Development of specific detection assays from environmental DNA of invasive diatom Cymbella janischii in Japan

The effect of invasive species on existing biological systems has become a major concern in recent years. Countermeasures for invasive species were among the goals of the United Nations Biodiversity Conference (COP15). Cymbella janischii is an alien diatom that recently invaded Japan. Its algal blooms not only spoil the scenery of the river but also raise concerns about its impact on the ecosystem, including damage to fish populations. To monitor the invasion of this species and employ river management, we developed the specific detection assays for this species from environmental DNA, using a polymerase that distinguishes a single base difference at the 3' end of the primer. Three assays for amplifying nuclear-encoded large-subunit rRNA genes (P1, P2, and P3) successfully detected C. janischii without false positives in benthic samples from 12 rivers in Japan. There were no significant differences among the results obtained using the three assays. The three assays showed significant positive correlations with the microscopic observation results, having the advantage of detecting species with low population density. These assays can be performed using inexpensive equipment because they do not necessarily require real-time PCR. However, it can enhance specificity by adding a fluorescent probe to the internal sequence, if necessary. These detection assays can apply to a wide range of environmental samples and are expected to help elucidate the invasive characteristics and ecology of this species.