Molecular diet studies of water mites reveal prey biodiversity

identity and 93% query coverage to Paratanytarsus sp. (Chironomidae) and non-mite DNA from Lebertia 2-BHL022317 had 99% identity though only 75% query coverage to Paratany- tarsus sp. Unexpectedly, non-mite DNA from the other three Lebertia had closest matches to oligochaetes (Slavina and Nais, respectively, but with only 88% sequence match) and another non-insect (e.g., Diaphanosoma, albeit with only 42% query coverage), which have not previ- ously been associated with Lebertia diets. The sequence chromatograms were often complex, possibly reflecting overlapping sequences from multiple prey organisms. Therefore, we next used Illumina high-throughput sequencing to identify multiple individual prey DNA sequences in freshly collected Lebertia. Next-generation sequencing of non-mite DNA in Lebertia and other water mites The next-generation sequencing results show a more diverse diet of Lebertia than any previous study. Neighbor-joining trees of sequences from representative water mites show specimens in which OTUs were predominantly chironomids (Fig 3 from L. davidcooki and Fig 4, from L. quinquemaculosa) and others with a mix of chironomids and oligochaetes (Fig 5 from L. quinquemaculosa). Chironomid DNA in Lebertia The diet profile of a representative Lebertia davidcooki water mite that was feeding primarily on chironomids is shown in Fig 3. Most of the 45 OTUs that matched chironomid sequences in Fig 3 had sequence identities to species in GenBank that were greater than 97%, including

identities of 97.9%, 97.2%, 98.2%, 98.2%, 98.8%, and 98.5% obtained for Chironomus riparius, Dicrotendipes sp., Cricotopus trifasciatus, Psectrocladius sp., Paratanytarsus sp., and Cryptochir- onomus sp., respectively. In addition to the 45 OTUs that matched chironomids, this L. david- cooki specimen also had one oligochaete, one cladocera and one non-chironomid dipteran OTU. The diet profile of a Lebertia quinquemaculosa specimen in Fig 4 also contains many Chironomids but most identifications beyond 97% were only up to the subfamily level of Chir- onominae. In addition to 30 chironomid OTUs, this specimen also included 10 Culex pipiens, 1 Culex conspirator, 1 oligochaete and 1 Lebertia OTU. The percent identities for the many dif- ferent Chironomid genera found in all specimens of L. davidcooki and L. quinquemaculosa are summarized in Table 3. Fig 2. Lebertia quinquemaculosa preying upon organisms in laboratory feeding experiments. A, Chironomus riparius; B, Culex pipiens, and C, Drosophila melanogaster. Calibration bars = 1 mm. https://doi.org/10.1371/journal.pone.0254598.g002 PLOS ONE Prey DNA diversity in water mites PLOS ONE | https://doi.org/10.1371/journal.pone.0254598 July 29, 2021 7 / 18