DOI: 10.5061/dryad.dv41ns23x
title: A next-generation sequencing study of arthropods in the diet of Laysan Teal (Anas laysanensis)

description: The critically endangered Laysan Teal Anas laysanensis (known as koloa
 pōhaka in the Hawaiian language) in the Northwestern Hawaiian Islands has
 wild populations on Kamole (Laysan Island), Kuaihelani (Midway Atoll NWR),
 and Hōlanikū (Kure Atoll). The Laysan Teal faces a new risk on Sand
 Island, Kuaihelani: non-target poisoning via a pending House Mouse Mus
 musculus eradication. After mice were observed attacking and depredating
 Laysan Albatross Phoebastria immutabilis (mōlī) in 2015, plans to
 eradicate mice were developed to protect this seabird species. However,
 this approach risks poisoning the Laysan Teal. To reduce exposure, teal
 will be translocated during mouse eradication. Even so, there is a
 potential risk of secondary poisoning for teal by ingesting arthropods
 that feed on mouse bait. We therefore used next-generation sequencing
 (NGS) to identify which arthropods teal consume. From August 2019 to
 February 2020, we collected 71 fresh teal faecal samples on Sand Island,
 and successfully extracted DNA from 21 samples. Via NGS, we found that
 teal most frequently consume cockroaches (order: Blattodea), freshwater
 ostracods (Cyprididae), midges (Chironomidae), and isopods
 (Porcellionidae). To a lesser degree, teal also eat spiders (Araneae),
 moths (Lepidoptera), beetles (Coleoptera), springtails (Entomobryomorpha),
 thrips (Thysanoptera), and crabs (Decapoda). Notably, Sand Island’s teal
 consume entirely different arthropods from teal on Kamole, which mainly
 eat flies (Diptera) and brine shrimp (Anostraca, Artemia sp.). Our study
 serves as a model for risk mitigation during invasive rodent eradications.
description: From August 2019 to February
 2020, we collected 71 fresh faecal samples from Laysan Teal across six
 locations on the island. Faecal sample collection occurred weekly and
 involved 30 min surveys in areas where teal aggregate and faecal samples
 could be collected with minimal contamination (i.e. from concrete or
 asphalt surfaces with scant vegetation). Faecal samples were collected immediately
 after an individual was directly observed defecating. Upon collection,
 faecal samples were individually stored in vials of 70% isopropyl alcohol,
 labelled, and placed in a -20°C freezer until shipment to the mainland for
 further preparation. Our sampling pool consisted of 33 females, 31 males,
 and seven teal whose sex we could not visually confirm; most of the
 samples (55) came from individuals one year or older (AHY = after hatch
 year, SY = second year, ASY = after second year; 77.5%), 10
 from hatch-years (HY;
 14.1%), and six from teal of unknown age (8.5%).
 For DNA extraction, we used the QIAamp
 PowerFecal DNA Kit and modified the second step to remove inhibitors in
 the faecal samples. Here, we rinsed the samples with water after removing
 them from storage media and incubated them overnight during the first
 refrigeration step (instead of the recommended 5 min). We prepared our DNA
 library using a two-step polymerase chain reaction (PCR) approach. Faecal
 DNA samples were first amplified with the ZBJ-ArtF1c/ZBJ-ArtR2c primers
 that target a 157 bp region of cytochrome c oxidase I (COI) of the
 <em>MT-CO1</em> gene, modified with adapters for the
 Illumina MiSeq
 system. After the first round of PCR, the purified
 products were amplified in a second PCR containing Nextera XT v2 indexes,
 with each sample receiving a unique combination of forward and reverse
 indexes. 
 Bioinformatics was performed by the UIC Research
 Informatics Core in the Research Resources Center using the DADA2
 pipeline with a 97%
 identity (sequence similarity) threshold to increase accuracy of taxonomic
 assignment and exclude chimeric sequences. A nucleotide BLAST
 search was used to match
 sequences to references through nt, the NCBI Genbank non-redundant
 nucleotide database. The
 resulting data were exported into a table of amplicon sequence variants
 (ASVs). We filtered out all ASVs assigned to any phylum other than
 Arthropoda and provided the most precise level of taxonomic identity as
 possible. In addition, we cross-referenced ASVs with arthropod records
 from Kuaihelani. Molecular data includes Laysan Teal faecal
 samples as rows and raw read counts of arthropod ASVs as columns. Each
 column is an ASV, and the assigned taxonomy is included in the first row.
 Data have not been normalized by total read counts per sample. The ASV
 tables contain data from teal faecal samples, from which DNA was
 extracted, amplified, and sequenced. In addition, there are several
 columns of metadata for each teal faecal sample, including the sample
 collection date, location, substrate, as well as the sex and age of the
 teal.
description: <strong>For ASV data</strong>: For arthropod ASVs,
 we filtered out all ASVs assigned to
 any phylum other than Arthropoda and provided the most precise level of
 taxonomic identity as possible. Data have not been normalized
 by total read counts per sample.