A Quantitative PCR Assay to Detect Planorbella trivolvis and Biomphalaria havanensis From Catfish Pond Water Samples

Celene M. Slifka1, Geoffrey C. Waldbieser2, Cynthia Ware3, Bradley M. Richardson2, Monica Wood2, Sujita Balami3, Divya Rose3, Charles M. Walker4, Caitlin E. Older2, Charles C. Mischke5, David J. Wise4, Matt J. Griffin3 and Thomas G. Rosser1 

1Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762; 2USDA-ARS Warmwater Aquaculture Research Unit, Thad Cochran National Warmwater Aquaculture Center, Stoneville, Mississippi 38776; 3Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, Mississippi 38776; 4Mississippi Agriculture and Forestry Experiment Station, Thad Cochran National Warmwater Aquaculture Center, Delta Research and Extension Center, Mississippi State University, Stoneville, MS 38776; 5Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, P.O. Box 197, Stoneville, MS 38776

ABSTRACT

Bolbophorus damnificus is a digenean trematode attributed to significant economic losses and farm closures in Mississippi catfish aquaculture. First recognized as a threat in the 1990’s, the parasite causes significant reductions in feeding activity and drastically limits growth. In addition, severe outbreaks can cause direct losses due to mortality in the fingerling and stocker stages of production.  The life cycle involves three hosts: the American White Pelican (Pelecanus erythrorhynchos), a snail intermediate host (Planorbella trivolvis or Biomphalaria havanensis) and an Ictalurid catfish.  Previous work indicates that channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel x blue catfish hybrids are equally susceptible to infection by Bolbophorus damnificus. Control methods involving bird harassment are logistically challenging and labor intensive.  As a result, current management practices focus on chemical eradication of the snail host through pondside applications of copper sulfate or hydrated lime. However, both of these chemotherapeutants have limitations. Copper sulfate can be highly toxic to fish at high temperatures and the phytotoxicity of copper can kill beneficial algal blooms, leading to catastrophic oxygen depletions.  Similarly, hydrated lime is limited in efficacy as it requires snail contact to be effective and has little effect on snails not present in the pond margins at the time of application.  As a result, alternative molluscicides are currently being explored.  Together, researchers at the Mississippi State University College of Veterinary Medicine and scientists at the USDA Warmwater Aquaculture Research Unit have sequenced the mitochondrial genomes of these two problem snails (P. trivolvis and B. havanensis) and identified unique regions suitable for discriminatory quantitative PCR analysis (qPCR). Primers and probes have been developed to target the cytochrome oxidase subunit 1 region for each species. The end goal is to use an eDNA/qPCR approach to estimate snail densities in catfish ponds and identify optimal treatment regimens (dose/# of applications/etc.) for current therapeutics (copper sulfate and lime).  Another application of this assay will evaluate the efficacy and utility of copper and lime alternatives.  Lastly, this assay also provides a molecular confirmatory test to support snail identification, which is an invaluable research tool.  This assay will greatly aid current research developing best management practices to mitigate the negative impacts of B. damnificus and other digenetic trematodes on catfish production in the southeastern United States.