Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV-irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV-dosage. Since UV energy crosslinks both intra- and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

The polychaete Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of H. elegans. A full-length CaM cDNA was successfully cloned from H. elegans (He-CaM) and it contained an open reading frame of 450 bp, encoding 149 amino acid residues. It was highly expressed in 12h post-metamorphic juveniles, and remained high in adults. In situ hybridization conducted in competent larvae and juveniles revealed that He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of H. elegans.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): Role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV‐irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV‐dosage. Since UV energy crosslinks both intra‐ and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

Proteomic profiling during the pre-competent to competent transition of the biofouling polychaete Hydroides elegans

The polychaete, Hydroides elegans, is a tube-building worm that is widely distributed in tropical and subtropical seas. It is a dominant fouling species and thus a major target organism in antifouling research. Here, the first high-throughput proteomic profiling of pre-competent and competent larvae of H. elegans is reported with the identification of 1,519 and 1,322 proteins, respectively. These proteins were associated with a variety of biological processes. However, a large proportion was involved in energy metabolism, redox homeostasis, and microtubule-based processes. A comparative analysis revealed 21 proteins that were differentially regulated in larvae approaching competency.     

Life history trade-offs and response to selection on egg size in the polychaete worm Hydroides elegans

In order to examine the genetic relationships among life-history traits in a hermaphroditic species we used artificial selection for increased egg size and measured correlated responses across the life cycle of the serpulid polychaete Hydroides elegans, a protandrous sequential hermaphrodite. We recorded sex ratios across generations, and measured egg size, egg energy, larval volume at two time points, juvenile tube length, adult dry weight and fecundity after selection. Selection for larger eggs produced positive correlated responses in egg energy, fecundity and larval size at competence. Selection for increased egg size was also manifested by earlier sex change and this resulted in selected individuals spending less time as males relative to controls. We propose that egg size is negatively correlated with duration of andromorphy, that is, that female fitness trades off with male fitness.     

Effect of biofilm formation by Pseudoalteromonas spongiae on induction of larval settlement of the polychaete Hydroides elegans

The effects of culture conditions and chloramphenicol treatment on the induction of the marine bacterium Pseudoalteromonas spongiae to larval settlement of Hydroides elegans were investigated. The results showed that P. spongiae cells grown in the medium containing both yeast extract and peptone (YP-grown P. spongiae) was highly inductive to larval settlement, whereas P. spongiae cells grown in the medium containing only peptone (P-grown P. spongiae) or YP-grown P. spongiae cells treated with chloramphenicol at the onset of biofilm development (YPC-grown P. spongiae) did not induce larval settlement. Analysis of biofilm formation, biofilm structure, and the surface protein profile indicated that only the induction-capable YP-grown P. spongiae formed a well-developed biofilm, while the P-grown P. spongiae and the YPC-grown P. spongiae did not. We report here for the first time that bacterial biofilm formation was associated with its induction of larval settlement.     

Characterization of six polymorphic microsatellites for the polychaete tubeworm Hydroides elegans and cross‐species amplification in the congener Hydroides hexagonus

We isolated and characterized six polymorphic microsatellite loci for the polychaete tubeworm, Hydroides elegans. Two additional loci were not reliably scorable and estimates of heterozygosity were obtained for the other six. In addition, cross‐species amplification was successful for two loci using the congener H. hexagonus. Given that few microsatellite loci are available for polychaetes, these markers will be useful in assessing dispersal and gene flow in H. elegans and probably also other polychaetes.     

Insights into the composition,morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus

To date, the calcareous tubes of serpulid marine worms have not been studied extensively in a biomineralization context. The structure and composition of the tube shell and adhesive cement of the marine tubeworm Hydroides dianthus were studied using a variety of characterization techniques, including powder XRD, FTIR, SEM, EDX, and AFM. The tube and cement were determined to be inorganic–organic composite materials, consisting of inorganic aragonite (CaCO₃) and Mg-calcite ((Ca_0.8Mg_0.2)CO₃) crystals, and both soluble and insoluble organic matrices (SOM and IOM). SEM imaging revealed a variety of crystal morphologies. AFM nanoindentation of the inorganic components yielded Young’s moduli of ～20 GPa in the wet state, and ～50 GPa in the dry state. Amino acid analysis of the SOM indicated substantial amounts of acidic and non-polar neutral amino acids. Part of the insoluble organic tube lining was identified as being composed of collagen-containing fibres aligned in a criss-crossed structure. The SOM and organic tube lining were found to contain carboxylated and sulphated polysaccharides. In an artificial seawater solution, the SOM and the organic tube lining mediated CaCO₃ mineralization in vitro.     

Waterborne compounds from the green seaweed Ulva reticulata as inhibitive cues for larval attachment and metamorphosis in the Polychaete Hydroides elegans

Field observations in Hong Kong waters have shown the marine macroalga Ulva reticulata Forsskal (Chlorophyta) to be free of fouling. In this study, the presumptive antifouling mechanism in this alga was investigated and attempts were made to distinguish between possible mechanisms. Waterborne algal compounds were analyzed in larval behavioral bioassays with the marine polychaete Hydroides elegans, a major fouling organism in tropical waters around the world. Larval attachment and metamorphosis in this sessile species is induced by specific bacterial strains in natural bio‐organic films. In bioassays with alga‐conditioned seawater, levels of larval metamorphosis were significantly reduced in comparison to controls. The results demonstrate that the inhibitive effect was caused by direct larval deterrence. Following a bio‐assay‐guided isolation procedure, an inhibitive fraction for larval metamorphosis was purified from U. reticulata‐conditioned seawater. Preliminary chemical analysis of the biologically active fraction pointed to polysaccharides, proteins or glycoconjugates with a molecular weight > 100 kD.     

Cyclic AMP concentration and protein kinase A (PKA) gene expression at different developmental stages of the polychaete Hydroides elegans

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed inductive effect on larval settlement of the polychaete Hydroides elegans. It has been suggested that IBMX triggers larval settlement by elevating the cellular adenosine 3',5'-cyclic monophosphate (cAMP) level in this species. To test this hypothesis, we first examined cAMP-level changes in both the competent (CL) and attached larvae (AL) and then characterized the cAMP-dependent protein kinase in H. elegans, which is the major mediator of cAMP action. Tissue extracts of the larvae were assayed for cAMP by enzyme immunoassay; the results showed that IBMX increased cAMP production up to approximately two-folds in the CL. However, there was no significant difference in the cAMP concentration between the CL and AL that were not treated with IBMX. The catalytic subunit of protein kinase A gene from H. elegans (designated HePKAc) was cloned, and its expression in different developmental stages of H. elegans was examined using quantitative real-time polymerase chain reaction. The gene expression level in the pre-competent trochophore larvae was the lowest, increased in the CL, reached the highest in the larvae undergoing normal and IBMX-induced metamorphosis, and then decreased in the adult stage. In situ hybridization results showed that HePKAc expressed mainly around eye regions and along body fragments of the CL and AL. Our results indicated that the IBMX-induced cAMP changes and the cAMP-dependent protein kinase gene may mediate larval development and settlement of H. elegans.     

Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans

Hydroides elegans is a major fouling organism in tropical waters around the world, including Pearl Harbor, Hawaii. To determine the importance of initial surface characteristics on biofilm community composition and subsequent colonization by larvae of H. elegans, the settlement and recruitment of larvae to biofilmed surfaces with six different initial surface wettabilities were tested in Pearl Harbor. Biofilm community composition, as determined by a combined approach of denaturing gradient gel electrophoresis and fluorescence in situ hybridization, was similar across all surfaces, regardless of initial wettability, and all surfaces had distinct temporal shifts in community structure over a 10 day period. Larvae settled and recruited in higher numbers to surfaces with medium to low wettability in both May and August, and also to slides with high wettability in August. Pearl Harbor biofilm communities developed similarly on a range of surface wettabilities, and after 10 days in Pearl Harbor all surfaces were equally attractive to larvae of Hydroides elegans, regardless of initial surface properties.     

Growth patterns during segmentation in the two polychaete annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages

Many animals generate new body segments sequentially from a posterior growth zone, and this is generally thought to be the case for the annelids. Most annelids, including polychaetes, have an indirect life cycle and generate their earliest segments during larval life. We have characterized the nature of the growth zone in two polychaetes, Hydroides elegans and Capitella sp. I, during both larval and juvenile stages of segment formation by examining cell division patterns with 5-bromo-2'-deoxyuridine incorporation. Cell division patterns show commonalities between the two species, even though they have distinct body plans and life history characteristics. In both polychaetes, larval segments arise from a field of dividing cells located in lateral regions of the body, rather than from a localized posterior growth zone. Circumferential expansion of the forming segmental tissue is particularly pronounced in Capitella sp. I. Post-metamorphic segments, in contrast, originate from a classical posterior growth zone, with the exception of four posterior thoracic segments of H. elegans, which appear to arise from an area in the middle of the body, indicating plasticity of segment-generating mechanisms present in different annelid life histories. The distinct nature of larval versus juvenile growth zones in H. elegans and Capitella sp. I raises the question of the mechanistic relationship between these two growth zones. The results of this study increase our understanding of the cellular origins of segments in annelids, and serve as a basis for interpretation of molecular expression patterns associated with segment formation in polychaetes.     

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide,Irgarol 1051

Abstract

The impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide, Irgarol 1051

AbstractThe impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans

Development in marine invertebrate species can take place through a variety of modes and larval forms, but within a species, developmental mode is typically uniform. Poecilogony refers to the presence of more than one mode of development within a single species. True poecilogony is rare, however, and in some cases, apparent poecilogony is actually the result of variation in development mode among recently diverged cryptic species. We used a phylogenetic approach to examine whether poecilogony in the marine polychaete worm, Pygospio elegans, is the result of cryptic speciation. Populations of worms identified as P. elegansooded, and intermediate larvae; these modes are found both within and among populations. We examined sequence variation among partial mitochondrial cytochrome c oxidase subunit I sequences obtained for 279 individual worms sampled across broad geographic and environmental scales. Despite a large number of unique haplotypes (121 haplotypes from 279 individuals), sequence divergence among European samples was low (1.7%) with most of the sequence variation observed within populations, relative to the variation among regions. More importantly, we observed common haplotypes that were widespread among the populations we sampled, and the two most common haplotypes were shared between populations differing in developmental mode. Thus, our results support an earlier conclusion of poecilogony in P elegans. In addition, predominantly planktonic populations had a larger number of population-specific low-frequency haplotypes. This finding is largely consistent with interspecies comparisons showing high diversity for species with planktonic developmental modes in contrast to low diversity in species with brooded developmental modes.     

Physiological acclimation to decreased water temperature and the relative importance of water viscosity in determining the feeding performance of larvae of a serpulid polychaete

Ambient temperature exerts both physiological and mechanicaleffects on the rates of functional processes of small aquaticectotherms. Physiological effects of temperature result fromits influence on the rates of chemical reactions. Mechanicaleffects of temperature result from the inverse relationshipbetween the temperature of water and its dynamic viscosity.We measured the relative importance of these components of temperatureon the feeding performance of polychaete larvae. Cohorts oflarvae were reared for 24 h at 20°C and 10°C in treatmentswhere the physiological and mechanical effects of these temperatureswere separated. The feeding performance of these larvae wassubsequently measured in treatments where these components oftemperature were similarly partitioned. Cold-reared larvae displayedcomplete acclimation of feeding performance to the physiologicaleffects of decreased temperature: thus, increased viscositywas responsible for 100% of the difference in feeding performancebetween 20°C and 10°C. The physiological ability ofsmall aquatic ectotherms to acclimate functional processes totemperature variation may be greater than previously thought,and these results have implications for understanding the responsesof aquatic ectotherms’ to global temperature change.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.