Molecular diet studies of water mites reveal prey biodiversity

(date = 3 June, 2018) to identify the closest identity in that database, using self-written Perl scripts to identify the top 5 hits in GenBank and only the hits with query coverage >50% and identity >80% were kept. In some cases where the top 5 hits were only at family level, BLAST analysis was repeated manually to determine if a specific genus or species could be identified within one or two percent identity of the top hit. Bioinformatics assistance and advice was pro- vided by Wayne State University’s Applied Genomics Technology Center (https:// genomesciencescore.wayne.edu/). MEGA6 [28] was used to compare various sequences, including alignments, pairwise comparisons, and construction of neighbor-joining trees. CD-HIT clusters of various sizes were sorted and graphed in pie charts on Excel© spreadsheets, which were also used for histogram analysis. Mann-Whitney non-parametric statistical test calculations used an on-line calculator (https://www.socscistatistics.com/tests/mannwhitney/ default2.aspx). Mann-Kendall and Yuen’s test calculations were performed by S. Sawilowsky (College of Education, Wayne State University, using R and Minitab1 18, respectively). Code availability The code used in this study was deposited in GitHub at https://github.com/GeoCoderL/mite- diet. Reference sequences and sequence data availability The high throughput nucleotide sequence data was uploaded to GenBank as Accession IDs MW605229—MW615071 administered by the National Center for Biotechnology Information

(https://submit.ncbi.nlm.nih.gov/subs/sra/) and tohttps://digitalcommons.wayne.edu/physio_ frp/2. Genus and species names, their authorities and years, and their family and subfamily identities were verified by reference to https://www.catalogueoflife.org/ and through the use of the Web of Science reference list (https://clarivate.com/webofsciencegroup/solutions/web-of- science/) and NCBI taxonomy browser (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/ wwwtax.cgi). Results Lebertia water mite species from Blue Heron Lagoon, Detroit Blue Heron Lagoon is home to at least two species of Lebertia water mites which we have reported in previous studies [20] and further confirmed in this work by DNA barcoding using the “universal metazoan” barcode primers (HCO2198 and LCO1490) of Folmer et al. [25, 29]. We identified the barcodes of L. quinquemaculosa and L. davidcooki (Fig 1A, 1B, and 1C, 1D, respectively; GenBank accession IDs MG773261.1 and MG773262.1, respectively). L. quinque- maculosa and L. davidcooki COI barcodes differed from one another by a pairwise difference of 16%. L. davidcooki differs from all previous Lebertia barcodes by > 11% and is close in mor- phology to L. inaequalis [30], whose barcode (HQ919893.1) differs from L. davidcooki by 13%. Laboratory-fed Lebertia and preliminary DNA analysis of freshly caught specimens DNA extracted from mites that had recently ingested chironomid or mosquito prey in the lab- oratory reliably yielded DNA sequences that matched the prey (Table 2). In control experi-

ments, DNA extracted from legs of Lebertia (therefore, not including the gut region) amplified as expected with HCO2198 and LCO1490 primers, yielding the expected water mite sequences. Leg DNA amplified by mLep/LCO1490 primers yielded no PCR product, as expected. Tests of mLep/LCO1490 primers with fish DNA were also negative. PLOS ONE Prey DNA diversity in water mites PLOS ONE | https://doi.org/10.1371/journal.pone.0254598 July 29, 2021 5 / 18