The influence of natural surface microtopographies on fouling

Multiple antifouling strategies of marine organisms may consist of combinations of physical, chemical and mechanical mechanisms. In this study, the role of surface microtopography (< 500 microns) of different marine organisms, such as Cancer pagurus, Mytilus edulis, Ophiura texturata and the eggcase of Scyliorhinus canicula, has been investigated as a possible component of their defence systems. High resolution resin replicates of these natural surface structures were exposed to natural fouling in field experiments. Abundances of recruits were determined and compared to those on untextured, but otherwise identical, control surfaces to quantify the influence of the different microtopographies on fouling rates. Antifouling effects of microtopographies varied with type of microtopography and coloniser species. The surface microtopography of C. pagurus significantly rejected macrofoulers. The surface structures of the eggcase and O. texturata had repellent effects on microfoulers. Barnacle settlement was temporarily reduced on surface microtopographies of M. edulis and the eggcase. These results emphasise the promising nontoxic antifouling properties of microtextured surfaces.     

Nontoxic piperamides and their synthetic analogues as novel antifouling reagents

Bioassay-guided isolation of an acetone extract from a terrestrial plant Piper betle produced four known piperamides with potent antifouling (AF) activities, as evidenced by inhibition of settlement of barnacle cypris larvae. The AF activities of the four piperamides and 15 synthesized analogues were compared and their structure–activity relationships were probed. Among the compounds, piperoleine B and 1-[1-oxo-7-(3′,4′-methylenedioxyphenyl)-6E-heptenyl]-piperidine (MPHP) showed strong activity against settlement of cyprids of the barnacle Balanus amphitrite, having EC₅₀ values of 1.1?±?0.3 and 0.5?±?0.2?μg?ml^?1, respectively. No toxicity against zebra fish was observed following incubation with these two compounds. Besides being non-toxic, 91% of piperoleine B-treated cyprids and 84% of MPHP-treated cyprids at a concentration of 100?μM completed normal metamorphosis in recovery bioassays, indicating that the anti-settlement effect of these two compounds was reversible. Hydrolysis and photolysis experiments indicated that MPHP could be decomposed in the marine environment. It is concluded that piperamides are promising compounds for use in marine AF coatings.     

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.     

The extraction of glycogen from marine invertebrate tissues

Summary 1. It is shown that the extraction by 5% trichloracetic acid of glycogen from the tissues of some typical marine invertebrates(Balanus balanoides, Nephtys hombergi, Carcinus maenas, Mytilus edulis) is a function of the homogenization procedure.2. In agreement with work on rat tissues, glycogen fractions cannot be differentiated by their solubility in cold 5% trichloracetic acid. Total glycogen only need be determined.

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Die Glykogenextraktion aus dem Gewebe mariner Invertebraten

Kurzfassung Es wird der Nachweis geführt, daß die Glykogenextraktion aus den Geweben einiger mariner Invertebraten(Balanus balanoides, Nephtys hombergi, Carcinus maenas, Mytilus edulis) durch 5% Trichloressigsäure von dem Homogenisierungsverfahren abhängt. In Übereinstimmung mit Untersuchungen an Rattengewebe können die Glykogenfraktionen nicht durch ihre Löslichkeit in kalter 5% Trichloressigsäure unterschieden werden. Daher ist nur die Bestimmung des Gesamt-Glykogengehaltes angebracht.

     

Phylogeography and historical ecology of the North Atlantic intertidal

Abstract Recent glaciation covered the full extent of rocky intertidal habitat along the coasts of New England and the Canadian Maritimes. To test whether this glaciation in fact caused wholesale extinction of obligate rocky intertidal invertebrates, and thus required a recolonization from Europe, we compared American and European populations using allelic diversity and techniques adapted from coalescent theory. Mitochondrial DNA sequences were collected from amphi-Atlantic populations of three cold-temperate obligate rocky intertidal species (a barnacle, Semibalanus balanoides , and two gastropods, Nucella lapillus and Littorina obtusata ) and three cold-temperate habitat generalist species (a seastar, Asterias rubens ; a mussel, Mytilus edulis , and an isopod, Idotea balthica ). For many of these species we were able to estimate the lineage-specific mutation rate based on trans-Arctic divergences between Pacific and Atlantic taxa. These data indicate that some obligate rocky intertidal taxa have colonized New England from European populations. However, the patterns of persistence in North America indicate that other life-history traits, including mechanisms of dispersal, may be more important for surviving dramatic environmental and climatic change.     

Cypris larvae (Cirripedia: Balanomorpha) display auto-fluorescence in nearly species-specific patterns

The identification of balanomorph larvae plays an important role in ecological study and for protection against biofouling. However, it is difficult to identify species of cyprids (settling larvae) of balanomorph barnacles, as they show remarkably similar morphology. Some authors have suggested distinguishing cyprids of different species by carapace length, pigmentation, and fine carapace detail. However, such criteria are only applicable to a narrow range of balanomorph species. Recently, we were have serendipitously found species-specific distribution of fluorescent substances in cultured cyprids obtained from adult balanomorph barnacles, collected near the coast of Japan. Fluorescent patterns (FPs) of cyprids from 11 species were classified into five major groups. Cyprids specimens collected from the field were estimated, based on the FPs and other morphological characteristics (pigmentation and carapace length), after which their species were identified using the following two criteria: the morphology of adults derived from the field cyprids that adhered to a culture dish, and cyprid 12S rRNA gene sequence. The results of species estimation by FPs largely corresponded to the correct species identification. Other FP groups were found in the field cyprids. This study of FPs should be helpful for identification of cyprid species.     

The prevention of microfouling and macrofouling on hydrogels impregnated with either Arquad 2C-75 or benzalkonium chloride

Optically clear, surfactant loaded poly (2-hydroxyethyl methacrylate) (pHEMA) hydrogels can be used to prevent fouling on optical windows of marine underwater sensors. To act successfully in this capacity, hydrogels need to prevent both microfouling and macrofouling. Panel trials were conducted using four different materials: unloaded hydrogels, hydrogels containing either benzalkonium chloride (BAC) or dicocodimethylammonium chloride (Arquad 2C-75) and PMMA coupons. Three panels were deployed at staggered intervals (2, 4 and 6 weeks) before the main settlement season of Semibalanus balanoides and Mytilus edulis in the Firth of Clyde, Scotland. Panels were left for a total period of 10, 12 and 14 weeks respectively. Results showed that no sample completely resisted fouling, but Arquad 2C-75 hydrogels were extremely effective at preventing both microfouling and macrofouling. The most heavily fouled materials were unloaded hydrogels and PMMA, despite differences in initial hydrophilicities. Arquad 2C-75 hydrogels were equally effective at preventing larval settlement, for up to 14 weeks.     

Chemical defence in mussels: antifouling effect of crude extracts of the periostracum of the blue mussel Mytilus edulis

Shells of the blue mussel Mytilus edulis remain free of fouling organisms as long as they possess an intact periostracum, and a multiple antifouling defence that comprises a ripple-like microtopography and the production of chemical antifouling compounds has been suggested previously. This study investigates the chemical defence strategy of blue mussels for the first time. Six crude extracts of the periostracum of intact shells were made using solvents of increasing polarity. These extracts were tested against common fouling organisms in laboratory based bioassays. Non-polar and moderately polar fractions showed the highest activities: the diethyl ether fraction strongly inhibited attachment of Balanus amphitrite cyprids and the marine bacteria Cobetia marina and Marinobacter hydrocarbonoclasticus. Attachment of the benthic diatom Amphora coffeaeformis was significantly reduced by the dichloromethane extract, whereas both ethyl acetate and diethyl ether fractions slowed diatom growth. These results provide the first evidence of surface bound compounds that may moderate surface colonisation.     

A molecular phylogeny of the marine mussel genus Perna (Bivalvia: Mytilidae) based on nuclear (ITS1&2) and mitochondrial (COI) DNA sequences

A molecular phylogeny is presented for marine mussels of the genus Perna, based on nuclear (ITS1,ITS2) and mitochondrial (COI) DNA sequence data. The three generally recognised species (Perna viridis, Perna perna and Perna canaliculus) and one putative species (Perna picta) were each sampled from several locations within their known geographic distributions. A range of phylogenetic analyses was used to investigate the current taxonomic assignments, evolutionary relationships and the biogeographical history of the genus. The different analyses produced similar, well supported topologies and verified the monophyly of the genus with respect to five mytilid outgroup species. P. perna (Atlantic), P. viridis (Indo-West Pacific), and P. canaliculus (New Zealand) each formed distinct clades, confirming their specific status. Putative P. picta from North Africa clustered within the P. perna clade and is not regarded as a separate species. P. perna and P. canaliculus were the most closely related of the three species. Possible biogeographic explanations for the present species distributions are evaluated.     

藤壶金星幼虫附着变态机制

藤壶属节肢动物门(Arthropoda)甲壳亚门(Crustacea)蔓足下纲(Cirripedia)围胸总目(Thoracica), 具备特殊的形态结构、生活史和种群生态特征,是最主要的海洋污损生物。其幼虫阶段通常经历6期无节幼体和1期不摄食的金星幼虫,从浮游的金星幼虫附着变态成固着的稚体是藤壶生活史中的一个关键环节。外界化学和生物因子中成体提取物、水溶性信息素、足迹、神经递质、激素、生物膜等均影响藤壶金星幼虫的附着变态;内在因子即金星幼虫的生理状态(能量储量和年龄)决定了其对外界因子的反应程度。概括了近年来藤壶附着变态生理机制和分子机制研究的进展,可为深入了解藤壶金星幼虫附着变态机制提供参考,也为开发新型、高效、环保的防污剂提供理论指导。     

Isethionic acid and floridoside isolated from the red alga, Grateloupia turuturu, inhibit settlement of Balanus amphitrite cyprid larvae

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml(-1)).     

Terpenes from the Red Alga <Emphasis Type="Italic">Sphaerococcus coronopifolius</Emphasis> Inhibit the Settlement of Barnacles

In this study, we screened eight terpenes isolated from the organic extract of Sphaerococcus coronopifolius for their antifouling activity in order to find possible new sources of non-toxic or less toxic bioactive antifoulants. The anti-settlement activity (EC₅₀) and the degree of toxicity (LC₅₀) of S. coronopifolius metabolites was evaluated using larvae of the cirriped crustacean Amphibalanus (Balanus) amphitrite (cyprids and nauplii) as model organism. For five of eight tested metabolites EC₅₀ was lower than 5 mg/L. The most promising results were observed for bromosphaerol (3), which expressed an EC₅₀ value of 0.23 mg/L, in combination with low toxicity levels (LC₅₀ > 100 mg/L). The therapeutic ratio—an index used to estimate whether settlement inhibition is due to toxicity or other mechanisms—is also calculated and discussed.     

Effect of cyprid age on the settlement of balanus amphitrite darwin in response to natural biofilms

Larval settlement of the barnacle Balanus amphitrite Darwin (Cirripedia, Balanidae) is influenced by natural biofilms. In previous work by others, discriminatory settlement of aged cyprids has been observed in response to biofilms of different age. This study extends prior work by considering the effect of the age of cyprids on the outcome of settlement assays. Settlement was investigated with 0‐day‐old (newly metamorphosed) and 5‐day‐old cyprids. Biofilms under investigation were developed in the field for periods of 5 d and 1 month, and were subsequently included in laboratory settlement assays with a choice between a filmed and an unfilmed substratum. The bioassay was modified from the conventional horizontal dish design in order to generate a low water surface‐to‐volume ratio, which served to suppress larval entrapment in an organic layer on the water surface. Irrespective of cyprid age, a clear discrimination between a filmed and an unfilmed substrata was observed, and the preference for filmed or unfilmed substratum was dependent on the age of the cyprids. Settlement of 0‐day‐old cyprids was inhibited by a biofilmed substratum whereas induction occurred with aged cyprids. This pattern of settlement was independent of biofilm age. Bacterial abundance on unfilmed substrata in treatments and controls was significantly lower than that on biofilmed surfaces, confirming that bacterial contamination did not change the qualitative option during the assay.     

Size and settling behaviour in male and female cypris larvae of the parasitic barnacle Sacculina carcini Thompson (Crustacea: Cirripedia: Rhizocephala)

Twenty-one broods from different externae of Sacculina carcini Thompson were cultured to the cypris stage. The size of the cyprids was measured and the larvae subjected to settling upon unparasitized crabs (Garcinus maenas (L.)) and small juvenile externae. The cyprids occur in two sizes that may appear singly or together in the same brood. Small cyprids are of the female sex that settle upon crabs and are infective, while large cyprids are of the male sex and only settle upon juvenile externae. These results are in agreement with other well-studied rhizocephalans.     

An improved barnacle attachment inhibition assay

The Balanus amphitrite attachment inhibition assay, developed by Rittschof et al. (1992), has been widely used for screening antifouling compounds. One shortcoming of this assay, however, is the low (often < 40%) attachment rate of cyprids, including in the controls that contain seawater only. In this study, trapping of cyprids at the air-water interface was found to be a main contributor to the low attachment rate. Procedures to eliminate the air-water interface were thus introduced. With the improved bioassay, a much higher cyprid attachment rate (>70%) was attained. To further illustrate the usefulness of the improved assay (ie eliminating the air-water interface), the effects of the length of cyprid storage and the effect of a reference biocide, tributyltin chloride, on the survival and attachment rate of the cyprids were examined. The length of cyprid storage was important, with newly molted cyprids, 3- to 9-day old cyprids and 12-day old cyprids having an attachment rate of 43%,>75% and 36%, respectively. The low attachment rate in the newly molted cyprids was due to a high percentage of cyprids that still swam at the end of exposure period, whereas the low attachment rate in the 12-day old cyprids was due to a high mortality rate. The cyprids showed an EC50 of 22 microg l(-1) for attachment inhibition and LC50 of 25 microg l(-1) for mortality. It is concluded that the air-water interface has an important confounding effect on cyprid attachment rate in the conventional B. amphitrite attachment assay. By eliminating the air-water interface more robust quantitative assay results were obtained.     

Isethionic Acid and Floridoside Isolated from the Red Alga,Grateloupia turuturu,Inhibit Settlement of Balanus amphitrite Cyprid Larvae

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-α-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml^?1).     

Effect of the physiological condition of cyprids and laboratory-mimicked seasonal conditions on the metamorphic successes of Balanus amphitrite Darwin (Cirripedia; Thoracica)

In order to understand the interaction between extrinsic and intrinsic factors on the metamorphic success of Balanus amphitrite cyprids, we investigated the effect of cyprids energy reserves and physiological age (physiological condition of cyprids) on the metamorphic success under five different combinations of water temperature and salinity mimicking different seasons in subtropical Hong Kong: 18 °C/30‰ (winter), 22 °C/34‰ (spring), 28 °C/30‰ (summer), 28 °C/20‰ (rainy season), and 25 °C/32‰ (autumn). The energy reserves of the cyprids were quantified by the ratio of triacylglycerols (TAG) to deoxyribonucleic acid (DNA). The metamorphic success of cyprids was distinctly influenced by their physiological condition and laboratory-mimicked seasons. There was a significant interaction between these factors. In clear contrast to young cyprids with low-TAG/DNA ratio, there was no difference in the metamorphic success of aged cyprids with high-TAG/DNA ratio among seasons. Our results suggest that the physiological condition of cyprids and seasonal variation in water temperature and salinity may jointly determine the metamorphic success of cyprids, and consequently, modulate the population dynamics of B. amphitrite in subtropical waters.     

Effect of ultrasound on cyprids and juvenile barnacles

Settlement inhibition of barnacle (Amphibalanus amphitrite) cypris larvae resulting from exposure to ultrasound was measured at three frequencies (23, 63, and 102 kHz), applied at three acoustic pressure levels (9, 15, and 22 kPa) for exposure times of 30, 150, and 300?s. The lowest settlement was observed for 23?kHz, which also induced the highest cyprid mortality. Cyprid settlement following exposure to 23?kHz at 22?kPa for 30?s was reduced by a factor of two. Observing surface exploration by the cyprids revealed an altered behaviour following exposure to ultrasound: step length was increased, while step duration, walking pace, and the fraction of cyprids exploring the surface were significantly reduced with respect to control cyprids. The basal area of juvenile barnacles, metamorphosed from ultrasound-treated cyprids was initially smaller than unexposed individuals, but normalised over two weeks' growth. Thus, ultrasound exposure effectively reduced cyprid settlement, yet metamorphosed barnacles grew normally.     

Quantifying the exploratory behaviour of Amphibalanus amphitrite cyprids

The behavioural response of cypris larvae from A. amphitrite (=Balanus amphitrite) exploring three model glass surfaces is quantified by close-range microscopy. Step length and step duration measurements reveal a response to both surface properties and flow. Without flow, 2-day-old cyprids took larger steps with shorter step duration on hydrophilic glass surfaces (bare and NH₂-treated) vs hydrophobic glass (CH₃-treated). These parameters suggest a more detailed, local inspection of hydrophobic surfaces and a more extensive exploration for hydrophilic surfaces. Cyprids under flow took longer steps and exhibited shorter probing times on hydrophobic glass. On hydrophilic glass, cyprids increased their step duration under flow. This active response is attributed to drag and lift forces challenging the cyprids' temporary anchoring to the substratum. Seven-day-old cyprids showed almost no discrimination between the model surfaces. Microscopic-scale observation of cyprid exploration is expected to provide new insights into interactions between cyprids and surfaces.