A “Snail” of a Tale: qPCR Detection of Two Intermediate Hosts of Bolbophorus damnificus From Mississippi Catfish Pond Water Samples

1Celene M. Slifka, 2Bradley M. Richardson, 2Monica Wood, 3Divya Rose, 3Sujita Balami, 3Cynthia Ware, 2Geoffrey C. Waldbieser, 2Caitlin E. Older, 4Charles M. Walker, 5Charles C. Mischke, 4David J. Wise, 3Matt J. Griffin and 1Thomas G. Rosser

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, MS 38776; 3Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS 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 trematode linked to major economic losses and farm closures in Mississippi catfish aquaculture. This calamitous parasite can lead to reductions in feeding activity and significantly limit growth. Severe outbreaks can cause direct losses in the fingerling and stocker stages of production. Three hosts are required in the indirect lifecycle: the American White Pelican (Pelecanus erythrorhynchos), a snail intermediate host (Planorbella trivolvis or Biomphalaria havanensis) and an ictalurid catfish. Control efforts through bird harassment are logistically challenging and labor intensive. Current management practices focus on chemical eradication of the snail host through pondside applications of copper sulfate or hydrated lime; but both have limitations. Copper sulfate can be highly toxic to fish at high temperatures and the copper phytotoxicity can kill beneficial algal blooms, leading to catastrophic oxygen depletions. Similarly, hydrated lime requires snail contact to be effective with little efficacy on snails not present in the pond margins at application time. 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). Sensitive and specific primers and probes have been developed to target the cytochrome c oxidase subunit 1 gene region for each species. The qPCR assays have been shown to be repeatable and reproducible. Both assays showed a linear dynamic range covering 6 orders of magnitude and a theoretical sensitivity limit of 30 copies of target DNA in a 30-mL reaction. Additionally, consistent amplification of both assays to their respective targets with no observed amplification from the non-target and naturally occurring snail, Physa sp., demonstrated the stability of the PCR target. Under the conditions used in the study, the clinical sensitivity is 8 snails in 250 gallons of water. In a 10-acre pond at 4 feet deep, this equates to 400,000 snails. The end goal is to use an eDNA/qPCR approach to estimate snail densities in catfish ponds and identify optimal treatment regimens (dosage and number of applications) for copper sulfate and lime. Additionally, this assay will be employed to provide a molecular confirmatory test for snail identification, an invaluable research tool. This assay will significantly aid current research in developing better management practices to mitigate the impacts of digenetic trematodes on catfish production in the southeastern United States.