**3. Results**

### *3.1. Responses of Rotifers Lorica to Commercial Bleach Treatment*

After treatment with commercial bleach at final concentrations of 20%, 10%, 5%, and 2.5%, the time before the loss of each rotifer's contents by lorica disintegration (five rotifers species tested, <sup>n</sup>=3) was measured. Every tested rotifer species, *Brachionus forficula*, *Keratella* sp., *Trichocerca* sp., *Polyarthra* sp., and *Asplanchna* sp. tended to have shorter times for tolerating treatment as the final concentration of commercial bleach increased. In particular, *Asplanchna* sp. having the weakest lorica showed the shortest time among tested rotifers regardless of treatment concentration. The lorica of *Asplanchna* disintegrated between 35 s and 240 s following exposure to different treatment solutions of various concentration, and its body contents including the gu<sup>t</sup> contents were released from the body. When *Asplanchna* was treated with 2.5% diluted commercial bleach, although it was observed to withstand up to 300 s of exposure, its lorica began to suffer disintegration after 240 s. Therefore, for preservation of its gu<sup>t</sup> contents, the treatment time should be considered as less than 240 s. Other tested rotifer species also showed different duration times for lorica survival against treatment solution depending on its concentration. However, regardless of their lorica thickness and structure, they showed a range of endurance time from 300 to 450 s at 2.5% of commercial bleach (Table 3, Figure 2).


**Table 3.** The responses of rotifers lorica to commercial bleach treatment; the minimum time (s) before the loss of the rotifers contents by lorica disintegration of each rotifers species.

**Figure 2.** An example of a disintegration process; response of *Asplanchna* sp. lorica to commercial bleach treatment (2.5 %).

To prevent loss of gu<sup>t</sup> contents during pretreatment for extracellular DNA by disintegration of rotifers lorica, we should establish the conditions (concentration and time of chemical) under which extracellular DNA can be removed, and keep the rotifer lorica undamaged. Since high concentration treatments allowed very limited time available for a ffecting the elimination of extracellular DNA, we decided to use the lowest concentration of commercial bleach for the longest time on samples in order to minimize damage while maximizing external DNA removal. Therefore, based on the response time of *Asplanchna* sp. lorica to the lowest concentration of commercial bleach treatment and consequent its shortest duration time examined by the experiment, rotifer specimens for extracting gu<sup>t</sup> contents DNA were treated with 2.5% diluted commercial bleach for 210 s (Table 3). We observed each treatment process through a microscope, and confirmed that the gu<sup>t</sup> contents of rotifers were likely to be released from the body when their lorica began to disintegrate. Therefore, we judged that it would be appropriate to use commercial bleach for removal of the extracellular DNA up to 30 s before the time when rotifer loricas begin disintegrating. In addition, to maximize treatment time while minimizing internal e ffects of the treatment by fixing rotifers, we determined that preservation with 60% ethanol soon after the samples are collected and treatment of 2.5% diluted commercial bleach for 210 s was the most e ffective pretreatment (Figure 3). We, therefore, selected *Asplanchna* sp. for our further experiment. In addition, *Asplanchna sp.* have a typical omnivore feeding behavior according to Chang et al. (2010) [48]. Therefore, it is an ideal experiment creature for this study.

**Figure 3.** Experimental designs. ( **A**) Verifying applicability of pretreatment, (**B**) Application of pretreatment to rotifers.

### *3.2. Application and E*ff*ectiveness Verification of Set Pretreatment Concentration and Time*

When the electrophoresis results of raw water from reservoir (non-treated water sample), treated water and treated rotifers samples were compared, they showed different bands in each gel. In the non-treated water sample, the primers used to detect various regions of genetic sequences were all amplified and detected as bands in the electrophoresis gel (Figure 4A–E,N). As a result of identifying the dominant signal information of sequences by the direct capillary sequencing method through BLASTn, all dominant signal identified in non-treated water samples were of Chlorophyceae, Diatomea, Cyanobacteria, Bacteria, and Ciliophora, which are known as common food sources of rotifers (Table 4; Non-treated water sample).

**Figure 4.** Electrophoresis detection results, (**A**) 18s rRNA for detecting Chlorophyceae; (**B**) 18s rRNA; V4; (**C**) 16s rRNA; ITS; (**D**) 16s rDNA; (**E**) 18s rRNA for detecting Ciliophora and HNF, heterotrophic nanoflagellates; N: non-treated water sample (raw water; control); T: treated water sample by ethanol (60%) and 2.5% commercial bleach solution; A: treated *Asplanchna* samples (n=3); D: distilled water (negative control); first lane of each gel: Ladder using 100-bp molecular marker.

**Table 4.** The summary of detected dominant signal information based on the direct capillary sequencing and cloning (identity %).


\* The eukaryote universal primer, which was used to detect HNF (heterotrophic nanoflagellates), has detected Ciliophora (*Tintinnidium* sp.; 88%).

On the other hand, food sources identified in non-treated water sample, except for bacteria were not detected after the treatment of 2.5% diluted commercial bleach for 210 s, indicating their DNA fragments were eliminated by our selected treatment method (Figure 4A–E, T1~T2). Based on these results, we treated same process on *Asplanchna* specimen sorted from the reservoir for verifying if this pretreatment is proper to apply to rotifer species. Most identified species in non-treated water sample were not detected as gu<sup>t</sup> contents in *Asplanchna*. However, Choanoflagellates, fungi species (*Meira*

sp.), and bacteria species (*Bacillus* sp.) were detected in some individuals. These species identified in treated *Asplanchna* samples seemed to have been detected by eliminating the signals that were strongly captured from the various DNA fragments that existed before the pretreatment. It means that selected commercial bleach as a pretreatment chemical and specified its concentration and exposure time properly can facilitate the removal of extracellular DNA fragments simultaneously with preserving rotifer body tissue, and consequently this process can be applied for detecting DNA of rotifers gu<sup>t</sup> contents without fear of extracellular DNA contamination (Figure. 4A–E, A1~A3).

Unfortunately, it was difficult to interpret the detected band of bacteria in *Asplanchna* specimen as gu<sup>t</sup> contents, because bacteria were not completely eliminated by the treatment of 2.5% diluted commercial bleach. The sequences from detected bands in the electrophoresis gel let us know that bacteria in non-treated and treated samples are species included in genus *Bacillus* (Table 4; Treated *Asplanchna* sample).
