Abstract
Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5′-CGC GGC GCT ACA TAT TAG AC-3′, Reverse: 5′-GCA CTT AAC CGA CCG TCG AC-3′). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44–66% of cephalopod species, corresponding to about 310–460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268–271; Peters et al. 2015 Mar. Ecol. 36, 1428–1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.
Highlights
Cephalopods, the molluscan class to which squids, octopods, cuttlefish and vampire squids belong, occur in all the world’s oceans from the intertidal zone to the deep sea [1,2,3]
This study describes the development of a novel set of universal primers for cephalopods targeting the nuclear 18S rRNA region and describes its complementarity to the mitochondrial 16S rDNA primer set [41,42] in a multi-marker approach to study the diversity and distribution of cephalopods through metabarcoding of Environmental DNA (eDNA)
The developed Ceph18S primer pair amplifies an approximately 200 bp target sequence estimated to be able to identify about 310–460 cephalopod species
Summary
Cephalopods, the molluscan class to which squids, octopods, cuttlefish and vampire squids belong, occur in all the world’s oceans from the intertidal zone to the deep sea [1,2,3]. Their high protein content and large populations make them important in commercial fisheries and food–web interactions as both predator and prey [4,5,6,7,8]. One of the reasons for this paucity of information is that cephalopods are difficult to study with traditional sampling methods like net catches or video surveys [2]. The challenges associated with cephalopod sampling, in remote areas such as deep pelagic environments, raise an urgent need for novel monitoring methods
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