Natural Sampler DNA (nsDNA) refers to environmental DNA (eDNA) retained by filter-feeding organisms such as mussels or sponges. Recent studies have highlighted the potential of nsDNA metabarcoding as a cost-efficient tool for biodiversity monitoring, with sponges emerging as the most studied natural samplers. However, most research has focused on fish, while the study of invertebrate communities remains limited due to the overabundance of host DNA. In this study, communities associated with the sponge Aplysina aerophoba were assessed through an integrative approach, combining nsDNA metabarcoding and morphological analysis. Metazoan diversity was characterized by amplifying the universal COI barcode region. To reduce host DNA amplification, blocking primers specific to A. aerophoba were designed. The addition of an annealing-inhibiting blocking primer at a 1:10 ratio to the amplifying primer significantly improved the sequencing depth of non-host organisms, increasing OTU counts per sample by an average of 97%. Morphological analysis performed on the same samples revealed site-level patterns not captured molecularly, underscoring its complementary value. These findings demonstrate that blocking primers offer a straightforward solution to reduce host DNA amplification in nsDNA metabarcoding. Further validation is needed using other natural samplers to evaluate potential inhibitory effects on non-host DNA.
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