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.     

Inhibition of barnacle cyprid settlement using low frequency and intensity ultrasound

Low frequency, low intensity ultrasound was demonstrated as an effective inhibitor of barnacle cyprid settlement. When the same substratum vibration amplitude (10.05 nm) and acoustic pressure (5 kPa) were applied, ultrasound at a frequency of 23?kHz significantly reduced cyprid settlement. The mechanism appeared to differ from the ultrasonic cavitation induced inhibition previously reported as no increased mortality was observed, and no change in the exploratory behaviour of cyprids was observed when they were exposed to this continuous ultrasonic irradiation regime. The application of ultrasound treatment in an intermittent mode of '5?min on and 20?min off' at 20-25?kHz and at the low intensity of 5?kPa produced the same effect as the continuous application of 23?kHz. This energy efficient approach to the use of low frequency, low intensity ultrasound may present a promising and efficient strategy regarding irradiation treatment for antifouling applications.     

Antipredator behaviour of invasive geckos in response to chemical cues from snakes

Antipredator behaviours and the ability to appropriately assess predation risk contribute to increased fitness. Predator avoidance can be costly; however, so we expect prey to most strongly avoid predators that pose the greatest risk (i.e., prey should show threat sensitivity). For invasive species, effectively assessing the relative risk posed by predators in the new environment may help them establish in new environments. We examined the antipredator behaviour of introduced Asian house geckos, Hemidactylus frenatus (Schlegel), by determining if they avoided shelters scented with the chemical cues of native predatory snakes (spotted pythons, Antaresia maculosa [Peters]; brown tree snakes, Boiga irregularis [Merrem]; common tree snakes, Dendrelaphis punctulata [Grey]; and carpet pythons, Morelia spilota [Lacépède]). We also tested if Asian house geckos collected from vegetation vs. anthropogenic substrates (buildings) responded differently to the chemical cues of predatory snakes. Asian house geckos did not show a generalised antipredator response, that is, they did not respond to the chemical cues of all snakes in the same way. Asian house geckos avoided the chemical cues of carpet pythons more strongly than those of other snake species, providing some support for the threat‐sensitivity hypothesis. There was no difference in the antipredator behaviour of Asian house geckos collected from buildings vs. natural vegetation, suggesting that individuals that have invaded natural habitats have not changed their antipredator behaviour compared to urban individuals. Overall, we found some evidence indicating Asian house geckos are threat‐sensitive to some Australian predacious snakes.     

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.     

Inhibition of barnacle (Amphibalanus amphitrite) cyprid settlement by means of localized, pulsed electric fields

The increasing needs for environmental friendly antifouling coatings have led to investigation of new alternatives for replacing copper and TBT-based paints. In this study, results are presented from larval settlement assays of the barnacle Amphibalanus (= Balanus) amphitrite on planar, interdigitated electrodes (IDE), having 8 or 25 mum of inter-electrode spacing, upon the application of pulsed electric fields (PEF). Using pulses of 100 ms in duration, 200 Hz in frequency and 10 V in pulse amplitude, barnacle settlement below 5% was observed, while similar IDE surfaces without pulse application had an average of 40% settlement. The spacing between the electrodes did not affect cyprid settlement. Assays with lower PEF amplitudes did not show significant settlement inhibition. On the basis of the settlement assays, the calculated minimum energy requirement to inhibit barnacle settlement is 2.8 W h m(-2).     

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.     

The effect of gregariousness in cyprid settlement assays

The in vitro settlement assay using cyprids of Balanus. amphitrite is an important tool in basic and applied research. The aim of this study was to quantify the effect of gregariousness within these assays, and to determine the interaction between gregariousness and container size, and settlement promoters and inhibitors. Assays with 1, 5, 10, 15, 20, and 40 cyprids in containers with a log range of surface area to volume ratios were conducted in a fully factorial design. Assays with the same range of cyprid numbers and six concentrations of the settlement promoter 3-isobutyl-1-methylxanthine (IBMX) or the inhibitor phloroglucinol were also conducted in a fully factorial design. Percentage settlement was analysed by GLM ANOVA. Significant (p < 0.05) gregarious effects were detected at > or = 5 cyprids in a well. Surface area:volume ratio had a strong effect on cyprid settlement, but this effect could be masked by overcrowding in very small wells. Gregarious interactions between only five cyprids magnified the effect of IBMX by a factor of 10, whereas phloroglucinol had no effect without gregarious interactions. The cyprid settlement bioassay is a valuable tool for basic and applied research but must be used with care.     

Smelly feet are not always a bad thing: the relationship between cyprid footprint protein and the barnacle settlement pheromone

A critical phase in the life cycle of sessile benthic marine invertebrates is locating a suitable substratum for settlement. For barnacles, it is the lecithotrophic cypris larva that makes this plankto-benthic transition. In exploring possible substrata for settlement, the cyprid leaves behind 'footprints' of a proteinaceous secretion that reportedly functions as a temporary adhesive, and also acts as a secondary cue in larval-larval interactions at settlement. Here, we show that two polyclonal antibodies raised against peptides localized at the N- and C-terminal regions of the adult settlement cue--the settlement-inducing protein complex (SIPC)--could both detect 'temporary adhesive' indicating that the SIPC is either a component of this secretion or that they are the same protein.     

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.     

Larval behaviour and settlement cues of a brooding coral reef sponge

Patterns of larval release, dispersal and settlement in sponges are poorly understood despite their significance in explaining adult ecology. Time of release, swimming speeds, phototaxis and vertical migration were quantified for larvae of the dictyoceratid sponge Coscinoderma matthewsi. The influence of cues associated with biofilms and coral rubble on larval settlement and metamorphosis was also measured. C. matthewsi is a brooding sponge and releases tufted parenchymellae larvae during the day. Upon release, larvae (>90%) have no phototactic response, maintaining their position at the water surface for 80 min ± 0 (mean ± SE) regardless of a light cue (natural daylight) before exhibiting negative phototaxis. At 28 h post-release, the majority of larvae (94.7% ± 6.1) exposed to light from the surface migrated to the bottom and assumed a demersal phase. Without light, larvae occupied the surface for up to 28 h post-release (89.3% ± 1.8) before migrating to the bottom. Larvae did not settle gregariously and began to settle and metamorphose after 28 h post-release without a cue. Settlement and metamorphosis were faster in the presence of a biofilm (settlement = 15.0% ± 8.7 and metamorphosis = 12.5% ± 9.5 at 28 h post-release), while the addition of coral rubble accelerated metamorphosis further (settlement = 10.0% ± 4.1 and metamorphosis = 27.5% ± 10.3 at 28 h post-release) compared to controls (sterile surfaces) (settlement = 0% and metamorphosis = 0% at 28 h post-release). However, both biofilms and coral rubble decrease total metamorphosis (control = 92.5% ± 4.8, biofilms = 67.5% ± 7.5 and coral rubble = 55.0% ± 13.2) due to mortality after 76 h post-release.     

Correlative assays of barnacle cyprid behaviour for the laboratory evaluation of antifouling coatings: a study of surface energy components

Laboratory evaluation of antifouling coatings is underpinned by settlement studies with specific fouling organisms. Established methods provide insight into the likelihood of failure of a particular coating system, but can neglect the process of surface selection that often precedes attachment. The present approach for quantifying the exploratory behaviour of barnacle cypris larvae suggested that inspection behaviour can be a rapid and predictive proxy for settlement. Two series of xerogels with comparable total surface energy, but different dispersive and polar components, were evaluated. Settlement assays with three-day-old cyprids of Balanus improvisus demonstrated that while attachment was not linked directly to dispersive free energy, the composition of the xerogel was nevertheless significant. Behavioural analysis provided insight into the mechanism of surface rejection. In the case of a 50:50?PH/TEOS (phenyltriethoxysilane-based) xerogel vs a 50:50 TFP/TEOS (3,3,3-trifluoropropyltrimethoxysilane-based) xerogel, wide-searching behaviour was absent on the former.     

Nature and perception of barnacle settlement pheromones

It is now almost 50 years since the gregarious settlement of barnacles and its chemical basis was first described. Although originally noted for Elminius modestus, mechanistic studies of gregariousness have focused on two species, Semibalanus balanoides and Balanus amphitrite. By virtue of its ease of study and its economic importance as a fouling organism, the latter species has assumed increasing importance in recent years. This paper will provide an overview of studies on settlement pheromones and their perception. An adult glycoprotein, arthropodin (now known as settlement-inducing protein complex or SIPC), was once thought to be the sole pheromone involved in the induction of cypris larval settlement. At least two other pheromones are now known to be involved, a waterborne cue originating from the adult and the cypris temporary adhesive. The latter is related, immunologically, to SIPC. In keeping with many other examples of chemical communication, the available evidence suggests that barnacle settlement induction involves receptor-ligand interactions and a signal transduction pathway(s) that translates into attachment and metamorphosis. Similar findings have been reported for some, but not all, marine invertebrate larvae examined thus far and the implications for antifoulant development are discussed.     

Olfactorily-mediated cortisol response to chemical alarm cues in zebrafish Danio rerio

Chemical cues released as a by-product of predation mediate antipredator behaviour, but little is known about the physiological responses to olfactory detection of predation risk. In this study, zebrafish Danio rerio were exposed to either chemical alarm cues from conspecifics, or water (control). Compared with water controls, D. rerio exposed to alarm cues responded behaviourally with antipredator behaviours such as erratic dashing and an increase in time spent near the bottom of the test aquarium. Danio rerio were sacrificed 5 min after exposure to test cues (alarm cues or water). Enzyme-linked immunosorbent assay (ELISA) revealed whole-body levels of cortisol that were significantly higher for fish exposed to alarm cues (mean ± SE, 11.9 ± 3.4 ng g⁻¹) than control fish (1.5 ± 0.7 ng g⁻¹). These data provide a benchmark for future studies of the proximate mechanisms of olfactorily mediated antipredator responses, modelling effects on aquatic life in a changing climate and, as a model organism, Danio rerio can further our understanding of anxiety in humans.     

Larval development and settlement of a whale barnacle

Larval development and settlement of whale barnacles have not previously been described, unlike intertidal barnacles. Indeed, the mechanisms of the association between barnacles and whales have not been studied. Here we describe the larval development and settlement of the whale barnacle, Coronula diadema, and possible involvement of a cue from the host in inducing larval settlement. Eight-cell stage embryos were collected from C. diadema on a stranded humpback whale, incubated in filtered seawater for 7 days, and nauplius larvae hatched out. When fed with Chaetoceros gracilis, the nauplii developed to stage VI, and finally metamorphosed to the cypris stage. The larval development looked similar to that of intertidal barnacles with planktotrophic larval stages. The cyprids did not settle in normal seawater, but did settle in polystyrene Petri dishes when incubated in seawater with a small piece of skin tissue from the host whale. This strongly suggests the involvement of a chemical cue from the host whale tissue to induce larval settlement.     

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.     

Physical and chemical cues influencing the oviposition behaviour of Aphidius ervi

The oviposition behaviour of the aphid parasitoid Aphidius ervi Haliday is influenced by both chemical and physical cues. Oviposition attack responses were elicited by paint pigments sealed into the tip of a glass capillary tube. Parasitoids reacted to yellow pigments with repeated oviposition attack responses, but they did not react to green pigments. The spectrum of reflected light from the yellow pigments was very similar to that from the `green' natural host Acyrthosiphon pisum (Harris), with a high proportion of the total radiation energy being emitted in the yellow-orange wavebands (580–660 nm). Pea aphid cornicle secretion also elicited oviposition attack responses, which were not exclusively induced by its pale yellow-green colour. In fact, the oviposition attack response to capillary tips coated with cornicle secretion remained evident under red light conditions, which, in contrast, nearly completely suppressed the response to yellow pigments. Chemical compounds from cornicle secretion do not appear to be involved in parasitoid orientation, even though they stimulate intense oviposition attack responses. Olfactometer experiments showed that the putative kairomone involved acts only at very short range or on contact. Host exuviae, which also elicited strong and persistent oviposition reactions from A. ervi females, appear to be a good alternative source of ovipositional kairomone(s). This work confirms the existence of an aphid cuticular kairomone.     

Ontogenetic changes in responses to settlement cues by Anemonefish

Population connectivity for most marine species is dictated by dispersal during the pelagic larval stage. Although reef fish larvae are known to display behavioral adaptations that influence settlement site selection, little is known about the development of behavioral preferences throughout the larval phase. Whether larvae are attracted to the same sensory cues throughout their larval phase, or exhibit distinct ontogenetic shifts in sensory preference is unknown. Here, we demonstrate an ontogenetic shift in olfactory cue preferences for two species of anemonefish, a process that could aid in understanding both patterns of dispersal and settlement. Aquarium-bred na?ve Amphiprion percula and A. melanopus larvae were tested for olfactory preference of relevant reef-associated chemical cues throughout the 11-day pelagic larval stage. Age posthatching had a significant effect on the preference for olfactory cues from host anemones and live corals for both species. Preferences of olfactory cues from tropical plants of A. percula, increased by approximately ninefold between hatching and settlement, with A. percula larvae showing a fivefold increase in preference for the olfactory cue produced by the grass species. Larval age had no effect on the olfactory preference for untreated seawater over the swamp-based tree Melaleuca nervosa, which was always avoided compared with blank seawater. These results indicate that reef fish larvae are capable of utilizing olfactory cues early in the larval stage and may be predisposed to disperse away from reefs, with innate olfactory preferences drawing newly hatched larvae into the pelagic environment. Toward the end of the larval phase, larvae become attracted to the olfactory cues of appropriate habitats, which may assist them in identification of and navigation toward suitable settlement sites.     

Some observations on the behaviour of the sodium efflux in barnacle muscle fibres towards lithium ions

• 1.1. The behaviour of the Na efflux towards Li⁺ was studied using single barnacle muscle fibres as a preparation.
• 2.2. It is found that the Na efflux into Li⁺-ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na⁺-ASW.
• 3.3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li⁺-ASW.
• 4.4(a). The remaining Na efflux into Li⁺-ASW is further reduced by external application of 10⁻⁴ M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Na_e by Li⁺. However, some fibres show a rise rather than a fall.
• 5.5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Na_e is replaced by Li⁺. A similar but less pronounced response is often seen with ouabain-poisoned fibres.
• 6.6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Na_e by Li⁺ fails to bring about a fall in the remaining efflux.
• 7.7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li⁺. The relative size of this component is about one-fifth and not one-half of the Na efflux.

     

Larval settlement and metamorphosis in the slipper limpet Crepidula onyx (Sowerby) in response to conspecific cues and the cues from biofilm

In the marine environment, aggregated distribution in the genus Crepidula is a very common phenomenon. Works from Pechenik's group suggested that this is the result of gregarious settlement of larvae in response to cues associated with conspecific adults. In this study, we investigated the existence of larval metamorphic cues associated with adults of C. onyx, a slipper limpet introduced to Hong Kong from the U.S. in the 1970s, through a series of laboratory bioassays. The results showed that derived cues in adult C. onyx were waterborne and the waterborne cues were not derived from bacteria associated with the shell and soft body of the adult Crepidula. The natural biofilm also induced the larval metamorphosis of C. onyx. The cues from the biofilm were associated with the surface of the biofilm and were not waterborne. The aggregated distribution in nature of adult C. onyx may result from a selective larval settlement process. On a small scale in the water column near the conspecific adults, larvae of C. onyx initially detect the waterborne conspecific cues, which then lead to positive downward swimming or passive sinking. This activity increases the chances for larvae to make contact with the biofilm and to be exposed into the higher concentration of waterborne conspecific cues. This may eventually lead to the enhanced larval settlement pattern on or near the conspecific adults.