# Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers
## 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, 5, 6]. In addition, as the problem of eutrophication increases in aquatic ecosystems, the abundance of macrozooplanktondecreases 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 great 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; 9; 10; 11; 12]. These limitations were attributed to the absence of adequate analytical methodologies applicable to field sites due to difficulties in culturing, handling and identification of both prey and predator (rotifers) which have small sizes (usually rotifers body size\(\leq\) 1000 \(\upmu\)m; rotifers prey size spectrum\(<\)1-20 \(\upmu\)m) [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 gut 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; 18; 19; 20]. So far, however, the microscopic and DNA identification of food remains in the gut contents have been limited to large-size organisms such as fish and benthic macroinvertebrates as gut contents extraction is difficult 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 gut 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].