Larvae from deep-sea methane seeps disperse in surface waters.

Many species endemic to deep-sea methane seeps have broad geographical distributions, suggesting that they produce larvae with at least episodic long-distance dispersal. Cold-seep communities on both sides of the Atlantic share species or species complexes, yet larval dispersal across the Atlantic is expected to take prohibitively long at adult depths. Here, we provide direct evidence that the long-lived larvae of two cold-seep molluscs migrate hundreds of metres above the ocean floor, allowing them to take advantage of faster surface currents that may facilitate long-distance dispersal. We collected larvae of the ubiquitous seep mussel "Bathymodiolus" childressi and an associated gastropod, Bathynerita naticoidea, using remote-control plankton nets towed in the euphotic zone of the Gulf of Mexico. The timing of collections suggested that the larvae might disperse in the water column for more than a year, where they feed and grow to more than triple their original sizes. Ontogenetic vertical migration during a long larval life suggests teleplanic dispersal, a plausible explanation for the amphi-Atlantic distribution of "B." mauritanicus and the broad western Atlantic distribution of B. naticoidea. These are the first empirical data to demonstrate a biological mechanism that might explain the genetic similarities between eastern and western Atlantic seep fauna.

Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Quantitative proteomics identify molecular targets that are crucial in larval settlement and metamorphosis of Bugula neritina.

The marine invertebrate Bugula neritina has a biphasic life cycle that consists of a swimming larval stage and a sessile juvenile and adult stage. The attachment of larvae to the substratum and their subsequent metamorphosis have crucial ecological consequences. Despite many studies on this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 and 24 h postattachment using label-free quantitative proteomics. We identified more than 1100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins and an assay of their functions indicated that they may have key roles in metamorphosis of B. neritina. These findings not only provide molecular evidence with which to elucidate the substantial changes in morphology and physiology that occur during larval attachment and metamorphosis but also identify potential targets for antifouling treatment.

2D gel-based multiplexed proteomic analysis during larval development and metamorphosis of the biofouling polychaete tubeworm Hydroides elegans.

Larval settlement and metamorphosis of a common biofouling polychaete worm, Hydroides elegans, involve remarkable structural and physiological changes during this pelagic to sessile habitat shift. The endogenous protein molecules and post-translational modifications that drive this larval transition process are not only of interest to ecologists but also to the antifouling paint industry, which aims to control the settlement of this biofouling species on man-made structures (e.g., ship hulls). On the basis of our recent proteomic studies, we hypothesize that rapid larval settlement of H. elegans could be mediated through changes in phosphorylation status of proteins rather than extensive de novo synthesis of proteins. To test this hypothesis, 2D gel-based multiplexed proteomics technology was used to monitor the changes in protein expression and phosphorylation status during larval development and metamorphosis of H. elegans. The protein expression profiles of larvae before and after they reached competency to attach and metamorphose were similar in terms of major proteins, but the percentage of phosphorylated proteins increased from 41% to 49% after competency. Notably, both the protein and phosphoprotein profiles of the metamorphosed individuals (adult) were distinctly different from that of the larvae, with only 40% of the proteins phosphorylated in the adult stage. The intensity ratio of all phosphoprotein spots to all total protein spots was also the highest in the competent larval stage. Overall, our results indicated that the level of protein phosphorylation might play a crucial role in the initiation of larval settlement and metamorphosis.

Comparative proteome and phosphoproteome analyses during cyprid development of the barnacle Balanus (=Amphibalanus) amphitrite.

The barnacle Balanus amphitrite (=Amphibalanus amphitrite) is a major marine biofouling invertebrate worldwide. It has a complex life cycle during which the larva (called a nauplius) molts six times before transforming into the cyprid stage. The cyprid stage in B. amphitrite is the critical stage for the larval decision to attach and metamorphose. In this study, proteome and phosphoproteome alterations during cyprid development/aging and upon treatment with the antifouling agent butenolide were examined with a two-dimensional electrophoresis (2-DE) multiplexed fluorescent staining approach. Optimized protein separation strategies, including solution-phase isoelectric fractionation and narrow-pH-range 2-DE, were used in a proteomic analysis. Our results show that the differential regulation of the target proteins is highly dynamic on the levels of both protein expression and posttranslational modification. Two groups of proteins, stress-associated and energy metabolism-related proteins, are differentially expressed during cyprid development. Comparison of the control and treatment groups suggests that butenolide exerts its effects by sustaining the expression levels of these proteins. Altogether, our data suggest that proteins involved in stress regulation and energy metabolism play crucial roles in regulating larval attachment and metamorphosis of B. amphitrite.

Dependency on de novo protein synthesis and proteomic changes during metamorphosis of the marine bryozoan Bugula neritina.

BACKGROUND: Metamorphosis in the bryozoan Bugula neritina (Linne) includes an initial phase of rapid morphological rearrangement followed by a gradual phase of morphogenesis. We hypothesized that the first phase may be independent of de novo synthesis of proteins and, instead, involves post-translational modifications of existing proteins, providing a simple mechanism to quickly initiate metamorphosis. To test our hypothesis, we challenged B. neritina larvae with transcription and translation inhibitors. Furthermore, we employed 2D gel electrophoresis to characterize changes in the phosphoproteome and proteome during early metamorphosis. Differentially expressed proteins were identified by liquid chromatography tandem mass spectrometry and their gene expression patterns were profiled using semi-quantitative real time PCR. RESULTS: When larvae were incubated with transcription and translation inhibitors, metamorphosis initiated through the first phase but did not complete. We found a significant down-regulation of 60 protein spots and the percentage of phosphoprotein spots decreased from 15% in the larval stage to12% during early metamorphosis. Two proteins--the mitochondrial processing peptidase beta subunit and severin--were abundantly expressed and phosphorylated in the larval stage, but down-regulated during metamorphosis. MPPbeta and severin were also down-regulated on the gene expression level. CONCLUSIONS: The initial morphogenetic changes that led to attachment of B. neritina did not depend on de novo protein synthesis, but the subsequent gradual morphogenesis did. This is the first time that the mitochondrial processing peptidase beta subunit or severin have been shown to be down-regulated on both gene and protein expression levels during the metamorphosis of B. neritina. Future studies employing immunohistochemistry to reveal the expression locality of these two proteins during metamorphosis should provide further evidence of the involvement of these two proteins in the morphogenetic rearrangement of B. neritina.

Temperature and salinity, not acidification, predict near-future larval growth and larval habitat suitability of Olympia oysters in the Salish Sea.

Most invertebrates in the ocean begin their lives with planktonic larval phases that are critical for dispersal and distribution of these species. Larvae are particularly vulnerable to environmental change, so understanding interactive effects of environmental stressors on larval life is essential in predicting population persistence and vulnerability of species. Here, we use a novel experimental approach to rear larvae under interacting gradients of temperature, salinity, and ocean acidification, then model growth rate and duration of Olympia oyster larvae and predict the suitability of habitats for larval survival. We find that temperature and salinity are closely linked to larval growth and larval habitat suitability, but larvae are tolerant to acidification at this scale. We discover that present conditions in the Salish Sea are actually suboptimal for Olympia oyster larvae from populations in the region, and that larvae from these populations might actually benefit from some degree of global ocean change. Our models predict a vast decrease in mean pelagic larval duration by the year 2095, which has the potential to alter population dynamics for this species in future oceans. Additionally, we find that larval tolerance can explain large-scale biogeographic patterns for this species across its range.

Spawning, development, and the duration of larval life in a deep-sea cold-seep mussel.

We describe culturing techniques and development for the cold-seep mussel "Bathymodiolus" childressi, the only deep-sea bivalve for which development has been detailed. Spawning was induced in mature mussels by injection of 2 mmol l(-1) serotonin into the anterior adductor muscle. The mean egg diameter is 69.15 +/- 2.36 microm (+/-S.D.; n = 50) and eggs are negatively buoyant. Cleavages are spiral and at 7-8 degrees C occur at a rate of one per 3-9 h through hatching, with free-swimming blastulae hatching by 40 h and shells beginning to develop by day 12. When temperature was raised to 12-14 degrees C after hatching, larvae developed to D-shell veligers by day 8 without being fed. Egg size and larval shell morphology indicate that "B." childressi has a planktotrophic larva, but we did not observe feeding in culture. Wide distribution of this species throughout the Gulf of Mexico and amphi-Atlantic distributions of closely related congeners suggest that larvae may spend extended periods in the plankton. Duration of larval life was estimated for "B." childressi by comparing calculated settlement times to known spawning seasons. These estimates suggest variability in the larval duration, with individuals spending more than a year in the plankton.

Dispersal of deep-sea larvae from the intra-American seas: simulations of trajectories using ocean models.

Using data on ocean circulation with a Lagrangian larval transport model, we modeled the potential dispersal distances for seven species of bathyal invertebrates whose durations of larval life have been estimated from laboratory rearing, MOCNESS plankton sampling, spawning times, and recruitment. Species associated with methane seeps in the Gulf of Mexico and/or Barbados included the bivalve "Bathymodiolus" childressi, the gastropod Bathynerita naticoidea, the siboglinid polychaete tube worm Lamellibrachia luymesi, and the asteroid Sclerasterias tanneri. Non-seep species included the echinoids Cidaris blakei and Stylocidaris lineata from sedimented slopes in the Bahamas and the wood-dwelling sipunculan Phascolosoma turnerae, found in Barbados, the Bahamas, and the Gulf of Mexico. Durations of the planktonic larval stages ranged from 3 weeks in lecithotrophic tubeworms to more than 2 years in planktotrophic starfish. Planktotrophic sipunculan larvae from the northern Gulf of Mexico were capable of reaching the mid-Atlantic off Newfoundland, a distance of more than 3000 km, during a 7- to 14-month drifting period, but the proportion retained in the Gulf of Mexico varied significantly among years. Larvae drifting in the upper water column often had longer median dispersal distances than larvae drifting for the same amount of time below the permanent thermocline, although the shapes of the distance-frequency curves varied with depth only in the species with the longest larval trajectories. Even species drifting for >2 years did not cross the ocean in the North Atlantic Drift.

Toward an understanding of the molecular mechanisms of barnacle larval settlement: a comparative transcriptomic approach.

BACKGROUND: The barnacle Balanus amphitrite is a globally distributed biofouler and a model species in intertidal ecology and larval settlement studies. However, a lack of genomic information has hindered the comprehensive elucidation of the molecular mechanisms coordinating its larval settlement. The pyrosequencing-based transcriptomic approach is thought to be useful to identify key molecular changes during larval settlement. METHODOLOGY AND PRINCIPAL FINDINGS: Using 454 pyrosequencing, we collected totally 630,845 reads including 215,308 from the larval stages and 415,537 from the adults; 23,451 contigs were generated while 77,785 remained as singletons. We annotated 31,720 of the 92,322 predicted open reading frames, which matched hits in the NCBI NR database, and identified 7,954 putative genes that were differentially expressed between the larval and adult stages. Of these, several genes were further characterized with quantitative real-time PCR and in situ hybridization, revealing some key findings: 1) vitellogenin was uniquely expressed in late nauplius stage, suggesting it may be an energy source for the subsequent non-feeding cyprid stage; 2) the locations of mannose receptors suggested they may be involved in the sensory system of cyprids; 3) 20 kDa-cement protein homologues were expressed in the cyprid cement gland and probably function during attachment; and 4) receptor tyrosine kinases were expressed higher in cyprid stage and may be involved in signal perception during larval settlement. CONCLUSIONS: Our results provide not only the basis of several new hypotheses about gene functions during larval settlement, but also the availability of this large transcriptome dataset in B. amphitrite for further exploration of larval settlement and developmental pathways in this important marine species.