Molecular diet studies of water mites reveal prey biodiversity

more diverse ingested species beyond that shown in laboratory-based feeding experiments and thereby reveal the natural constituents of water mite diets. Two species of Lebertia were chosen as the focus of this molecular water mite diet studies because of their year-round seasonal availability, previous reports on their predation of aquatic Diptera larvae of ecological impor- tance, abundance and diversity in a local freshwater lagoon [7, 19–22]. Methods Ethics statement JLR is Director of the Belle Isle Aquarium research field lab and had a collecting permit from the Michigan Department of Natural Resources for Belle Isle. The sample collections did not involve any endangered or protected species. Water mite collection and DNA extraction Water mites were collected on multiple dates between July 2016 and March 2017 (see S1 Table) from Blue Heron Lagoon (latitude 42.344661, longitude -82.959324), on Belle Isle, an island park of the State of Michigan located in the Detroit River, by dragging a 200 μm net along the bottom and through emergent vegetation in depths of 0.5–1 m, washed on a 250 μm sieve, and transported to the Wayne State University Belle Isle Aquarium Field Research Laboratory within 30 minutes of collection. Live water mites were immediately picked on sorting trays and preserved by blanching followed by immersion in 70–90% ethanol and storage at 4 oC, as previously described [20]. Mites were sorted according to genus and species (Lebertia was our study model) and isolated from the rest of the sample as done in our

prior studies [20]. Each mite was washed with ethanol to avoid cross-contamination from other organisms. Mites that were damaged or pierced during sorting were not selected for this analysis. Whole mite DNA was extracted by puncturing individual water mites with sterile minutien pins to allow water mite lysate, including gut contents, to ooze out. DNA was obtained from the punctured mites by lysing and extracting with Proteinase K solution over- night (8–12 hours at 57 oC), and purifying DNA using a Qiagen DNA spin-column protocol (https://www.qiagen.com/us/resources/resourcedetail?id=6b09dfb8-6319-464d-996c- 79e8c7045a50&lang=en), followed by storage of the extracted DNA at -20 oC [20]. A voucher of the exoskeleton was retained for morphological analysis and is being held at our laboratory in the Physiology Department, Wayne State University School of Medicine. DNA extraction methods using the Qiagen DNA spin-column protocol were applied to various chironomids and mosquitoes without puncturing the organism. Their sequences were generated when needed for comparative purposes and positive controls. Molecular gut analysis of laboratory-fed water mites To validate our approach, DNA was extracted from Lebertia mites after feeding them insect larvae. Water mites (Lebertia) were placed in 6-well plates (Fisher Scientific, IL) and provided chironomid and mosquito larvae. Early instar fruit fly (Drosophila) larvae were also offered as prey to water mites by submerging the larvae in the well with the mite. Photos were taken of mites feeding on or attempting to feed on larvae.

After mites had fed on and released the prey item the mites were blanched and DNA was extracted as described above. DNA was then amplified with the mitochondrial cytochrome oxidase I (COI) primer pair mLep and LCO1490 (top of Table 1), previously used as “non- arachnid arthropod-specific COI primers.” These primers generate a product of 332 bp [13, 23, 24]. The more broad “metazoan” DNA barcoding primers; COI forward and reverse PLOS ONE Prey DNA diversity in water mites PLOS ONE | https://doi.org/10.1371/journal.pone.0254598 July 29, 2021 3 / 18