year Ophion parasitoid wasps. We tested this hypothesis by modelling a 35-year time series of Xestia and Ophion from Northern Finland. Additionally, we used DNA barcoding to ascertain the species diversity of Ophion and targeted amplicon sequencing of their gut contents to confirm their larval hosts.

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface.

Validation of COI metabarcoding primers for terrestrial arthropods.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated.

DNA metabarcoding allows non-invasive identification of arthropod prey provisioned to nestling Rufous hummingbirds ().

Hummingbirds consume sugars from nectar, sap and honeydew, and obtain protein, fat and minerals from arthropods. To date, the identity of arthropod taxa in hummingbird diets has been investigated by observation of foraging or examination of alimentary tract contents. Direct examination of nestling provisioning adds the extra complication of disturbance to the young and mother.

Metabarcoding a diverse arthropod mock community.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species.

A Sequel to Sanger: amplicon sequencing that scales.

Background: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.

Results: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes.

Molecular approaches for blood meal analysis and species identification of mosquitoes (Insecta: Diptera: Culicidae) in rural locations in southern England, United Kingdom.

Thirty-four species of Culicidae are present in the UK, of which 15 have been implicated as potential vectors of arthropod-borne viruses such as West Nile virus. Identification of mosquito feeding preferences is paramount to the understanding of vector-host-pathogen interactions which, in turn, would assist in the control of disease outbreaks. Results are presented on the application of DNA barcoding for vertebrate species identification in blood-fed female mosquitoes in rural locations.

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str.

Calibrating the taxonomy of a megadiverse insect family: 3000 DNA barcodes from geometrid type specimens (Lepidoptera, Geometridae).

It is essential that any DNA barcode reference library be based upon correctly identified specimens. The Barcode of Life Data Systems (BOLD) requires information such as images, geo-referencing, and details on the museum holding the voucher specimen for each barcode record to aid recognition of potential misidentifications. Nevertheless, there are misidentifications and incomplete identifications (e.

Rapid identification of the botanical and entomological sources of honey using DNA metabarcoding.

Honey is generated by various bee species from diverse plants, and because the value of different types of honey varies more than 100-fold, it is a target for fraud. This paper describes a protocol that employs DNA metabarcoding of three gene regions (ITS2, rbcLa, and COI) to provide an inexpensive tool to simultaneously deliver information on the botanical and entomological origins of honey. This method was used to examine seven varieties of honey: light, medium, dark, blended, pasteurized, creamed, and meliponine.

DNA Barcoding of Marine Metazoans.

The accumulation of DNA barcode sequences will provide an increasingly useful and comprehensive library for species identification and discovery of marine metazoans. Here we present a summary of protocols designed to obtain DNA barcodes of marine metazoans from diverse phyla.

DNA barcodes from century-old type specimens using next-generation sequencing.

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain.

Molecular species identification, host preference and detection of myxoma virus in the Anopheles maculipennis complex (Diptera: Culicidae) in southern England, UK.

Background: Determining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An.

DNA barcoding of Neotropical black flies (Diptera: Simuliidae): Species identification and discovery of cryptic diversity in Mesoamerica.

Although correct taxonomy is paramount for disease control programs and epidemiological studies, morphology-based taxonomy of black flies is extremely difficult. In the present study, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies from Mesoamerica was assessed. A total of 32 morphospecies were analyzed, one belonging to the genus Gigantodax and 31 species to the genus Simulium and six of its subgenera (Aspathia, Eusimulium, Notolepria, Psaroniocompsa, Psilopelmia, Trichodagmia).