Convenient Assay for Settlement Inducing Substances of Barnacles

A convenient assay method for estimation of barnacle, Balanus amphitrite, settlement inductive activity was developed. To avoid the inductive effect by comrades and laborious observation requirements, cyprid larvae were put individually into wells of a 96-well plate. The settlement ratio from the experiment without any inducers was quite low; therefore this assay allowed easy estimation of settlement inductive activities. Some known inductive agents, such as serotonin and barnacle extracts, clearly showed inductive activities. This assay method is proven to be suitable for estimation of barnacle settlement-inducing activities of both water-soluble and -insoluble compounds. Received October 31, 1997; accepted July 10, 1998.     

Seasonal changes in frequency of diseases in dab,Limanda limanda,from the southern North Sea

Infestation rates of flatfishLimanda limanda from the southern North Sea (German Bight) were investigated in 1981 and 1982. At three stations, including a dumping area of acid-iron wastes northwest of Helgoland, dabs were examined for the occurrence of lymphocystis and epidermal papillomas. Marked seasonal changes of infestation rates were observed with maxima of diseased fish in March and May (epidermal papillomas 2 % in 1981, 4.1 % in 1982; lymphocystis 7.5 % in 1981, 17.2 % in 1982). Infestation minima were found from June to October. Differences in incidence rates were observed between stations in winter and spring, but not in summer. Both diseases turned out to be size-dependent. Seasonality of the two diseases is discussed in relation to spawning cycle and water quality, including pollution.     

Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN)

Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase, i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules.     

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

In laboratory-based biofouling assays, the influence of physico-chemical surface characteristics on barnacle settlement has been tested most frequently using the model organism Balanus amphitrite (= Amphibalanus amphitrite). Very few studies have addressed the settlement preferences of other barnacle species, such as Balanus improvisus (= Amphibalanus improvisus). This study aimed to unravel the effects of surface physico-chemical cues, in particular surface-free energy (SFE) and surface charge, on the settlement of cyprids of B. improvisus. The use of well-defined surfaces under controlled conditions further facilitates comparison of the results with recent similar data for B. amphitrite. Zero-day-old cyprids of B. improvisus were exposed to a series of model surfaces, namely self-assembled monolayers (SAMs) of alkanethiols with varying end-groups, homogenously applied to gold-coated polystyrene (PS) Petri dishes. As with B. amphitrite, settlement of cyprids of B. improvisus was influenced by both SFE and charge, with higher settlement on low-energy (hydrophobic) surfaces and negatively charged SAMs. Positively charged SAMs resulted in low settlement, with intermediate settlement on neutral SAMs of similar SFE. In conclusion, it is demonstrated that despite previous suggestions to the contrary, these two species of barnacle show similar preferences in response to SFE; they also respond similarly to charge. These findings have positive implications for the development of novel antifouling (AF) coatings and support the importance of consistency in substratum choice for assays designed to compare surface preferences of fouling organisms.     

Influence of oil shale on intertidal organisms: Effect of oil shale extract on settlement of the barnacle Balanus balanoides (L.)

As part of a study to investigate the effect of oil seeps on intertidal organisms, oil extracts of Blackstone oil shale from Kimmeridge on the Dorset coast were used in laboratory experiments to test their effect on the settlement of the barnacle Balanus balanoides (L.). Thin films of oil extract painted on the surface of pits in slate panels had no effect on cyprid settlement when applied up to a surface density of 2.8 g · m^?2, representing a thickness of 3.3 μm. Larger surface densities of oil stimulated cyprids to settle in far greater numbers than on unoiled panels. The maximum effect was obtained at a surface density of between 14.0 and 56.0 g · m ^?2, representing a thickness of 16.5 μm and 66.0 μm. With higher concentration of oil in the pits, stimulation to settle was reduced although cyprid settlement was still encouraged at a surface density of oil of 112g · m^?2 or 132 μm thickness.The unfractionated crude oil shale extract was a less powerful stimulus for barnacle settlement than a partially purified solution of the integumental protein arthropodin, another strong settlement inducer for barnacle cyprids.     

Indole Derivatives as Potent Inhibitors of Larval Settlement by the Barnacle,Balanus amphitrite

A potent inhibitor of larval settlement by the barnacle, Balanus amphitrite, was isolated as 2,5,6-tribromo-1-methylgraimne from a marine invertebrate. In comparative tests on the activity of related compounds, such compounds as 2-methylgramine and 2-methyl-3-(morpholinomethyl)-indole exhibited potent inhibitory activity. The inhibitory activity toward larval settlement was found not to be due to toxicity but to a repellent effect.     

Some Effects of a Power Plant Effluent on Estuarine Epibenthic Organisms

A study using the submerged test panel method was conducted at the Oyster Creek Nuclear Generating Station near Barnegat Bay, New Jersey to investigate effects of the heated effluent on epibenthic communities. Test panels were placed in the intake and discharge canals and collected using SCUBA at 28, 84, 168 and 335-day intervals between October 1970 and October 1971. Exposed panels were replaced by clean panels upon collection. Faunal resemblance, both between stations during a sampling date and within stations from one sampling date to the next, was low. Numbers of species were higher at the discharge station than at the intake station from late fall to early summer and lower from midsummer to early fall. Overall differences in diversity at the two stations could not be detected. The optimum temperature for growth of Balanus sp. was approached more closely during winter in the discharge canal than in the intake canal where growth was retarded by cold water. Balanus eburneus appeared to have an extended breeding season. Because of dilution of the discharged water with cooler intake water through a plant bypass system during the summer, the effluent was not seriously harmful to amphipod reproduction. Season abundances of numerous invertebrates at both stations are reported and possible power plant effects are discussed.     

Metazooplankton dynamics in the Salton Sea, California, 1997–1999

The dynamics of metazooplankton populations were studied over 3 years at the saline (43 g l^–1) Salton Sea, California's largest lake. Total abundance was highest in summer following late winter/early spring phytoplankton blooms. At this time, metazooplankton consisted mostly of the copepod, Apocyclops dengizicus, and the rotifer, Brachionus rotundiformis. In August or September, severe crashes in the metazooplankton populations occurred each year in mid-lake due to strong wind events which increased mixing and caused low oxygen and high sulfide concentrations throughout the water column. Larvae of the polychaete worm, Neanthes succinea and the barnacle, Balanus amphitrite were present mostly in late winter and spring. Their scarcity in summer is due in part to persistent anoxic bottom conditions that decrease adult populations and in part to predation by tilapia, an omnivorous fish that has become abundant in the lake since the 1960s. Two Synchaeta species, rotifers not previously reported from the Sea, were abundant in winter and spring and predation on these may have permitted the copepod to persist at low levels through the winter. There were two major changes in metazooplankton dynamics since 1954–1956 in addition to the appearance of the two synchaetid rotifers in the fauna. First, there are now much lower densities of barnacle and polychaete larvae in the fall, probably due to the invasion of the zooplanktivorous fish, tilapia. Second the precipitous crashes now seen in metazooplankton densities, especially the copepod, in late summer-early fall did not occur in the 1950s possibly because fall overturn events did not result in such high sulfide levels.     

Primary productivity of the River Ely,South Wales,U.K.

The present study was carried out on the non-tidal reaches of the River Ely, South Wales, from October 1979 to October 1980. The seasonal variations of the chlorophyll-a content of the phytoplankton was unimodal with one maximum in May and a minimum in December. The chlorophyll-a content varied from 0.277 to 41.259 mg m^?3. The primary productivity showed a bimodal seasonal distribution with two maxima in May and September and lower values throughout the remainder of the year, particularly in winter. The value for the primary productivity varied from 0.269 to 24.302 mg C m^?3 h^?1. A positive correlation was obtained between chlorophyll-a content and primary productivity. The seasonal distribution of the dominant algal species and the saprobity of the River Ely were also studied. The diatom species almost showed a similar seasonal periodicity. Their concentrations were low during the winter months and high during most of the spring and summer months. Many of the dominant diatom species found in the phytoplankton populations were either considered by the other authors as saprobic (Nitzchia palea) or as inhabitants of eutrophic waters (Gomphonema parvulum, Navicula cryptocephala and Synedra ulna). Chlamydomonas spp. were the most abundant green algae followed by Chlorella vulgaris. The effect of sewage effluent disposal and cattle excreta at three of the sampling sites might partly explain the presence of high Chlamydomonas spp. populations at those sites. Physical factors (low flow rates, high transparency and water temperature) and organic pollution at some sampling sites seemed to play an important role in increasing the number of algal species during spring and summer. The non-tidal reaches of the River Ely were found to be β-mesosaprobic above and below Station 5 and α-mesosaprobic at the latter station and therefore, the river can be considered as polluted at Station 5 and mildly polluted at the others.     

Characterisation of the bacteria associated with barnacle,Balanus amphitrite,shell and their role in gregarious settlement of cypris larvae

The acorn barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) is a model organism to investigate pelago-benthic transitions in marine invertebrates. A driver for larval settlement in this organism is the need to attach close to conspecifics, to allow reproduction to take place. Adult barnacles are covered by microbial biofilms and the contribution of these biofilms to conspecific recognition is not fully understood. Little information is available on microbial communities associated with B. amphitrite. We compared biofilm communities from the barnacle shell surface with those from the surrounding rocks using the culture-independent methods of quantitative PCR and denaturing gradient gel electrophoresis. Quantification of the relative abundances of higher bacterial taxa showed that barnacles hosted a greater proportion of α-Proteobacteria compared to rock-associated biofilms (p < 0.01). Differences in relative abundances of other taxa were not observed but DGGE profiling suggested that differences were present at lower taxonomic levels. The capacity of these communities to influence larval settlement was assessed by growing multispecies biofilms on artificial medium, obtained by extracting nutrients from adult barnacles. Biofilms composed of shell-associated bacteria were capable of promoting conspecific settlement by 67% compared to control surfaces (p < 0.05), while rock-associated communities showed contrasting effects. A taxonomic comparison of settlement-stimulating and -inhibiting bacteria was performed by DGGE and band sequencing. All partial 16S rRNA genes sequenced were similar to members of the Vibrio and Pseudoalteromonas genera, suggesting that larvae can detect and respond to variations in the composition of microbial biofilms at low taxonomic levels. Our results indicate that barnacle larvae may be able to detect parentally-associated biofilms and use this information to settle close to members of its own species.     

The interplay of substrate nature and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement

The settlement of marine larvae is influenced by a wide range of physical and biological factors. It is still poorly known how the nature of substrate and the biofilm can interact in regulating settlement patterns of invertebrate larvae. Here we use laboratory experiments focused on settlement behaviour of the barnacle Balanus amphitrite. The aim of this work is to understand whether: (i) the nature of substratum can affect biofilm formation and its structure, (ii) the nature of substratum can affect B. amphitrite larval settlement, (iii) the age of the biofilms and the nature of substrate can interact in influencing larval settlement.Four kinds of substrata (marble, quartz, glass, and cembonit) were biofilmed under laboratory conditions for 5, 10 and 20 days at the temperature of 28 °C. Settlement response was investigated with 5-day-old cyprids. Biofilms were quantitatively and qualitatively analysed by scanning electron microscopy. The settlement of B. amphitrite larvae significantly differed among substrata; also, the patterns of development of biofilm assemblages changed with substrate. In addition, the larval attractiveness of different substrates tends to disappear with biofilm age.     

Spatial variation in larval concentrations as a cause of spatial variation in settlement for the barnacle,Balanus glandula

Summary Settlement rates of the high intertidal barnacle, Balanus glandula, were monitored at three sites in the rocky intertidal zone in Central California simultaneously with measurements of larval concentrations in the adjacent water column. In both 1983 and 1984, settlement rates onto vacant substrate differed among the sites by nearly two orders of magnitude. For all sampling dates, this spatial variation in settlement mirrored the spatial distribution of Balanus glandula cyprid concentration in the water column. A perfect rank correlation was found between cyprid concentrations near a site and subsequent settlement. A noteworthy observation was that the sites switched rank in their settlement rates from 1983 to 1984. This change in settlement rankings matched a switch in rankings for cyprid concentrations.Settlement itself appears to be an important cause of the spatial pattern of cyprid concentrations. Comparing the rates of settlement to estimates of the number of cyprids available at a site suggests that settlement causes a large drain on the cyprid population as a water mass passes over successive sites. No consistent spatial patterns were found in the distribution of other major plankton groups (calanoid copepods) that are similar in size to Balanus cyprids but do not settle.The large differences in settlement rates among these sites were previously shown to be a leading cause of large differences in the structure of benthic barnacle populations. The close correspondence shown here between these large differences in settlement and differences in larval concentrations suggests that nearshore oceanic processes affecting larval arrival contribute to the control of benthic community structure.     

Volatile substances from adult extracts induce larval settlement of the barnacle balanus amphitrite *

The volatile substance extracted from conspecific adults induces larval settlement of the barnacle Balanus amphitrite. The settlement inducing activity of the volatile fractions was checked monthly from June, 1997 to December, 1998. Both water soluble extracts and volatile fractions from the barnacle were prepared by the steam distillation. The active cue in the volatile fraction was always extracted with n‐pentane under acidic conditions, although settlement inducing activity varied with the sample. GC‐MS analysis of the active and inactive pentane fractions revealed 1, 2, 3‐trimethyl‐benzene as the settlement inducing substance. Commercially available 1, 2, 3‐trimethylbenzene also showed high settlement inducing activity at a concentration of 0.8 × 10^‐12 M (100 pg 1^‐1). This substance was detected at concentrations of more than 7 ng g^‐1 of wet barnacle (equivalent to 0.6 × 10^‐12M, equal to 70pg1^‐1) by GC analysis. These results indicate that 1,2,3‐trimethylbenzene in the volatile fractions acts as a chemical cue for larval settlement. Monthly variation in the settlement inducing activity was observed, which synchronized with the breeding season of the barnacle. This observation suggests that the barnacle produced the chemical cue in the gonad during maturation or accumulated it from the environment.     

Effect of the substratum in the recruitment and survival of the introduced barnacle Balanus glandula (Darwin 1854) in Patagonia, Argentina

The barnacle Balanus glandula was introduced in Argentina in the 1970s, and today it dominates the high intertidal level in most Argentinean rocky shores. The aim of this work was to evaluate the effect of the type of substrata and intertidal height on a population of Balanus glandula by conducting field surveys and one-year field experiments in which we combined different substrata (hardness: hard and soft, and texture: smooth and rough) at two intertidal heights (mid and high). In natural populations, the highest density of adults and recruits occurred on soft-rough substratum and in the high intertidal. The different textures were important only on the soft substrata and high intertidal, and the density of barnacles of the soft-rough substrata was higher than soft and smooth ones. The most suitable experimental substratum was the soft-rough of the high intertidal, which had the highest recruitment, survival and final density of barnacles at the end of the experiment. In contrast, the hard and smooth of the high and middle intertidal were the least suitable in all cases. Although the recruitment of B. glandula occurred throughout the year, it was higher in the high intertidal, and it showed a recruitment peak in the winter and a second in the summer. While most studies on this barnacle investigated the effects of granite or other volcanic hard substrata, our study also focused on soft substrata. The effects of soft substrata are particularly important because soft sedimentary rocks characterise the southern Atlantic coast of South America and the presence of soft rocks appears to optimize the success of Balanus glandula.     

Vicious circle in the intertidal: Facilitation between barnacle epibionts, a shell boring polychaete and trematode parasites in the periwinkle Littorina littorea

We studied interactions between three organisms associated with a common gastropod of northern Atlantic shores, the periwinkle Littorina littorea: barnacle epibionts Balanus crenatus, a shell boring polychaete Polydora ciliata, and tissue invading trematodes which use the periwinkles as first intermediate host. Snails collected shortly after barnacle settlement with > 50% cover of barnacles had significantly higher infestation of shell boring worms compared to unfouled snails, while trematode infestation was similar. The result was the same at two sites, and we conclude that the worm P. ciliata facilitates barnacle fouling on snails. The reverse was also the case. In an experiment with 14 weeks of exposure, snail treatments with barnacle epibionts had a significantly higher P. ciliata load than unfouled and cleaned snails. Again, trematode infestations were similar. The reciprocal positive interactions between barnacle epibionts and shell boring worms on snail houses is regarded as a case of facultative mutualism. On the other hand, for the snail basibiont, both barnacles and shell boring worms exert strongly negative effects by reducing fecundity, growth, and survival, resulting in a vicious circle for the snails. The combined effects of these associated organisms may rival in importance any competitive or predacious effects on the host L. littorea.     

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).     

Supercooling Ability, Water Content and Hardiness of Rhododendron Flower Buds during Cold Acclimation and Deacclimation

The relationship between supercooling ability and water contentand killing temperature of flower buds during cold acclimationand deacclimation were studied using R. kiusianum and R. x akebono.The occurrence of multiple floret exotherms and their shiftto a narrow range at lower subzero temperatures, as well asthe marked decrease of florets water content, were observedas the symptoms of cold acclimation occuring in flower budsfrom fall to winter, and vice versa in spring buds during deacclimation.In R. kiusianum, the fully acclimated period was from Novemberto March and two months longer than that of R. x akebono. Thesupercooling ability of the former was about –25°Cand about –20°C in the latter. Although the watermigration within bud tissues during the freezing process wasdetermined in the acclimated and deacclimated buds for R. xakebono, no significant water changes could be observed, evenin the acclimated buds. Thus, it is conceivable that deep supercoolingin florets may result not necessarily from water migration fromflorets and bud axes to scales in response to freezing, butfrom low water content in situ of cold-acclimated or artificiallydehydrated flower buds. (Received July 29, 1981; Accepted October 12, 1981)     

The Bromotyrosine Derivative Ianthelline Isolated from the Arctic Marine Sponge Stryphnus fortis Inhibits Marine Micro- and Macrobiofouling

The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described. All major stages of the fouling process are investigated. The effect of ianthelline on adhesion and growth of marine bacteria and microalgae is tested to investigate its influence on the initial microfouling process comparing with the known marine antifoulant barettin as a reference. Macrofouling is studied via barnacle (Balanus improvisus) settlement assays and blue mussel (Mytilus edulis) phenoloxidase inhibition. Ianthelline is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (minimum inhibitory concentration (MIC) values 0.1–10 μg/mL) and barnacle larval settlement (IC₅₀?=?3.0 μg/mL). Moderate effects are recorded on M. edulis (IC₅₀?=?45.2 μg/mL) and microalgae, where growth is more affected than surface adhesion. The effect of ianthelline is also investigated against human pathogenic bacteria. Ianthelline displayed low micromolar MIC values against several bacterial strains, both Gram positive and Gram negative, down to 2.5 μg/mL. In summary, the effect of ianthelline on 20 different representative marine antifouling organisms and seven human pathogenic bacterial strains is presented.     

The early development of the cement apparatus in the barnacle, Balanus balanoides (L.) (Crustacea: Cirripedia)

The development of the cement cells of Balanus balanoides (L.) has been followed over a 26-day post-settlement period. Cells of the cyprid cement glands de-differentiate to form part of the juvenile barnacle apparatus, but the majority of juvenile cement cells develop by differentiation of collecting duct cells. These latter cement cells are recognized by apical invaginations which are continuous with the collecting duct lumen. Collecting ducts are present in the cyprid. The cement duct system was not studied in detail, but preliminary observations of the arrangement in B. balanoides and Elminius modestus Darwin have shown them to differ, the system in E. modestus being simpler.