Molecular diet studies of water mites reveal prey biodiversity

insects, such as mosquitoes, as the insect adult emerges from its aquatic pupal stage. Water mites are one of the most diverse aquatic invertebrates, with over 10,000 described species; however, relatively little is known about water mites, especially in North America [1]. It is esti- mated that up to half of the species have not even been described, named [1] or barcoded [2]. A similar lack of knowledge exists about the identity and diversity of the microscopic or small macroscopic prey that water mites consume including oligochaetes which are reported here [3]. In contrast to other aquatic organisms such as fish, water mites have largely been ignored in food web depictions of aquatic ecosystems as a result of scientific neglect for freshwater inver- tebrates when compared to freshwater vertebrates [4]. For example, the current Great Lakes food web descriptions from the National Oceanic and Atmospheric Association (NOAA) are incomplete and do not include many important benthic predators such as water mites (https:// www.glerl.noaa.gov/res/projects/food_web/food_web.html). Water mites are considered “underappreciated” aquatic predators, and this work aims to raise the profile of water mite research, especially in North America [5]. Even the analysis of water mite diets has largely been limited in the past to testing what water mites will ingest in the laboratory [5–7]. Acarologists have previously listed a variety of water mite prey based on field observations and laboratory feeding experiments. While we can

easily show that various species of water mites in the laboratory will attack and consume larvae of chironomids, mosquitoes, and ostracods, that is far from revealing what they ingest in nature. Water mites are presumed to prey on a range of invertebrates available in the habitats they reside in. The list includes copepods, cladocerans, chironomids, mosquito larvae and oth- ers [5, 6, 8]. For example, Lebertia has been reported to feed on Diptera larvae [8]. Some stud- ies have suggested that mite predation are so great that they compete with fish for food [9] and may limit the spread of invasive species due to their parasitism [10]. Water mites are also para- sitic on adult aquatic insects including mosquitoes, dragonflies, chironomids and beetles but this is restricted to the larval stage [11]. While some work has revealed observational records of water mite adults feeding on mosquito larvae in small temporary ponds [12] the present study is the first comprehensive investigation of the diet diversity of water mites, and the only study to our knowledge that has used high throughput next-generation sequencing (NGS) for this purpose in water mites. Water mites are members of Arachnida, the large group of invertebrates that includes spi- ders, ticks and terrestrial mites. They all share a similar way of feeding, by external digestion that results in a liquefied meal. It is impossible to identify prey items by microscopy after lique- fication; therefore, this study applies a molecular method as the best means to identify the

water mite prey, by sequencing the partially digested DNA of ingested prey. Previous work on spiders’ diets from coastal habitats used a combination of molecular analysis of diet DNA and carbon isotopes to differentiate whether their prey was coming from a terrestrial or marine source [13]. In our study we chose to use the molecular approach since adult water mites are fully aquatic, microscopic, and difficult to observe directly in their natural habitat. NGS has become a standard way to study diet contents in other animals groups, especially when gut contents are too fragmented to study morphologically [14]. NGS has been used to study diets in fish, spiders, ticks, and parasitic wasps [13, 15–17]. Martin et al. [18] sequenced DNA extracted from the gut of Hygrobates water mites that had previously been eating a single species of Chironomid in laboratory-based-feeding experiments. Their experiments showed that Hygrobates mites, offered chironomid prey in a laboratory setting, fed on the chirono- mids, and subsequent PCR amplification with chironomid-specific 18S primers detected chi- ronomid DNA signals for at least 24 hours after feeding [18]. This study hypothesizes that using NGS technology on DNA from freshly collected water mites would detect the DNA of PLOS ONE Prey DNA diversity in water mites PLOS ONE | https://doi.org/10.1371/journal.pone.0254598 July 29, 2021 2 / 18 This work was also supported by a donation from Dr. David R. Cook to Wayne State University. Dr. Adrian Amelio Vasquez The funders had no role in study design, data collection and analysis, decision

to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.