Biofouling Communities Research Articles

How plastic litter sunk by biofouling recovers buoyancy - The role of benthic predation

Estimates suggest that the amount of plastic litter discarded in the ocean is several times greater than what remains floating at the sea surface, raising questions about the fate of this marine debris. Fouling-induced sinking of plastic litter is one of the proposed mechanisms responsible for this mass difference. While some of this ‘missing’ plastic mass may be explained by the effects of fouling, it has also been hypothesized that sinking litter may return to the surface after benthic organisms consume the biofouling. However, this hypothesis has never been tested. The present study evaluated the structure and biomass of the fouling community in response to benthic predation in both summer and winter seasons. Floating PVC plates were installed during winter and summer in central Chile (36°S) until the growing biofouling community caused them to sink. Plates were then moved to the seabed, where they were exposed to benthic predation, while control plates were maintained in a mesh cage impeding predator access. In summer, all plates recovered their buoyancy, while in the winter only 60 % recovered buoyancy. All caged control samples remained on the bottom in both seasons. The community structure differed both in the treatments and across the seasons, with plates that recovered buoyancy initially being dominated by Ulva sp. and Ciona robusta. Conversely, plates that did not refloat were mainly covered by species resistant to predation such as Pyura chilensis, Austromegabalanus psittacus, and Balanus laevis. Thus, fouling community structure influences how predation facilitates buoyancy recovery, because not all epibionts can be consumed by predators. While previous studies had shown how fouling organisms cause sinking of floating litter, this is the first study to provide experimental evidence that predation can reverse this process and allow litter to resurface and become again available as dispersal vectors for native and invasive species.

Life on every stone: Characterizing benthic communities from scour protection layers of offshore wind farms in the southern North Sea

The scour protection layer (SPL) is a layer of large stones placed around man-made structures in the marine environment, preventing sediment scouring while also providing new hard substrate and potentially increasing the structural complexity of the original environment. This fosters development of diverse benthic communities, supporting high abundance of organisms. Future SPLs are therefore a potential tool for the ecological enhancement of degrading marine habitats following the principles of nature-inclusive design. Yet, factors that shape the benthic communities on SPLs are poorly understood. Here, we analysed existing data from SPLs from offshore wind farms and a gas platform in the southern North Sea to determine how SPL characteristics affect the biofouling community structure. We combined this analysis with an in-situ experiment testing for the effects of habitat complexity on SPL communities. Our results demonstrate that abundant and diverse communities are present on all SPLs. On a regional scale, communities are mainly affected by depth and location. Increasing habitat complexity has significant and positive effects on species richness yet was non-significant for biomass and abundance of the biofouling community. If applied thoughtfully, nature-inclusive design of the SPL habitat, including manipulation of the physical complexity of the structure, can effectively promote biodiversity.

Development and Succession of Non-Indigenous and Cryptogenic Species over Two Different Substrates in the Port of Alicante (Western Mediterranean)

Artificial structures act as points of entry for non-indigenous species (NIS) in port areas and may support higher abundance and richness of them. The studies about NIS are increasing, but studies focusing on the variations in temporal recruitment and ecological mechanisms are still scarce. Thus, the aim of this work was to determine the colonization and development of non-indigenous sessile fouling species over two types of substrates (electrolytic carbonated and steel) during 12 months of immersion in the Alicante harbor. The biofouling communities of both substrates were analyzed in terms of abundance and species richness by status (native, cryptogenic, and NIS), and NIS assemblages of both substrates were studied by means of multivariate analyses. In total, 53 different species were identified, 38 in steel (six NIS and six cryptogenic) and 50 in the carbonated substrate (six NIS and 10 cryptogenic). Most NIS were more abundant and diverse after 9 months of immersion and had a preference for carbonated substrates. Furthermore, most of them were positively correlated in both substrates (mainly in steel) and it is noted that the number of NIS in the port of Alicante is increasing as new records have been detected.

Disentangling eukaryotic biodiversity patterns from man‐made environments (port and marina) and nearby coral reefs in the Red Sea: A focus on the surveillance of non‐indigenous species

AbstractMan‐made environments such as ports and marinas are gateways for many marine non‐indigenous species (mNIS) transported via shipping worldwide. These habitats are often the focus of biosecurity programs for the surveillance of mNIS, but there have been few studies investigating the distribution of biofouling communities in natural habitats and nearby artificial habitats. Here, following a DNA metabarcoding approach, we tested for differences in spatio‐temporal trends of biological pioneer communities established after one‐week colonization between a port, a marina, and coral reefs using two different matrices (PVC panels and water samples) in the central Red Sea. In addition, we aimed to investigate differences in the prevalence of mNIS between man‐made and natural habitats during the summer and winter seasons, and whether the number and patterns of mNIS differed between the port (an active historic port subjected to intense shipping traffic) and the marina (a small recently developed marina utilized by small and medium research vessels). The community structure from samples collected at the two coral reefs showed greater similarity to each other compared to that obtained from the port and the marina. A total of 29 mNIS were detected across all sites, of which 16 were reported in the Red Sea for the first time, with Jeddah port hosting 28 mNIS. By applying standardized methodological approaches, this study provides comparable data regarding the monitoring of introduced and invasive species, particularly within the data‐poor Red Sea, a major shipping corridor. This study serves as the foundation for implementing a robust and rapid baseline monitoring system to assess native biodiversity and facilitate the early detection of NIS.

Combatting microbial biofouling network associated with Chembarambakkam Lake water using Acalypha indica derived metabolites

AbstractThis study assessed the anti‐biofouling potential of Acalypha indica ethanolic extract against biofouling communities associated with Chembarambakkam Lake water under in vitro conditions. Water samples from the lake were collected at various seasons, and changes in various physicochemical parameters and microbial status were evaluated. Most significantly, the efficacy of the microflora associated with water‐forming biofouling or biofilm network on its susceptibility to the ethanolic extract of A. indica was investigated. Distinct changes were recorded in all the tested physico‐chemical and microbiological statuses in all the seasons. The efficacy of biofouling or biofilm network formation was found to be statistically high during the winter season. Biofouling or biofilm formation was susceptible to the ethanolic extract of A. indica. A. indica treatment brought about notable changes in the morphology of the biofouling networks. A. indica bioactive compounds such as 9,10‐dideutero octadecanal and cyclononasiloxane, octadecamethyl known for their antimicrobial and in vitro properties, effectively prevented biofilm and biofouling formation.

Differences in Aurelia coerulea and Nemopilema nomurai colony proliferation and biofouling invasion at various in situ polyp densities in Jiaozhou Bay, China

Scyphozoan polyp colonies are typically patchily distributed on substrate surfaces at varying densities in the natural environment. We investigated the differences in Aurelia coerulea and Nemopilema nomurai colony proliferation and biofouling invasion at high and low in situ polyp densities in Jiaozhou Bay, China, to explore potential polyp density-dependent effects. The study found that in situ A. coerulea polyp colonies on settling plates consistently survived at high initial density (24.4 ± 2.8 ind•cm−2) over a one-year experiment, but 60% of colonies disappeared at low initial density (0.6 ± 0.1 ind•cm−2). Polyp density in these colonies constantly fluctuated with a significant difference between the two treatments, decreasing by 71.5 ± 37.0% relative to the original level at high initial density by the end of the experiment, but increasing by 237.4 ± 662.7% at low initial density. In contrast, all N. nomurai polyps, whether at high or low initial density (1.9 ± 0.6 ind•cm−2 and 0.2 ± 0.0 ind•cm−2), died after approximately six weeks. Strobilation occurred in A. coerulea, but not in N. nomurai owing to the untimely die-off of polyp colonies. No significant differences in strobilation percentage, number of discs strobila−1 and the calyx diameter of polyps were observed between the two density treatments. Macro-fouling organisms (e.g., ascidians and bryozoans) invaded the settling plates one week after the start of the experiment. Their coverage percentage was significantly lower at high initial polyp density in A. coerulea in a year, but there was no significant difference between both treatments in N. nomurai polyps. Nudibranch predators were discovered in the biofouling communities during the summer, with no significant differences in their numbers between the two treatments for each polyp species. The results indicated that density-dependent effects may play an important role in regulating in situ polyp colony proliferation and combatting biofouling invasion in A. coerulea, but not in N. nomurai. High colony survival and low levels of biofouling invasion at high initial polyp densities in A. coerulea might favor their extensive colonization in natural environment over extended periods of time.

UV-C LED-induced cyclobutane pyrimidine dimer formation, lesion repair and mutagenesis in the biofilm-forming diatom, Navicula incerta

The use of ultraviolet-C (UV-C) irradiation in marine biofouling control is a relatively new and potentially disruptive technology. This study examined effects of UV-C exposure on the biofilm-forming diatom, Navicula incerta. UV-C-induced mutations were identified via Illumina HiSeq. A de novo genome was assembled from control sequences and reads from UV-C-exposed treatments were mapped to this genome, with a quantitative estimate of mutagenesis then derived from the frequency of single nucleotide polymorphisms. UV-C exposure increased cyclobutane pyrimidine dimer (CPD) abundance with a direct correlation between lesion formation and fluency. Cellular repair mechanisms gradually reduced CPDs over time, with the highest UV-C fluence treatments having the fastest repair rates. Mutation abundances were, however, negatively correlated with CPD abundance suggesting that UV-C exposure may influence lesion repair. The threshold fluence for CPD formation exceeding CPD repair was >1.27 J cm−2. Fluences >2.54 J cm−2 were predicted to inhibit repair mechanisms. While UV-C holds considerable promise for marine antifouling, diatoms are just one, albeit an important, component of marine biofouling communities. Determining fluence thresholds for other representative taxa, highlighting the most resistant, would allow UV-C treatments to be specifically tuned to target biofouling organisms, whilst limiting environmental effects and the power requirement.

Artificial reef biofouling community impacts on ecosystem metabolism and biogeochemical cycling in estuarine waters of the northern Gulf of Mexico

Artificial reefs increase hard bottom habitat and bolster shoreline protection in coastal waters. Reefs are rapidly colonized by biofouling communities which can potentially alter ecosystem metabolism. Despite the high biomass and ubiquitous nature of biofouling communities, little is known about their ecosystem metabolism or role in biogeochemical nutrient cycling. Using a combination of laboratory and field-based methods, this study focused on the metabolic activity of artificial reef biofouling communities and their potential to contribute to water column hypoxia in a shallow subtropical estuary. Study objectives aimed to (1) measure in situ dissolved oxygen (DO) concentrations in surface and bottom waters along reef transects during summer months when episodic hypoxic events typically occur, (2) estimate respiration rates in relation to nutrient regeneration in the absence versus presence of biofouling communities, and (3) examine seasonal shifts in trophic structure by relating elemental C:N ratios and stable nitrogen isotope (δ15N) values of the biofouling community across eight sample periods. In situ DO in bottom waters was low near high profile reefs. Water column respiration and nutrient regeneration rates were comparable to other estuaries. Respiration and regeneration rates of ammonium and phosphate were all manifestly higher in the presence of a biofouling community. Biofouling metabolic-driven processes were higher at low profile reefs than at high profile reefs. Seasonal shifts in biofouling elemental composition indicated by higher C:N and lower δ15N during cooler months and lower C:N and higher δ15N during warmer months reflected a transition from a more autotrophic community in winter to a more heterotrophic community during the rest of the year. Our results demonstrate that artificial reef biofouling communities can potentially contribute to hypoxic conditions in coastal waters of the northern Gulf of Mexico.

Distribution of functionally distinct native and non‐indigenous species within marine urban habitats

AbstractAimTrait‐based approaches are powerful to examine the processes associated with biological invasions. Functional comparison among native and non‐indigenous species (NIS) can notably infer whether novel assemblages result from neutral or niche‐based assembly rules. Applying such a framework to biofouling communities, our study aimed to elucidate their distributions within two marine urban habitats (namely floating vs. nonfloating habitats).LocationSoutheast Pacific—Central Chilean coastline.MethodsHere, we examined the distribution of 12 functional traits in fouling communities established on settlement plates, after 3 and 13 months of deployment in the two habitats and across ports in Central Chile. Based upon previously described differences of assemblages and NIS contribution across habitats, we hypothesized that nonindigenous, cryptogenic and native taxon pools would be functionally distinct (and trait biased), and that functional diversity and structure would vary across habitats and successional stages.ResultsOur results show, as anticipated, that nonindigenous (13 taxa), cryptogenic (12) and native (18) taxon pools are functionally distinct, though overlapping in the trait space. Non‐indigenous species are rather related to colonizing traits, while native species are more related to competitive traits. Only one widespread NIS was functionally similar to the late successional and most competitive native species, including taxa elsewhere invasives. Despite differences in taxonomic composition between habitats, we did not observe functional differences between them. In contrast, temporal variations across colonization stages were detected along with an increased contribution in large and long‐lived taxa, together with site‐specific trajectories.Main ConclusionsWe conclude that the functional distinctness among nonindigenous, cryptogenic and native taxa occupying artificial habitats in ports reflects niche‐based processes. Site‐specific trajectories indicate that scale‐dependent assembly processes, such as dispersal and species interactions, are at play.

Latitudinal distribution of skeleton shrimps (Amphipoda, Caprellidae) in artificial habitats throughout the eastern South Pacific coast of Peru

ABSTRACT Biodiversity tends to decrease poleward and increase towards the equator. However, these patterns remain unknown for many invertebrate taxa throughout Peru's eastern South Pacific coast, particularly for caprellid crustaceans which are conspicuous in artificial habitats. Three biogeographical units have been proposed for the Peruvian coast: Panamic and Humboldt provinces with a transition zone in between, but a few invertebrates support this classification. We evaluated if the diversity of caprellids decreases towards high latitudes and if the assemblages of caprellids depicted the biogeographic zonation proposed for the Peruvian coast. From 15th to 31st October 2021, caprellids were collected from biofouling communities colonizing boat hulls, buoys, and ropes throughout the Peruvian coast (3.74°–17.99° S). The caprellid assemblage consisted on six species: Caprella penantis [sensu lato], C. penantis f. gibbosa, C. equilibra, C. scaura, Paracaprella pusilla, and Deutella venenosa. Overall, caprellids' abundance, species richness, and diversity decreased southwards. Specific caprellid assemblages showed affinities for each of the three biogeographical units. The dissimilarity of the caprellid assemblages increased at approximately every one degree of latitude. Although artificial substrates can alter biodiversity patterns, caprellids showed the canonical pattern of high diversity towards the equator and resembled the biogeographic zonation proposed for the Peruvian coast.

Measuring the recruitment and growth of biofouling communities using clear recruitment panels

Ecological monitoring has been recognized as a key tool for guiding biofouling management practices. A two-year study was designed to collect comprehensive data on the biofouling community progression at Port Canaveral, Florida, using clear recruitment panels and a scanner to directly observe organisms attached to the surface. This method allowed for minimal disruption to the natural community development and aided the collection of a suite of metrics to explore environmental relationships. Seasonal changes in community composition and biofouling pressure, especially at earlier stages, were related to abiotic conditions. Interannual variation within seasonal communities was also observed. The type of dominant organism present impacted the rate at which surfaces were covered (e.g. fastest cover with tunicates) and the overall biomass accumulation (e.g. highest rate with tubeworms). Results highlight that understanding the influence of the time of year and the dominant organism identity is ecologically vital for improving biofouling management.

A conceptual basis for surveying fouling communities at exposed and protected sites at sea: Feasible designs with exchangeable test bodies for in-situ biofouling collection

The enhanced inertia load caused by biofouling on device components, such as the foundations of wind turbines or other structures at sea, modifies the hydrodynamic properties, and increases the stress to structures, predominantly in upper water layers with high impact from wave dynamics. This compromises the stability, functioning, operation as well as the durability of these devices especially in exposed environments. A main challenge is the quantification of the impact of hydrodynamic forces on irregular bodies being overgrown by soft- and hard-bodied biofouling organisms. Therefore, test bodies from the upper 1–5 m water depth and thus exposed to the strongest wave actions close to the surface shall be overgrown by biofouling and used in measurement trials in a wave and current flume. These measurements shall shed light on the varying roughness and its influence on the load bearing capacity of foundation piles. Consequently, the main aims of the present work were the development of two independent test stations as holding devices for artificial test bodies for the collection of biofouling organisms during field studies: a carrying unit floating at the surface in an exposed area (System A) and a sampling device with access from a land-based facility (System B). Both systems are relatively easy to access, exhibit straightforward handling, and are reasonable cost-effective. A Test Body Support Unit (TBSU, System A) was designed and mounted on a spare buoy to carry the test bodies (cylinders), which serve as substrate for the fouling. The system was sufficiently robust to withstand several periods of rough sea conditions over the first two years. This system can only be accessed by vessels. System B (MareLift) provided the robustness and functionality needed for areas exhibiting harsh conditions but can be operated from land. The here used test bodies (steel panels) exhibited a sound basis for the monitoring of succession processes in the biofouling development. System B offered the possibility to analyse two habitats (intertidal and subtidal) and revealed clear differences in the composition and development of their fouling communities.Overall, both systems provide advantages in obtaining standardized biofouling samples compared to previous approaches. Such test stations play an important role in the risk management of marine sectors as they could help characterising biofouling communities over different geographical areas. System A and B provide a sound basis for biofouling research but potentially also for other potential research approaches in exposed areas as they provide space for future developments.

Multispecies biofilms on reverse osmosis membrane dictate the function and characteristics of the bacterial communities rather than their structure

The main reason for the deterioration of membrane operation during water purification processes is biofouling, which has therefore been extensively studied. Biofouling was shown to reduce membrane performance reflected by permeate flux decline, reduced selectivity, membrane biodegradation, and consequently, an increase in energy consumption. Studies of biofouling focused on the identification of the assembled microbial communities, the excretion of extracellular polymeric substances (EPS), and their combined role in reduced membrane performance and lifetime. However, the link between the structure and function of biofouling communities has not been elucidated to date. Here, we provide a novel insight, suggesting that bacterial functions rather than composition control biofouling traits on reverse osmosis (RO) membranes. We studied the potential activity of RO biofilms at metatranscriptome resolution, accompanied by the morphology and function of the biofouling layer over time, including microscopy and EPS composition, adhesion, and viscoelastic properties. To that end, we cultivated natural multispecies biofilms in RO membranes under treated wastewater flow and extracted RNA to study their taxonomies and gene expression profiles. Concomitantly, the biofilm structure was visualized using both scanning electron microscopy and laser scanning confocal microscopy. We also used quartz crystal microbalance with dissipation to characterize the affinity of EPS to membrane-mimetic sensors and evaluated the viscoelasticity of the Ex-Situ EPS layer formed on the sensor. Our results showed that different active bacterial taxa across five taxonomic classes were assembled on the RO membrane, while the composition shifted between 48 and 96 h. However, regardless of the composition, the maturation of the biofilm resulted in the expression of similar gene families tightly associated with the temporal kinetics of the EPS composition, adhesion, and viscoelasticity. Our findings highlight the temporal selection of specific microbial functions rather than composition, featuring the adhesion kinetics and viscoelastic properties of the RO biofilm.

Seasonal Variability of Biofouling Community Structure in the Gulf of Mannar, Southeast Coast of India: A Multivariate Approach

Seasonal Variability of Biofouling Community Structure in the Gulf of Mannar, Southeast Coast of India: A Multivariate Approach

The impact of ascidian biofouling on the farmed Mediterranean mussel Mytilus galloprovincialis physiology and welfare, revealed by stress biomarkers

In biofouling communities, ascidians are among the most damaging species, presenting severe threats, such as depressed growth rates and decreased chances of lower survival, to shellfish aquaculture. However, little is known concerning the fouled shellfish physiology. In an effort to obtain information for the magnitude of stress caused by ascidians to farmed Mytilus galloprovincialis, five seasonal samplings took place in a mussel aquaculture farm suffering from ascidian biofoulants, in Vistonicos Bay, Greece. The dominant ascidian species were recorded and several stress biomarkers, including Hsp gene expression at both mRNA and protein levels, as well as MAPKs levels, and enzymatic activities of intermediate metabolism were examined. Almost all investigated biomarkers revealed elevated stress levels in fouled mussels compared to non-fouled. This enhanced physiological stress seems to be season-independent and can be attributed to the oxidative stress and/or feed deprivation caused by ascidian biofouling, thus illuminating the biological impact of this phenomenon.

Long-Term Succession on Offshore Wind Farms and the Role of Species Interactions

The presence of biofouling communities in very large densities in offshore wind farms (OWFs) generates broad effects on the structure and functioning of the marine ecosystem, yet the mechanisms behind the temporal development of these communities remain poorly understood. Here, we use an 11-year series on biofouling fauna from OWFs installed in Belgian waters to determine succession patterns and to unravel the role of biological interactions in shaping community development. Our analysis shows that biological interactions, besides age and location, affect diversity patterns in OWFs. The abundance of foundation species, predators, and space occupiers was significantly related to richness and/or diversity. The trends in richness, diversity, and community composition suggest that no permanent stable climax is reached after 11 years, which can be linked to the dynamic and disturbance-prone environment of offshore fouling communities.

Vertical and seasonal variations in biofilm formation on plastic substrates in coastal waters of the Black Sea

Plastic contamination of the marine environment is an increasing concern worldwide. Therefore, it is important to understand the kinetics of biofilms on plastics to study their behavior, fate, and transport pathways in the ocean. In this study, the vertical and seasonal variations in biofouling formation on transparent polyethylene terephthalate (PET) plastic fragments in the Southwest Crimea coastal waters of the Black Sea were investigated. Biofilms were identified in the transient light as ‘dark spots’ on the plastic surface, for which the numbers, size, and area were measured using specialized software. The rate of biofouling in the surface water layer was lower than those found in the middle and near-bottom water column, which could be due to a damaging effect of turbulent mixing on the biofilm. The highest rates of biofouling and diverse community were observed during the summer. The epibiotic assembly was represented by diatoms (11 taxa), dinoflagellates (3 taxa), green algae, filamentous cyanobacteria, small flagellates, and ciliates. Significant differences between the biofouling rates observed in different seasons made it difficult to estimate the period of time the plastic substrate has been in the marine environment. It was proposed to use the green alga Phycopeltis arundinacea (Montgn) De Tender et al., 2015 as a bioindicator to study the age of the biofouling community. Discoid thalli were identified at all stages of colonization of the plastic fragments in different seasons. Results obtained in this study demonstrate that biofouling organisms may be good model organisms in revealing age of biofilm formation and longevity of plastic debris in the ocean. Consequently, it is proposed that such biofouling organisms could be used as target species to monitor the biodegradation of plastic debris.

Multi-Year Succession of Biofouling Communities on Subsea Engineering Structures in the Aphotic Zone of the Sea of Okhotsk

Multi-Year Succession of Biofouling Communities on Subsea Engineering Structures in the Aphotic Zone of the Sea of Okhotsk

Nature-based solutions in a sandy foreshore: A biological assessment of a longline mussel aquaculture technique to establish subtidal reefs

Climate-induced natural disasters such as floods and storms are expected to become more frequent and intense in the coming years. From 2017 till 2020, a pilot project was set up where mussel reefs were established as a nature-based solution to mitigate coastal erosion off the Belgian Coast. These subtidal mussel reefs were created by applying a longline mussel aquaculture technique consisting of filamentous and non-filamentous dropper lines of different materials. In this study, several biological aspects of the Nature-based Solution (NbS) set-up were examined: the suitability of the set-up as a whole, the suitability of the seven dropper line types, distribution of biofouling organism on the dropper lines and the spatio-temporal evolution of the mussel reef and its impact on the underlying benthic communities. Only during the summer months, a mussel reef could be established under the dropper line structure. Filamentous and non-filamentous dropper lines harbored different mussel densities and sizes, leading to a possible different effect on the underlying mussel reef. The biofouling community on the dropper lines was also defined in this study with amphipod species such as Jassa sp. and Monocorophium sp. as the dominant species. The impact of the NbS set-up on the macrofaunal communities remained low for the study period. First indications of a shift towards a less diverse and oligochaete dominated community was observed, although longer time series and an optimized sampling protocol will be needed. This pilot study showed the application of using a mussel longline technique in order to establish a subtidal mussel reef in a soft-sediment environment.

Eukaryotic diversity of marine biofouling from coastal to offshore areas

Marine biofouling communities, including biofilms, are composed of many eukaryotes with high taxonomic and functional diversities. However, molecular characterization of eukaryotic diversity of marine biofouling has been barely developed due to the only recent interest in research areas such as marine renewable energies, antifouling technologies, or plastic pollution. The aim of this study was to compare the diversity and taxonomic composition of biofouling through different metabarcoding approaches used to detect the widest range of taxa from samples collected in several contrasted marine environments (French Atlantic and Mediterranean coasts). Thus, we assessed four DNA extraction methods and six primers pairs targeting the 18S rDNA gene (including the V1-V2, V4TAR, V4UNI, V7 and V9 regions) and the COI gene, the latter with two databases (BOLD and MIDORI). In addition the influence of primers selection was analyzed at three sites to compare geographic variations in eukaryotic diversity. Although none of the extraction methods greatly altered the community diversity or composition. we have observed that eukaryotic biofouling community diversity and structure varied depending on primers pairs, reference databases and sites. 18S rDNA regions allowed the detection of more taxa at the species level, including microeukaryotes, while the COI recovered more ASVs, but with a large proportion that remained taxonomically unassigned probably because BOLD and MIDORI specifically targeted metazoans. Interestingly, the spatial pattern obtained with both COI and 18S rDNA markers were similar showing that spatial selection occurred throughout a wide diversity of eukaryotic taxa. These results encouraged the use of these two complementary markers for future metabarcoding investigations but also highlighted the relevance of completing databases to enhance the identification of biofouling eukaryotes.