Lernaea cyprinecea, Linnaeus, 1758

Welicky, Rachel L., Swardt, Jurgen De, Gerber, Ruan, Netherlands, Edward C. & Smit, Nico J., 2017, Drought-associated absence of alien invasive anchorworm, Lernaea cyprinacea (Copepoda: Lernaeidae), is related to changes in fish health, International Journal for Parasitology: Parasites and Wildlife 6 (3), pp. 430-438 : 431-432

2.2. Molecular identi fi cation of Lernaea cyprinecea

Ethanol-preserved samples (n = 2) were used for molecular work. Genomic DNA was extracted from the samples using a rapid DNA extraction method as detailed in the KAPA Express Extract Kit (Kapa Biosystems, Cape Town, South Africa). Polymerase chain reactions (PCR) were used to amplify a 900 nt fragment of the 18S rRNA gene using the primer sets 1F and 5R (see Giribet et al., 1996). PCR was performed with volumes of 25 M l, using 12.5 M l Thermo Scientific DreamTaq PCR master mix (2×) (2×DreamTaq buffer, 0.4 mM of each dNTP, and 4 mM MgCl2), 1.25 M l of each primer, and 1 M l DNA. The final reaction volume was made up with PCR-grade nuclease free water (Thermo Scientific, Vilnius, Lithuania). The PCR reactions were carried out using a ProFlex™ PCR thermal cycler (applied biosystems by life technologies), following the PCR conditions as detailed in Boyer et al. (2007). PCR products were sent to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa) for purification and sequencing in both directions. Resultant sequences were assembled, and chromatogram-based contigs were generated and trimmed using Geneious Ver. 9.1.

Based on molecular evidence, 18S rDNA sequences were identified as Lernaea cyprinacea using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast). Uncorrected pair-wise distances (p-distance) and base pair differences were determined with the MEGA7 bioinformatics software program (http://www.megasoftware.net). The sequences obtained in the current study were aligned and compared to all available 18S rDNA sequences of L. cyprinacea [Genbank: DQ107554 - DQ107557; KM281816; KP235363; KX258625] ( Table 1). The analysis involved eight nucleotide sequences with an alignment length of 618 nt. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. A single sequence was deposited in the NCBI GenBank database under the accession number: KY435939.