The reproductive system of Osedax (Annelida,Siboglinidae): ovary structure,sperm ultrastructure,and fertilization mode

Osedax is a genus of siboglinid annelids in which the females live on dead vertebrate bones on the seafloor. These females have a posterior end that lies within the bone and contains the ovarian tissue, as well as the “roots” involved with bone degradation and nutrition. The males are microscopic and live as “harems” in the lumen of the gelatinous tube that surrounds the female trunk, well away from the ovary. Females are known to spawn fertilized primary oocytes, suggesting internal fertilization. However, little is known about sperm transfer, sperm storage, or the location of fertilization, and the morphology of the female reproductive system has not been described and compared with the reproductive systems of other siboglinids. A 3D‐reconstruction of the ovisac of Osedax showed ovarian tissue with multiple lobes and mature oocytes stored in a “uterus” before being released through the single oviduct. The oviduct emerges as a gonopore on the trunk and travels along the trunk to finally open to the seawater as a thin cylindrical tube among the crown of palps. Light and transmission electron microscopy of mature Osedax sperm revealed elongate heads consisting of a nucleus with helical grooves occupied by mitochondria. In contrast to other Siboglinidae, Osedax sperm are not packaged into spermatophores or spermatozeugmata, and Osedax females lack a discrete region for sperm storage. Transmission electron microscopy and fluorescence microscopy allowed detection of sperm associated with ovarian tissue of the female ovisac of four different Osedax species. This provides the first evidence for the site of internal fertilization in Osedax. A heart body was found in the circulatory system, as seen in other siboglinids and some other annelids. The possible presence of nephridia in the anterior ovisac region was also documented. These morphological features provide new insights for comparing the regionalization of Osedax females in relation to other siboglinids.     

Trace fossil evidence of predation upon bone‐eating worms on a baleen whale skeleton from the Oligocene of New Zealand

The osteophagous worm Osedax (Annelida: Siboglinidae) colonizes vertebrate bones in deep‐sea environments globally. Osedax bioerosion of modern bones suggests a potentially destructive agent in the marine vertebrate fossil record, but the dearth of published reports of abundant Osedax traces suggests an uncertain taphonomic influence of this organism. This study reports Osedax traces (Osspecus boreholes, pockmarks and collapsed galleries) in an Oligocene baleen whale (Cetacea: Eomysticetidae) from New Zealand, which constitute the first record of fossil Osedax traces from the southern hemisphere. Some Osedax traces are cross‐cut by linear biogenic scrape marks, implying that sharks or bony fish fed upon Osedax worms, a process which compounds or potentially accelerates worm‐inflicted damage to vertebrate bones in marine environments.     

Evidence of Osedax worm borings in Pliocene (∼3 Ma) whale bone from the Mediterranean

Osedax worms subsist entirely on vertebrate skeletons on the seafloor, using root-like tissues to bore into and degrade the bones. Paleontologists have only recently begun to appreciate the possible destructive effect that these worms may have had on the marine vertebrate fossil record and little is known of their evolutionary history. Using microcomputed tomography, we document Osedax-like borings in a fossil whale bone from the Pliocene of Italy and present new data on the borings of extant Osedax worms. The fossil borings are distinguished from those of other known borers and identified as traces of Osedax activity based on diagnostic features. Our results suggest that it is necessary to isolate individual borings for the confident identification of Osedax traces. This is only the second paleogeographic occurrence of Osedax in the fossil record and indicates that by the Pliocene these worms had colonised a large portion of the world's oceans. This is the first evidence for Osedax in the Mediterranean, past or present, and suggests that more species await discovery in this region.     

Molecular taxonomy of Osedax (Annelida: Siboglinidae) in the Southern Ocean

We report the discovery of three new species of Osedax in the deep Southern Ocean, expanding the diversity and geographical range of this genus of bone‐eating worms. Osedax rogersi sp. n. and Osedax crouchi sp. n. were found on a whale skeleton at 1444 m in the Kemp Caldera in the East Scotia Sea during the Chemosynthetic Ecosystems of the Southern Ocean (ChEsSo) project. The recently described species, Osedax antarcticus, found on whale bones implanted at a depth of 550–650 m off Smith Island in the Bransfield Strait, was also found inhabiting the whale skeleton in the Kemp Caldera. Osedax crouchi as well as another new species Osedax nordenskjoeldi sp. n. have also been found on the implanted whale bones off Smith Island. These two localities are approximately 1800 km apart demonstrating a wide distribution of species within the genus. We describe the three new species, O. rogersi, O. crouchi and O. nordenskjoeldi and report the second record of O. antarcticus. We also present a new phylogenetic analysis for Osedax, including data examining genetic connectivity between the Scotia Arc and the Bransfield Strait.     

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

An unusual symbiosis, first observed at ∼3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. ¹³C tracer experiments confirmed the assimilation of glycine/proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of ‘free-living'' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.     

Traces of the bone-eating annelid Osedax in Oligocene whale teeth and fish bones

The range of substrates that the bone-eating marine worm Osedax is able to consume has important implications for its evolutionary history, especially its potential link to the rise of whales. Once considered a whale specialist, recent work indicates that Osedax consumes a wide range of vertebrate remains, including whale soft tissue and the bones of mammals, birds and fishes. Traces resembling those produced by living Osedax have now been recognized for the first time in Oligocene whale teeth and fish bones from deep-water strata of the Makah, Pysht and Lincoln Creek formations in western Washington State, USA. The specimens were acid etched from concretions, and details of the borehole morphology were investigated using micro-computed tomography. Together with previously published Osedax traces from this area, our results show that by Oligocene time Osedax was able to colonize the same range of vertebrate remains that it consumes today and had a similar diversity of root morphologies. This supports the view that a generalist ability to exploit vertebrate bones may be an ancestral trait of Osedax.     

Species identification and likely catch time period of whale bones from South Georgia

Skeletal remains of baleen whales killed during the onset of 20th century commercial whaling lie scattered across the shores and abandoned whaling stations of the subantarctic island of South Georgia. Here we report on genetic species identification of whale bones collected from South Georgia using standard historical DNA protocols. We amplified and sequenced short fragments of the mitochondrial DNA (mtDNA) control region from 281 available bone samples. Of these, 231 provided mtDNA sequences of sufficient quality and length (174–194 bp) for species identification: 158 bones were identified as humpback whale (Megaptera novaeangliae), 51 bones were identified as fin whale (Balaenoptera physalus), 18 bones were identified as blue whale (B. musculus), two bones were identified as sei whale (B. borealis), one bone was identified as a southern right whale (Eubalaena australis), and one bone was identified as a southern elephant seal (Mirounga leonina). The prominence of humpback, fin, and blue whale bones in the sample collection corresponds to the catch record of the early years of whaling on the island of South Georgia (pre‐1915), prior to the depletion of these populations.     

Genetic Diversity and Potential Function of Microbial Symbionts Associated with Newly Discovered Species of Osedax Polychaete Worms

We investigated the genetic diversity of symbiotic bacteria associated with two newly discovered species of Osedax from Monterey Canyon, CA, at 1,017-m (Osedax Monterey Bay sp. 3 “rosy” [Osedax sp. MB3]) and 381-m (Osedax Monterey Bay sp. 4 “yellow collar”) depths. Quantitative PCR and clone libraries of 16S rRNA gene sequences identified differences in the compositions and abundances of bacterial phylotypes associated with the newly discovered host species and permitted comparisons between adult Osedax frankpressi and juveniles that had recently colonized whalebones implanted at 2,891 m. The newly discovered Osedax species hosted Oceanospirillales symbionts that are related to Gammaproteobacteria associated with the previously described O. frankpressi and Osedax rubiplumus (S. K. Goffredi, V. J. Orphan, G. W. Rouse, L. Jahnke, T. Embaye, K. Turk, R. Lee, and R. C. Vrijenhoek, Environ. Microbiol. 7:1369-1378, 2005). In addition, Osedax sp. MB3 hosts a diverse and abundant population of additional bacteria dominated by Epsilonproteobacteria. Ultrastructural analysis of symbiont-bearing root tissues verified the enhanced microbial diversity of Osedax sp. MB3. Root tissues from the newly described host species and O. frankpressi all exhibited collagenolytic enzyme activity, which covaried positively with the abundance of symbiont DNA and negatively with mean adult size of the host species. Members of this unusual genus of bone-eating worms may form variable associations with symbiotic bacteria that allow for the observed differences in colonization and success in whale fall environments throughout the world's oceans.     

The simplicity of males: Dwarf males of four species of Osedax (Siboglinidae; Annelida) investigated by confocal laser scanning microscopy

Dwarf males of the bone‐eating worms Osedax (Siboglinidae, Annelida) have been proposed to develop from larvae that settle on females rather than on bone. The apparent arrest in somatic development and resemblance of the males to trochophore larvae has been posited as an example of paedomorphosis. Here, we present the first investigation of the entire muscle and nervous system in dwarf males of Osedax frankpressi, O. roseus, O. rubiplumus, and O. “spiral” analyzed by multistaining and confocal laser scanning microscopy. Sperm shape and spermiogenesis, the sperm duct and internal and external ciliary patterns were likewise visualized. The males of all four species possess morphological traits typical of newly settled siboglinid larvae: a prostomium, a peristomium with a prototroch, one elongate segment and a second shorter segment. Each segment has a ring of eight long‐handled hooked chaetae. The longitudinal muscles are distributed as evenly spaced strands forming a grid with the thin outer circular muscles. Oblique protractor and retractor muscles are associated with each of the chaetal sacs. The nervous system comprises a cerebral ganglion, a prototroch nerve ring, paired dorsolateral longitudinal nerves, five ventral longitudinal nerves with paired, posterior ganglia and a terminal commissure, as well as a net of fine peripheral transverse plexuses surrounding the first segment. Internal ciliation occurs as paired ventrolateral bands along the first segment. The bands appear to lead the free mature sperm to a ciliated duct and seminal vesicle lying just behind the prototroch region. A duct then runs from the seminal vesicle into the dorsal part of the prostomium. The similarity of Osedax males to the larvae of Osedax and other siboglinid annelids as well as similarities shown here to the neuromuscular organization seen in other annelid larvae supports the hypothesis of paedomorphosis in males of Osedax. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Microscopic anatomy of the integument in the ribbon worm Micrura bella (Stimpson, 1857) (Pilidiophora,Nemertea)

The integument of ribbon worms in the order Heteronemertea is distinct from the integuments in the other taxa of nemerteans due to the presence of a special subepidermal glandular layer, the cutis. Among heteronemerteans, the ultrastructure of the cutis has been studied only in the Lineus ruber species complex. In the current study, ultrastructural (transmission electron microscopy) and histochemical studies of the epidermis and the cutis of Micrura bella from the basal Lineage A of the family Lineidae were performed. The epidermis consisted of ciliated and serous gland cells and is separated from the cutis by a layer of the subepidermal extracellular matrix; the basal lamina was not detected. The cutis comprised musculature, two types of mucous and four types of granular gland cells, and pigment cells with four types of granules. In the cutis of juvenile worms, type II granular gland cells and type II mucous cells were not observed. The integument of the caudal cirrus consisted of ciliated and serous gland cells and two intraepidermal lateral nerve cords; the cutis was absent. The compositions of the integument glands of M. bella and the L. ruber species complex are similar, except for the presence of type IV granular gland cells with narrow rod-shaped and lamellated granules exhibiting an alternating dark and light transverse layers and type II mucous cells found only in M. bella.     

Evolutionary innovation: a bone-eating marine symbiosis

Symbiotic associations between microbes and invertebrates have resulted in some of the most unusual physiological and morphological adaptations that have evolved in the animal world. We document a new symbiosis between marine polychaetes of the genus Osedax and members of the bacterial group Oceanospirillales, known for heterotrophic degradation of complex organic compounds. These organisms were discovered living on the carcass of a grey whale at 2891 m depth in Monterey Canyon, off the coast of California. The mouthless and gutless worms are unique in their morphological specializations used to obtain nutrition from decomposing mammalian bones. Adult worms possess elaborate posterior root-like extensions that invade whale bone and contain bacteriocytes that house intracellular symbionts. Stable isotopes and fatty acid analyses suggest that these unusual endosymbionts are likely responsible for the nutrition of this locally abundant and reproductively prolific deep-sea worm.     

Is the trophosome of <Emphasis Type="Italic">Ridgeia piscesae</Emphasis> monoclonal?

The hydrothermal vent tubeworm Ridgeia piscesae relies on intracellular chemolithoautotrophic symbionts for its nutrition. Yet, little is known about symbiont diversity within and between individual worms. We report several lines of molecular evidence for multiple genotypes of very closely related symbionts within the trophosome of the R. piscesae. We examined the distribution of genotypic variants (insertions, deletions, and substitutions) in whole genome shotgun sequences of symbionts from the trophosome of a unique individual R. piscesae and the pooled sequences of five other tubeworms of the same species. Observed heterogeneity is unlikely to be the result of recent point or structural mutations of a monoclonal symbiont lineage. To assess inter-host diversity we examined single nucleotide polymorphisms (SNPs) in pyrosequences of the highly variable regions V1 to V3 of the symbiont 16S rRNA gene across 53 individual hosts from two vent sites. Most of the identified SNPs were found in more than one individual, and one seemed to be region specific. Two of the identified SNPs were also present in metagenomic data generated from high-throughput sequencing of trophosome material from an individual R. piscesae. Finally, we observed compositional and structural variations of CRISPR spacers within a CRISPR array.     

Ultrastructure of the rotifer integument: peculiarities of Sinantherina socialis (Monogononta: Gnesiotrocha)

The rotifer integument is a well‐described syncytium that contains an apical intracytoplasmic lamina (ICL) that functions for both skeletal support and muscle insertion. To date, there is limited information on the structure of the integument in species of Gnesiotrocha, a diverse subclade of Monogononta that consists of solitary, colonial, sessile, and planktonic species. In this study, we examined the ultrastructure of the integument in the colonial rotifer Sinantherina socialis to determine how it corresponds to that of other monogononts. The integument of S. socialis was broadly similar to that of other rotifers, consisting of a thickened glycocalyx, multilaminate ICL, and syncytial epidermis. However, it was different in several regards. The ICL consisted of three distinct layers from apical to basal: layer 1 consisted of at least two electron‐dense laminae; layer 2 was a thickened matrix of amorphous, electron‐dense material or was fibrous; and layer 3 was an electron‐dense lamina of varying thickness that covered the underlying syncytium. Significantly, layers 1 and 2 formed a ridge‐and‐groove like system of finger‐like projections across the trunk surface that has not been observed in other rotifers. A voluminous syncytial cytoplasm (up to 3 μm thick) was present beneath the ICL and was mostly electron lucent and with few organelles. Bundles of potential microtubules were scattered throughout the syncytium. We hypothesize that the voluminous cytoplasm with microtubules serves as skeletal support for the rotifer's sessile lifestyle, while the external ridges may function as a texture‐based deterrent to predators, or serves to trap secretions from the species' defensive glands. Basally, the epidermis was highly folded and bordered by a thin basal lamina that separated the plasmalemma from the blastocoel. Membrane‐bound vesicles were present throughout the integument's cytoplasm and are hypothesized to function in the secretion of extracellular matrix and in the maintenance of the ICL.     

Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae)

Abstract. The trophosome of adults of Riftia pachyptila (Vestimentifera) was reinvestigated using 3-dimensional ultrastructural reconstruction and quantitative morphological analysis. The symbionts make up 24.1%, the symbiont-containing cells (bacteriocytes) are 70.5% of the trophosome's volume. The trophosome is composed of lobules that have a central axial blood vessel surrounded by a myoepithelium containing bacteriocytes, in turn surrounded by an apolar tissue of bacteriocytes. Part of the splanchnic peritoneum lining the trunk coelom encases the bacteriocytes and forms a ramifying network of peripheral blood vessels. Based on the morphology and ultrastructure of the adult, we hypothesize a mesodermal rather than endodermal origin of trophosome and its constitute bacteriocytes. Some of the central bacteriocytes are part of the myoepithelium surrounding the axial blood vessel and act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone. Lysis of cocci occurs in the degenerative zone.     

Bone-eating Osedax worms lived on Mesozoic marine reptile deadfalls

We report fossil traces of Osedax, a genus of siboglinid annelids that consume the skeletons of sunken vertebrates on the ocean floor, from early-Late Cretaceous (approx. 100 Myr) plesiosaur and sea turtle bones. Although plesiosaurs went extinct at the end-Cretaceous mass extinction (66 Myr), chelonioids survived the event and diversified, and thus provided sustenance for Osedax in the 20 Myr gap preceding the radiation of cetaceans, their main modern food source. This finding shows that marine reptile carcasses, before whales, played a key role in the evolution and dispersal of Osedax and confirms that its generalist ability of colonizing different vertebrate substrates, like fishes and marine birds, besides whale bones, is an ancestral trait. A Cretaceous age for unequivocal Osedax trace fossils also dates back to the Mesozoic the origin of the entire siboglinid family, which includes chemosynthetic tubeworms living at hydrothermal vents and seeps, contrary to phylogenetic estimations of a Late Mesozoic–Cenozoic origin (approx. 50–100 Myr).     

The Osedax trophosome: organization and ultrastructure

The polychaete family Siboglinidae, which is currently construed as comprising the Frenulata, Monilifera (composed of Sclerolinum), Vestimentifera, and Osedax, has become known for its specialized symbiont-housing organ called the trophosome. This organ replaced the digestive system of the worms and is located in the elongated trunk region in Frenulata, Sclerolinum, and Vestimentifera. Currently two types of trophosomes have been described: in the taxa Frenulata and Sclerolinum the bacteriocytes originate from endoderm, and in Vestimentifera they originate from mesoderm. In Osedax, a trophosome was described as lacking (Rouse et al., 2004), but bacteriocytes are located in Osedax's characteristic root tissue. Here, we argue for a consistent name for the symbiont-housing tissue, namely trophosome, as in other siboglinids. In this study we provide morphological evidence that in Osedax the bacteriocytes are derived from somatic mesoderm. We show that the trophosome in Osedax is an apolar tissue composed of bacteriocytes and nonsymbiotic cells. As in vestimentiferans, a specific cell cycle was identified; however, in this case it is directed from the posterior to the anterior end of the worms instead of from the center toward the periphery. Comparison of all siboglinid trophosomes and re-evaluation of their body regions allows us to discuss whether the trophosomes are homologous and to hypothesize about the organization of the last common ancestor of Siboglinidae.     

Bone-boring worms: characterizing the morphology, rate, and method of bioerosion by Osedax mucofloris (Annelida, Siboglinidae)

Osedax worms possess unique "root" tissues that they use to bore into bones on the seafloor, but details of the boring pattern and processes are poorly understood. Here we use X-ray micro-computed tomography to investigate the borings of Osedax mucofloris in bones of the minke whale (Balaenoptera acutorostrata), quantitatively detailing their morphological characteristics for the first time. Comparative thin-sections of the borings reveal how the bone is eroded at the sub-millimeter level. On the basis of these results we hypothesize a model of boring that is dependent on the density and microstructure of the bone. We also present evidence of acidic mucopolysaccharides in the mucus of the root tissue, and hypothesize that this plays an important role in the boring mechanism. We discuss the utility of these new data in evaluating Osedax trace fossils and their relevance for O. mucofloris ecology. Measured rates of bone erosion (6% per year) and evidence of enhanced sulfide release from the borings indicate that Osedax worms are important habitat modifiers in whale-fall communities.     

Myzostomida: A review of the phylogeny and ultrastructure

Myzostomids are minute, soft-bodied, marine worms associated with echinoderms since the Carboniferous. Due to their long history as host-specific symbionts, they have acquired a highly derived body plan that obscures their phylogenetic affinities to other metazoans. Because certain organs are serially arranged a closer relationship between polychaetes and myzostomids has repeatedly been discussion. We presented here a review on the ultrastructure of myzostomids with the most recent analyses that concern their phylogenetic position. The ultrastructure of the integument, digestive system, excretory system and nervous system are summarized. Unpublished information on the gametogenesis and reproductive systems of myzostomids are also exposed with a view on their reproductive process.