Working Primers and qPCR Protocols for Rapid eDNA Identification of Four Aquatic Invasive Species Found in the Lower Great Lakes with High Potential for Ballast Transport to Lake Superior

Reliable, timely and economical target organism detection in harbors and ballast water is urgently needed to prevent the spread of aquatic invasive species (AIS) by commercial ships in the North American Great Lakes (NAGL). Inter-Great Lake ships (Lakers) transport large volumes (ca. 52 million metric tons. annually) of untreated lake water between lakes, with over 50% transported against the natural flow from the lower lakes to Western Lake Superior ports. The transport of ballast water is the number one threat of AIS spread throughout the NAGL. A relatively new tool to fight the spread of AIS is the use of eDNA for rapid detection and identification of target organisms. This technology opens doors for advancing control of ballast-mediated AIS through rapid detection. To that end, we have developed species-specific, reliable eDNA primers to target specific detection of four AIS in water samples along with qPCR protocols. Target organisms were selected based on the following criteria: (1) they are known to be invasive in the lower NAGL, (2) they are established in the lower NAGL but not in Superior, (3) they are biodegradable, and (4) they are obtainable, morphologically distinct and have existing DNA sequence information. Working primers, qPCR protocols and detection limits are provided for three invertebrate species and one alga species. These species are Daphnia lumholtzi (a water flea), Cercopagis pengoi (the fishhook water flea), Echinogammarus ishnus (a scud) and Nitellopsis obtusa (Starry Stonewort).