Evaluation and optimization of an eDNA metabarcoding assay for detection of freshwater myxozoan communities

Abstract

The environmental DNA (eDNA) metabarcoding approach has become a useful tool for detecting the species diversity of different animal groups, including parasites.Myxozoa (Malacosporea and Myxosporea) represent a unique group of morphologically simplified endoparasites that mainly infest fish and whose diversity remains largely unexplored. Metabarcoding of DNA from the aquatic environment is a promising non-invasive method that allows us to assess myxozoan biodiversity at a given site. This is essential not only for describing myxozoan communities, but also for the development of disease control methods. Using an alignment of 330 sequences, we employed in silico PCR to score primer pairs, designed to amplify the V4 region of the SSU rDNA of different myxosporean sublineages comprising the entire diversity of oligochaete-infecting (freshwater) myxosporeans. We selected eight clade-specific primer sets for metabarcoding, avoiding amplification of DNA from other organisms present in eutrophic freshwaters. The metabarcoding approach used in the analysis of eDNA sediment samples detected a high myxosporean diversity even in small sample volumes (in total 44 OTUs). Furthermore, metabarcoding analysis of myxosporeans in fish tissue samples selected for primer testing revealed 91 different myxosporean OTUs, more than double the number obtained by classical PCR screening and Sanger sequencing with general myxozoan primers and almost seven times higher detection than by microscopic examination. Our results further suggest quantitative sampling requirements for realistic future diversity estimates by comparing OTUs from fish tissue metabarcoding and eDNA samples. The use of specific primer sets enabled the detection of a high proportion of myxosporean reads (63-100%) in all datasets, even in highly eutrophic habitats. This shows our metabarcoding approach as an excellent tool for non-invasive and sensitive detection of myxosporean biodiversity in aquatic sediments, potentially useful for monitoring myxozoan disease agents that threaten economically important fish in aquaculture.

Diversity of myxozoans (Cnidaria) infecting Neotropical fishes in southern Mexico

“…This method increased the myxozoan prevalence detection up to 6 ×, representing 3.7 × more species detected by PCR screening than by microscopy; this represents nearly 1.3 × higher than a recent similar screening of fish tissues from the Czech Republic (2.8 ×). In this same study, a newly developed eDNA metabarcoding assay was shown to capture up to 2.5 × more diversity than PCR and Sanger sequencing in fish tissue 44 . The molecular screening carried in this study represents a useful tool for myxozoan diversity exploration and can be used for preliminary screening of fish tissue, representing a less intensive labor method than microscopy screening and a more accessible/less complex methodology than eDNA metabarcoding.…”

“…However, eDNA metabarcoding assays of myxozoans may provide a closer to reality biodiversity estimation, especially when using fish tissues. Lisnerová et al 44 also demonstrated the effectivity of these assays when applied to sediment samples, as a non-invasive (no fish sacrifice) technique. This could represent an excellent alternative for future myxozoan diversity estimation in Mexico, where many fish species are rare, threatened or endangered.…”