2.2 Pathogen identification
Soon after the second event started, we collected 15 moribund sea urchins in a very early stage of infection from two localities off the southeastern coast of Tenerife (Figure 2b). The sea urchins were brought to the laboratory of Ciencias Marinas, Universidad de La Laguna, for analysis. Both amoeba and Vibrio tests were performed. To isolate amoebae, coelomic fluid (up to 5 ml) was extracted from the sea urchins after draining the seawater using an 18G × 1.5’ needle and sterile syringe, which was inserted through the peristomal membrane at an angle that avoided contact with the lantern area (Dykova et al. 2011). A few drops of these samples were cultured on Non-Nutrient seawater agar prepared from seawater collected from the same place as the sea urchins. The plates were cultured at 20°C and checked daily for the presence of amoebae. Primary isolate colonies were obtained following this procedure. For the bacteria test, the samples of coelomic fluid were also added to the surface of specific media to isolate Vibriospp. (TCBS agar, Merck, Spain), Aeromonas spp. (Aeromonas agar, Oxoid, UK) and Pseudomonas spp. (Cetrimide agar, Scharlab, Germany) and incubated at 37°C for 48 h. Additionally, we conducted DNA extraction and PCR amplification of amoebic isolates. DNA from cultures identified as positive for amoebae by microscopy was extracted (Reyes-Batlle et al. 2016) using a Maxwell® 16 Tissue DNA Purification Kit sample cartridge (Promega, Madrid, Spain) and a Maxwell® 16 Instrument (Promega). DNA yield and purity were determined using a NanoDrop 1000 spectrophotometer (Fisher Scientific, Madrid, Spain). PCR amplification of the 18S rDNA gene was carried out using a universal primer pair FLA-F and FLA-R (Reyes-Batlle et al. 2016). The PCR products were sequenced using the MACROGEN sequencing service (Madrid, Spain), and the sequences were aligned using the Mega 5.0 software program (Pennsylvania State University, USA).