Whole-body integration of gene expression and single-cell morphology.

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets.

The cellular 3D printer of a marine bristle worm-chaetogenesis in Platynereis dumerilii (Audouin & Milne Edwards, 1834) (Annelida).

Annelid chaetae are extracellular chitinous structures that are formed in an extracellular epidermal invagination, the chaetal follicle. The basalmost cell of this follicle, the chaetoblast, serves like a 3D-printer as it dynamically shapes the chaeta. During chaetogenesis apical microvilli of the chaetoblast form the template for the chaeta, any structural details result from modulating the microvilli pattern. This study describes this process in detail in the model organism Platynereis dumerilii and clarifies some aspects of chaetogenesis in its close relative Nereis vexillosa, the first annelid in which the ultrastructure of chaetogenesis had been described. Nereid species possess compound chaetae characteristic for numerous subgroups of errant annelids. The distal most section of these chaetae is movable; a hinge connects this part of the chaeta to the shaft. Modulation of the microvilli and differences in their structure, diameter and number of microvilli, and their withdrawal and reappearance determine the shape of these compound chaetae. Chaetal structure and pattern also change during life history. While larvae possess a single type of chaeta (in addition to internal aciculae), juveniles and adults possess two types of chaetae that are replaced by large paddle-shaped chaetae in swimming epitokous stages. Chaetogenesis is a continuous process that lasts during the entire lifespan. The detailed developmental sequence of chaetae and their site of formation are very similar within species and species groups. We expect that similarity results from a conserved gene regulatory network making this an optimal system to test the phylogenetic affinity of taxa and the homology of their chaetae.

Concentric Inclusions in the Intestinal Epithelium of Echiura Bonellia viridis: Structure and Possible Function.

The study of the anatomy and fine structure of Echiura is of great importance for understanding the biology of these animals, which lead a secretive life and dominate in various benthic communities. The first data on the organization of the siphonal part of the midgut of female Bonellia viridis were obtained by the methods of scanning and transmission electron microscopy. Unusual concentric inclusions similar in the ultrastructure to those described in other animals, e.g., in the gut of many nematode species and in the tegument of some cestodes, were first found in the cells of the midgut. It is known that, in these animals, the concentric inclusions play an important role in the binding of chemical agents inherent in redox environments. Interestingly, the individuals of B. viridis studied were found on the surface of a substrate devoid of redox environment signs. New results indicate the presence in B. viridis and, possibly, in all spoon worms, of preadaptations to life in redox environments. New data on the structure and composition of concentric inclusions will shed light on their origin and function.

Delimitation of cryptic species drastically reduces the geographical ranges of marine interstitial ghost-worms (Stygocapitella; Annelida, Sedentaria).

The recognition of cryptic species concealed in traditionally established species may reveal new biogeographical patterns and alter the understanding of how biodiversity is geographically distributed. This is particularly relevant for marine ecosystems where the incidence of cryptic species is high and where species distribution data are often challenging to collect and interpret. Here, we studied specimens of the 'cosmopolitan' interstitial meiofaunal annelid Stygocapitella subterranea Knöllner, 1934 (Parergodrilidae, Orbiniida), obtaining data from four coastlines in the Northern hemisphere. Using phylogenetic tools and several species-delimitation methods (haplotype networks, GMYC, bPTP, maximum likelihood, posterior probability and morphology) we describe eight new Stygocapitella species. With one exception, all species are present along a single coastline, ultimately challenging the idea that Stygocapitella subterranea has a cosmopolitan distribution. We found evidence for several oceanic transitions having occurred in the past as well as a recent translocation, potentially due to human activity. No diagnostic characters were found, and qualitative and quantitative morphological data do not allow an unequivocal differentiation of the identified cryptic species. This suggests that (i) neither traditional diagnostic features nor quantitative morphology suffice to recognise species boundaries in cryptic species complexes, such as the Stygocapitella species complex; and that (ii) the recognition and description of cryptic species is of seminal importance for biodiversity assessments, biogeography and evolutionary biology.

Photosome membranes merge and organize tending towards rhombohedral symmetry when light is emitted.

Polynoid worm elytra emit light when mechanically or electrically stimulated. Specialized cells, the photocytes, contain light emitting machineries, the photosomes. Successive stimulations induce light intensity variations and show a coupling within and between photosomes. Here, we describe, using electron tomography of cryo-substituted elytra and freeze-fracturing, the structural transition associated to light emission: undulating tubules come closer, organize and their number forming photosomes increases. Two repeating undulating tubules in opposite phase compose the photosome. Undulations are located on three hexagonal layers that regularly repeat and are equally displaced, in x y and z. The tubule membranes within layers merge giving rise to rings that tend to obey to quasi-rhombohedral symmetry. Merging may result either from close-association, hemifusion (one leaflet fusion) or from fusion (two leaflets fusion). Although the resolution of tomograms is not sufficient to distinguish these three cases, freeze-fracturing shows that hemifusion is a frequent process that leads to an reversible anastomosed membrane complex favoring communications, appearing as a major coupling factor of photosome light emission.

Developmental studies provide new insights into the evolution of sense organs in Sabellariidae (Annelida).

BACKGROUND: Sabellarids, also known as honeycomb or sandcastle worms, when building their tubes, produce chemical signals (free fatty acids) that are responsible for larval settlement and the formation of three-dimensional aggregations. The larval palps and the dorsal hump (becoming the median organ in adults) are presumed to participate in such a substrate selection during settlement. Notably, the sabellariid median organ is an apparently unique organ among annelids that has been attributed with a sensory function and perhaps with some affinities to the nuchal organs of other polychaetes. Nevertheless, detailed investigations of this prominent character complex including ultrastructural examinations are lacking so far. RESULTS: Our comprehensive investigations provide data about the anterior sensory organs in Sabellariidae and inform about their transformation during pelagic larval development. We used a comparative approach including immunostaining with subsequent confocal laser scanning microscopy (clsm), histological sections as well as electron microscopy in a range of larval and adult stages of two sabellariid species. We find that the neuronal innervation as well as the ultrastructure of the sabellariid ciliary structures along the median organ are highly comparable with that of nuchal organs known from other polychaetes. Furthermore, the myoinhibitory protein (MIP) - a protein known to be also involved into chemo-sensation - was detected in the region of the larval median organ. Moreover, we reveal the presence of an unusual type of photoreceptor as part of the median organ in Idanthyrsus australiensis with a corrugated sensory membrane ultrastructure unlike those observed in the segmental ocelli of other polychaetes. CONCLUSIONS: We are describing for the first time the nuchal organ-like structures in different developmental stages of two species of Sabellariidae. The external morphology, neuronal innervation, developmental fate and ultrastructure of the newly-discovered median organ-based ciliary pits are comparable with the characteristics known for annelid nuchal organs and therefore indicate a homology of both sensory complexes. The presence of myoinhibitory peptide (MIP) in the respective region supports such a hypothesis and exhibits the possibility of an involvement of the entire sabellariid median organ complex, and in particular the prominent ciliated pits, in chemo-sensation.

Molecular phylogeny, morphology, and distribution of Polygordius (Polychaeta: Polygordiidae) in the Atlantic and Mediterranean.

Low morphological diversity among interstitial taxa makes it difficult to delimit species and their geographic boundaries based solely on morphology and molecular data often reveal cryptic species. Polygordius (Annelida, Polygordiidae) have low morphological diversity, but are unusual among interstitial species in their comparatively large size due to their elongated form, high fecundity, and potential for long-distance dispersal via a planktotrophic larval stage. Polygordius species collected from 14 localities in the Northwest Atlantic, Mediterranean Sea, and Southwest Atlantic including several of the respective type localities were analysed. This study presents the first phylogeny of the genus Polygordius and combines molecular data, sequences of COI, 16S and ITS1/2 genes, and morphological data for a systematic re-evaluation focusing on Atlantic species, with an emphasis on populations from European waters. Phylogenetic analyses recovered six valid species (P. appendiculatus, P. lacteus, P. neapolitanus, P. triestinus, P. jouinae, and P. eschaturus) and their distinctness is confirmed by haplotype network analyses. Thus, molecular data supported the validity of the previously recognized morpho-species and no new species were present. P. erythrophthalmus and P. villoti are invalid species being synonymous with P. lacteus. Subtle differences in head and pygidial morphology and larval type (endolarva vs. exolarva), were useful characters for discrimination. Yet seemingly significant variation in characters among individuals in some species was not diagnostic (e.g., number of pygidial cirri). Highly similar species based on adult morphology were shown to be sister taxa occurring in allopatry. Present day distribution patterns of species are summarized in light of this study.

Microbiota associated with tubes of Escarpia sp. from cold seeps in the southwestern Atlantic Ocean constitutes a community distinct from that of surrounding marine sediment and water.

As the depth increases and the light fades in oceanic cold seeps, a variety of chemosynthetic-based benthic communities arise. Previous assessments reported polychaete annelids belonging to the family Siboglinidae as part of the fauna at cold seeps, with the 'Vestimentifera' clade containing specialists that depend on microbial chemosynthetic endosymbionts for nutrition. Little information exists concerning the microbiota of the external portion of the vestimentiferan trunk wall. We employed 16S rDNA-based metabarcoding to describe the external microbiota of the chitin tubes from the vestimentiferan Escarpia collected from a chemosynthetic community in a cold seep area at the southwestern Atlantic Ocean. The most abundant operational taxonomic unit (OTU) belonged to the family Pirellulaceae (phylum Planctomycetes), and the second most abundant OTU belonged to the order Methylococcales (phylum Proteobacteria), composing an average of 21.1 and 15.4% of the total reads on tubes, respectively. These frequencies contrasted with those from the surrounding environment (sediment and water), where they represent no more than 0.1% of the total reads each. Moreover, some taxa with lower abundances were detected only in Escarpia tube walls. These data constitute on the first report of an epibiont microbial community found in close association with external surface of a cold-seep metazoan, Escarpia sp., from a chemosynthetic community in the southwestern Atlantic Ocean.

Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population.

Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

The Syllis gracilis species complex: A molecular approach to a difficult taxonomic problem (Annelida, Syllidae).

Syllis gracilis is an emblematic member of the subfamily Syllinae (Syllidae, Annelida), which inhabits shallow, temperate coastal waters and can be found on algae, coral rubble, and sponges. Their distinctive ypsiloid chaetae, usually found in specimens from populations all around the world, led to the consideration of the species as cosmopolitan, even though four other species have similar chaetae: Syllis magellanica, S. picta, S. mayeri and S. ypsiloides. The discovery of deeply divergent lineages in the Mediterranean Sea, that were morphologically similar, questioned the cosmopolitanism of S. gracilis and suggested the possibility of it being a species complex. In order to assess the speciation patterns within the putative S. gracilis complex, we undertook species delimitation and phylogenetic analyses on 61 specimens morphologically ascribed to Syllis gracilis and closely related species using a multilocus molecular dataset (two mitochondrial and two nuclear markers). Our results suggest high levels of genetic differentiation between the S. gracilis populations analyzed, some of which have morphologically distinctive features. Five to eight distinct lineages (depending on the analysis) were identified, all with geographically restricted distributions. Although the presence of ypsiloid chaetae has been traditionally considered the main character to identify S. gracilis, we conclude that this feature is homoplastic. Instead, we propose that characters such as the degree of fusion of blades and shafts in chaetae, the morphology of the posterior chaetae or the animal color pattern should be considered to differentiate lineages within the S. gracilis species complex. Our study does not support the cosmopolitanism of S. gracilis, and instead provides morphological and molecular evidence of the existence of a complex of pseudo-cryptic species.

Fine structure of agglutinated tubes of polychaetes of the family Terebellidae (Annelida).

The results of the first study on the fine structure of agglutinated tubes of the family Terebellidae are presented.

Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans.

The serpulid tubeworm, Hydroides elegans, is an ecologically and economically important species whose biology has been fairly well studied, especially in the context of larval development and settlement on man-made objects (biofouling). Nevertheless, ontogenetic changes associated with calcareous tube composition and structures have not yet been studied. Here, the ultrastructure and composition of the calcareous tubes built by H. elegans was examined in the three early calcifying juvenile stages and in the adult using XRD, FTIR, ICP-OES, SEM and Raman spectroscopy. Ontogenetic shifts in carbonate mineralogy were observed, for example, juvenile tubes contained more amorphous calcium carbonate and were predominantly aragonitic whereas adult tubes were bimineralic with considerably more calcite. The mineral composition gradually shifted during the tube development as shown by a decrease in Sr/Ca and an increase of Mg/Ca ratios with the tubeworm's age. The inner tube layer contained calcite, whereas the outer layer contained aragonite. Similarly, the tube complexity in terms of ultrastructure was associated with development. The sequential appearance of unoriented ultrastructures followed by oriented ultrastructures may reflect the evolutionary history of serpulid tube biominerals. As aragonitic structures are more susceptible to dissolution under ocean acidification (OA) conditions but are more difficult to be removed by anti-fouling treatments, the early developmental stages of the tubeworms may be vulnerable to OA but act as the important target for biofouling control.

A chemosynthetic weed: the tubeworm Sclerolinum contortum is a bipolar, cosmopolitan species.

BACKGROUND: Sclerolinum (Annelida: Siboglinidae) is a genus of small, wiry deep-sea tubeworms that depend on an endosymbiosis with chemosynthetic bacteria for their nutrition, notable for their ability to colonise a multitude of reducing environments. Since the early 2000s, a Sclerolinum population has been known to inhabit sediment-hosted hydrothermal vents within the Bransfield Strait, Southern Ocean, and whilst remaining undescribed, it has been suggested to play an important ecological role in this ecosystem. Here, we show that the Southern Ocean Sclerolinum population is not a new species, but more remarkably in fact belongs to the species S. contortum, first described from an Arctic mud volcano located nearly 16,000 km away. RESULTS: Our new data coupled with existing genetic studies extend the range of this species across both polar oceans and the Gulf of Mexico. Our analyses show that the populations of this species are structured on a regional scale, with greater genetic differentiation occurring between rather than within populations. Further details of the external morphology and tube structure of S. contortum are revealed through confocal and SEM imaging, and the ecology of this worm is discussed. CONCLUSIONS: These results shed further insight into the plasticity and adaptability of this siboglinid group to a range of reducing conditions, and into the levels of gene flow that occur between populations of the same species over a global extent.

Weakening mechanisms of the serpulid tube in a high-CO2 world.

Many benthic marine organisms produce calcium carbonate (CaCO3) structures for mechanical protection through a biologically controlled calcification process. However, the oceans are becoming unfavorable for calcification because of the stress associated with ocean acidification (OA) and associated chemical changes such as declining saturation state of CaCO3 and decreasing seawater pH. This work studies the impacts of OA-driven decreased pH on the calcareous tubes produced by the serpulid tubeworm Hydroides elegans. Tubes grown under control and OA experimental conditions were measured for structural and mechanical properties, and their mechanical properties were further interpreted using finite element analysis (FEA). The near-future predicted pH value of 7.8 altered tube ultrastructure, volume, and density and decreased the mean tube hardness and elasticity by ∼ 80 and ∼ 70%, respectively. The crushing force required for breaking the tube was reduced by 64%. The FEA results demonstrated how a simulated predator attack may affect the structure with different structural and mechanical properties and consequently shift the stress development and distribution in the tubes, causing a more concentrated stress distribution and therefore leading to a lower ability to withstand attacks.

The regulation of copper stress response genes in the polychaete Nereis diversicolor during prolonged extreme copper contamination.

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their conspecifics.

Segment regeneration in the vestimentiferan tubeworm, Lamellibrachia satsuma.

The ability to regenerate missing body parts varies among species. To elucidate the evolution of regenerative capability, an understanding of the regeneration mechanisms of diverse organisms is required. We focus on vestimentiferan tubeworms, which have a body plan that is unique among annelids. We found that the vestimentiferan Lamellibrachia satsuma is able to regenerate its posterior body parts, but not its anterior body parts. Based on observations of live specimens, we defined five stages in the process of posterior regeneration. The morphogenesis was observed in detail by a series of sections and scanning electron microscopy. The most posterior domain of the opisthosome differentiated from the blastema, while the anterior domain of the opisthosome regenerated from the remaining trunk region. We also examined the expression pattern of the engrailed gene during regeneration, and found that engrailed was expressed in the mesodermal cells of each segment.

The genus Exogone (Polychaeta: Syllidae) from the Brazilian coast, with the description of a new species.

A new species of Exogone, E. gigas sp. n., is described herein, together with E. africana, E. arenosa, E. dispar, E. naidinoides, E. rolani, and E. simplex, collected along the Brazilian coast, including new morphological details. The distribution of E. breviantennata is expanded to include the states of Espírito Santo, Paraíba and Pernambuco. Exogone gigas sp. n. is characterized by having a large median antenna, the absence of dorsal cirri on chaetiger 2, and having a triangular process and minute spines on the shaft of the spiniger-like chaetae on chaetiger 2. The Brazilian specimens are compared to the morphologically most similar congeners. A key for the species described in this paper is provided.

Occurrence and distribution of Pseudoscalibregma and Scalibregma (Annelida, Scalibregmatidae) in the deep Nordic Seas, with the description of Scalibregma hanseni n. sp.

Until recent years, only a few scalibregmatid species have been known from the Nordic Seas, largely from shelf and coastal waters. Access to a large collection from deep areas has made it possible to provide more knowledge on the diversity of this group in the area. Pseudoscalibregma parvum (Hansen, 1879) is here redescribed. The species has a wide geographic distribution in the Nordic Seas, the Barents Sea, and the Kara Sea. Type specimens of Eumenia longisetosa Théel, 1879 were found to be similar to specimens of P. parvum, confirming the synonymy of the species. A new species, Scalibregma hanseni n. sp., is described from specimens found on the continental slope. It is particularly characterised by having three pairs of rather simple branchiae. Both P. parvum and S. hanseni have small spines in the most anterior chaetiger(s), resembling spines reported from a few other Pseudoscalibregma and Scalibregma species and supporting the need to emend the genus diagnosis of Pseudoscalibregma. Scalibregma abyssorum Hansen, 1879 was reassessed and considered to be a nomen dubium. Scalibregma inflatum, which has a wide distribution along the Norwegian coast and continental shelf, is found to be restricted to depths above about 900 m. Depths from 600-800 m on the continental slope represent a transition zone with fluctuations between temperate North Atlantic water (about 7°C) and cold Norwegian Sea water (below 0°C). The three species coexist in this zone, whereas P. parvum and S. hanseni n. sp. extend down to 1700 and 1200 m, respectively, on the slope at temperatures below 0°C.

Ultrastructure of the Jurassic serpulid tubes-phylogenetic and paleoecological implications.

The ultrastructural diversity of the Middle and Late Jurassic serpulid tubes from the Polish Basin has been investigated. The inspection of 12 taxa representing the two major serpulid clades allowed for the identification of three ultrastructure types-irregularly oriented prismatic structure (IOP), spherulitic prismatic structure (SPHP), and simple prismatic structure (SP). Six of the studied species are single-layered and six species possess two distinct layers. Ultrastructural diversity corresponds to certain serpulid clades. The members of Filograninae have single-layered tube walls composed of possibly plesiomorphic, irregularly oriented prismatic structure (IOP). Two-layered tubes occur solely within the clade Serpulinae, where the external, denser layer is built of either the ordered spherulitic (SPHP) or simple prismatic microstructure (SP), and the internal layer is composed of irregularly oriented prismatic structure (IOP). Apart from phylogenetic signals provided by the tube ultrastructure, it can be used in analyzing paleoecological aspects of tube-dwelling polychaetes. Compared to the more primitive, irregularly oriented microstructures of Filograninae, the regularly oriented microstructures of Serpulinae need a higher level of biological control over biomineralization. The advent of the dense outer protective layer (DOL) in serpulids, as well as the general increase in ultrastructure diversity, was likely a result of the evolutionary importance of the tubes for serpulids. Such diversity of the tube ultrastructural fabrics allowed for maximizing functionality by utilizing a variety of morphogenetic programs. The biomineralization system of serpulids remains more complex compared to other tube-dwelling polychaetes. Physiologically more expensive tube formation allows for mechanical strengthening of the tube by building robust, strongly ornamented tubes and firm attachment to the substrate. Contrary to sabellids, which perform a fugitive strategy, an increased tube durability allows serpulids a competitive advantage over other encrusters.

Dynamic microvilli sculpt bristles at nanometric scale.

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.