Development of a quantitative polymerase chain reaction assay for the detection of skin fluke Neobenedenia girellae larvae from environmental water

Skin fluke Neobenedenia girellae infection is a chronic problem in marine finfish aquaculture. Current control measures rely on bath treatments, which are labor intensive and cause significant stress to fish. Although there is an urgent need to develop a strategy to prevent this infection, little is known about the distribution of the larval stage of the skin fluke around culture cages. We aimed to develop a Neobenedenia-specific real-time quantitative polymerase chain reaction (qPCR) assay to detect and quantify N. girellae larvae in environmental water. New PCR primers targeting mitochondrial DNA were designed and showed specificity for N. girellae among five capsalid monogeneans known to occur in Japanese waters. A single N. girellae oncomiracidium is estimated to carry 2.2 million copies of mitochondrial DNA, and the qPCR assay reliably detects DNA equivalent to half of that. We used this qPCR to quantify N. girellae larvae from a fish culture site. Analyses of water samples collected inside shaded culture cages of Seriola dumerili revealed lower larval density than that in the unshaded cage. The results indicate the usefulness of this newly developed qPCR assay for monitoring skin fluke larval density and distribution in fish farms.