Structure Analysis Of Eukaryotic Community On The Surface Of Micro-nano Antifouling Coating And Design Of Specific Primers

The problem of marine biofouling has been regarded as an important obstacle to marine related structures and industries.There are many reports on the comprehensive antifouling technology based on the surface micro-nano structure and physical and chemical properties in recent years.The preliminary study of the research group found that the more seriously the diversity of eukaryotic microbial community was destroyed in the biofilm samples on the surface of the micro-nano modified coating,the comprehensive score of its antifouling performance was higher.It is speculated that nano-coating can influence the attachment or growth of fouling larvae by affecting the diversity of eukaryote communities on the surface,reshaping the interaction and alternation of various communities in the biofilm.Therefore,this paper further studies the structural composition and differences of the eukaryotic communities in the early fouling of biofilms on the surface of different matrix modified coatings with different antifouling properties.According to the previous research of the research group,7 kinds of nano-materials with good and bad antifouling properties were selected and mixed with chlorinated rubber iron red antirust paint and acrylate paint respectively to prepare 1%(w/v)micro-nano modified coatings,a total of 32 coatings were prepared with the addition of negative controls with OTU nanomaterials.The steel plate was observed and the biofilm samples were collected by hanging plate in the ocean.The samples of biofilm with the growth time of 4 weeks were selected for the extraction of metagenomic DNA and high-throughput whole-length sequencing,the analysis of 18 S r RNA and ITS(Internal Transcribed Spacer)sequences was used to analyze the community structure of eukaryotes and fungi in the biofilm on the surface of micro-nano modified coatings.At the same time,using ITS sequences information and SAP(Simple Allele-discriminating Polymerase Chain Reaction)theory to design,screen and verify the specific primers.Finally,the primers can be used to detect and quantify specific strains in the samples.By analyzing the Alpha diversity index of high throughput sequencing data,it was found that the addition of nano-materials could decrease the diversity of eukaryotic community,the abundance of eukaryotic community and the diversity of eukaryotic community.For the fungal community,the nano-materials in the chlorinated rubber iron red paint could decrease the number of OTU in the biofilm on the coating surface.However,the number of OTU in the biofilm was increased when the anti-fouling nano-materials were added into the acrylate,while the number of OTU in the biofilm was lower than that of the negative control when the anti-fouling nano-tubes were added into the coatings.No matter what kind of film-forming paint is added with poor anti-fouling performance of nano-material coating surface biofilm fungus abundance and spectrum diversity is low.In general,regardless of antifouling performance,Acineta is the dominant genus in the bacterial community.The relative content of Acine in the iron red negative control group is 10% higher than that in the modified coating with good anti-fouling performance of micro-nano-iron red and 10% lower than that in the modified coating with poor anti-fouling performance.In the acrylate modification group,the content of Acine increased from 15% to 83% with the addition of good antifouling nanomaterials,and to 66% with the addition of poor antifouling nanomaterials.The addition of nanomaterials with different antifouling properties has greatly increased the content of Acineta,which is different in growth rate but consistent in trend.For fungi,the differences in fungal community structure in biofilms on several different coating surfaces are relatively small.The dominant species was an unclassified fungi,which was more than 80% in all samples.According to the multi-parameter clustering analysis,it can be seen that,in addition to the large influence of the film-forming material itself on the eukaryotic community,the effect of the nanomaterials added to different film-forming materials is also different.The difference in community structure caused by the addition of nano-materials with different antifouling properties to the chlorinated rubber iron red anti-rust paint is relatively large,while the difference in the community structure of the addition to the acrylic hydrophobic paint is relatively small.Therefore,when designing marine antifouling coatings,it is necessary to consider the role of nanomaterials and film-forming materials and make design choices.The ITS sequence information obtained by high-throughput sequencing was used to determine SNP and specific primer design was carried OTU in conjunction with SAP theory.A total of 27 primer pairs were designed,covering 13 OTUs.Finally,12 pairs of specific primers were successfully designed,covering 5 OTUs.The success rate of specific primer design reached 44%,and nearly half of the primers had good specificity.It can be used to detect and quantify Ciona intestinalis,Botrylloides and Bougainvillia.