When similar beginnings lead to different ends: Constraints and diversity in cirripede larval development

Summary

Cirripedes are fascinating models for studying both functional constraints and diversity in larval development. Adult cirripedes display an amazing variation in morphology from sessile suspension feeders that still retain many crustacean characters to parasites that have lost virtually all arthropod traits. In contrast, cirripede larval development follows a common scheme with pelagic larvae comprising a series of nauplii followed by a cyprid. Variations are mostly concerned with whether or not the nauplii are feeding and the degree of abbreviation of development, culminating in species where the larvae hatch as cyprids. The cypris larvae are very similar among the ingroups of the Cirripedia, but interesting variations occur in structures used for substrate location and attachment. The cyprid is specialized to both swim through the water and actively explore the substratum by walking on the antennules and using an array of sensory organs in search for a suitable site to attach. This unique morphology and behavior of the cyprid have enabled the Cirripedia to colonize widely different habitats ranging from hard rock to soft animal tissue. Yet, the cyprid can metamorphose into juveniles as different as a setose feeding barnacle and the vermiform stages of the parasitic forms. This emphasizes the importance of the cyprid as one of the key features for the evolutionary success of the Cirripedia.     

Barnacle Settlement on Hydrogels

Settlement of cultured Balanus amphitrite cyprid larvae was tested on different non-solid hydrogel surfaces. Gels consisting of alginate (highly anionic), chitosan (highly cationic), polyvinyl alcohol substituted with light-sensitive stilbazolium groups (PVA-SbQ; very low cationic) and agarose (neutral) were applied in cell culture multi-well plates. Polystyrene served as a solid surface reference. Preliminary experiments were performed to determine whether any substances leaching out of the gels could inhibit barnacle settlement. Whilst leachate from the gels revealed no toxicity towards Artemia salina nauplius larvae, PVA-SbQ in solution at and above a concentration of 0.4 ppm inhibited B. amphitrite cyprid settlement. Gels were therefore washed to avoid such effects during further testing, and toxicity and settlement tests with B. amphitrite nauplii and cyprids, respectively, applied to verify that washing was effective. Settlement was tested directly on the different test materials, followed by a quality test of non-settled larvae. All gels inhibited barnacle settlement compared to the polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed promising antifouling properties. Although some settlement occurred on 2.5% PVA-SbQ gels, metamorphosis was clearly inhibited. Only 10% of the larvae had settled on 0.5% agarose gels after 8 d. Less than 40% settlement occurred on alginate gels, as well as on 2% chitosan gels. Quality testing showed that the majority of remaining non-settled larvae in all gel experiments were able to settle when offered a suitable solid substratum.     

Assessment of a trap for measuring larval supply of intertidal barnacles on wave-swept, semi-exposed shores

Larval supply and settlement of Semibalanus balanoides (L.) on a semi-exposed wave-swept shore in E Scotland were assessed by means of a new larval trap design. Two midshore sites were utilised throughout the peak of the 2001 settlement season (May 11–June 3). Larval supply was quantified by 176 ml baffled cylinder traps filled with a 4-M killing solution of urea in seawater. Larval trap washout of urea typically ranged from 25% per day under minimal wave action to a maximum of 39% under heavy wave action during an onshore gale. The maximum capture for a single trap was 334 cyprids over one tide. Significant and consistent positional effects, both on urea retention and larval capture, were found for replicate traps separated by 9 cm. These indicated fine-scale variations in hydrodynamic flow and larval supply over the substratum which will have implications for the quantification of spatial heterogeneity of larval input to the benthos. The retention of urea and larval capture efficiency generally were not compromised by traps being serviced daily, as opposed to tidally, except perhaps for captures at very low larval densities. Larval supply measured on a tidal basis was strongly correlated between sites (r=0.975), but supply at Site C was 5× that at Site T: the reduction at Site T could not be explained by its slightly shorter immersion time and hence ‘availability’ of cyprids alone. At Site C settlement was quantified for 5×5 cm quadrats of natural substratum cleared daily. Settlement on these clearances (subject to possible grazing by limpets) was quantified only from May 20–June 3, after the peak of larval supply on May 17, and the variation in larval supply explained 65% of the variance in settlement. The larval supply/settlement relationship at Site T was quantified using grooved acrylic panels. A high correlation coefficient (0.961) was obtained for the daily supply/settlement relationship for these panels over the period May 15–June 3, which included the peak of supply. Larval supply varied up to 15-fold within a site on consecutive tides and up to 100-fold between sites on the same tide. Although further improvements to both the larval trap and the settlement panels can be made, this larval trap does appear capable of providing high-resolution data on tidal or daily larval supply over a wide range of wave conditions and larval concentrations.     

Field-based Behavioural Bioassays for Testing the Efficacy of Antifouling Coatings

Improving non-toxic coatings using immersion trials is time consuming and requires more rigorous data analysis than is necessary for toxic coatings. The aim of this study was to determine if the exploratory behaviour of barnacle cyprids on coatings deployed in the field can be used as a bioassay for coating efficacy. A standard tin-based coating, and a silicone and unknown non-toxic coatings were videoed remotely underwater using a high resolution camera. Exploratory behaviours of the coatings by cyprids were digitised. There was a significant difference in larval behaviour between coatings (MANOVA, p <0.001 ). Canonical discriminant analysis clearly separated the three coatings and enabled 100% prediction of group membership. Thus the behaviour of exploring cyprids can be used to discriminate between the coatings. This study has demonstrated how a simple field-based bioassay could be suitable for the testing of antifouling coatings and may therefore offer some scope for rapidly pre-screening coatings prior to full-scale immersion trials. However, several disadvantages with the bioassay are noted, including variable illumination in the field, the high cost of the equipment used, the long time required to manually analyse recorded video, and the need for a predictable larval supply.     

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).     

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.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

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.     

Roles of larval supply and behavior in determining settlement of barnacles in a temperate mangrove forest

Recruitment is often a major influence on the spatial distribution of populations of benthic marine invertebrates, but the contributions of different components of recruitment are not well known, with the added complication that the relative importance of various life-history processes may be scale-dependent. Previously, we have shown that over a large scale across a mangrove (Avicennia marina) forest in southeastern Australia, settlement of the barnacle Elminius covertus explained its patterns of recruitment, which in turn explained the distribution of adults on mangrove pneumatophores. Post-settlement mortality had little influence on this pattern. In contrast, small-scale vertical distributions of adult barnacles along individual pneumatophores were determined by the pattern of recruitment, which differed from the pattern of settlement, so post-settlement mortality determined the vertical patterns of adults.

In this study, we tested whether larval supply and/or settlement behavior influence the observed settlement patterns of E. covertus across a forest (from seaward to landward zones). We also tested whether larval supply could explain the vertical settlement patterns along the pneumatophores. A pumping system was used to collect cypris larvae from seaward, mid and landward zones of a mangrove forest and an adjacent, unvegetated shore and from three heights above the sediment surface. We also used transplantation of wooden stakes bearing microbial films and barnacle recruits between horizontal zones of the forest to determine whether settlement was influenced by these films or recruits.

Both cyprid supply and cyprid behavior were important factors in determining the patterns of settlement of E. covertus across the forest. Cyprid supply was a result of three-fold differences in immersion times of different (landward, mid and seaward) zones across the forest and a decrease in density of cyprids in the water column from the seaward zone of the forest to the landward sections. In the absence of mangroves immediately adjacent to the forest, there was no temporally consistent difference in cyprid density across the shore and even the differences in immersion time did not produce consistent differences in cyprid supply across the shore. Wooden substrata that had been immersed at seaward sections of the forest attracted consistently more settlers than substrata immersed initially at other sections of the forest and settlement could be induced beyond the normal distribution of adults of E. covertus by stakes transplanted from the seaward zone.

The vertical settlement pattern could not be explained by the supply of cyprids, suggesting that larval behavior must determine the vertical settlement pattern.     

Larval development of the pedunculate barnacles Octolasmis angulata Aurivillius 1894 and Octolasmis cor Aurivillius 1892 (Cirripedia: Thoracica: Poecilasmatidae) from the gills of the mud crab,Scylla tranquebarica Fabricius, 1798

Detailed studies of larval development of Octolasmis angulata and Octolasmis cor are pivotal in understanding the larval morphological evolution as well as enhancing the functional ecology. Six planktotrophic naupliar stages and one non-feeding cyprid stage are documented in details for the first time for the two species of Octolasmis. Morphologically, the larvae of O. angulata and O. cor are similar in body size, setation patterns on the naupliar appendages, labrum, dorsal setae-pores, frontal horns, cyprid carapace, fronto-lateral gland pores, and lattice organs. Numbers of peculiarities were observed on the gnathobases of the antennae and mandible throughout the naupliar life-cycle. The setation pattern on the naupliar appendages are classified based on the segmentation on the naupliar appendages. The nauplius VI of both species undergoes a conspicuous change before metamorphosis into cyprid stage. The cyprid structures begin to form and modify beneath the naupliar body towards the end of stage VI. This study emphasises the importance of the pedunculate barnacle larval developmental studies not only to comprehend the larval morphological evolution but also to fill in the gaps in understanding the modification of the naupliar structures to adapt into the cyprid life-style.