# Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers
## 3 Results  
### 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, n=3) was measured. Every tested rotifer species, _Brachionus forficula_, _Keratella_ sp., _Trichocerca_ sp., _Polyarthra_ sp., and _Asplanchua_ sp. tended to have shorter times for tolerating treatment as the final concentration of commercial bleach increased. In particular, _Asplanchua_ sp. having the weakest lorica showed the shortest time among tested rotifers regardless of treatment concentration. The lorica of _Asplanchua_ disintegrated between 35 s and 240 s following exposure to different treatment solutions of various concentration, and its body contents including the gut contents were released from the body. When _Asplanchua_ 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 gut 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). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Food source** & **Primer** & **Sequence (5’-3)** & **Base Pair** & **Ref.** \\ \hline Cyanobacteria & 16S27F & AGA GTT TGA TCC TGG CTC AG & 422 bp & [44] \\ (165 rRNA; ITS *) & 23S30R & CTT GGC CTC TGT GCTG CCT AGG T & [44] \\  & 94 \({}^{\circ}\)C, 5 min -> 10 cycles [94 \({}^{\circ}\)C, 45 \(\rightarrow\) 57 \({}^{\circ}\)C **, 45s -> 68 \({}^{\circ}\)C, 2 min] & \\  & -> 25 cycles [92 \({}^{\circ}\)C, 45s -> 54 \({}^{\circ}\)C **, 45s -> 68 \({}^{\circ}\)C, 2 min] & -> 68 \({}^{\circ}\)C, 7 min & \\ Bacteria & Forward & GAG TTG GAT CGT GGC TGA G & About \\ (165 rRNA; nearly & Reverse & AGG GAGG GAGG ATG CGC CC & 200 bp & [45,46] \\ full-length) & 95 \({}^{\circ}\)C, 3 min -> 35 cycles [94 \({}^{\circ}\)C, 1 min -> 60 \({}^{\circ}\)C *, 1 min -> 72 \({}^{\circ}\)C, 2 min] -> 72 \({}^{\circ}\)C, 3 min & \\  & Cil F & TGG TAG TGT ATT GGA WCA CCA & \\ Ciliophora & 1 & TCT GAT CGT CTT TGA TCC CTT & 600–670 bp & [47] \\ (18S rRNA) & Cil R- & 2 & TCT RAT CGT CTT TGA TCC CFT & \\  & 3 & TCT GAT TGT TGA TCC CCT & \\  & 95 \({}^{\circ}\)C, 5 min -> 35 cycles [94 \({}^{\circ}\)C, 45 s -> 55 \({}^{\circ}\)C *, 1 min -> 72 \({}^{\circ}\)C, 1 min] -> 72\({}^{\circ}\)C, 10 min & \\ Heterotrophic & EukA *** & AAC CIG GTT GAT CGT GCC AGT & 800–900 bp & [38,39] \\ nanotoflagellates & EukB *** & TGA TCC TTC TGC AGG TTC TAC & & [38,39] \\ (18s rRNA) & 95 \({}^{\circ}\)C, 2 min -> 35 cycles [95 \({}^{\circ}\)C, 30s -> 55 \({}^{\circ}\)C *, 30s -> 72 \({}^{\circ}\)C, 2 min] -> 72 \({}^{\circ}\)C, 7 min & \\ \hline \hline \multicolumn{4}{l}{* Internal transcribed spacer; ** Underlined temperature: annealing temperature of each primer; *** EukA and EukB} \\ \multicolumn{4}{l}{are universal primer for eukaryote.} \\ \end{tabular}
\end{table} 
Table 2: _Cont._ \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Concentration** & \multicolumn{5}{c}{**Rotifer Species**} \\ \cline{2-5}
**(Final) and** & \multicolumn{2}{c}{_Brachionus Forficula_} & _Keratella_ sp.** & _Trichocerca_ sp.** & _Polyarthra_ sp.** & _Asplanchua_ sp.** \\ \hline
20\% & 60 s & 90 s & 120 s & 60 s & 35 s \\
10\% & 210 s & 180 s & 150 s & 90 s & 45 s \\
5\% & 300 s & 240 s & 300 s & 210 s & 120 s \\
2.5\% & 450 s & 300 s & 450 s & 300 s & 240 s \\ \hline \hline \end{tabular}
\end{table} 
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.