Microanatomy of the trophosome region of <Emphasis Type="Italic">Paracatenula</Emphasis> cf. <Emphasis Type="Italic">polyhymnia</Emphasis> (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.     

Molecular identification of an endosymbiotic bacterium associated with pederin biosynthesis in Paederus sabaeus (Coleoptera: Staphylinidae)

Biosynthesis of the structurally complex hemolymph toxin pederin is an eminent character of Paederus females. For that capability, however, they rely on endosymbiotic bacteria that are lacking in aposymbiotic females. The bacterial inhabitants of the two phenotypes in Paederus sabaeus are evaluated in a PCR-based analysis of 16S rDNA. A certain fragment, which is not found in aposymbiotic females, is highly dominant in the other, biosynthesizing females and thus identifies the endosymbiont. Its DNA sequence reveals a member of the gamma subdivision of the Proteobacteria that is clustered within the genus Pseudomonas (sensu stricto) as it is most closely related to Pseudomonas aeruginosa. These bacteria appear as the hypothesized common producers of pederin and the pederin family of analogs from marine sponges.     

Endosymbiotic Microflora of the Vestimentiferan Tubeworm (<Emphasis Type="Italic">Lamellibrachia</Emphasis> sp.) from a Bathyal Cold Seep

Gutless vestimentiferan tubeworms are known to harbor endosymbiotic bacteria in a specialized tissue, the trophosome, which consists of lobules. The endosymbionts of vestimentiferans inhabiting sulfide-rich hydrothermal vents are monospecific for their host. In contrast, previous studies suggest that vestimentiferas of methane-rich seeps may host multispecific symbionts. Phylogenetic analysis and dot-blot hybridization of 16S ribosomal RNA genes (16S rDNA) detected 4 operational taxonomic units (OTUs) in the trophosome of the vestimentifera Lamellibrachia species from a bathyal methane-seep. The OTUs were closely related to 16S rDNA of the species belonging to -Proteobacteria (Sulfitobacter), -Proteobacteria (Janthinobacterium), and -Proteobacteria (Acinetobacter and Pseudomonas). Localizations of the 4 OTUs within the trophosome were confirmed by in situ hybridization (ISH). ISH signals of the -proteobacterial OTU were observed in the innermost zone of the trophosome lobules. In contrast, ISH signals of the - and -proteobacterial OTUs were observed at the periphery of the lobules; however, whether they occur inside or outside the lobules remains unclear. These results support the possibility that the studied methane-seep tubeworm has a microflora composed of multispecific endosymbionts. Present address (Yukimasa Higashide): Noto Marine Center, 3-47 Uchiura, Suzu, Ishikawa 927-0552, Japan Present address (Hiroyuki Kimura): Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki 305-8566 Japan     

Enzymatic defenses against oxygen toxicity in the hydrothermal vent animals Riftia pachyptila and Calyptogena magnifica

Two deep-sea hydrothermal vent organisms, the tube worm Riftia pachyptila and the clam Calyptogena magnifica, contain superoxide dismutase, dianisidine peroxidase, and glutathione peroxidase. The tube worm trophosome exhibits an iron-containing superoxide dismutase, ordinarily associated with prokaryotes and not previously seen in an animal tissue, in accord with the presence of symbiotic bacteria in this tissue. The enzymes which provide a defense against oxygen toxicity are thus present in these animals.     

Accelerated evolution in bacterial endosymbionts of aphids.

When compared with free living bacteria, it is proposed that there are at least two endosymbiotic processes in aphids based on the A + T content as well as the increased evolutionary rate of the beta-subunit of the F-ATPase complex in different endosymbiotic bacteria. The first well established process corresponds to the integration of Buchnera aphidicola more than 150 million years ago. The other is postulated to correspond to new endosymbiotic processes in which the bacteria involved contain less A + T and show a lower increase of evolutionary rates when compared with B. aphidicola. It is proposed, therefore, that endosymbioses are active processes in aphid evolution.     

Actual distribution of bacteriocytes in the trophosome of a beard worm (Oligobrachia mashikoi, Siboglinidae, Annelida): clarification using whole-mount in situ hybridization

Beard worms (Siboglinidae, Polychaeta) lack a mouth and a digestive tract and harbour chaemosynthetic bacteria in the bacteriocytes of the trophosome. Since beard worms depend on the organic compounds produced by the bacteria for nourishment, the bacteriocytes should be efficient in exchanging various substances with body fluids. For this reason, it is important to determine how the bacteriocytes are organized in the trophosome. As the first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections. Second, visualization of the actual distribution of the bacteriocytes was attempted using whole‐mount in situ hybridization with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin & eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient‐deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole‐mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three‐dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.     

Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. They are successful invaders due to their preference for disturbed habitats, high reproductive rates, and the ability to feed on a wide variety of food items (omnivorous). Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of endosymbiotic bacteria possibly involved in nutrient distributions. Our study utilized 16S rDNA sequencing to describe the composition of the bacterial community in fourth-instar ant larvae in order to identify possible endosymbiotic bacteria present therein. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. Our results did not detect the presence of endosymbiotic bacteria in the guts of RIFA larvae among the colonies. In addition, minimal species overlap was found when bacterial inventories were compared among colonies. Thus, bacteria coadapted with red imported fire ant larvae were not detected. Identified bacteria were not closely affiliated with endosymbiotic bacteria common in other insect species. Bacteria communities appeared to be unique to each geographical location and were determined by the foods consumed by the ants.     

Contribution of the bacterial endosymbiont to the biosynthesis of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.     

Characterization of carbonic anhydrases from Riftia pachyptila,a symbiotic invertebrate from deep-sea hydrothermal vents

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.     

The morphology and anatomy of the vestimentiferan worm Oasisia alvinae Jones, 1985 (Annelida: Siboglinidae). III. Coelomic cavity, trophosome and blood, excretory and reproductive systems

This study deals with the organization of the coelom in the all the parts of the body of the vestimentiferan Oasisia alvinae. The localization of the dissepiment between the vestimental and trunk regions in males and females differs. The histological structure and anatomy of the trophosome and the vascular, excretory, and reproductive systems is described. Three cell types are distinguishable in each trophosome lobe. Up to four bacteriocytes could be found in a row from the central blood vessel to the surface of the organ. The main blood vessels in almost all parts of the body have a well-developed muscle sheath and an endothelium that is expressed in varying degrees. In the trunk region, an intravasal body, which is represented by two modifications, is observed in the dorsal blood vessel. The excretory tree is located under the brain. The reproductive system is symmetrical in males and dissymmetrical in females. The gonoducts of females are extensive; in males, they are short and open in the front part of the reproductive coelom. The genital coeloms of both sexes perform the functions of gamete production and storage.     

Endosymbiotic microorganisms in Adelges (Sacchiphantes) viridis (Insecta,Hemiptera, Adelgoidea: Adelgidae): Molecular characterization,ultrastructure and transovarial transmission

The aim of this paper was to identify endosymbiotic microorganisms living in the body cavity of a Polish population of an aphid, Adelges (Sacchiphantes) viridis, as well as to describe their ultrastructure and mode of transmission between generations. Molecular data (amplification and sequencing of 16S rRNA genes) indicated that endosymbionts of A. (S.) viridis are Betaproteobacteria of the species “Candidatus Vallotia virida”. Endosymbiotic bacteria are rod-shaped and localized in the cytoplasm of specific cells, termed bacteriocytes, of host insects. Endosymbionts sharing the same bacteriocytes differ in the density of their cytoplasm. There are two morphotypes of endosymbiotic bacteria: with electron-dense cytoplasm and electron-translucent cytoplasm. Since only bacteria containing electron-dense cytoplasm were observed in the binary fusion stage, differences in density of the cytoplasm are probably due to changes in the cytoskeleton of bacteria during division. Endosymbionts of A. (S.) viridis are transovarially (i.e. via oocytes) transmitted from the mother to the offspring.     

Ethidium bromide: a fast fluorescent staining procedure for the detection of symbiotic partnership of flagellates and prokaryotes

The hindgut of 'lower' termites harbors a dense population of flagellates and bacteria. The flagellates possess ecto- and endosymbiotic prokaryotes. Most of them are hardly visible in the phase contrast microscope. Staining with the DNA-intercalating agent ethidium bromide visualizes the nuclei of the flagellates as well as the ecto- and endosymbiotic bacteria as red objects. Furthermore, it is possible to distinguish between endosymbiotic methanogens and other bacteria. Following UV excitation, the blue-green autofluorescence of the methanogenic bacteria eclipses the red fluorescence light of the intercalated ethidium bromide. The dye facilitates the observation of symbiotic bacteria and helps identify the number, shape, localization, and dividing status of the nuclei. Thus, it is a powerful tool for the examination of microorganisms in complex habitats, which are rich in strongly autofluorescent material, like wood.     

Endosymbiosis,cell evolution,and speciation

In 1905, the Russian biologist C. Mereschkowsky postulated that plastids (e.g., chloroplasts) are the evolutionary descendants of endosymbiotic cyanobacteria-like organisms. In 1927, I. Wallin explicitly postulated that mitochondria likewise evolved from once free-living bacteria. Here, we summarize the history of these endosymbiotic concepts to their modern-day derivative, the “serial endosymbiosis theory”, which collectively expound on the origin of eukaryotic cell organelles (plastids, mitochondria) and subsequent endosymbiotic events. Additionally, we review recent hypotheses about the origin of the nucleus. Model systems for the study of “endosymbiosis in action” are also described, and the hypothesis that symbiogenesis may contribute to the generation of new species is critically assessed with special reference to the secondary and tertiary endosymbiosis (macroevolution) of unicellular eukaryotic algae.     

DNA base composition and genome size of the prokaryotic symbiont in Riftia pachyptila (Pogonophora)

Abstract It has been proposed that Riftia pachyptila , a pogonophoran tube worm abundant at hydrothermal deep sea vents, metabolizes solely via a chemoautotrophic symbiosiols. The symbionts resemble sulfur oxidizing bacteria and form the specific 'trophosome' tissue. Samples of DNA purified from trophosome and vestimentum (muscle) tissues of R. pachyptila were comparatively characterized by thermal denaturation studies, and by analysis of renaturation kinetics. The results show that the great majority of trophosome DNA is homogeneous and prokaryotic with a base ratio of approx. 58 mol% G + C. Its genome size (genetic complexity) is typical of free-living bacteria. Approx. 5% of trophosome DNA appears to be invertebrate DNA equivalent to that found in the vestimentum tissue which lacks symbionts.     

Arginine metabolism in the deep sea tube worm Riftia pachyptila and its bacterial endosymbiont

The present study describes the distribution and properties of enzymes involved in arginine metabolism in Riftia pachyptila, a tubeworm living around deep sea hydrothermal vents and known to be engaged in a highly specific symbiotic association with a bacterium. The results obtained show that the arginine biosynthetic enzymes, carbamyl phosphate synthetase, ornithine transcarbamylase, and argininosuccinate synthetase are present in all of the tissues of the worm and in the bacteria. Thus, Riftia and its bacterial endosymbiont can assimilate nitrogen and carbon via this arginine biosynthetic pathway. The kinetic properties of ornithine transcarbamylase strongly suggest that neither Riftia nor the bacteria possess the catabolic form of this enzyme belonging to the arginine deiminase pathway, the absence of this pathway being confirmed by the lack of arginine deiminase activity. Arginine decarboxylase and ornithine decarboxylase are involved in the biosynthesis of polyamines such as putrescine and agmatine. These activities are present in the trophosome, the symbiont-harboring tissue, and are higher in the isolated bacteria than in the trophosome, indicating that these enzymes are of bacterial origin. This finding indicates that Riftia is dependent on its bacterial endosymbiont for the biosynthesis of polyamines that are important for its metabolism and physiology. These results emphasize a particular organization of the arginine metabolism and the exchanges of metabolites between the two partners of this symbiosis.     

Endosymbionts of Siboglinum fiordicum and the phylogeny of bacterial endosymbionts in Siboglinidae (Annelida)

Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known-vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of gamma-proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.     

From extracellular to intracellular: the establishment of mitochondria and chloroplasts.

Paracoccus and Rhodopseudomonas are unusual among bacteria in having a majority of the biochemical features of mitochondria; blue-green algae have many of the features of chloroplasts. The theory of serial endosymbiosis proposes that a primitive eukaryote successively took up bacteria and blue-green algae to yield mitochondria and chloroplasts respectively. Possible characteristics of transitional forms are indicated both by the primitive amoeba, Pelomyxa, which lacks mitochondria but contains a permanent population of endosymbiotic bacteria, and by several anomalous eukaryotic algae, e.g. Cyanophora, which contain cyanelles instead of chloroplasts. Blue-green algae appear to be obvious precursors of red algal chloroplasts but the ancestry of other chloroplasts is less certain, though the epizoic symbiont, Prochloron, may resemble the ancestral green algal chloroplast. We speculate that the chloroplasts of the remaining algae may have been a eukaryotic origin. The evolution or organelles from endosymbiotic precursors would involve their integration with the host cell biochemically, structurally and numerically.     

Mutation rate and genome reduction in endosymbiotic and free-living bacteria

Genome reduction has been considered the hallmark of endosymbiotic bacteria, such as endocellular mutualists or obligatory pathogens until it was found exactly the same in several free-living bacteria. In endosymbiotic bacteria genome reduction is mainly attributed to degenerative processes due to small population size. These cannot affect the free-living bacteria with reduced genomes because they are known to have very large population sizes. It has been proposed that selection for simplification drove genome reduction in these free-living bacteria. For at least one of them (Prochlorococcus), genome reduction is associated with accelerated evolution and we suggest an alternative hypothesis based on increase in mutation rate as the primary cause of genome reduction in free-living bacteria.