Mioduchowska Monika, Czyż Michał Jan, Gołdyn Bartłomiej, Kilikowska Adrianna, Namiotko Tadeusz, Pinceel Tom, Łaciak Małgorzata, Sell Jerzy
Department of Genetics and Biosystematics, Faculty of Biology, University of Gdansk, Gdansk, Poland.
Research Centre of Quarantine, Invasive and Genetically Modified Organisms, Institute of Plant Protection-National Research Institute, Poznan, Poland.
PeerJ. 2018 Dec 14;6:e6039. doi: 10.7717/peerj.6039. eCollection 2018.
Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.
水生无脊椎动物的细菌内共生体仍未得到充分研究。这至少部分是由于缺乏合适的鉴定技术和引物。我们设计了一对非简并引物，能够从各种已知的细菌内共生体物种中扩增出约500 bp的16S rRNA基因片段。通过这种方法，我们在来自广泛地理区域的临时水域中的7种分类学上不同的淡水甲壳类宿主中鉴定出4种细菌内共生体、2种内寄生虫和1种未培养细菌。我们新的WOLBSL和WOLBSR引物扩增细菌16S rRNA基因的总体效率为100%。然而，如果同时扩增一个样本中的不同细菌物种，尽管PCR产物质量良好，但序列仍难以辨认。因此，我们建议在细菌内共生体鉴定的第一阶段使用我们的引物。随后，推荐使用属特异性引物。总体而言，在下一代测序时代，我们的方法可以作为一种简单且低成本的初步方法，通过简单的桑格测序来鉴定与淡水甲壳类相关的潜在微生物共生体。以这种方式检测各种无脊椎动物宿主中细菌共生体的潜力将有助于宿主 - 共生体相互作用和协同进化的研究。
