**1. Introduction**

It is important to understand the role and function of interactions in the microbial food web of aquatic ecosystems. The key biological interaction in the aquatic microbial food web is matter cycling mediated by predation, and predation often works as a regulating factor for energy pathways, as well as determining species composition [1]. In particular, rotifers are critical components linking microorganisms with larger predatory organisms such as crustaceans and fish within the grazing food chain: bacteria, heterotrophic nano-flagellates, rotifers/copepods/cladocerans, larval fish, mature fish [2,3]. Consequently, they function as a channel for the flux of organic matter within diverse organism assemblages organized in an intermediate position between the two different food webs, and transfer nutrients and energy from the microbial loop to higher trophic levels [4–6]. In addition, as the problem of eutrophication increases in aquatic ecosystems, the abundance of macrozooplankton decreases and consequently the contribution of rotifers in energy flow of aquatic food web becomes greater [2]. As a result, rotifer-focused biological interactions, especially rotifer feeding behaviors in microbial food web, are receiving a grea<sup>t</sup> attention to understand not only the interrelated biological relationships but also the structure and function in aquatic food webs [7].

However, the comprehensive understanding of rotifers feeding characteristics has not been well-elucidated in comparison to their importance, because most previous studies were conducted at the lab-scale with limited environmental conditions over a short time period, limited to common and dominant species as the tested species, and therefore have not been verified in the field [8–12]. These limitations were attributed to the absence of adequate analytical methodologies applicable to field sites due to di fficulties in culturing, handling and identification of both prey and predator (rotifers) which have small sizes (usually rotifers body size≤ 1000 μm; rotifers prey size spectrum<1–20 μm) [13]. In order to overcome the methodological limitations for the analysis of rotifer feeding behaviors, the introduction and application of appropriate techniques are required.

In recent years, genomic technologies have developed rapidly and been applied to ecological research. DNA barcoding techniques have increased the reliability of identifying specific taxonomic groups of organisms at both species and genus level [14], and environmental DNA techniques have enabled the detection of elusive species in various environments [15,16]. Genomic approaches have also been used to understand trophic ecology, particularly biological interaction, for both aquatic habitat environments and food webs by collecting information from food material found in gu<sup>t</sup> contents and the excrement of various organisms and this helps to overcome the existing limitations of food source analyses, which were usually based on visual analysis [17–20].

So far, however, the microscopic and DNA identification of food remains in the gu<sup>t</sup> contents have been limited to large-size organisms such as fish and benthic macroinvertebrates as gu<sup>t</sup> contents extraction is di fficult to perform. In the case of zooplankton, crustaceans, with relatively large body size (usually larger than 1 mm) and a hard exoskeleton structure, such as a carapace, which covers the digestive organs, have been the main target for food source analysis. Their morphological characteristics allow physical and chemical treatments, as well as dissection to extract gu<sup>t</sup> contents, avoiding DNA fragments from microorganisms attached to their bodies and DNA from the predator itself. In practice, diets analyses of copepods (small crustaceans) using the DNA-based methods were conducted in both freshwater and ocean ecosystems [21,22]. On the other hand, since small rotifers (usually < 0.5 mm) are relatively soft-bodied, it is di fficult to apply similar physical and chemical treatments as for other zooplankton, and there are no proper methodologies and su fficient information of rotifer food sources as results [23]. For a wide range of applications of DNA technology in food source identification, it is necessary to develop a method for separating gu<sup>t</sup> content items from an object by minimizing other possible DNA contaminants, no matter how small the target size is.

For applications of DNA technology to the identification of rotifers food sources, the most critical part of methodology is to distinguish the DNA in the rotifers gu<sup>t</sup> contents from contamination sources that can be attached to the outside of the rotifers lorica and exist in the sample water (so-called 'extracellular DNA'). Since the detection of extracellular DNAs can cause confusion in the interpretation of the results of the rotifers gu<sup>t</sup> contents analysis, treatment for eliminating them (so-called 'pretreatment') is necessary to obtain the more accurate results of rotifers gu<sup>t</sup> contents analysis. However, unlike crustacean zooplankton, which have a solid carapace, the rotifer body is covered by a lorica, which is relatively softer than the carapace. In addition, lorica hardness is di ffers by species [24].

In this study, we focused on the establishment of an appropriate method for detecting the DNA of gu<sup>t</sup> contents, which is applicable for soft-bodied rotifers. In this analysis procedure, it is important to eliminate the cells and DNA fragments of microorganisms attached to rotifers in order to extract and analyze only those food sources included in the gu<sup>t</sup> to eliminate extra-cellular DNA contamination. Therefore, we selected chemicals for eliminating di fferent types of extracellular DNA and tested their effects on the lorica of rotifers under di fferent concentration treatments to find the most e ffective

concentration and time for both the preservation of the rotifers and removal of different types of extracellular DNA. Following this, we tested the applicability of gu<sup>t</sup> content analysis to rotifers using DNA technology by verifying whether the DNA fragments of rotifer food sources were eliminated or not when the prescribed treatment method was used.
