Exploration of structure-antifouling relationships of capsaicin-like compounds that inhibit zebra mussel (Dreissena polymorpha) macrofouling

Abstract

Macrofouling of aquatic man-made structures by zebra mussels (Dreissena polymorpha) poses significant economic burdens on commercial freshwater shipping and facilities utilising raw water. The negative environmental impact of some current antifouling technologies has limited their use and prompted investigation of non-organometallic and non-oxidising antifoulants as possible environment-friendly alternatives. The plant-derived natural product capsaicin and 18 other compounds with one or more capsaicin-like structural features were tested for their potential to inhibit zebra mussel byssal attachment at a single high concentration of 30 μM. Of these, three compounds displaying the highest levels of attachment inhibition where selected for further concentration-response testing. This testing revealed that capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide), N-vanillylnonanamide, and N-benzoylmonoethanolamine benzoate all inhibited byssal attachment with potency values (EC₅₀) in the micromolar range. None of these compounds were lethal to adult specimens of the water flea, Daphnia magna, at concentrations that inhibited mussel byssal attachment.     

Novel Proteins Identified in the Insoluble Byssal Matrix of the Freshwater Zebra Mussel

The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.     

Putative identification of expressed genes associated with attachment of the zebra mussel (Dreissena polymorpha)

Because of its aggressive growth and firm attachment to substrata, the zebra mussel (Dreissena polymorpha) has caused severe economic and ecological problems since its invasion into North America. The nature and details of attachment of this nuisance mollusc remains largely unexplored. Byssus, a special glandular apparatus located at the root of the foot of the mussel produces threads and plates through which firm attachment of the mollusc to underwater objects takes place. In an attempt to better understand the adhesion mechanism of the zebra mussel, the suppression subtractive hybridization (SSH) assay was employed to produce a cDNA library with genes unique to the foot of the mussel. Analysis of the SSH cDNA library revealed the presence of 750 new expressed sequence tags (ESTs) including 304 contigs and 446 singlets. Using BLAST search, 365 zebra mussel ESTs showed homology to other gene sequences with putative functions. The putative functions of the homologues included proteins involved in byssal thread formation in zebra and blue mussels, exocrine gland secretion, host defence, and house keeping. The generated data provide, for the first time, some useful insights into the foot structure of the zebra mussel and its underwater adhesion.     

Putative identification of expressed genes associated with attachment of the zebra mussel (Dreissena polymorpha)

Because of its aggressive growth and firm attachment to substrata, the zebra mussel (Dreissena polymorpha) has caused severe economic and ecological problems since its invasion into North America. The nature and details of attachment of this nuisance mollusc remains largely unexplored. Byssus, a special glandular apparatus located at the root of the foot of the mussel produces threads and plates through which firm attachment of the mollusc to underwater objects takes place. In an attempt to better understand the adhesion mechanism of the zebra mussel, the suppression subtractive hybridization (SSH) assay was employed to produce a cDNA library with genes unique to the foot of the mussel. Analysis of the SSH cDNA library revealed the presence of 750 new expressed sequence tags (ESTs) including 304 contigs and 446 singlets. Using BLAST search, 365 zebra mussel ESTs showed homology to other gene sequences with putative functions. The putative functions of the homologues included proteins involved in byssal thread formation in zebra and blue mussels, exocrine gland secretion, host defence, and house keeping. The generated data provide, for the first time, some useful insights into the foot structure of the zebra mussel and its underwater adhesion.     

Zebra mussel adhesion: structure of the byssal adhesive apparatus in the freshwater mussel, Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well.     

LIGHT, GRAVITY AND CONSPECIFICS AS CUES TO SITE SELECTION AND ATTACHMENT BEHAVIOUR OF JUVENILE AND ADULT DREISSENA POLYMORPHA PALLAS, 1771

Dreissena polymorpha, the zebra mussel, is one of the mostimportant components of the aquatic ecosystems it inhabits,due to high densities and filtration rates of this fouling organism.This laboratory study deals with several physical and biologicalfactors influencing zebra mussel juveniles (<10 mm) and adults(>10 mm) site selection behaviour and their byssal attachmentto substrate. Mussels preferred black substrate rather thanwhite. This preference was found to be stronger in smaller individuals.Furthermore, all mussels, independently of their size, selectedshadowed sites and avoided illuminated ones. Large mussels attachedmore often in darkness than in light, while for small specimenslight conditions made no difference. Large mussels tended toattach to lower parts of vertical test-tubes, while the numbersof small specimens in both halves of the test-tubes were similar.If initial conditions, used by mussels to select their attachmentsite, were changed by turning the test-tubes upside down, smallindividuals detached and looked for a new site more frequentlythan large ones. Large mussels attached more often in higherdensities of conspecifics, but physical contacts among conspecificsand their size composition seemed to have no impact on attachment.This study shows that many environmental stimuli influence locomotionand attachment of post-metamorphic zebra mussels. Therefore,the behaviour of this group may impact upon adult distributionobserved in field. Preferences exhibited by mussels lead tothe creation of dense aggregations in dark, deep places, providing protectionagainst dessication, predators and excessive water flow. (Received 18 April 2000; accepted 5 October 2000)     

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.     

Inhibition of the reattachment of young adult zebra mussels by single-species biofilms and associated exopolymers

AIMS: To determine the effects of single-species bacterial films and their associated extracellular products on the reattachment of young adult zebra mussels. MATERIALS AND RESULTS: Ten strains of bacteria were isolated from surfaces where adult zebra mussels can be found attached in nature. Single-species biofilms were developed on both glass and polystyrene using these bacteria. The reattachment of zebra mussels (i.e. with byssal threads) was compared between surfaces with and without films. Although no differences were observed in mussel reattachment between glass surfaces with and without films (P > 0.05, anova), a reduction in mussel reattachment between polystyrene surfaces with and without films was observed for seven of the 10 strains (P < or = 0.05 to <0.001, anova). Bacterial extracellular products (BEP) were isolated from five bacterial films and tested for their effects on mussel reattachment. Four of the five sets of isolated extracellular products evoked the same effects as their respective intact biofilms. CONCLUSIONS: We conclude that depending on the substratum, individual strains of bacteria in biofilms can inhibit the reattachment of adult zebra mussels. In some cases, BEP were the source of the inhibitory effects. SIGNIFICANCE AND IMPACT OF THE STUDY: The nature of the substratum on which the biofilms develop affects properties of the biofilm and its extracellular components, which subsequently influences zebra mussel reattachment.     

Mussel attachment on rocky shores: the effect of flow on byssus production

Mussels rely on a strong byssal attachment to persist in a range of habitats with differing rates of water flow. Recent studies, however, suggest that the ability of one mussel species to sense and respond adaptively to the flow in its environment is limited under even modest flow conditions because the process of byssal thread formation is disrupted. This study extends these findings to four mussel species, Mytilus trossulus, M. galloprovincialis, M. californianus, and Modiolus modiolus. Collectively, the response of byssal thread formation decreased with rates of flow above ～25 cm/s and the critical flow threshold was estimated to be <50 cm/s. How can mussels persist on shores where flow is an order of magnitude higher? Using a combination of techniques for measuring flow, velocity profiles were obtained above and within mussel aggregations in the laboratory and in the field. Flow was greatly reduced within mussel aggregations, ranging from 0.1% to 10% of free-stream velocity. These results suggest one key to the success of mussels in habitats with high rates of flow is the ability to form aggregations that ameliorate flows to a level that is conducive to byssal thread formation.     

Attachment strength of an adhesive nuisance mussel,limnoperna fortunei,against water flow

The attachment strength of the freshwater mussel Limnoperna fortunei against water flow was studied. Newton's expression successfully described the hydrodynamic drag force acting on the mussel with a drag coefficient value of 1.03. The drag‐resistant force (defined as hydrodynamic drag force at mussel detachment) was smaller than the detachment force measured using a tensile load test. A fairly good correlation was obtained between the drag‐resistant force and the number of secreted threads. The drag‐resistant force divided by the number of threads increased with shell size, suggesting that byssal thread strength increased with mussel growth. For the mussel specimens obtained from a water transmission pipe, thread width increased with shell size. However, thread width was not dependent on current velocity. There was no correlation between the number of secreted threads and shell length, which indicated that the number of secreted threads did not change with mussel size. Therefore, the water velocity needed to detach mussels increases with shell size of the mussel when the number of secreted threads is constant. The increases in the water velocity to detach mussels with larger shells suggests that the mussel becomes more resistant to water flow as it grows. It is estimated that a flow velocity of around lms^‐1 is critical for attachment/detachment of a juvenile mussel with a shell length of a few millimeters and one hundred byssal threads.     

The ecomechanics of mussel attachment: from molecules to ecosystems

One aspect of the physiological ecology of intertidal organismsis their mechanical design, which can be explored at many hierarchicallevels, from molecules to ecosystems. Mechanical structures,as with any other physiological feature, require energy to constructand maintain, are subject to manufacturing and evolutionaryconstraints, and influence ecological performance. This contributionfocuses on the ecomechanics of mussel attachment, which contributesto the competitive dominance of mussels on many wave-swept shores.Examples are presented to illustrate the hierarchical natureof mussel attachment, how levels of the hierarchy are interrelated,and where gaps in our knowledge remain. For example, water motiongenerates forces that mechanically deform byssal threads, butmay also enhance the rate at which threads subsequently restoretheir original toughness. Furthermore, the ability of musselsto sense and respond to changes in their flow environment byproducing a stronger attachment may be subject to physiologicalconstraints, which in turn may have important consequences forthe ecological response of mussels to shifts in wave climate.Thus an integrative approach to the study of byssal attachmentis needed to fully understand this important aspect of the physiologicalecology of mussels on rocky intertidal shores.     

A comparative study of byssogenesis on zebra and quagga mussels: the effects of water temperature, salinity and light-dark cycle

The quagga mussel (Dreissena rostriformis bugensis) and zebra mussel (Dreissena polymorpha) are invasive freshwater bivalves in Europe and North America. The distribution range of both Dreissena species is still expanding and both species cause major biofouling and ecological effects, in particular when they invade new areas. In order to assess the effect of temperature, salinity and light on the initial byssogenesis of both species, 24 h re-attachment experiments in standing water were conducted. At a water temperature of 25°C and a salinity of 0.2 psu, the rate of byssogenesis of D. polymorpha was significantly higher than that of D. rostriformis bugensis. In addition, byssal thread production by the latter levelled out between 15°C and 25°C. The rate of byssogenesis at temperatures<25°C was similar for both species. Neither species produced any byssal threads at salinities of 4 psu or higher. At a salinity of 1 psu and a water temperature of 15°C, D. polymorpha produced significantly more byssal threads than D. rostriformis bugensis. There was no significant effect of the length of illumination on the byssogenesis of either species. Overall, D. polymorpha produced slightly more byssal threads than D. rostriformis bugensis at almost all experimental conditions in 24 h re-attachment experiments, but both species had essentially similar initial re-attachment abilities. The data imply that D. rostriformis bugensis causes biofouling problems identical to those of D. polymorpha.     

Effects of artificial epibionts on byssogenesis,attachment strength,and movement in two size classes of the blue mussel,Mytilus edulis

Blue mussels (Mytilus edulis) can alter the strength of byssal attachment and move between and within mussel aggregations on wave‐swept shores, but this movement ability may be limited by epibiont fouling. We quantified the effects of artificial epibiont fouling on the production of byssal threads, attachment strength, and movement in two size classes of blue mussels. In a factorial experiment, large epibiont‐covered mussels produced more functional byssal threads (i.e., those continuous from animal to substrate) after 24 h than large unfouled and small fouled mussels, but not more than small unfouled mussels. Small unfouled mussels formed and released more byssus bundles compared to any other treatment group, which indicates increased movement. Conversely, epibiont fouling resulted in decreased numbers of byssus bundles shed, and therefore reduced movement in small mussels. Epibiont‐covered mussels started producing byssal threads sooner than unfouled mussels, while small mussels began producing byssal threads earlier compared to large mussels. Mean attachment strength from both size classes increased by 9.5% when mussels were artificially fouled, and large mussels had a 34% stronger attachment compared to small mussels. On the other hand, a 2.3% decrease in attachment strength was found with increasing byssus bundles shed. Our results suggest that fouling by artificial epibionts influences byssal thread production and attachment strength in large mussels, whereas epibionts on small mussels impact their ability to move. Mussels are able to respond rapidly to fouling, which carries implications for the dynamics of mussel beds in their intertidal and subtidal habitats, especially in relation to movement of mussels within and among aggregations.     

Zebra Mussel Antifouling Activity of the Marine Natural Product Aaptamine and Analogs

Several aaptamine derivatives were selected as potential zebra mussel (Dreissena polymorpha) antifoulants because of the noteworthy absence of fouling observed on Aaptos sponges. Sponges of the genus Aaptos collected in Manado, Indonesia consistently produce aaptamine-type alkaloids. To date, aaptamine and its derivatives have not been carefully evaluated for their antifoulant properties. Structure–activity relationship studies were conducted using several aaptamine derivatives in a zebra mussel antifouling assay. From these data, three analogs have shown significant antifouling activity against zebra mussel attachment. Aaptamine, isoaaptamine, and the demethylated aaptamine compounds used in the zebra mussel assay produced EC₅₀ values of 24.2, 11.6, and 18.6 μM, respectively. In addition, neither aaptamine nor isoaaptamine produced a phytotoxic response (as high as 300 μM) toward a nontarget organism, Lemna pausicostata, in a 7-day exposure. The use of these aaptamine derivatives from Aaptos sp. as potential environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a possible control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses.     

Preferences of the Ponto-Caspian amphipod Dikerogammarus haemobaphes for living zebra mussels

A Ponto-Caspian amphipod Dikerogammarus haemobaphes has recently invaded European waters. In the recipient area, it encountered Dreissena polymorpha , a habitat-forming bivalve, co-occurring with the gammarids in their native range. We assumed that interspecific interactions between these two species, which could develop during their long-term co-evolution, may affect the gammarid behaviour in novel areas. We examined the gammarid ability to select a habitat containing living mussels and searched for cues used in that selection. We hypothesized that they may respond to such traits of a living mussel as byssal threads, activity (e.g. valve movements, filtration) and/or shell surface properties. We conducted the pairwise habitat-choice experiments in which we offered various objects to single gammarids in the following combinations: (1) living mussels versus empty shells (the general effect of living Dreissena ); (2) living mussels versus shells with added byssal threads and shells with byssus versus shells without it (the effect of byssus); (3) living mussels versus shells, both coated with nail varnish to neutralize the shell surface (the effect of mussel activity); (4) varnished versus clean living mussels (the effect of shell surface); (5) varnished versus clean stones (the effect of varnish). We checked the gammarid positions in the experimental tanks after 24 h. The gammarids preferred clean living mussels over clean shells, regardless of the presence of byssal threads under the latter. They responded to the shell surface, exhibiting preferences for clean mussels over varnished individuals. They were neither affected by the presence of byssus nor by mussel activity. The ability to detect and actively select zebra mussel habitats may be beneficial for D. haemobaphes and help it establish stable populations in newly invaded areas.     

Habitat Shift in Invading Species: Zebra and Quagga Mussel Population Characteristics on Shallow Soft Substrates

Unexpected habitat innovations among invading species are illustrated by the expansion of dreissenid mussels across sedimentary environments in shallow water unlike the hard substrates where they are conventionally known. In this note, records of population characteristics of invading zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels from 1994 through 1998 are reported from shallow (less than 20m) sedimentary habitats in western Lake Erie. Haphazard SCUBA collections of these invading species indicated that combined densities of zebra and quagga mussels ranged from 0 to 32,500 individuals per square meter between 1994 and 1998, with D. polymorpha comprising 75–100% of the assemblages. These mixed mussel populations, which were attached by byssal threads to each other and underlying sand-grain sediments, had size–frequency distributions that were typical of colonizing populations on hard substrates. Moreover, the presence of two mussel cohorts within the 1994 samples indicated that these species began expanding onto soft substrates not later than 1992, within 4 years of their initial invasion in western Lake Erie. Such historical data provide baselines for interpreting adaptive innovations, ecological interactions and habitat shifts among the two invading dreissenid mussel species in North America.     

Reciprocal Effects between Burying Behavior of a Larval Dragonfly (Odonata: Macromia illinoiensis) and Zebra Mussel Colonization

Invasive zebra mussels (Dreissena polymorpha) often colonize dragonfly larvae, especially spawling species whose survivorship to emergence as terrestrial predators is consequently reduced. Using individuals of the sprawler, Macromia illinoiensis, as their own controls, we compared the burying behavior of penultimate instar larvae before (i.e. baseline) and after their colonization by zebra mussels under ambient conditions. Individuals that took longer to bury themselves when mussel-free had a higher rate of colonization by mussels over a five-day period compared to those that buried faster. In contrast, the depth at which individuals buried when mussel-free was not predictive of subsequent colonization rate. Although mean bury time did not differ between baseline and when an individual carried one or more mussels, colonized larvae buried more shallowly than when mussel-free. Moreover, attached mussels increased the risk of subsequent colonization by zebra mussels. After naturally losing all of their attached mussels, bury time and depth of individuals did not differ from their baseline behavior, indicating that the changes in the behavior of colonized individuals were due to mussel loads and not their time in captivity. Under natural conditions, the positive feed-back between mussel attachment and increasing vulnerability to colonization helps explain how mussel loads, which are lost at molting, can accumulate quickly over the duration of the final larval stadium. Because zebra mussel attachment decreases the crypsis that that a M. illinoiensis gains from burying, the invasive mussel may also make dragonfly larvae more detectable to visual predators.     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

Rapid range expansion of the invasive quagga mussel in relation to zebra mussel presence in The Netherlands and Western Europe

Since its appearance in 2006 in a freshwater section of the Rhine–Meuse estuary (Hollandsch Diep, The Netherlands), the non-indigenous quagga mussel has displayed a rapid range expansion in Western Europe. However, an overview characterising the spread and impacts of the quagga mussel in this area is currently lacking. A literature study, supplemented with field data, was performed to gather all available data and information relating to quagga mussel dispersal. Dispersal characteristics were analysed for rate and direction and in relation to hydrological connectivity and dispersal vectors. To determine ranges of conditions suitable for quagga mussel colonisation, physico-chemical characteristics of their habitats were analysed. After its initial arrival in the freshwater section of the Rhine-Meuse estuary and River Danube, the quagga mussel demonstrated a rapid and continued range expansion in Western Europe. Quagga mussels have extended their non-native range to the network of major waterways in The Netherlands and in an upstream direction in the River Rhine (Germany), its tributaries (rivers Main and Moselle) and the River Meuse (Belgium and France). The calculated average quagga mussel dispersal rate in Europe was 120 km year^?1 (range 23–383 km year^?1). Hydrological connectivity is important in determining the speed with which colonisation occurs. Dispersal to water bodies disconnected from the freshwater network requires the presence of a suitable vector e.g. pleasure boats transferred over land. Upstream dispersal is primarily human mediated through the attachment of mussels to watercraft. The relative abundance of quagga mussel to zebra mussel has greatly increased in a number of areas sampled in the major Dutch rivers and lakes and the rivers Main and Rhine and the Rhine–Danube Canal leading to a dominance shift from zebra mussels to quagga mussels. However, evidence for displacement of the zebra mussel is limited due to the lack of temporal trends relating to the overall density of zebra and quagga mussel.