A Molecular Detecting Method To Quantify Early Attachment Of Marine Macrofouler Larvae

Marine fouling has brought great distress to the human, but the development of environmental-friendly antifouling technology and product is just in its beginning. One of the starting points of the new antifouling technology is based on surface micro-nano structure of nanomaterials filled coatings. Many of this kind of coatings can effectively inhibit the attachment of macrofoulers’ larvae that are invisible by our naked eyes. The aim of this study is to establish a molecular detection method by 18 S ribosomal RNA gene to rapidly quantify the amount of attached lavae in the early fouling stages.This project collected several main types of marine fouling eukaryotes in Weihai area, analysed their known 18 S r RNA gene sequences and looked for the sites of species-specific SNPs(Single Nucleotide Polymorphisms).Thespecific primers were designed by the SAP(Simple Allele-discriminating PCR) technology, to achieve single nucleotide discrinimation in 18 S gene among fouling species.Ten pairs of primers were targeted to the main macrofoulers as Botryllus schlosseri, Styela clava, Callopora lineata, Bugula neritina, Mytilus edulis, Crassostrea gigas, B.improvisus, Fistulobalanus albicostatus, Amphibalanus cirratus, and Hydroides ezoensis.The specificity of designed primers was checked out with the following steps:(1) DNA sequencing of the amplicons using the main macrofouler(pure species) as template;(2) BLAST analysis of the above sequences to confirm that any primer only amplify targeted species;(3) Silva database screening to list the coverage of each primer. Ten pairs of primers have good specificity at and above the family-level.The specificity of the primers was further confirmed as follow:(1) PCR amplification specificity using mixed pure templates;(2) PCR amplification specificity using biofilm samples obtained from surfaces of nanomaterials-filled coatings in field tests. The relative attachment amount of macrofouler lavae was measured using agarose-gel data and Gel-Pro software;(3) QPCR quantification of attached macrofouler lavae in nanomaterials-filled coating surfaces using the best two pairs of primers, TH1 for barnacle and Bug1 for Bryozoa, respectively.The conclusion and the novelty of this research are:(1) The above 18 S r DNA and SAP-based primer design approach is good enough for primer specificity; and(2) this project repoprted a molecular detection method for early stage attachment quantification of each marine macrofouler based on 18 S r RNA with high discrimination potential in the field of surface macro-nano structure-based antifouling technology development.