18S metabarcoding of the copepod Pseudocalanus newmani and environment seawaters
Eukaryotic parasites and preys of the copepod Pseudocalanus newmani were investigated using 18S metabarcoding. Zooplankton samples were collected using NORPAC net with a 335 Âµm mesh size at the Okhotsk Tower, located 1 km off the coast of Mombetsu, northeastern Hokkaido, Japan. The samples were immediately preserved in RNAlater. DNA was extracted from each individual of adult female P. newmani using AllPrep DNA/RNA Micro Kit (Qiagen). Seawater samples were collected from the sea surface using a bucket and filtered on 0.22 Âµm Sterivex filters. Genomic DNA of the filter samples was extracted using 5% Chelex buffer. The 18S V7-V9 region (approximately 500 bp) was amplified using KOD Plus version 2 (Toyobo) and the universal primer pair 18S-V7F and 18S-V9R, according to Sildever et al. (2019). For copepod samples, Peptide Nucleic Acid (PNA) was used to block the amplification of the host sequences in the first PCR. All PCR product libraries were sequenced using the MiSeq Reagent Kit v3 (600 cycles) on an Illumina MiSeq, and 300 bp paired-end sequence reads were obtained.