**2. Materials and Methods**

### *2.1. Selection of Chemicals for Eliminating Extracellular DNAs*

In order to select the appropriate chemicals to remove the extracellular DNA fragments in detecting DNA of rotifers gu<sup>t</sup> contents, we reviewed the different treatment methods and their procedures found in the literature (Table 1, Table A1).


**Table 1.** Summary of previous treatment processes for decontamination in DNA analyses.

\* Underlined treatment and procedure: the case of the application of bleach for decontamination

Most previous published treatment methods were not suitable for the selective elimination of external DNA, which is required for gu<sup>t</sup> content analysis of rotifers, due to their soft lorica. Therefore, we selected ethanol and commercial bleach to remove extracellular DNA from the rotifers, while maintaining the internal gu<sup>t</sup> content DNA composition inside the rotifers. Ethanol (Ethyl alcohol; CAS No. 64-17-5) was used for preservation and sterilization of the raw water sample. Accordingly, the raw water sample was fixed with ethanol to a final concentration of 60% (typical concentrations for disinfection and sterilization: 60–95% [34]). Preserving rotifers with ethanol would limit the exposure to damage of the rotifer lorica by the action of commercial bleach, which has previously been used for DNA elimination and extraction of gu<sup>t</sup> contents in zooplankton, only externally [33,35]. Yuhan-Clorox (Yuhanrox regular) (Yuhan Co, Ltd., Korea) composed of 4%–6% NaClO (CAS No. 7681-52-9) and 0.1%–0.5 % NaOH (CAS No. 1310-73-2) was used for the chemical wash treatment.

### *2.2. Responses of Rotifers Lorica to Bleach*

To find a suitable treatment time and concentration of commercial bleach for extracellular DNA removal without affecting rotifer lorica and gu<sup>t</sup> contents, we measured the response time of different rotifer species, which were collected from a water reservoir, to different exposure concentrations. We minimized contamination by separating each sample, using bleach sterilized gloves, and instruments sterilized by autoclave and ethanol. As testing the response time and range of commercial bleach concentrations requires multiple individuals of each rotifer species, we targeted large species in which at least three individuals can be gathered by sorting, *Brachionus forficula*, *Keratella* sp., *Trichocerca* sp., *Polyarthra* sp., and *Asplanchna* sp. having variable lorica characteristics; from species having soft lorica (e.g., *Asplanchna* sp.) to hard lorica (e.g., *Keratella* sp. and *Polyarthra* sp.) [36]. Each rotifer species was treated using commercial bleach with final concentrations of 20%, 10%, 5%, and 2.5%. We measured the time until lorica disintegration of the three individuals per rotifer species through microscopic inspection (OLYMPUS CKX41). These time results were used as baselines to determine the concentration and time of removing extracellular DNA without damaging the rotifer individuals (Figure 1).

**Figure 1.** Diagram of necessity of proper pretreatment process in rotifers gu<sup>t</sup> contents analysis; (**A**) Without treatment for removing contaminants and other detectable DNA including extracellular DNA, there is possibility to be confused that detected DNA is from the rotifers gu<sup>t</sup> contents or not, (**B**) Through proper treatment, contaminants and other detectable DNA can be removed without damaging the rotifer individuals, (**C**) and (**D**) Rotifer individuals can be damaged and their gu<sup>t</sup> contents can be overflowed by excessive treatment according to their lorica characteristics.

### *2.3. Application and E*ff*ectiveness Verification of Set Treatment Concentration and Time*

We collected water samples from a eutrophic reservoir (Shin-gal reservoir, Korea; N 37.241536, E 127.0929190) in fall (4th November 2018). Rotifers dominated the zooplankton community of the reservoir during this season. We repeatedly filtered 10 L of surface water (nĄ20) into a 60 μm mesh sized zooplankton net and obtained a 1 L filtered water sample. From the collected sample, all organisms were removed by hand using a microscope (OLYMPUS CKX41) and 0.5 mL of subsample was extracted to micro-tubes. For every sample taken, we made a negative control to prevent cross contamination.

For the application and effective verification of a set treatment concentration and time on the DNA fragments of rotifer food sources (Chlorophyceae, Diatomea, Cyanobacteria, Bacteria, Ciliophora, and Heterotrophic nanoflagellates) [9], we compared both treated and non-treated samples with 0.5 mL filtered water samples. In the case of treated sample, after set treatment time, we poured and filtered the sample immediately by washing with distilled water to prevent further effects. Non-treated sample was also filtered in order to proceed with the same DNA extraction process as the treated sample.

To confirm detection of DNA in the rotifers gu<sup>t</sup> without extracellular DNA using the suggested treatment method, we collected a rotifer species, *Asplanchna* sp., from reservoir water, and applied this method to the treatment. We sorted *Asplanchna* specimens from the water sample and transferred them to distilled water several times until pure rotifer individuals were collected without other visible particles, particularly phytoplankton cells. We then checked for removal of particles under the microscope and selected clean individuals without attached particle or microorganisms (one individual per a sample, 3 replicates). As with water samples, rotifers samples were filtered after pretreatment for extraction of their DNA. The 47mm diameter cellulose nitrate filters with a pore size of 0.45μm pore size (NC 45 ST, WhatmanTM) were used to filter DNA fragments [37].
