All sampling and filtering equipment was exposed to a 10% bleach solution for at least 30 min before use. For water samplings in the aquarium, approximately 10 l of seawater was collected from the surface using multiple casts of an 8 l polyethylene bucket fastened to a 10 m rope. The bucket was thoroughly prewashed with tank water. The sampling was conducted between 10.00 and 13.00 before daily feeding on two consecutive days (2 and 3 June 2014). The sampled water was stored in a valve-equipped 10 l book bottle and immediately brought to the laboratory before subsequent filtering. For water samples from the coral reefs near the aquarium, 10 l of seawater was collected in a similar manner on 4 June and 7 November 2014.
One to three 2 l lots of seawater from the 10 l samples were vacuum-filtered onto 47 mm diameter glass-fibre filters (nominal pore size, 0.7 μm; Whatman, Maidstone, UK). Each filter was wrapped in commercial aluminium foil and stored in −20°C before eDNA extraction. Two litres of Milli-Q water was used as the negative control and treated identically to the eDNA samples, to monitor contamination during the filtering and subsequent DNA extraction.
DNA was extracted from the filters using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) in combination with a spin column (EZ-10; Bio Basic, Markham, Ontario, Canada). After removing the attached membrane from the spin column (EZ-10), the filter was tightly folded into a small cylindrical shape and placed in the spin column. The spin column was centrifuged at 6000g for 1 min to remove redundant seawater for DNA extraction. The column was then placed in a new 2 ml tube and subjected to lysis using proteinase K. Before lysis, Milli-Q water (400 μl), proteinase K (20 μl) and buffer AL (180 μl) were mixed and the mixed solution was gently pipetted onto the folded filter in the spin column. The column was then placed on a 56°C preheated aluminium heat block and incubated for 30 min. The spin columns were covered with commercial aluminium foil and a clean blanket for effective incubation at the specified temperature. After the incubation, the spin column was centrifuged at 6000g for 1 min to collect the DNA. In order to increase DNA yields from the filter, 300 μl of sterilized TE buffer was gently pipetted onto the folded filter and the spin column was again centrifuged at 6000g for 1 min. The collected DNA solution (ca 900 μl) was purified using the DNeasy Blood and Tissue Kit following the manufacture's protocol.
One to three 2 l lots of seawater from the 10 l samples were vacuum-filtered onto 47 mm diameter glass-fibre filters (nominal pore size, 0.7 μm; Whatman, Maidstone, UK). Each filter was wrapped in commercial aluminium foil and stored in −20°C before eDNA extraction. Two litres of Milli-Q water was used as the negative control and treated identically to the eDNA samples, to monitor contamination during the filtering and subsequent DNA extraction.
DNA was extracted from the filters using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) in combination with a spin column (EZ-10; Bio Basic, Markham, Ontario, Canada). After removing the attached membrane from the spin column (EZ-10), the filter was tightly folded into a small cylindrical shape and placed in the spin column. The spin column was centrifuged at 6000g for 1 min to remove redundant seawater for DNA extraction. The column was then placed in a new 2 ml tube and subjected to lysis using proteinase K. Before lysis, Milli-Q water (400 μl), proteinase K (20 μl) and buffer AL (180 μl) were mixed and the mixed solution was gently pipetted onto the folded filter in the spin column. The column was then placed on a 56°C preheated aluminium heat block and incubated for 30 min. The spin columns were covered with commercial aluminium foil and a clean blanket for effective incubation at the specified temperature. After the incubation, the spin column was centrifuged at 6000g for 1 min to collect the DNA. In order to increase DNA yields from the filter, 300 μl of sterilized TE buffer was gently pipetted onto the folded filter and the spin column was again centrifuged at 6000g for 1 min. The collected DNA solution (ca 900 μl) was purified using the DNeasy Blood and Tissue Kit following the manufacture's protocol.