# Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers
## 3 Results
### Application and Effectiveness 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).  
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 _Asplanchma_ 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 gut contents in _Asplanchma_. However, Choanoflagellates, fungi species (_Meira  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Samples.** & **18s rRNA** & **18s rRNA; V4** & **165 rRNA; ITS** & **165 rDNA** & **185 rRNA** \\ \hline \multirow{3}{*}{Non-treated} & _Chlamyakumous nivials_ & _Aulacoseira granulate_ & & & \\  & (99\%) & (100\%) & _Chlorophyta sp._ & _Bacillus cereus_ & _Tintrinidium fircuilite_ \\  & _Vitrochlamps_ & _Aulacoseira amliguau_(99\%) & (98\%) & (81\%) & (90\%) \\ \hline \multirow{3}{*}{Treated} & _Chlamyakalte_ & & & & \\  & Not-detected & (97\%) & Not-detected & _Bacillus thuringiensis_ & Not-detected \\ \cline{1-1}  & & (99\%) & & (85\%) & Not-detected \\ \hline \hline \end{tabular}  
* The eukaryote universal primer, which was used to detect HNF (heterotrophic nanoflagellates), has detected Ciliophora (_Tintrinidium_ sp.; 88%).  
\end{table}
Table 4: The summary of detected dominant signal information based on the direct capillary sequencing and cloning (identity %).  
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 _Asplanchma_ samples (n=3); D: distilled water (negative control); first lane of each gel: Ladder using 100-bp molecular marker.  
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 gut 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 gut 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).