Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers

Abstract

We designed an experiment to analyze the gut 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 gut 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 gut contents analysis of more diverse rotifers species and better DNA analysis techniques by supplementing more rigorous limitations.

Application of Next-Generation Sequencing for the Determination of the Bacterial Community in the Gut Contents of Brackish Copepod Species (Acartia hudsonica, Sinocalanus tenellus, and Pseudodiaptomus inopinus)

“…Individuals of A. hudsonica , S. tenellus , and P. inopinus were sorted under a dissecting microscope and stored separately in glass vials filled with 60% ethanol to prevent cross-contamination among individuals ( n = 3, respectively). The exoskeleton of each copepod individual was exposed to commercial bleach diluted 2.5% for 2 min and then washed three times with distilled water to avoid any effects from the bleach remaining on the skeleton [ 22 ]. Pre-treated copepod individuals were stored individually in 2 mL microtubes.…”

“…This method has been used for large species such as Calanus (2–3 mm of prosomal length); however, it is difficult to apply it to small-sized species [ 20 , 21 ]. Although the DNA analysis methods of the gut contents of small zooplankton (rotifers, under 400 μm) have been proposed [ 22 ], zooplankton species-specific differences, such as exoskeleton hardness, must be considered, and methods that can be applied to small-sized copepod species (700 μm–1 mm of prosomal length) are still required [ 23 ].…”

Changes in Antibiotic-Resistance Genes Induced by the Grazing Effect in Three Cladoceran Species

“…Each was stored separately in glass vials filled with 60% ethanol to prevent cross-contamination among individuals before DNA extraction to eliminate extracellular DNA attached to the exoskeleton. The exoskeleton of each individual was exposed to commercial bleach diluted 2.5% for 2 min and then washed three times with distilled water to avoid any effects from the remaining bleach on the skeleton [ 22 ]. Pretreated cladoceran individuals were stored individually in 2 mL microtubes (total: 80 samples).…”

First Gut Content Analysis of 4th Instar Midge Larvae (Diptera: Chronomidae) In Large-Scale Weirs Using a DNA Meta-Barcoding Approach

“…However, most gut content-analysis studies based on MI have the following disadvantages: (1) ambiguous prey specimen identification because of extensive digestion, (2) the presence of unidentified partial tissues, (3) identification failure due to a lack of expert knowledge, and 4) low-level identification resolution (identification of levels only higher than the family or order level). In addition, MI is unsuitable for tiny predators, such as a Chironomidae larvae and rotifers [17,18]. Applying DNA sequence-based techniques to gut-content identification has recently increased identification resolution, particularly in marine ecosystems [19][20][21][22][23][24].…”