The goal of this research is to develop a multiplex qPCR assay to detect the se three protozoans in the water column of aquarium environments. The ciliate, Cryptocaryon irritans, is a common fish parasite that causes marine white spot disease, otherwise known as “Marine Ich”, in both natural and aquarium environments. The life cycle of C. irritans involves three separate stages that allow for quick infection and mortality of aquarium fish. The most notable stage involves the trophont, which produces the signature “white spot” on infected fish. The theront, a free-swimming stage of C. irritans , can survive for approximately 48 hours in the water column. After this time, it either dies or infects a host fish. Uronema marinum is an opportunistic parasite that causes infection and mortality in both natural and aquarium environments. Infection of U. marinum causes ulceration of the skin and gills, and eventually infiltration of the musculature, skin, and central nervous system. Infection with the dinoflagellate Amyloodinium ocellatum is visible by brown spots on the skin and gills of affected fish. This parasite has caused significant losses of aquarium fish in both public and home aquaria. Amyloodiniosis (the disease caused by A. ocellatum) is commonly referred to as “Marine Velvet”. This protozoan has a similar life cycle to that of C. irritans, differing only with the length of time that the dinospores (equivalent to theronts) can remain free-swimming (can survive for 15 days without a host).
All three protozoans have a stage in which they are free-swimming in the water column. Identifying increases in abundance of these parasites in the water column of aquarium tanks would provide time for preventative/treatment methods to occur before morbidity and mortality occurs . Currently, a successful TaqMan qPCR assay has been developed for C. irritans using previously designed primers from Taniguchi et al (2011) and a newly designed fluorescent probe. This specific assay has been able to produce an estimated organism number for each sample tested . A TaqMan qPCR assay for U. marinum is in development using newly designed, validated primers and a fluorescent probe. Primers are currently in development for A. ocellatum for use in the multiplex TaqMan qPCR assay with the other two protozoans. These primers and probes will be added to the C. irritans qPCR for simultaneous detection of the three parasites in the water column of aquarium systems.