**Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers**

### **Hye-Ji Oh 1, Paul Henning Krogh 2, Hyun-Gi Jeong 3, Gea-Jae Joo 4, Ihn-Sil Kwak 5,6, Sun-Jin Hwang 1, Jeong-Soo Gim 4, Kwang-Hyeon Chang 1,\* and Hyunbin Jo 6,\***


Received: 29 December 2019; Accepted: 26 January 2020; Published: 5 February 2020

**Abstract:** We designed an experiment to analyze the gu<sup>t</sup> content of Rotifera based on DNA barcoding and tested it on *Asplanchna* sp. in order to ensure that the DNA extracted from the rotifer species is from the food sources within the gut. We selected ethanol fixation (60%) to minimize the inflow effects of treated chemicals, and commercial bleach (the final concentration of 2.5%, for 210 s) to eliminate the extracellular DNA without damage to the lorica. Rotifers have different lorica structures and thicknesses. Therefore, we chose a pretreatment method based on *Asplanchna* sp., which is known to have weak durability. When we used the determined method on a reservoir water sample, we confirmed that the DNA fragments of Chlorophyceae, Diatomea, Cyanobacteria, and Ciliophora were removed. Given this result, Diatomea and cyanobacteria, detected from *Asplanchna*, can be considered as gu<sup>t</sup> contents. However, bacteria were not removed by bleach, thus there was still insufficient information. Since the results of applying commercial bleach to rotifer species confirmed that pretreatment worked effectively for some species of rotifers food sources, in further studies, it is believed to be applicable to the gu<sup>t</sup> contents analysis of more diverse rotifers species and better DNA analysis techniques by supplementing more rigorous limitations.

**Keywords:** gu<sup>t</sup> content of Rotifera; eliminate the extracellular DNA; commercial bleach; pretreatment
