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).