Molecular diet studies of water mites reveal prey biodiversity

20% trimming, the generally accepted default), showed that the distribution of non-mite sequences between L. davidcooki and L. quinquemaculosa was significantly different for both chironomid (Ty = 15.34, df = 6.275, p = 0.00001) and oligochaete (Ty = 12.02, df = 5.18, p = 0.0002) sequences. Application of the seasonal Mann-Kendall test [32] for trend determined that the higher percentage of oligochaetes in the diet of L. quinquemaculosa in the fall was sig- nificant (p = 0.031) while seasonal trends in the diet of L. davidcooki were not significant. Discussion To our knowledge, this study is the first investigation of the diet of water mites based on the analysis of DNA in the gut contents of water mites from nature using next-generation sequencing. This work shows that two species of Lebertia have highly diverse diets as evi- denced by DNA from multiple types of organisms associated with each mite. The method used here, next-generation sequencing analysis, expands on the work that used PCR with 18S prim- ers and lab-fed mites [18] to show the retention of prey DNA by mites after ingestion. The use of next-generation sequencing with mLep/LCOI primers in the present study enabled amplifi- cation of non-mite DNA in mites not only from chironomids, which were the sole target of the studies of Martin et al. [18], but also from oligochaetes, other dipterans including mosqui- tos, and crustaceans. The data provide evidence for seasonal variation in water mite diets. Fur- thermore, the presence of predation on mosquitoes, combined with the previously well

studied parasitic associations with emerging adult mosquitoes indicates that impacts on mos- quitoes may be a potentially important ecosystem service provided by water mites. The hypothesis upon which the present work is based is that non-mite DNA amplified from field-collected water mites represents DNA from organisms that the mite has ingested. Diet studies in other organisms have shown that molecular technologies such as next-genera- tion sequencing can help unravel previously unknown trophic complexities [14]. Studies of spiders (a terrestrial relative of water mites) using molecular sequencing methods revealed prey choices from terrestrial or marine sources [13]. Studies of wild octopus revealed up to 122 molecular taxonomic units (potentially different prey items) that would not have been possible to identify by morphological examination [33]. The results of the next-generation sequencing analysis revealed great prey biodiversity for both L. quinquemaculosa and L. davidcooki. Many mites had high proportions and high spe- cies identity of DNA barcodes to multiple species of chironomids (see Figs 3 and 4). Recent studies using correlative data on water mite prey complexity demonstrated that copepod, cla- doceran and chironomid densities correlated with water mite abundance and species richness [34, 35]. However, this was based on correlation and not on direct observation of water mites feeding or DNA studies, the latter being unique to this present study. As stated in Pozojevic,

Jursic [34], molecular studies would increase our understanding of water mite food web func- tional contributions such as the presence of Oligochaetes in water mite diet which was not reported in the Pozojevic, Jursic [34] study. The results seen here helps us understand some of the ecological significance of the high biodiversity of chironomids revealed in previous studies of the region [36]. For example, our barcode study of chironomids in Maumee Bay and Mau- mee River (Toledo, OH) revealed up to 45 chironomid operational taxonomic units [36], many of which are similar or identical to those we found with NGS in water mite diets. Our laboratory continues to expand this list of chironomid species in the Great Lakes region (unpublished work), identifying many new barcodes that may be represented in water mite diets. This work is the first to show evidence of oligochaetes as a significant part of the diet of water mites. We have found only one mention, without supporting evidence, that water mites PLOS ONE Prey DNA diversity in water mites PLOS ONE | https://doi.org/10.1371/journal.pone.0254598 July 29, 2021 13 / 18