Primer Design
Specimens of C. fleckeri , were collected from Horseshoe Bay (19.1333° S, 146.8500° E), Magnetic Island, Queensland (1 November 2018), with other species having been collected either off the coast of Magnetic Island, or Cairns, Queensland (Supplementary materials). Genomic DNA (gDNA) was extracted using a Qiagen DNeasy Blood and Tissue Kit (Venlo, The Netherlands). A 584 bp fragment of the mitochondrial 16S rRNA gene was amplified via PCR using the “universal jellyfish primers”, Forward-16SL, (Bayha et al . 2010) Reverse-Aa_H16S_15141H (Ender & Schierwater 2003) and thermocycling conditions reported for the forward and reverse primers respectively. Purified PCR product was then sent to the Australian Genome Research Facility (AGRF, Brisbane, Australia) to be Sanger sequenced. Returned sequences were then edited and aligned in Geneious (Biomatters, Auckland, New Zealand) and put into a n-Blast search downloaded from the NCBI website to confirm their taxonomy and to identify likely polymorphic sites among species suitable for the design of species-specific eDNA primers (Appendix S1, Supporting information).
Species-specific eDNA primers were designed for C. fleckeri, C. sivickisi , C. xaymacana and C. barnesi using Geneious (Biomatters, Auckland, New Zealand) based on alignment of sequences and identification of unique primer sequences for each species within the mtDNA 16s rRNA gene. The specificity of species-specific primers was then assessed against the genomic DNA of all other cubozoan taxa from waters surrounding Magnetic Island, Queensland.
Using Real-Time PCR, technical replicates for each species were performed. Amplicons were visualised on a 1.5% agarose electrophoresis gel at 80 V for 40 min against other cubozoan species (Appendix S2, Supporting information). The triplicate technical replicates of each species were repeated for qPCR reactions. To deem specificity of primers, exclusive amplification of the target species was completed.