Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

Running Head: Surveillance of protected fish

Kayla M. Fast¹, Anakela Popp², Patrick E. O’Neil³, Stuart W. McGregor³, Michael W. Sandel¹

¹Department of Biological and Environmental Sciences, the University of West Alabama, Livingston, Alabama, USA 

²Georgia Department of Natural Resources, Wildlife Resources Division, Social Circle, GA, USA

³Geological Survey of Alabama, Tuscaloosa, AL, USA

Correspondence: Kayla M. Fast and Michael W. Sandel, Department of Biological and Environmental Sciences, the University of West Alabama, Livingston, Alabama, USA. Email: kfast@uwa.edu (KMF) and msandel@uwa.edu (MWS)

ABSTRACT

Environmental DNA (eDNA) has increasingly been used in the surveillance of imperiled aquatic species. Despite recent efforts in drawing genetic material from the environment, there are still pitfalls surrounding this field. We created a novel protocol which implements loop-mediated isothermal amplification (LAMP) to detect target DNA. Our methods are applied here in the surveillance of Etheostoma trisella , the Trispot Darter, a freshwater fish that recently received protection under the U.S. Endangered Species Act. Water samples (n = 256) were collected at sites in Alabama and Georgia to determine whether E. trisella still occupies historic sites and whether it inhabits previously unknown areas. We found evidence of E. trisellapresence in 69 water samples while 187 were negative. Our LAMP protocol is capable of amplifying low quantities of DNA in the water, and is a robust technique for freshwater species surveillance. Verification of positive results from eDNA experiments is essential to confirm reaction reliability. Application of methods such as ours are necessary for recognizing species under threat that require conservation.

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