Cyprid Settlement Research Articles

Structure-Functional Activity of Pyrone Derivatives for Inhibition of Barnacle Settlement and Biofilm Formation.

Naturally occurring 6-pentyl-2H-pyran-2-one and its synthetic analogues greatly inhibit the settlement of Amphibalanus amphitrite cyprids and the growth and biofilm formation of marine bacteria. To optimize the antifouling activities of pyrone derivatives, this study designed pyrone analogues by modifying functional groups, such as the benzyl group, cyclopentane, and halides, substituted on both sides of a pyrone. The antifouling effects of the synthesized pyrone derivatives were subsequently evaluated against five marine biofilm-forming bacteria, Loktanella hongkongensis, Staphylococcus cohnii, S. saprophyticus, Photobacterium angustum, and Alteromonas macleodii, along with barnacle cyprids of Amphibalanus amphitrite. Substituting nonpolar parts-such as the aliphatic, cyclopentyl, or phenyl moieties on C-5 or the furan moieties on C-3-not only increased antibacterial activity and inhibited biofilm formation but also inhibited barnacle cyprid settlement when compared to 6-pentyl-2H-pyran-2-one.

New genes helped acorn barnacles adapt to a sessile lifestyle.

Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.

Rapid discovery of a new antifoulant: From in silico studies targeting barnacle chitin synthase to efficacy against barnacle settlement

Due to the adverse environmental impacts of toxic heavy metal–based antifoulants, the screening of environmentally friendly antifoulants has become important for the development of marine antifouling technology. Compared with the traditional lengthy and costly screening method, computer-aided drug design (CADD) offers a promising and efficient solution that can accelerate the screening process of green antifoulants. In this study, we selected barnacle chitin synthase (CHS, an important enzyme for barnacle settlement and development) as the target protein for docking screening. Three CHS genes were identified in the barnacle Amphibalanus amphitrite, and their encoded proteins were found to share a conserved glycosyltransferase domain. Molecular docking of 31,561 marine natural products with AaCHSs revealed that zoanthamine alkaloids had the best binding affinity (−11.8 to −12.6 kcal/mol) to AaCHSs. Considering that the low abundance of zoanthamine alkaloids in marine organisms would limit their application as antifoulants, a marine fungal–derived natural product, mycoepoxydiene (MED), which has a similar chemical structure to zoanthamine alkaloids and the potential for large-scale production by fermentation, was selected and validated for stable binding to AaCHS2L2 using molecular docking and molecular dynamics simulations. Finally, the efficacy of MED in inhibiting cyprid settlement of A. amphitrite was confirmed by a bioassay that demonstrated an EC50 of 1.97 μg/mL, suggesting its potential as an antifoulant candidate. Our research confirmed the reliability of using AaCHSs as antifouling targets and has provided insights for the efficient discovery of green antifoulants by CADD.

Evaluation of natural resveratrol multimers as marine antifoulants

In the current study we investigate the antifouling potential of three polyphenolic resveratrol multimers (–)-hopeaphenol, vaticanol B and vatalbinoside A, isolated from two species of Anisoptera found in the Papua New Guinean rainforest. The compounds were evaluated against the growth and settlement of eight marine microfoulers and against the settlement and metamorphosis of Amphibalanus improvisus barnacle cyprids. The two isomeric compounds (–)-hopeaphenol and vaticanol B displayed a high inhibitory potential against the cyprid larvae metamorphosis at 2.8 and 1.1 μM. (–)-Hopeaphenol was also shown to be a strong inhibitor of both microalgal and bacterial adhesion at submicromolar concentrations with low toxicity. Resveratrol displayed a lower antifouling activity compared to the multimers and had higher off target toxicity against MCR-5 fibroblasts. This study illustrates the potential of natural products as a valuable source for the discovery of novel antifouling leads with low toxicity.

Antifouling activity of isonitrosoacetanilides against microfouling and macrofouling.

Biofouling is responsible for structural and economic damage to man-made surfaces. Antifouling paints with biocides have been applied to structures to avoid organism adhesion; however, they have high toxicity and are not able to prevent all biofouling processes, necessitating the periodic mechanical removal of organisms and paint reapplication. Thus, there is an urgent demand for novel, effective, and environmentally friendly antifouling alternatives. As isonitrosoacetanilide is the precursor for many compounds with antibacterial activity, we believe that it could have antifouling activity against microfouling and, consequently, against macrofouling. The aim of this work was to investigate the antifouling potential of six isonitrosoacetanilide compounds and their toxicity. The compounds were employed at different concentrations (0.625-1.25-2.5-5-10µgmL-1) in this study. The biofilm and planktonic bacteria inhibition and biofilm eradication potential were evaluated by crystal violet assay, while Amphibalus amphitrite barnacle settlement was evaluated by cyprid settlement assay. Toxicity evaluation (LC50 and EC50) was performed with A. amphitrite nauplii II and cyprid larvae. At least one of the tested concentrations of 4-Br-INA, 4-CH3-INA, and 2-Br-INA compounds showed nontoxic antifouling activity against microfouling (antibiofilm) and macrofouling (antisettlement). However, only 4-CH3-INA and 2-Br-INA also showed biofilm eradication potential. These compounds with antibiofilm activity and nontoxic effects could be combined with acrylic base paint resin or added directly into commercial paints in place of toxicant biocides to cover artificial structures as friendly antifouling agents.

The movement and settlement behaviour of cyprids of Balanus reticulatus on the surfaces of the titanium alloys

The motion paths of Balanus reticulatus cyprids were similar on all the titanium alloys surfaces. On the parallel grinding surfaces, the temporary attachment duration and the settlement ratio of the cyprids were influenced by the roughness and the composition of the surfaces and correlated positively. The surface roughness could also change the contact area and the numbers of the attachment points of the cyprids in the similar pattern. Consequently, the roughness and the composition of the surfaces regulated the cyprid settlement by the temporary attachment duration. The cross grinding increased the temporary attachment duration but drastically decreased the settlement ratio to 0 compared to the parallel grinding, possibly due to the voids and the drastic decrease of the contact area and the numbers of the attachment points of the cyprids on the cross grinding surface, respectively. The cross grinding therefore significantly reduced the cyprid settlement compared to the parallel grinding.

Faint chemical traces of conspecifics delay settlement of barnacle larvae

Larval settlement drives population and community dynamics and evolution of marine invertebrates. While it is well known that larvae sense a variety of biological and environmental factors, there is limited information on how they react to specific factors so as to locate settlement sites. Proteinaceous Waterborne Settlement Pheromone (WSP) was purified from adult barnacles, Amphibalanus amphitrite, in a previous study, but it is unclear how WSP guides barnacle cyprids to the source. Here, a series of settlement assays were conducted to investigate the concentration dependence of recombinant WSP. We report that low concentrations of barnacle WSP decrease the probability of settlement of conspecific cyprids, whereas high concentrations have the opposite effect. We suggest that this is because weak WSP informs cyprids that suitable settlement sites with adult barnacles are distant, inducing them to extend the larval phase. The present study clearly shows that larvae use WSP concentration, rather than presence/absence information. In barnacles, several other conspecific chemical/visual cues also attract larvae. This study opens the door to future research to determine how these cues influence larval behavior in nature.

Natural polyketide 6-pentyl-2H-pyrone-2-one and its synthetic analogues efficiently prevent marine biofouling

Biofouling is a widespread phenomenon in oceans worldwide. With increasing human development and activities in open and coastal waters, and due to the environmental impact of AF organotins and copper-based paint, the demand for nontoxic antifouling (AF) paints is increasing. Various bioassays for antimicrobial activity, anti-biofilm formation and anti-barnacle settlement were established to evaluate the possibility of using marine natural products as AF agents. A series of natural products, isolated from the marine-derived fungi Trichoderma atroviride and T. reesei, were evaluated for their AF activity. One pyrone-type compound (1) demonstrated significant inhibitory activities toward barnacle cyprid settlement. Furthermore, a series of pyrone analogues (S1–S6) were synthesized, and their bioactivities were evaluated in the established systems. The results showed that compounds S5 and S6 exhibited a broad spectrum of bioactivities, such as anti-barnacle settlement, anti-biofilm formation and antimicrobial activities.

Transcriptome analysis provides insights into a molecular mechanism of histamine response in the cyprid larvae of Amphibalanus amphitrite

Barnacles are notorious marine fouling creatures; their planktonic cyprid larvae attach to material substrates and metamorphose. Histamine has shown great importance in regulating cyprid settlement and metamorphosis. This study aimed to investigate the mechanisms of histamine-induced larval settlement. Cyprids were exposed to histamine or loratadine, an anti-histamine compound. The percent larval settlements of the histamine- and anti-histamine-treated cyprids were significantly higher and lower, respectively, than the control group. Transcriptomic analyses showed that histamine-treated cyprids had 18498 differentially expressed genes (DEGs, 14531 up-regulated, 3967 down-regulated) and the anti-histamine group had 18055 DEGs (17237 up-regulated, 818 down-regulated) in comparison to untreated controls. In both treatment groups, significant enrichment of DEGs involved in the mitogen-activated protein kinase signaling pathway was observed. Based on the results of larval settlement bioassays, we set 4 filter conditions to perform DEG analyses, and 19 DEGs were selected as functional genes related to cyprid settlement. The functional categories of these genes included structural proteins, spider silk proteins, energy metabolism proteins, cement proteins, glycosyl proteins, and multifunctional proteins. The energy metabolism protein AdipoR was significantly up-regulated in the histamine-treated cyprids but significantly down-regulated in the anti-histamine group. The activity of adenosine monophosphate-activated protein kinase, a downstream signaling protein of AdipoR, increased in the histamine-treated group and decreased in the anti-histamine-treated group. Our results provide new insights into the molecular mechanisms underlying the histamine-induced settlement of barnacle cyprids and identify AdipoR as an important gene that can affect the settlement of cyprids, likely through regulating cyprid energy metabolism.

Nonfreezable Water and Polymer Swelling Control the Marine Antifouling Performance of Polymers with Limited Hydrophilic Content.

Zwitterionic chemical groups have well-documented resistance to marine fouling species when presented as homogeneous polymer brushes. These model formulations are not, however, suitable for practical fouling-control applications. It is presently unknown if a uniform film of zwitterions is required to elicit nonfouling character via the binding of interfacial water or if the incorporation of zwitterionic functionality into a more practical bulk polymer system will suffice. Here, copolymers of n-butyl methacrylate were synthesized with low incorporation levels (up to 20 mol %) of hydrophilic functionality, including zwitterionic moieties. Their antifouling (AF) properties were evaluated using barnacle cyprids (Balanus improvisus), diatom cells (Navicula incerta), and a multispecies biofilm. The laboratory assays revealed higher resistance of ionic copolymers toward cyprid settlement, which was attributed to their swelling and the presence of nonfreezable water molecules bound tightly to the polymer chains. Additionally, cells of N. incerta and the multispecies biofilm were removed more effectively on polymers containing sulfobetaine methacrylate and sulfopropyl methacrylate moieties. The results indicate that the presence of tightly bound interfacial water is not limited to model systems of pure hydrophilic homopolymers, but that this mechanism can also reduce the settlement and adhesion of fouling species via bulk copolymer systems with limited hydrophilic content. The swelling of polymers with hydrophilic content may also contribute to their AF efficacy, and such materials may therefore represent a route to translation of the well-documented nonfouling character of zwitterions into practical, industrially relevant coating formulations.

Microbiome acquisition during larval settlement of the barnacle Semibalanus balanoides.

Barnacles are conspicuous members of rocky intertidal communities and settlement of the final larval stage, the cyprid, is influenced by the presence of biofilms. While modulation of cyprid settlement by biofilms has been studied extensively, the acquisition of a specific microbiome by the settling larva has not. This study investigated settlement in the field of Semibalanus balanoides in two consecutive years when the composition of the benthic bacterial community differed. In both years, settling cyprids adopted a specific sub-set of benthic bacteria that was distinct from the planktonic cyprid and the benthos. This microbiome was consistent, regardless of annual variability in the benthic community structure, and established within hours of settlement. The results imply that a natural process of selection occurs during the critical final transition of S. balanoides to the sessile form. The apparent consistency of this process between years suggests that optimal growth and survival of barnacles could depend upon a complex inter-kingdom relationship, as has been demonstrated in other animal systems.

Brush Swelling and Attachment Strength of Barnacle Adhesion Protein on Zwitterionic Polymer Films as a Function of Macromolecular Structure.

The exceptional hydration of sulfobetaine polymer brushes and their resistance toward nonspecific protein absorption allows for the construction of thin films with excellent antibiofouling properties. In this work, swollen sulfobetaine brushes, prepared by surface-initiated atom transfer radical polymerization of two monomers, differentiated by the nature of the polymerizable group, are studied and compared by a liquid-cell atomic force microscopy technique and spectroscopic ellipsometry. Colloidal AFM-based force spectroscopy is employed to estimate brush grafting density and characterize nanomechanical properties in salt water. When the ionic strength-induced swelling behaviors of the two systems are compared, the differences observed on the antipolyelectrolyte response can be correlated with the stiffness variation on brush compression, likely to be promoted by solvation differences. The higher solvation of amide groups is proposed to be responsible for the lower adhesion force of the barnacle cyprid’s temporary adhesive proteins. The adhesion results provide further insights into the antibiofouling activity against barnacle cyprid settlement attributed to polysulfobetaine brushes.

Quantitative analysis of the complete larval settlement process confirms Crisp's model of surface selectivity by barnacles.

For barnacle cypris larvae at the point of settlement, selection of an appropriate surface is critical. Since post-settlement relocation is usually impossible, barnacles have evolved finely tuned surface-sensing capabilities to identify suitable substrata, and a temporary adhesion system for extensive surface exploration. The pattern of exploratory behaviour appears complex and may last for several hours, imposing significant barriers to quantitative measurement. Here, we employ a novel tracking system that enables simultaneous analysis of the larval body movement of multiple individuals over their entire planktonic phase. For the first time, to our knowledge, we describe quantitatively the complete settlement process of cyprids as they explore and select surfaces for attachment. We confirm the 'classic' behaviours of wide searching, close searching and inspection that comprise a model originally proposed by Prof. Dennis Crisp FRS. Moreover, a short-term assay of cyprid body movement has identified inspection behaviour as the best indicator of propensity to settle, with more inspection-related movements occurring in conditions that also promote higher settlement. More than half a century after the model was first proposed by Crisp, there exists a precise method for quantifying cyprid settlement behaviour in wide-ranging investigations of barnacle ecology and applied studies of fouling management.

Use of extract from adults of the triangle barnacle, Balanus trigonus, for reducing fouling in mussel farms

Many millions of dollars are lost each year in the green-lipped mussel (Perna canaliculus) aquaculture industry in New Zealand due to fouling with the triangle barnacle (Balanus trigonus). A novel approach to reducing the numbers of barnacle cyprids settling on the cultured mussels would be to promote their settlement onto sacrificial materials placed in the vicinity of the mussels. An initial field experiment found settlement plates treated with an extract from adult B. trigonus (AE) increased the settlement of conspecific cyprids by 4.6 times. Subsequently, AE-treated material placed near cultured mussels reduced the overall number of barnacles on the mussels by around 4.5 times and increased the number of barnacle-free mussels by almost a third. These results suggest this novel approach to using natural settlement cues to remove barnacle cyprids from the vicinity of valuable shellfish may have potential in controlling these major biofouling pests in shellfish aquaculture.

Chlorine dioxide as an alternative antifouling biocide for cooling water systems: Toxicity to larval barnacle Amphibalanus reticulatus (Utinomi)

Chlorine dioxide (ClO2) is seen as an effective alternative to chlorine, which is widely used as an antifouling biocide. However, data on its efficacy against marine macrofoulants is scanty. In this study, acute toxicity of ClO2 to larval forms of the fouling barnacle Amphibalanus reticulatus was investigated. ClO2 treatment at 0.1mg/L for 20min elicited 45–63% reduction in naupliar metamorphosis, 70% inhibition of cyprid settlement and 80% inhibition of metamorphosis to juveniles. Increase in concentration to 0.2mg/L did not result in any significant difference in the settlement inhibition or metamorphosis. Treatment with 0.2mg/L of ClO2 elicited substantial reduction in the settlement of barnacle larvae compared to control. The study indicates the possibility of using ClO2 as an alternative antifouling biocide in power plant cooling water systems. However, more work needs to be done on the environmental effects of such switchover, which we are currently undertaking.

Effects of pharmacological compounds on the barnacle larval development, metabolism and settlement

Effects of select pharmacological compounds, with known mode of action in vertebrates, were evaluated on the development, metabolism and settlement of larvae of the common fouling barnacle, Amphibalanus amphitrite. Atrovastatin, a lipid-regulating compound, cetrizine hydrochloride, an anti-histamine, atenolol, a β-blocker, and amlodipine, a calcium-channel blocker were the compounds studied. Nauplii treated with these compounds took more days to reach the cypris stage when compared with the control. These compounds also inhibited the settlement of cyprids on Petri dishes. While exposure to these compounds led to a decrease in the metabolic activity of stage III nauplii, it increased the respiratory rate of cyprids. The results emphasize the role of neurotransmitters and lipids in the development and settlement of barnacle larvae on hard surfaces.

Prevention of Settlement of Cyprids to the Bottom of a Spherical Surface by Small-Bubble Flow

Prevention of Settlement of Cyprids to the Bottom of a Spherical Surface by Small-Bubble Flow

Influence of elevated temperature, pCO2, and nutrients on larva-biofilm interaction: Elucidation with acorn barnacle, Balanus amphitrite Darwin (Cirripedia: Thoracica)

Selection of optimal habitat by larvae of sessile organism is influenced by cues offered by the biofilm. Ocean warming and acidification are likely to enforce changes in the biofilm community and inturn influence the settlement process. Hence, we evaluated the influence of biofilm (multispecies and unialgal) and diet-mediated changes on the settlement of Balanus amphitrite cyprids (presettlement non-feeding larval stage) under different combinations of temperature (28, 30, 32 and 34 °C), pCO2 (400, 750 and 1500 μatm) and nutrient (unenriched and f/2 enriched). Nutrient enrichment enhanced the diatom and bacterial abundance at ambient temperature (30 °C) and pCO2 (400 μatm), which inturn increased larval settlement. Elevated pCO2 (750 and 1500 μatm) had no direct effect but a variable cascading effect on the settlement via biofilm-mediated changes was observed, depending on the type of biofilm. In contrast, elevated temperature (32 and 34 °C), either individually or in combination with elevated pCO2 had direct negative effect on settlement. However, biofilm-mediated changes compensated this negative effect. The larval settlement was also influenced by changes in the larval diet. Under elevated temperature and pCO2, cyprids raised with a feed (Chaetoceros calcitrans) from ambient temperature and pCO2 were of poor quality (lower RNA:DNA ratio, lower protein synthetic capacity) and yielded lower settlement. However, cyprids raised with a feed from elevated temperature and pCO2 were of better quality (higher RNA:DNA ratio, higher protein synthetic capacity) and yielded higher settlement. Overall, the observations from the present study provide insights into the significance of biotic interactions on the coastal biofouling communities under future climatic scenario and emphasise the need for future experiments on these aspects.

Antifouling, Antimicrobial, and Antibiocorrosion Multilayer Coatings Assembled by Layer-by-layer Deposition Involving Host–Guest Interaction

Layer-by-layer (LBL) deposition of polyethylenimine-β-cyclodextrin (PEI-β-CD) and ferrocene-modified chitosan (Fc-CHT) via host–guest interaction chemistry was developed for the fabrication of low-fouling, antimicrobial, and biocorrosion inhibition multilayer coatings on stainless steel. An electrochemical method is proposed to monitor and determine the LBL deposition process. The resulting multilayer coatings exhibit good resistance to bacterial adhesion, including Pseudomonas sp. and Staphylococcus aureus, and microalgal attachment of Amphora coffeaeformis. Settlement of barnacle cyprids is also significantly reduced on the bilayer-coated substrates. As the number of assembled host–guest bilayers increases, the antifouling efficacy, antimicrobial efficiency, and antibiocorrosion performance also improve progressively. The stability and durability of the multilayer coatings were ascertained after a 30-day immersion treatment in seawater. Thus, the multilayer coatings deposited via LBL method are potentia...

Hydrogel brushes grafted from stainless steel via surface-initiated atom transfer radical polymerization for marine antifouling

Crosslinked hydrogel brushes were grafted from stainless steel (SS) surfaces for marine antifouling. The brushes were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-methacryloyloxyethyl phosphorylcholine (MPC) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) respectively with different fractions of crosslinker in the feed. The grafted layers prepared with different thickness were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), ellipsometry and water contact angle measurements. With the increase in the fraction of crosslinker in the feed, the thickness of the grafted layer increased and the surface became smooth. All the brush-coated SS surfaces could effectively reduce the adhesion of bacteria and microalgae and settlement of barnacle cyprids, as compared to the pristine SS surface. The antifouling efficacy of the PEGMA polymer (PPEGMA)-grafted surface was higher than that of the MPC polymer (PMPC)-grafted surfaces. Furthermore, the crosslinked hydrogel brush-grafted surfaces exhibited better fouling resistance than the non-crosslinked polymer brush-grafted surfaces, and the antifouling efficacy increased with the crosslinking density. These hydrogel coatings of low toxicity and excellent anti-adhesive characteristics suggested their useful applications as environmentally friendly antifouling coatings.