European flat oysters (Ostrea edulis ) are a commercially valuable species that have a 90% mortality rate when infected by the parasite Bonamia ostreae. Oysters infected with B. ostreae were relayed briefly in the Limfjorden in Denmark , which was followed by a series of ice winters between 1980 and 1990 and subsequently by an increase in oyster populations. S urvey programs conducted in the Limfjorden declared it free of B. ostreae from 1996 to 2014 . Similar phenomena were observed in USA and Norway, where a low prevalence of the parasite was seen following below zero temperatures .
To understand how extreme low temperatures affect B. ostreae, a temperature challenge experiment was conducted. This study aimed to assess the efficacy of non-invasive e nvironmental DNA (eDNA) sampling for parasite detection in close water systems and to measure the amount of B. ostreae released at ice winter temperatures.
Sets of Bonamia -positive and Bonamia-negative oysters were individually incubated at either -1°C or 12°C for a period of one, two or three months. Each set was then incubated in groups at 20°C for two months to stimulate larvae production. Hemolymph , water samples and passive e DNA sensors were collected for the quantification of B. ostreae with qPCR.
5.56% (two individuals ) of the infected oysters from the -1°C treatment survived , in contrast to 0% in the infected 12°C treatment groups . These surviv ing oysters also showed no detectable levels of B. ostreae at the end of the experiment. B. ostreae was detectable in water samples and passive sensors deployed in the tanks of the oysters. Uninfected oysters in the 1-month cold treatment group produced the highest number of larvae amongst all the groups.
These findings offer an insight to the host-parasite dynamics between flat oysters and B. ostreae in the context of a warming climate. T he eDNA quantification techniques can be applied in other hatcheries and be further tested o n open sea sites.