New metagenomic procedure for the investigation of the eukaryotes present in the digestive gland of Mytillus galloprovincialis

Mussels' aquaculture is one of the most important cultures in Europe, with a high level of self-sufficiency. In Spain, the main cultivated species is M. galloprovincialis, whose production takes place primarily in Galicia. In that region, a Protected Designation of Origin certification exists to certify the high quality of the product and its sustainability. This species is also present in other regions making the control of its traceability complicated. In order to distinguish Galician mussels from the others, the eukaryotes present in its digestive gland could be a useful tool. However, to use them as biomarkers, having an adapted protocol to recover the diversity present in the digestive gland is essential. In this work, we have selected a new protocol to optimize the study of the diversity of eukaryotes associated with the digestive gland of M. galloprovincialis. This protocol is based on a combination of a pre-treatment and a commercial DNA extraction kit, the amplification of DNA with two newly designed primer sets to avoid the DNA from the mussel, and the use of next-generation sequencing. The results showed a significant reduction of mussel DNA amplification and enough DNA quality for the subsequent sequencing, increasing the number of sequences recovered from other eukaryotes. This new protocol offers then a good chance to determine the diversity of eukaryotes present in the digestive gland of M. galloprovincialis, and it can be used in future studies to examinate the seasonal variation of the present species and to evaluate its potential as geographical traceability tool.