Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers

Hye-Ji Oh,Paul Henning Krogh,Gea-Jae Joo,Hyunbin Jo,Jeong-Soo Gim,Kwang-Hyeon Chang,Ihn-Sil Kwak,Sun-Jin Hwang,Hyun-Gi Jeong

doi:10.3390/app10031064

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.

Highlights

It is important to understand the role and function of interactions in the microbial food web of aquatic ecosystems
The lorica of Asplanchna 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
It was found that each rotifer species showed different response times when treated with commercial bleach at the same concentration, and the duration time for lorica survival differed by its characteristics (Table 3); the shortest time was for Asplanchna sp., having the softest lorica and the longest time for Trichocerca sp. and Brachionus sp. having lorica that are not damaged [49]

Summary

Introduction

It is important to understand the role and function of interactions in the microbial food web of aquatic ecosystems. Rotifers are critical components linking microorganisms with larger predatory organisms such as crustaceans and fish within the grazing food chain: bacteria, heterotrophic nano-flagellates, rotifers/copepods/cladocerans, larval fish, mature fish [2,3]. They function as a channel for the flux of organic matter within diverse organism assemblages organized in an intermediate position between the two different food webs, and transfer nutrients and energy from the microbial loop to higher trophic levels [4,5,6].

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Conclusion