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.     

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 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.     

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.     

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.     

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.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

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.     

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.     

Biofouling likely serves as a major mode of dispersal for the polychaete tubeworm Hydroides elegans as inferred from microsatellite loci

The polychaete tubeworm Hydroides elegans (Haswell) is a biofouling species with relatively limited larval dispersal. Four highly polymorphic microsatellite loci were used to make inferences about the migration and global population structure of 137 individuals from seven sub-populations located in the Atlantic, Pacific, and Indian Oceans and in the Mediterranean Sea. The results of the genetic analyses suggest minimal population sub-structure (F(st) = 0.09). Estimates of pairwise F(st) and migration rates using the coalescent-based method of MIGRATE suggest that there is little genetic differentiation between certain populations. Variation in relatedness among pairs of populations is consistent with a suite of local and global factors. The most likely explanation for close genetic relatedness among certain populations over such vast distances is the regular and consistent transport of adults and larvae on the hulls and in the ballast water of ships, respectively.     

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.     

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.     

Effect of biofilm age on settlement of Mytilus edulis

Biofilm ageing is commonly assumed to improve mussel settlement on artificial substrata, but the structure and taxonomic composition of biofilms remains unclear. In the present study, multi-species biofilms were characterized at different ages (1, 2, and 3 weeks) and their influence on settlement of the blue mussel, Mytilus edulis, was tested in the field. As biofilms can constitute a consistent food resource for larvae, the lipid quality, defined as the proportion of related essential fatty acids, may be a selection criterion for settlement. Overall mussel settlement increased on biofilms older than 1 week, and the enhanced settlement corresponded to the abundance and composition of the biofilm community, rather than to essential fatty acid levels. However, during a pulse of phytoplankton, the positive influence of biofilm was not detected, suggesting that pelagic cues overwhelmed those associated with biofilms. The influence of biofilms on mussel settlement could be more crucial when planktonic resources are limited.     

The importance of larval eyes in the polychaete Capitella teleta: effects of larval eye deletion on formation of the adult eye

In many marine invertebrates with biphasic life cycles, juvenile/adult traits begin to develop before metamorphosis. For structures that are present at multiple developmental stages, but have distinct larval and adult forms, it is unclear whether larval and adult structures have shared or distinct developmental origins. In this study, we examine the relationship between the larval and adult eyes in the polychaete Capitella teleta. In addition, we describe a novel marker for larval and juvenile photoreceptor cells. Infrared laser deletion of individual micromeres in early embryos suggests that the same micromeres at the eight‐cell stage that are specified to generate the larval eyes also form the adult eyes. Direct deletion of the larval eye, including the pigment cell and the corresponding photoreceptor cell, resulted in a lack of shading pigment cells in juveniles and adults, demonstrating that this structure does not regenerate. However, a sensory photoreceptor cell was present in juveniles following direct larval eye deletions, indicating that larval and adult photoreceptors are separate cells. We propose that the formation of the adult eye in juveniles of C. teleta requires the presence of the pigment cell of the larval eye, but the adult photoreceptor is either recruited from adjacent neural tissue or arises de novo after metamorphosis. These results are different from the development and spatial orientation of larval and adult eyes found in other polychaetes, in which two scenarios have been proposed: larval eyes persist and function as adult eyes; or, distinct pigmented adult eyes begin developing separately from larval eyes prior to metamorphosis.     

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.     

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.