A next-generation sequencing study of arthropods in the diet of Laysan Teal (Anas laysanensis)
Data files
Jun 07, 2023 version files 20.22 KB
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Arthropod_ASV_Table.xlsx
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README.md
Abstract
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.
Methods
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 MT-CO1 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.
Usage notes
For ASV data: 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.