Development of Molecular Markers for Environmental DNA Detection of the Invasive African Jewel Fish (Hemichromis letourneuxi): A New Monitoring Tool for Loxahatchee National Wildlife Refuge

Species-specific primers and a probe for eDNA detection via traditional PCR agarose gel visualization and qPCR were developed for Hemichormis letourneuxi. Theoretical qPCR detection threshold levels based on standard curve for this species were approximately 0.0002 ng/uL (R2 = 0.90) at a PCR cycling threshold of 28.5-29. There was a positive and significant relationship between fish density and eDNA detection with detection probabilities ranging from 0.32-1.00 depending on fish density. Environmental DNA persisted in controlled tank experiments for up to 24 days post removal of H. letourneuxi with minimal degradation, but between 24 and 31 days DNA concentration and 260/280 optical density readings decreased significantly. The only significant (P = 0.0299) factor influencing DNA persistence in controlled tank experiments for the eight estimated abiotic parameters held over an eight day period was temperature.  Degradation of DNA occurred between 25°C and 33°C. The use of qPCR for eDNA detection along with confirmation from direct sequencing of positive PCR reactions as well as the detection of the first positive sample from natural environments where the species had been reported demonstrate that qPCR detection with the designed primers and probe is a reliable method for the detection of H. letourneuxi when their densities are greater than threshold values and PCR inhibition is minimized.