# Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers
## 4 Discussion  
As a chemical for pretreatment to remove external DNA on the lorica, we selected commercial bleach, which can be used on samples through the proper combination of bleach concentration and exposure duration time (seconds). Commercial bleach has been used mainly to prevent or eliminate contamination in DNA analyses (Table 1), but at the same time, it affects the body tissue of zooplankton, which can lead to the disintegration of rotifer loricas, and thus the release of rotifers' gut contents [33; 35]. In this study, it was found that each rotifer species showed different response times when treated with commercial bleach at the same concentration, and the duration time for lorica survival differed by its characteristics (Table 3); the shortest time was for _Asplanchna_ sp., having the softest lorica and the longest time for _Trichocrea_ sp. and _Bnachionus_ sp. having lorica that are not easily damaged [49]. In the case of the genus _Keratella_, although its lorica has been suggested as a hard cover, which can be protective against mechanical interference by daphnids and predation by invertebrate predators [36], the loss of inner contents occurred through the mouth parts and not through lorica disintegration following the commercial bleach treatment. Therefore, based on the response time observed for _Asplanchna_ sp. showing the shortest response time for disintegration after treatment with 2.5% diluted commercial bleach, we set the pretreatment time to 210 s, as this is an appropriate standard pretreatment method universally applicable to all rotifer taxa. After applying this pretreatment method to raw water samples from the reservoir, it was confirmed that DNA fragments of rotifer food sources detected in non-treated samples were completely removed; Chlorophyceae, Diatomea, Cyanobacteria, and Ciliophora, except for bacteria (Figure 3, N, T1, T2). Further sequencing analyses indicated that the bacteria detected were mainly _Bacillus_ sp. which is known to be tolerant and survive various removal treatments such as disinfection [50] (Table 4). _Bacillus_ sp., gram-positive bacterium, has commonly been found in soil and other environments. It has been reported that _Bacillus_ plays important roles in the lysis of bloom-forming blue-green alga and the control of their biomass in aquatic ecosystems [51; 52]. Therefore, when we applied the pretreatment determined from this study to DNA analysis of gut content of rotifers, we cannot distinguish the source origins of bacteria detected in rotifer species, whether they came from contamination, water samples, or rotifer gut content, like the results of _Asplanchna_ (Figure. 4D, A1-3). Since bacteria is one possible main food source for rotifers [53], a suitable pretreatment method for eliminating extracellular bacterial DNA should additionally be developed. In the results based on DNA analysis, we used each group-specific primer for detection of targeted groups to confirm their presence/absence. As far as we know, there is no information about HNF-specific primer [54], so we applied instead a universal primer set for eukaryotes (Euk-A and Euk-B) which has been used to detect HNF (Table 2). We, therefore, carried out an additional experiment to define the applicability of the HNF primers set to rotifers. As a result, this primer set amplified all possible rotifer species from our study site except for _Asplanchna_ sp. (Figure A1). These results provide a proper explanation for why the primer set did not work for all our samples (Figure 4F). The usage of the primer sets that act specifically for each targeted biological community can help in improving detection accuracy for a targeted species group. However, there remain some limitations in verifying the effectiveness of a determined pretreatment on biological communities where specific primers have not yet been developed, such as HNF. In spite of these limitations, the results of the applied pretreatment method to _Asplanchua_ sp. showed that specific food sources were detected in the gut content.