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.     

Oxygen-depleted surfaces: a new antifouling technology

A novel, non-toxic strategy to combat marine biofouling is presented. The technology is paint with additions of up to 43% of industrial protein. Through microbial degradation of the protein component, an oxygen-depleted layer rapidly forms in a 0.2 mm layer close to the paint surface. With the present paint formulations, a stable, O₂-depleted layer can persist for 16 weeks. Barnacle larvae (cyprids) did not settle on panels where oxygen saturation was <20%, and cyprids were killed when exposed to O₂-free water for more than 1 h. It is also shown that the O₂-depleted layer will rapidly reform (within 15 min) after exposure to turbulent flow. Field exposure of panels for 16 weeks showed that paint with protein reduced fouling by barnacles and bryozoans by 80% and close to 100%, respectively. The results suggest that this novel technology may be developed into a non-toxic alternative to copper-based antifouling paints, especially for pleasure boats in sensitive environments. There is clearly potential for further development of the paint formulation, and a full-scale test on a boat-hull suggested that service-life under realistic operations needs to be improved.     

Probing the settlement signals of Amphibalanus amphitrite

To achieve their reproductive potential, barnacles combine tactile exploration of surface structural properties and integration of cellular signals originating from their antennular sensory setae within a developmentally defined, temporally narrow window of settlement opportunity. Behavioural assays with cyprids coupled with biometric analysis of scanning electron microscopy-acquired images in the presence of specific chemical compounds were used to investigate how settlement on a substratum is altered in response to the presence of these compounds. Impeding tactile exploration was shown which altered cellular signalling and/or induced malformation of anatomical features of the antennular sensory setae, which disrupted the settlement behaviour of the model barnacle species Amphibalanus amphitrite. It is concluded that surface exploration by the cyprids relies on mechanical and nociception-related and calcium-mediated signals while a protein kinase C signalling cascade controls the timely metamorphosis of the cyprids to sessile juveniles.     

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.     

Copper and iron concentrations in Ascophyllum nodosum (Fucales, Phaeophyta) from different sites in Ireland and after culture experiments in relation to thallus age and epiphytism

In laboratory experiments, copper concentrations in plants of Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaeophyta) increased with the concentrations in the culture media and were highest in younger, meristematic thallus parts. After initial accumulation in high-copper medium and subsequent transfer to clean seawater for 5 days, no release of copper could be detected. Iron concentrations in A. nodosum tissue were not related to concentrations in the culture medium. Differences between copper concentrations in plants from different sites in areas with high yachting activity in Strangford Lough, Northern Ireland, could be explained by differences in water motion and human activity, in particular the application of copper-releasing antifouling paints to leisure boats. Iron concentrations were also highest in plants from the sheltered, polluted site but did not differ significantly between the other two sites. No differences in copper nor iron concentrations were found between different-aged thallus parts of plants from any site. X-ray microanalysis revealed that most of the iron detected was located in epiphytic pennate diatoms on the A. nodosum surface. In thallus areas without diatoms, iron levels were below the detection limit for X-ray microanalysis. Mapping for copper indicated that most of the accumulated copper was located in cells near and immediately below the thallus surface. "Epidermis"-shedding occurred in plants from the culture experiments and also in freshly-collected material and may have resulted in a loss of metal ions accumulated by surface cells and by epiphytic diatoms. The results suggest that A. nodosum could be used as a biological indicator for copper but not for iron, and that young, apical plant parts are most sensitive to changes in metal concentrations in the water.     

Combined effect of cyprid age and lipid content on larval attachment and metamorphosis of Balanus amphitrite darwin

Various algal diets of different lipid content and composition were used to rear batches of naupliar larvae of Balanus amphitrite. The cyprids in these larval batches differed in lipid content and were used to investigate the combined effect of cyprid lipid content and cyprid age on attachment and metamorphosis. For this purpose, cyprids were aged for 0,3 and 6 d at 8°C prior to utilization in laboratory attachment assays. The percentage attachment of cyprids with similar lipid content differed significantly among the three age categories. A strong and a weak positive relationship between cyprid lipid content and attachment were monitored in young and old cyprids, respectively. A significant interaction (two‐way ANOVA) between cyprid age and lipid content was observed, indicating that these factors jointly affect larval attachment and metamorphosis in B. amphitrite from the beginning of the cyprid stage.     

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。     

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.     

Antennulary sensory organs in cyprids of Octolasmis and Lepas (Crustacea: Thecostraca: Cirripedia: Thoracica): a scanning electron microscopic study

Cypris larvae of the pedunculate barnacles Octolasmis angulata (Poecilasmatidae), Lepas australis, L. pectinata, and Dosima fascicularis (Lepadidae) were studied with scanning electron microscopy, focusing on the sensory setae and the attachment disc on the antennules. The antennules of O. angulata did not exhibit any remarkable trait, but carry the same number of setae as seen in most other thoracicans. The third segment is bell-shaped and quite distinct from the second and its attachment disc is surrounded by a skirt. We found several potential synapomorphies in antennulary morphology between cyprids of the lepadid species but none of them were shared with the cyprids of Octolasmis; the list of unique lepadid characters includes: one additional, preaxial seta on the second segment; multiple similar (up to eight) postaxial setae (PS3) on the third segment, unlike all other thoracicans, where there is only a single PS3; the third segment consists almost entirely of the attachment disc, which is distended and surrounded by two parallel rows of radial setae; on the fourth segment the terminal seta E is diminutive. We found no traits in cyprids of Octolasmis that seem to be adaptations to their attachment site within the branchial chamber of swimming crabs and, in particular, no similarities with cyprids of rhizocephalan barnacles, many of which also attach in the gill chamber. The synapomorphies between cyprids of the lepadid species may be adaptations to their life in the neuston.     

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.     

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 distribution and orientation of epizoic barnacles on crabs

The distribution and orientation of Balanus crenatus on Carcinus maenas is described. Elminius modestus was also found on C. maenas but it was not common. Both species of barnacle were also found on Cancer pagurus, although neither species of barnacle appeared to be as common on Cancer as on Carcinus. Possible reasons for this are discussed.
Treating the carapaces of similar sized C. maenas as sampling units it is clear that the distribution of B. crenatus over these sampling units is not random but is aggregated. This probably arises because of the gregarious nature of the cyprids when they settle out but other possibilities are considered. B. crenatus on the carapace of C. maenas is found exclusively in the grooves and depressions on the carapace and this is because the cyprids preferentially settle in concavities.
The orientation of B. crenatus also shows a consistent pattern, with the cirral nets facing predominantly backwards. This pattern could arise because the barnacle cyprids orientate to water currents, generated by the exhalant respiratory currents, flowing forwards over the carapace.     

The relation between cypris ultrastructure and metamorphosis in male and female Sacculina carcini (Crustacea,Cirripedia)

Summary To elucidate current controversies on sex in rhizocephalan barnacles, broods of Sacculina carcini, infesting the shore crab Carcinus maenas, were raised to cyprids in the laboratory and followed through settlement and metamorphosis. Free-swimming cyprids were studied by transmission electron microscopy and occur in male and female morphological types, which differ in the structure of carapace cuticle, antennular cuticle, antennular glands, and the cells suspected of being the stem cells during metamorphosis. These dissimilarities are in addition to the already known differences in cypris size, in number of antennular sense organs, and in substrata settled on by morphological males and females. Metamorphosing males (trichogons) and females (kentrogons) are illustrated in interference phase-contrast micrographs. The morphological differences between male and female cyprids are directly related to their dissimilar metamorphosis. Hence, cyprids of male morphology are anatomically incapable of metamorphosing into kentrogons, while cyprids of female morphology cannot metamorphose into trichogons. The determination of sex in rhizocephalan barnacles is discussed.The results refute the hypothesis that sex in Sacculina carcini is determined environmentally, e.g., by the substratum encountered by the cyprids at settlement. It is concluded that sex is determined already in the free-swimming larvae and, most probably, already in the ovary. This agrees with the mode of sex determination in other species of the Rhizocephala Kentrogonida.     

How whale and dolphin barnacles attach to their hosts and the paradox of remarkably versatile attachment structures in cypris larvae

How larvae of whale and dolphin epibionts settle on their fast-swimming and migrating hosts is a puzzling question in zoology. We successfully reared the larvae of the whale and dolphin barnacle Xenobalanus globicipitis to the cyprid stage. We studied the larval developmental ecology and antennular morphology in an attempt to assess whether an epibiotic lifestyle on this extreme substratum entails any unique larval specializations. Morphological parameters were compared with five other barnacle species that also inhabit extreme substrata. We found no larval specializations to a lifestyle associated with marine mammals. The external morphology of the antennules in Xenobalanus cyprids is morphologically similar to species from strikingly different substrata. We found variation only in the structures that are in physical contact with the substratum, i.e., the third segments carrying the villi-covered attachment disc. The third segments of the Xenobalanus cyprid antennules are not spear-shaped as in the stony coral barnacles, which are here used to penetrate the live tissue of their hosts. The presence of a cyprid cement gland implies that Xenobalanus uses cement protein when attaching to its cetacean host. Naupliar instars developed outside of the mantle cavity, indicating dispersal is planktonic. Our results militate against the idea that the cyprids settle during ocean migrations of their hosts. We suggest cyprids settle during coastal aggregations of the cetacean hosts. We conclude that the ecological success of barnacles has ultimately depended on a larva that with little structural alteration possesses the ability to settle on an amazingly wide array of substrata, including cetaceans.

     

Inhibitory Effects of Mediterranean Sponge Extracts and Metabolites on Larval Settlement of the Barnacle Balanus amphitrite

One of the most promising alternative technologies to antifouling paints based on heavy metals is the development of coatings whose active ingredients are compounds naturally occurring in marine organisms. This approach is based on the problem of epibiosis faced by all marine organisms and the fact that a great number of them cope with it successfully. The present study investigated the antifouling activity of a series of extracts and secondary metabolites from the epibiont-free Mediterranean sponges Ircinia oros, I. spinosula, Cacospongia scalaris, Dysidea sp., and Hippospongia communis. Antifouling efficacy was evaluated by the settlement inhibition of laboratory-reared Balanus amphitrite Darwin cyprids. The most promising activity was exhibited by the metabolites 2-[24-acetoxy]-octaprenyl-1-4-hydroquinone (8a), dihydrofurospongin II (10), and the alcoholic extract of Dysidea sp.     

Gregarious settlement in cypris larvae:the effects of cyprid age and assay duration

Gregarious behaviour of marine larvae is perhaps most clearly associated with finding a suitable habitat in a changeable or restricted environment, or with finding other conspecifics with which to mate. Prior work has shown that in settlement assays using cypris larvae of the barnacle Balanus amphitrite, gregarious interactions significantly affected the interpretation of experiments testing the activity of organic settlement promoters and inhibitors. Other studies have also shown effects of cyprid age and pheromone concentration on settlement behaviour. However, the effects of interactions between gregariousness and these two factors are not known. The aim of this study was to test the hypotheses that i) as cyprids age the effects of gregariousness become less apparent, and ii) as the duration of the experiment increases gregarious effects become more apparent, using cypris larvae of B. amphitrite and Balanus improvisus. Three age classes of cyprids were used at six densities in a fully factorial design. For B. improvisus cyprids significant gregarious effects occurred between 3 or more larvae, and although larval age and experiment duration had significant main effects, there were no interactions between these important factors and gregariousness. For B. amphitrite cyprids significant gregarious effects also occurred with 3 larvae per well, though this effect was strongly dependent upon experiment duration. B. amphitrite cyprid sensitivity to conspecific cues does not change with age, although increasing experiment duration and age interact to increase settlement. Differences between species may be due to different thresholds to conspecific larval cues, or B. improvisus cyprids release much more larval temporary adhesive during exploration.     

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.     

Modulation of barnacle (Balanus amphitrite Darwin) cyprid settlement behavior by sulfobetaine and carboxybetaine methacrylate polymer coatings

Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.     

Amelioration by copper supplementation of mutant gene effects in the crinkled mouse.

The possibility of a relationship between the autosomal recessive mutant gene crinkled in mice and copper metabolism was investigated by examining the effect of copper supplementation during pregnancy and lactation on the expression of the gene in homozygous mutant young. Survival of mutant mice to 30 days of age was doubled by feeding their mothers a high copper diet (500 ppm copper) during pregnancy and lactation, as compared with controls (6-11 ppm dietary copper). High dietary copper also prevented the lag in pigment development characteristic of the mutants. Furthermore, skin and epidermal thickness and hair bulb development were nearly normal in the high copper group, in contrast to thin skin and paucity of hairs in controls. Supplementation with manganese did not have these effects. Scanning electron micrographs showed the presence of three types of hair abnormalities in crinkled mutants, monilethrix, pili torti, and possibly trichorrhexis nodosa. The results show that increased availability of copper favorably altered the expression of the mutant gene, and demonstrate the interaction of a gene and a trace metal in development.