Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium.

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1-3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, 'Candidatus Celerinatantimonas neptuna', that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.

Coming together-symbiont acquisition and early development in deep-sea bathymodioline mussels.

How and when symbionts are acquired by their animal hosts has a profound impact on the ecology and evolution of the symbiosis. Understanding symbiont acquisition is particularly challenging in deep-sea organisms because early life stages are so rarely found. Here, we collected early developmental stages of three deep-sea bathymodioline species from different habitats to identify when these acquire their symbionts and how their body plan adapts to a symbiotic lifestyle. These mussels gain their nutrition from chemosynthetic bacteria, allowing them to thrive at deep-sea vents and seeps worldwide. Correlative imaging analyses using synchrotron-radiation based microtomography together with light, fluorescence and electron microscopy revealed that the pediveliger larvae were aposymbiotic. Symbiont colonization began during metamorphosis from a planktonic to a benthic lifestyle, with the symbionts rapidly colonizing first the gills, the symbiotic organ of adults, followed by all other epithelia of their hosts. Once symbiont densities in plantigrades reached those of adults, the host's intestine changed from the looped anatomy typical for bivalves to a straightened form. Within the Mytilidae, this morphological change appears to be specific to Bathymodiolus and Gigantidas, and is probably linked to the decrease in the importance of filter feeding when these mussels switch to gaining their nutrition largely from their symbionts.

Chemosynthetic symbioses.

Symbioses between chemosynthetic bacteria and eukaryotic hosts can be found almost everywhere in the ocean, from shallow-water seagrass beds and coral reef sediments to the deep sea. Yet no one knew these existed until 45 years ago, when teeming communities of animals were found thriving at hydrothermal vents two and a half kilometers below the sea surface. The discovery of these lightless ecosystems revolutionized our understanding of the energy sources that fuel life on Earth. Animals thrive at vents because they live in a nutritional symbiosis with chemosynthetic bacteria that grow on chemical compounds gushing out of the vents, such as sulfide and methane, which animals cannot use on their own. The symbionts gain energy from the oxidation of these reduced substrates to fix CO2 and other simple carbon compounds into biomass, which is then transferred to the host. By associating with chemosynthetic bacteria, animals and protists can thrive in environments in which there is not enough organic carbon to support their nutrition, including oligotrophic habitats like coral reefs and seagrass meadows. Chemosymbioses have evolved repeatedly and independently in multiple lineages of marine invertebrates and bacteria, highlighting the strong selective advantage for both hosts and symbionts in forming these associations. Here, we provide a brief overview of chemosynthesis and how these symbioses function. We highlight some of the current research in this field and outline several promising avenues for future research.

Morphology of obligate ectosymbionts reveals Paralaxus gen. nov.: A new circumtropical genus of marine stilbonematine nematodes.

Stilbonematinae are a subfamily of conspicuous marine nematodes, distinguished by a coat of sulphur-oxidizing bacterial ectosymbionts on their cuticle. As most nematodes, the worm hosts have a relatively simple anatomy and few taxonomically informative characters, and this has resulted in numerous taxonomic reassignments and synonymizations. Recent studies using a combination of morphological and molecular traits have helped to improve the taxonomy of Stilbonematinae but also raised questions on the validity of several genera. Here, we describe a new circumtropically distributed genus Paralaxus (Stilbonematinae) with three species: Paralaxus cocos sp. nov., P. bermudensis sp. nov. and P. columbae sp. nov. We used single worm metagenomes to generate host 18S rRNA and cytochrome c oxidase I (COI) as well as symbiont 16S rRNA gene sequences. Intriguingly, COI alignments and primer matching analyses suggest that the COI is not suitable for PCR-based barcoding approaches in Stilbonematinae as the genera have a highly diverse base composition and no conserved primer sites. The phylogenetic analyses of all three gene sets, however, confirm the morphological assignments and support the erection of the new genus Paralaxus as well as corroborate the status of the other stilbonematine genera. Paralaxus most closely resembles the stilbonematine genus Laxus in overlapping sets of diagnostic features but can be distinguished from Laxus by the morphology of the genus-specific symbiont coat. Our re-analyses of key parameters of the symbiont coat morphology as character for all Stilbonematinae genera show that with amended descriptions, including the coat, highly reliable genus assignments can be obtained.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).

Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2.

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3-6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host's internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11-13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan's nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

Chemosynthetic symbiont with a drastically reduced genome serves as primary energy storage in the marine flatworm Paracatenula.

Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant'Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29-4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca R. santandreae's versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts-serving as the primary energy storage for its animal host.

Characterization of the First "Candidatus Nitrotoga" Isolate Reveals Metabolic Versatility and Separate Evolution of Widespread Nitrite-Oxidizing Bacteria.

Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus "Candidatus Nitrotoga" are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure "Ca Nitrotoga" culture was available. Here we obtained the first "Ca Nitrotoga" isolate from activated sludge. This organism, "Candidatus Nitrotoga fabula," prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous "Ca Nitrotoga" enrichments, which were described as cold-adapted NOB. "Ca Nitrotoga fabula" also showed an unusually high tolerance to nitrite (activity at 30 mM NO2-) and nitrate (up to 25 mM NO3-). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of "Ca Nitrotoga fabula" were reconstructed from the closed genome. "Ca Nitrotoga fabula" possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in "Ca Nitrotoga" and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, "Ca Nitrotoga fabula" uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable "Ca Nitrotoga fabula" to survive nitrite depletion and colonize new niches.IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus "Candidatus Nitrotoga" and performed a detailed physiological and genomic characterization of this organism ("Candidatus Nitrotoga fabula"). Differences between key phenotypic properties of "Ca Nitrotoga fabula" and those of previously enriched "Ca Nitrotoga" members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that "Ca Nitrotoga" are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in "Ca Nitrotoga fabula" expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.

Host-Polarized Cell Growth in Animal Symbionts.

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.

Asynchronous division by non-ring FtsZ in the gammaproteobacterial symbiont of Robbea hypermnestra.

The reproduction mode of uncultivable microorganisms deserves investigation as it can largely diverge from conventional transverse binary fission. Here, we show that the rod-shaped gammaproteobacterium thriving on the surface of the Robbea hypermnestra nematode divides by FtsZ-based, non-synchronous invagination of its poles-that is, the host-attached and fimbriae-rich pole invaginates earlier than the distal one. We conclude that, in a naturally occurring animal symbiont, binary fission is host-oriented and does not require native FtsZ to polymerize into a ring at any septation stage.

A specific and widespread association between deep-sea Bathymodiolus mussels and a novel family of Epsilonproteobacteria.

Bathymodiolus mussels dominate animal communities at many hydrothermal vents and cold seeps. Essential to the mussels' ecological and evolutionary success is their association with symbiotic methane- and sulfur-oxidizing gammaproteobacteria, which provide them with nutrition. In addition to these well-known gammaproteobacterial endosymbionts, we found epsilonproteobacterial sequences in metatranscriptomes, metagenomes and 16S rRNA clone libraries as well as by polymerase chain reaction screening of Bathymodiolus species sampled from vents and seeps around the world. These epsilonproteobacterial sequences were closely related, indicating that the association is highly specific. The Bathymodiolus-associated epsilonproteobacterial 16S rRNA sequences were at most 87.6% identical to the closest cultured relative, and 91.2% identical to the closest sequences in public databases. This clade therefore represents a novel family within the Epsilonproteobacteria. Fluorescence in situ hybridization and transmission electron microscopy showed that the bacteria are filamentous epibionts associated with the gill epithelia in two Bathymodiolus species. In animals that host highly specific symbioses with one or a few types of endosymbionts, other less-abundant members of the microbiota can be easily overlooked. Our work highlights how widespread and specific associations with less-abundant microbes can be. Possibly, these microbes play an important role in the survival and health of their animal hosts.

Improved ultrastructure of marine invertebrates using non-toxic buffers.

Many marine biology studies depend on field work on ships or remote sampling locations where sophisticated sample preservation techniques (e.g., high-pressure freezing) are often limited or unavailable. Our aim was to optimize the ultrastructural preservation of marine invertebrates, especially when working in the field. To achieve chemically-fixed material of the highest quality, we compared the resulting ultrastructure of gill tissue of the mussel Mytilus edulis when fixed with differently buffered EM fixatives for marine specimens (seawater, cacodylate and phosphate buffer) and a new fixative formulation with the non-toxic PHEM buffer (PIPES, HEPES, EGTA and MgCl2). All buffers were adapted for immersion fixation to form an isotonic fixative in combination with 2.5% glutaraldehyde. We showed that PHEM buffer based fixatives resulted in equal or better ultrastructure preservation when directly compared to routine standard fixatives. These results were also reproducible when extending the PHEM buffered fixative to the fixation of additional different marine invertebrate species, which also displayed excellent ultrastructural detail. We highly recommend the usage of PHEM-buffered fixation for the fixation of marine invertebrates.

Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

Size-independent symmetric division in extraordinarily long cells.

Two long-standing paradigms in biology are that cells belonging to the same population exhibit little deviation from their average size and that symmetric cell division is size limited. Here, ultrastructural, morphometric and immunocytochemical analyses reveal that two Gammaproteobacteria attached to the cuticle of the marine nematodes Eubostrichus fertilis and E. dianeae reproduce by constricting a single FtsZ ring at midcell despite being 45 µm and 120 µm long, respectively. In the crescent-shaped bacteria coating E. fertilis, symmetric FtsZ-based fission occurs in cells with lengths spanning one order of magnitude. In the E. dianeae symbiont, formation of a single functional FtsZ ring makes this the longest unicellular organism in which symmetric division has ever been observed. In conclusion, the reproduction modes of two extraordinarily long bacterial cells indicate that size is not the primary trigger of division and that yet unknown mechanisms time the localization of both DNA and the septum.

Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota.

A mesophilic, neutrophilic and aerobic, ammonia-oxidizing archaeon, strain EN76(T), was isolated from garden soil in Vienna (Austria). Cells were irregular cocci with a diameter of 0.6-0.9 µm and possessed archaella and archaeal pili as cell appendages. Electron microscopy also indicated clearly discernible areas of high and low electron density, as well as tubule-like structures. Strain EN76(T) had an S-layer with p3 symmetry, so far only reported for members of the Sulfolobales. Crenarchaeol was the major core lipid. The organism gained energy by oxidizing ammonia to nitrite aerobically, thereby fixing CO2, but growth depended on the addition of small amounts of organic acids. The optimal growth temperature was 42 °C and the optimal pH was 7.5, with ammonium and pyruvate concentrations of 2.6 and 1 mM, respectively. The genome of strain EN76(T) had a DNA G+C content of 52.7 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain EN76(T) is affiliated with the recently proposed phylum Thaumarchaeota, sharing 85% 16S rRNA gene sequence identity with the closest cultivated relative 'Candidatus Nitrosopumilus maritimus' SCM1, a marine ammonia-oxidizing archaeon, and a maximum of 81% 16S rRNA gene sequence identity with members of the phyla Crenarchaeota and Euryarchaeota and any of the other recently proposed phyla (e.g. 'Korarchaeota' and 'Aigarchaeota'). We propose the name Nitrososphaera viennensis gen. nov., sp. nov. to accommodate strain EN76(T). The type strain of Nitrososphaera viennensis is strain EN76(T) ( = DSM 26422(T) = JMC 19564(T)). Additionally, we propose the family Nitrososphaeraceae fam. nov., the order Nitrososphaerales ord. nov. and the class Nitrososphaeria classis nov.

Phylogenetic confirmation of the genus Robbea (Nematoda: Desmodoridae, Stilbonematinae) with the description of three new species.

The Stilbonematinae are a monophyletic group of marine nematodes that are characterized by a coat of thiotrophic bacterial symbionts. Among the ten known genera of the Stilbonematinae, the genus Robbea Gerlach 1956 had a problematic taxonomic history of synonymizations and indications of polyphyletic origin. Here we describe three new species of the genus, R. hypermnestra sp. nov., R. ruetzleri sp. nov. and R. agricola sp. nov., using conventional light microscopy, interference contrast microscopy and SEM. We provide 18S rRNA gene sequences of all three species, together with new sequences for the genera Catanema and Leptonemella. Both our morphological analyses as well as our phylogenetic reconstructions corroborate the genus Robbea. In our phylogenetic analysis the three species of the genus Robbea form a distinct clade in the Stilbonematinae radiation and are clearly separated from the clade of the genus Catanema, which has previously been synonymized with Robbea. Surprisingly, in R. hypermnestra sp. nov. all females are intersexes exhibiting male sexual characters. Our extended dataset of Stilbonematinae 18S rRNA genes for the first time allows the identification of the different genera, e.g. in a barcoding approach. http://zoobank.org/urn:lsid:zoobank.org:pub:D37C3F5A-CF2B-40E6-8B09-3C72EEED60B0.

Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae).

Barnacles produce a proteinaceous adhesive called cement to attach permanently to rocks or to other hard substrata. The stalked barnacle Dosima fascicularis is of special interest as it produces a large amount of foam-like cement that can be used as a float. The morphology of the cement apparatus and of the polymerized cement of this species is almost unknown. The current study aims at filling these gaps in our knowledge using light and electron microscopy as well as x-ray microtomography. The shape of the cement gland cells changes from round to ovoid during barnacle development. The cytoplasm of the gland cells, unlike that of some other barnacles, does not have distinct secretory and storage regions. The cement canals, which transport the cement from the gland cells to the base of the stalk, end at different positions in juvenile and mature animals. With increasing size of the cement float, the exit of the cement canals shift from the centrally positioned attachment disk of the vestigial antennules to more lateral positions on the stalk. The bubbles enclosed in the foam-like float are most likely filled with CO(2) that diffuses from the hemolymph into the cement canal system and from there into the cement.

Growth in width and FtsZ ring longitudinal positioning in a gammaproteobacterial symbiont.

Rod-shaped bacteria usually grow in length and place their FtsZ ring and division site at midcell, perpendicular to their long axis [1,2]. Here, we provide morphometric and immunocytochemical evidence that a nematode-associated gammaproteobacterium [3,4] grows in width, sets a constricting FtsZ ring parallel to its long axis, and divides longitudinally by default. Remarkably, the newly described FtsZ ring appears to be not only 90° shifted with respect to model rods, but also elliptical and discontinuous. This reveals an unexpected versatility of the gammaproteobacterial cytokinetic machinery.

Bacterial symbiosis maintenance in the asexually reproducing and regenerating flatworm Paracatenula galateia.

Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.

Microanatomy of the trophosome region of Paracatenula cf. polyhymnia (Catenulida, Platyhelminthes) and its intracellular symbionts.

Marine catenulid platyhelminths of the genus Paracatenula lack mouth, pharynx and gut. They live in a symbiosis with intracellular bacteria which are restricted to the body region posterior to the brain. The symbiont-housing cells (bacteriocytes) collectively form the trophosome tissue, which functionally replaces the digestive tract. It constitutes the largest part of the body and is the most important synapomorphy of this group. While some other features of the Paracatenula anatomy have already been analyzed, an in-depth analysis of the trophosome region was missing. Here, we identify and characterize the composition of the trophosome and its surrounding tissue by analyzing series of ultra-thin cross-sections of the species Paracatenula cf. polyhymnia. For the first time, a protonephridium is detected in a Paracatenula species, but it is morphologically reduced and most likely not functional. Cells containing needle-like inclusions in the reference species Paracatenula polyhymnia Sterrer and Rieger, 1974 were thought to be sperm, and the inclusions interpreted as the sperm nucleus. Our analysis of similar cells and their inclusions by EDX and Raman microspectroscopy documents an inorganic spicule consisting of a unique magnesium-phosphate compound. Furthermore, we identify the neoblast stem cells located underneath the epidermis. Except for the modifications due to the symbiotic lifestyle and the enigmatic spicule cells, the organization of Paracatenula cf. polyhymnia conforms to that of the Catenulida in all studied aspects. Therefore, this species represents an excellent model system for further studies of host adaptation to an obligate symbiotic lifestyle. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00435-011-0135-y) contains supplementary material, which is available to authorized users.