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Gardebrecht, A, Markert S, Sievert SM, Felbeck H, Thurmer A, Albrecht D, Wollherr A, Kabisch J, Le Bris N, Lehmann R, Daniel R, Liesegang H, Hecker M, Schweder T.  2012.  Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics. ISME Journal. 6:766-776.   10.1038/ismej.2011.137   AbstractWebsite

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments. The ISME Journal (2012) 6, 766-776; doi: 10.1038/ismej.2011.137; published online 20 October 2011

Markert, S, Gardebrecht A, Felbeck H, Sievert SM, Klose J, Becher D, Albrecht D, Thurmer A, Daniel R, Kleiner M, Hecker M, Schweder T.  2011.  Status quo in physiological proteomics of the uncultured Riftia pachyptila endosymbiont. Proteomics. 11:3106-3117.   10.1002/pmic.201100059   AbstractWebsite

Riftia pachyptila, the giant deep-sea tube worm, inhabits hydrothermal vents in the Eastern Pacific ocean. The worms are nourished by a dense population of chemoautotrophic bacterial endosymbionts. Using the energy derived from sulfide oxidation, the symbionts fix CO(2) and produce organic carbon, which provides the nutrition of the host. Although the endosymbionts have never been cultured, cultivation-independent techniques based on density gradient centrifugation and the sequencing of their (meta-) genome enabled a detailed physiological examination on the proteomic level. In this study, the Riftia symbionts' soluble proteome map was extended to a total of 493 identified proteins, which allowed for an explicit description of vital metabolic processes such as the energy-generating sulfide oxidation pathway or the Calvin cycle, which seems to involve a reversible pyrophosphate-dependent phosphofructokinase. Furthermore, the proteomic view supports the hypothesis that the symbiont uses nitrate as an alternative electron acceptor. Finally, the membrane-associated proteome of the Riftia symbiont was selectively enriched and analyzed. As a result, 275 additional proteins were identified, most of which have putative functions in electron transfer, transport processes, secretion, signal transduction and other cell surface-related functions. Integrating this information into complex pathway models a comprehensive survey of the symbiotic physiology was established.

Muller, F, Brissac T, Le Bris N, Felbeck H, Gros O.  2010.  First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat. Environmental Microbiology. 12:2371-2383.   10.1111/j.1462-2920.2010.02309.x   AbstractWebsite

Archaea may be involved in global energy cycles, and are known for their ability to interact with eukaryotic species (sponges, corals and ascidians) or as archaeal-bacterial consortia. The recently proposed phylum Thaumarchaeota may represent the deepest branching lineage in the archaeal phylogeny emerging before the divergence between Euryarchaeota and Crenarchaeota. Here we report the first characterization of two marine thaumarchaeal species from shallow waters that consist of multiple giant cells. One species is coated with sulfur-oxidizing gamma-Proteobacteria. These new uncultured thaumarchaeal species are able to live in the sulfide-rich environments of a tropical mangrove swamp, either on living tissues such as roots or on various kinds of materials such as stones, sunken woods, etc. These archaea and archaea/bacteria associations have been studied using light microscopy, transmission electron microscopy and scanning electron microscopy. Species identification of archaeons and the putative bacterial symbiont have been assessed by 16S small subunit ribosomal RNA analysis. The sulfur-oxidizing ability of the bacteria has been assessed by genetic investigation on alpha-subunit of the adenosine-5'-phosphosulfate reductase/oxidase's (AprA). Species identifications have been confirmed by fluorescence in situ hybridization using specific probes designed in this study. In this article, we describe two new giant archaeal species that form the biggest archaeal filaments ever observed. One of these species is covered by a specific biofilm of sulfur-oxidizing gamma-Proteobacteria. This study highlights an unexpected morphological and genetic diversity of the phylum Thaumarchaeota.

Garcia-Esquivel, Z, Felbeck H.  2009.  Comparative performance of juvenile red abalone, Haliotis rufescens, reared in laboratory with fresh kelp and balanced diets. Aquaculture Nutrition. 15:209-217.   10.1111/j.1365-2095.2008.00585.x   AbstractWebsite

Juvenile Haliotis rufescens were reared in the laboratory in order to investigate the extent to which fresh kelp and formulated feeds with 250 g kg(-1) (25P) and 380 g kg(-1) protein content (38P) affected their growth rate, gut residence time (GRT), food consumption (C), food conversion ratio (FCR) and digestibility. Abalone from 38P attained the highest growth rate (70.5 +/- 4.2 mu m day(-1); 98.3 +/- 6.95 mu g day(-1)), followed by 25P (47.9 +/- 2.79 mu m day(-1); 67.4 +/- 2.82 mu g day(-1)) and kelp (23.6 +/- 3.36 mu m day(-1); 28.2 +/- 4.11 mu g day(-1)). No significant differences were observed in consumption rate among treatments (0.61-0.68% body weight per day), yet kelp-fed abalone exhibited higher FCR (2.44), protein efficiency ratio (4.42), and apparent digestibility of dry matter (69.5%), protein (69.8%) and gross energy (79.2%) than 38P organisms (59.8, 62.4 and 62.2%, respectively). They also showed longer GRT (23.1 +/- 0.93 h). This study demonstrated that formulated diets with 250 g kg(-1) and 380 g kg(-1) protein inclusion can sustain higher growth rates of juvenile H. rufescens than fresh algae. These differences seem to be due to the amount of dietary protein. Kelp meal appears to improve the consumption and digestibility of balanced diets, and its inclusion in formulated diets is recommended.

Arndt-Sullivan, C, Lechaire JP, Felbeck H.  2008.  Extreme tolerance to anoxia in the Lucinoma aequizonata symbiosis. Journal of Shellfish Research. 27:119-127.   10.2983/0730-8000(2008)27[119:ettait];2   AbstractWebsite

Our study describes the extraordinary capability of the endosymbiont-bearing bivalve Lucinoma aequizonata to tolerate environmental anoxia. The clam survives without oxygen for 262 days (50% mortality). The total quantity of glycogen in a specimen does not decrease significantly after long-term anoxia (10.5 mo). Common glycogen-derived anaerobic products (opines, lactate, succinate, acetate, and propionate) are only produced in minor quantities. This indicates either severe metabolic depression or the utilization of alternative energy sources. We have found indications that the endosymbiotic bacteria might function as an important carbon source for the bivalve. Transmission electron microscopy studies showed that the symbionts are largely degraded after L. aequizonata was incubated anoxically for 10.5 mo. Polyphosphates detected in symbiont granules by energy dispersive X-ray spectrometry (EDX) might represent an alternative energy source for the clam's metabolism under this stress situation.

Robidart, JC, Bench SR, Feldman RA, Novoradovsky A, Podell SB, Gaasterland T, Allen EE, Felbeck H.  2008.  Metabolic versatility of the Riftia pachyptila endosymbiont revealed through metagenomics. Environmental Microbiology. 10:727-737.   10.1111/j.1462-2920.2007.01496.x   AbstractWebsite

The facultative symbiont of Riftia pachyptila, named here Candidatus Endoriftia persephone, has evaded culture to date, but much has been learned regarding this symbiosis over the past three decades since its discovery. The symbiont population metagenome was sequenced in order to gain insight into its physiology. The population genome indicates that the symbionts use a partial Calvin–Benson Cycle for carbon fixation and the reverse TCA cycle (an alternative pathway for carbon fixation) that contains an unusual ATP citrate lyase. The presence of all genes necessary for heterotrophic metabolism, a phosphotransferase system, and dicarboxylate and ABC transporters indicate that the symbiont can live mixotrophically. The metagenome has a large suite of signal transduction, defence (both biological and environmental) and chemotaxis mechanisms. The physiology of Candidatus Endoriftia persephone is explored with respect to functionality while associated with a eukaryotic host, versus free-living in the hydrothermal environment.

Markert, S, Arndt C, Felbeck H, Becher D, Sievert SM, Hugler M, Albrecht D, Robidart J, Bench S, Feldman RA, Hecker M, Schweder T.  2007.  Physiological proteomics of the uncultured endosymbiont of Riftia pachyptila. Science. 315:247-250.   10.1126/science.1132913   AbstractWebsite

The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data, we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute similar to 12% of the total cytosolic proteome, which highlights the essential role of these enzymes for the symbiont's energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid cycle in addition to the previously identified Calvin cycle for CO2 fixation.

Garcia-Esquivel, Z, Felbeck H.  2006.  Activity of digestive enzymes along the gut of juvenile red abalone, Haliotis rufescens, fed natural and balanced diets. Aquaculture. 261:615-625.   10.1016/j.aquaculture.2006.08.022   AbstractWebsite

Two carbohydrases (cellulase, lysozyme), three proteases (trypsin, aminopeptidase and non-specific protease), a non-specific lipase, and semiquantitative tests of 19 digestive enzymes were assayed in different gut sections of juvenile red abalone, Haliotis rufescens, in order to identify the regions where digestion takes place and investigate the extent to which diet composition can modify the digestive capacity of abalone. The abalone were fed either fresh kelp (K) or balanced diets containing 25 or 38% crude protein for 6 months. Enzyme assays were carried out on different sections of the abalone's gut at the end of this period. On a weight-specific basis, the digestive gland was the site containing most of the enzymes. On a protein-specific basis, two main digestion regions were identified: the digestive gland-stomach region that is characterized by high activities of cellulase and lysozyme, chymotrypsin and protease, and the mouth-intestine region with a typically high activity of lipase and amino peptidase. Significant dietary effects were observed on the activity of enzymes, especially in the digestive gland. Abalone fed with 25 and 38% crude protein diets exhibited higher cellulase (39.8 +/- 4.6 and 14.2 +/- 0.8 mU mg(-1) protein, respectively) and lysozyme activities (88.0 +/- 20.4 and 56.6 +/- 15.7 U, respectively) than those fed with fresh kelp (5.5 +/- 0.7 mU mg-1 protein and 17.1 +/- 1.8 U). In contrast, higher protease activity was found in kelp-fed organisms (234.1 +/- 20.4 mu g product/mg protein) than those fed the 25 and 38% crude protein diets (109.5 +/- 20.7 and 119.5 +/- 20.5 mu g product/mg protein, respectively). Semiquantitative API ZYM assays resulted in no clear food-specific effects on the activity of carbohydrases, proteases, ester hydrolases or phosphohydrolases, yet organ-specific differences were conspicuous in various cases, and generally agreed with quantitative results. It is suggested that the increased carbohydrase activity exhibited by organisms fed the balanced diets resulted from a combination of an increased number of resident bacteria in the abalone's gut and facilitated contact between dietary substrates and digestive cells. The present results indicate that H. rufescens can adjust their enzyme levels in order to maximize the acquisition of dietary protein and carbohydrates. This characteristic can be advantageously used to search for suitable diets in abalone aquaculture. Published by Elsevier B.V.

Dufour, SC, Felbeck H.  2006.  Symbiont abundance in thyasirids (Bivalvia) is related to particulate food and sulphide availability. Marine Ecology-Progress Series. 320:185-194.   10.3354/meps320185   AbstractWebsite

Many bivalve species with chemoautotrophic symbionts have mixotrophic diets and some of their nutritional requirements are met by particulate feeding. The symbionts require reduced compounds (such as sulphide) for their autotrophic production. As the concentration of both particulate food and sulphide can vary in their habitat, it has been suggested that symbiont numbers may vary in response. To address whether symbiont abundance can be influenced by the external medium, we compared symbiont. abundance (1) in specimens kept with or without particulate food, and (2) in specimens kept in sediments with low or high dissolved sulphide content. The relative surface area occupied by symbionts on semi-thin gill sections was determined for Thyasira flexuosa (and, in the sulfide experiment, for Parathyasira equalis and T sarsi) kept for up to 3 wk in experimental microcosms, where the quantity of particulate food or dissolved sulphide was manipulated. Symbiont abundance declined with time; the decline was greater in particle-starved thyasirids and in specimens kept in low sulphide. In the latter conditions, T flexuosa may have also had a greater symbiont digestion rate. The observed patterns may be due to a combination of increased symbiont uptake by the bivalve host, and reduced symbiont vigour under the imposed conditions. The flexible feeding mode of chemosymbiotic thyasirids may help them to survive in fluctuating environments.

Felbeck, H, Bartlett DH.  2006.  Third International Symposium on Hydrothermal Vent and Seep Biology : La Jolla, USA, September 12-16, 2005. :147., Roscoff: Station biologique de Roscoff Abstract
Hahlbeck, E, Pospesel MA, Zal F, Childress JJ, Felbeck H.  2005.  Proposed nitrate binding by hemoglobin in Riftia pachyptila blood. Deep-Sea Research Part I-Oceanographic Research Papers. 52:1885-1895.   10.1016/j.dsr.2004.12.011   AbstractWebsite

Riftia pachyptila lives in the unstable environment at hydrothermal vent sites along oceanic spreading zones in the Eastern Pacific. The tubeworm has a symbiosis with intracellular carbon-fixing and sulfide-oxidizing bacteria. Nitrate is the main source of nitrogen available from their habitat. This compound serves as a substrate either for nitrate respiration or for biosynthesis after transformation into ammonia. Very high nitrate (up to 3.2 mM) and nitrite (up to 0.8 mM) concentrations in vascular blood of R. pachyptila indicate a novel uptake mechanism. The dialysis experiments reported here demonstrate the binding and transport of nitrate to the symbionts by high molecular weight components in the blood, most likely hemoglobin. The extent to which nitrate is bound differed markedly between blood from different animals. In addition, a strong inverse correlation was found between the concentrations of sulfide and nitrate in vascular blood, as well as between the sulfur content of trophosome and the nitrate content of vascular blood. Specimens with low sulfur stores showed much lower nitrate levels than those with pale green trophosome due to high levels of elemental sulfur. (c) 2005 Elsevier Ltd. All rights reserved.

Duplessis, MR, Dufour SC, Blankenship LE, Felbeck H, Yayanos AA.  2004.  Anatomical and experimental evidence for particulate feeding in Lucinoma aequizonata and Parvilucina tenuisculpta (Bivalvia : Lucinidae) from the Santa Barbara Basin. Marine Biology. 145:551-561.   10.1007/s00227-004-1350-6   AbstractWebsite

Previous nutritional models for adults of the lucinid bivalve Lucinoma aequizonata contend that symbiotic chemoautotrophic bacteria provide most of the organic carbon for the host. The existence of this symbiosis, coupled with the host's distinctive anatomical features, shaped the impression that particulate feeding was not a significant part of L. aequizonata nutrition. Here, we use several techniques to show that particulate feeding is a consistent and important part of the L. aequizonata nutritional strategy. Histological and scanning electron microscopy observations reveal that the gills of L. aequizonata, like those of the lucinid Parvilucina tenuisculpta, have functional mucociliary epithelia, able to transport captured particles to the mouth. Observations of gut content and radiolabeled feeding experiments indicate that L. aequizonata does ingest and assimilate carbon from particulate organic matter. Furthermore, molecular identification of a broad spectrum of organisms in the guts of native adult specimens demonstrates that L. aequizonata is non-selective when ingesting organic material, and has a mixotrophic diet.

Felbeck, H, Arndt C, Hentschel U, Childress JJ.  2004.  Experimental application of vascular and coelomic catheterization to identify vascular transport mechanisms for inorganic carbon in the vent tubeworm, Riftia pachyptila. Deep-Sea Research Part I-Oceanographic Research Papers. 51:401-411.   10.1016/j.dsr.2003.10.012   AbstractWebsite

Maintaining deep sea animals in in situ conditions has always been technically difficult because of the high-pressure requirements. Even more difficult are any attempts in manipulating or sampling these organisms while keeping them alive in high-pressure aquaria. We present a technique to withdraw blood samples by vascular catheterization which allows withdrawal of samples of during maintenance of specimens under high-pressure conditions. We have developed this technique to answer a long debated question, how carbon dioxide is transported from the ambient sea water to the bacterial symbionts inside the trophosome of the hydrothermal vent tubeworm. Riftia pachyptila. Our results indicate that the carbon supply to the symbionts is mainly through inorganic CO2 while its incorporation into malate and succinate may serve storage functions at periods Of low CO2 availability in the environment. (C) 2003 Elsevier Ltd. All rights reserved.

Duplessis, MR, Ziebis W, Gros O, Caro A, Robidart J, Felbeck H.  2004.  Respiration strategies utilized by the gill endosymbiont from the host lucinid Codakia orbicularis (Bivalvia : Lucinidae). Applied and Environmental Microbiology. 70:4144-4150.   10.1128/aem.70.7.4144-4150.2004   AbstractWebsite

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.

Childress, JJ, Fisher CR, Felbeck H, Girguis P.  2004.  On the edge of a deep biosphere: real animals in extreme environments. The subseafloor biosphere at mid-ocean ridges. ( Wilcock WSD, Delong EF, Kelley DS, Baross JA, Cary SC, Eds.).:41-49., Washington, DC: American Geophysical Union Abstract
Gros, O, Liberge M, Heddi A, Khatchadourian C, Felbeck H.  2003.  Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment). Applied and Environmental Microbiology. 69:6264-6267.   10.1128/aem.69.10.6264-6267.2003   AbstractWebsite

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.

Dufour, SC, Felbeck H.  2003.  Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves. Nature. 426:65-67.   10.1038/nature02095   AbstractWebsite

In a symbiotic association between an invertebrate host and chemoautotrophic bacteria, each partner has different metabolic requirements, and the host typically supplies the bacteria with necessary reduced chemicals ( sulphide or methane). Some combination of anatomical, physiological and behavioural adaptations in the host often facilitates uptake and transport of reduced chemicals to the symbionts(1-4). We have studied five species of bivalve molluscs of the family Thyasiridae ( that is, thyasirids) three of which harbour chemoautotrophic bacteria. Here we show that the symbiotic bivalves extend their feet to form elongated and ramifying burrows in the sediment, most probably to gain access to reduced sulphur. Closely related bivalves ( including some thyasirid species) without bacterial symbionts show no comparable foot extension behaviour. The length and number of burrows formed by chemosymbiotic thyasirids are related to the concentration of hydrogen sulphide in the sediment. The burrows are formed by the foot of each bivalve, which can extend up to 30 times the length of the shell, and may be the most extreme case of animal structure elongation documented to date.

Gros, O, Liberge M, Felbeck H.  2003.  Interspecific infection of aposymbiotic juveniles of Codakia orbicularis by various tropical lucinid gill-endosymbionts. Marine Biology. 142:57-66.   10.1007/s00227-002-0921-7   AbstractWebsite

Previous molecular phylogenetic analyses have shown that five tropical lucinid species living in or near Thalassia testudinum seagrass beds are colonized by the same bacterial symbiont species. In addition, a new Iucinid species belonging to the genus Anodontia, which inhabits reducing sediment found near seagrass beds and in mangrove swamps, has been included in the present study. Endosymbiosis in Anodontia alba was examined according to symbiont phylogenetic and gill ultrastructural analysis. Phylogenetic analysis showed that partial 16S rDNA sequences of A. alba- and Codakia orbicularis-symbionts were 100% identical at all nucleotide positions determined, suggesting that A. alba also harbors the same symbiont species as C. orbicularis (and, consequently, as C. orbiculata, C. pectinella, Linga pensylvanica and Divaricella quadrisulcata). Based on light and electron microscopy, the cellular organization of the gill filament appeared similar to those already described in other lucinids. The most distinctive feature is the lack of "granule cells" in the lateral zone of A. alba gill filaments. In order to confirm the single-species hypothesis, purified fractions of gill bacterial symbionts obtained from the gills of each of the six tropical lucinids cited above were used to infect aposymbiotic juveniles of C. orbicularis. In each case, aposymbiotic juvenile batches were successfully infected by the gill-endosymbiont fractions, whereas, during the experiments, juveniles from the negative control were still uninfected. These experimental data confirm the phylogenetic data and also demonstrate that chemoautotrophic bacterial endosymbionts from their host cells can colonize aposymbiotic juveniles. The conclusion also follows that intracellular gill-endosymbionts still have the capacity to recognize and colonize new host generations. Lucinids provide a unique model for the study of sulfide-oxidizing symbiosis, even if symbionts remain unculturable.

Bright, M, Arndt C, Keckeis H, Felbeck H.  2003.  A temperature-tolerant interstitial worm with associated epibiotic bacteria from the shallow water fumaroles of Deception Island, Antarctica. Deep-Sea Research Part Ii-Topical Studies in Oceanography. 50:1859-1871.   10.1016/s0967-0645(03)00095-x   AbstractWebsite

A prominent not previously identified species of Monocelidae (Platyhelminthes, Proseriata) was found in the vicinity of fumarole activity at Fumarole Bay. The distribution of this animal and the metazoan meiobenthos in the vicinity of this area suggests that this species constitutes the most abundant species and the bulk of the biomass at these shallow water fumaroles. In contrast to the other metazoan meiofauna, the distribution of this species is positively correlated with the water temperature and gas emissions, indicating a preference for the areas around fumaroles. The range of temperature tolerated by this animal was determined in in vivo experiments to be at least 30-40degreesC. The outer surface the animals is colonized by apparently symbiotic bacteria, which are usually rod-like and approximately 0.68 mum wide and 2.07 mum long. The results of this study revealed a remarkable difference between shallow-water and deep-sea hydrothermal vent meiobenthic communities. Generalists capable of tolerating extreme abiotic conditions appear to dominate shallow-water vents, whereas endemism seems to be the rule in the deep-sea vents. (C) 2003 Elsevier Science Ltd. All rights reserved.

Garcia-Esquivel, Z, Bricelj VM, Felbeck H.  2002.  Metabolic depression and whole-body response to enforced starvation by Crassostrea gigas postlarvae. Comparative Biochemistry and Physiology a-Molecular and Integrative Physiology. 133:63-77.   10.1016/s1095-6433(02)00112-5   AbstractWebsite

Physiological and biochemical measurements were performed on six oyster (Crassostrea gigas) cohorts, in order to: (a) investigate the whole-body response (growth, energy content, metabolic and excretion rates) of 2-week-old postlarvae (spat) to enforced (0-8 days) starvation, and (b) test the potential use of three aerobic enzyme systems as indices of physiological condition. Starvation resulted in exponential reduction of postlarval metabolic and excretion rates, as well as a linear decrease in enzyme activity. These response mechanisms effectively limited the loss of endogenous reserves after 2 days of starvation and maintained the oyster's functional integrity over prolonged (8 days) starvation. Proteins appeared to be selectively conserved during short-term (2 days) starvation, as suggested by a decrease in total protein content, while maintaining constant weight-specific enzyme activity. Postlarvae starved for 2 days exhibited relatively higher lipid losses, lower mortality and lower metabolism than metamorphosing stages, thus suggesting a greater buffering capacity to starvation in the former. The activity of the electron transport system may be a useful indicator of long-term stress or developmental condition of oyster postlarvae, while citrate synthase and cytochrome oxidase could be used as indicators of growth rate. None of these enzyme systems is recommended as an index of aerobic metabolism during short-term starvation. (C) 2002 Elsevier Science Inc. All rights reserved.

Arndt, C, Gaill F, Felbeck H.  2001.  Anaerobic sulfur metabolism in thiotrophic symbioses. Journal of Experimental Biology. 204:741-750. AbstractWebsite

Hydrogen sulfide is generally accepted to be the energy source for the establishment of sulfur-oxidizing symbiotic communities. Here, we show that sulfur-storing symbioses not only consume but also produce large amounts of hydrogen sulfide. The prerequisite for this process appears to be the absence of oxygen, Anaerobic sulfide production is widespread among different thiotrophic symbioses from vent and non-vent sites (Riftia pachyptila, Calyptogena magnifica, Bathymodiolus thermophilus, Lucinoma aequizonata and Calyptogena elongata), The extent of H2S generation correlates positively with the amount of elemental sulfur stored in the symbiont-bearing tissues of the hosts. Sulfide production starts a few hours after anoxia sets in, with H2S initially accumulating in the circulatory system before it is excreted into the surrounding environment. We propose that not sulfate but the elemental sulfur deposited in the symbionts serves as a terminal electron acceptor during anoxia and is reduced to sulfide. In anoxia-tolerant symbioses such as L. aequizonata, anaerobic sulfur respiration may be important for producing maintenance energy to help the species survive several months without oxygen, The increased levels of cysteine in the gills of L. aequizonata may be caused by a lack of reoxidation due to the absence of oxygen.

Hentschel, U, Millikan DS, Arndt C, Cary SC, Felbeck H.  2000.  Phenotypic variations in the gills of the symbiont-containing bivalve Lucinoma aequizonata. Marine Biology. 136:633-643.   10.1007/s002270050723   AbstractWebsite

The marine bivalve Lucinoma aequizonata (Lucinidae) maintains a population of sulfide-oxidizing chemoautotrophic bacteria in its gill tissue. These are housed in large numbers intracellularly in specialized host cells, termed bacteriocytes. In a natural population of L. aequizonata, striking variations of the gill colors occur, ranging from yellow to grey, brown and black. The aim of the present study was to investigate how this phenomenon relates to the physiology and numbers of the symbiont population, Our results show that in aquarium-maintained animals, black gills contained fewer numbers of bacteria as well as lower concentrations of sulfur and total protein. Nitrate respiration was stimulated by sulfide (but not by thiosulfate) 33-fold in homogenates of black gills and threefold in yellow gill homogenates. The total rates of sulfide-stimulated nitrate respiration were the same. Oxygen respiration could be measured in animals with yellow gills but not in animals with black gills. The cumulative data suggest that black-gilled clams maintained in the aquarium represent a starvation state. When collected from their natural habitat black gills contain the same number of bacteria as yellow gills. Also, no significant difference in glycogen concentrations of the host tissues was observed. Therefore, starvation is unlikely the cause of black gill color in a natural population. Alternative sources of nutrition to sulfur-based metabolism are discussed. Denaturing gradient gel electrophoresis (DGGE) performed on the different gill tissues, as well as on isolated symbionts, resulted in a single gill symbiont amplification product, the sequence of which is identical to published data. These findings provide molecular evidence that one dominant phylotype is present in the morphologically different gill tissues. Nevertheless, the presence of other phylotypes cannot formally be excluded. The implications of this study are that the gill of L. aequizonata is a highly dynamic organ which lends itself to more detailed studies regarding the molecular and cellular processes underlying nutrient transfer, regulation of bacterial numbers and host-symbiont communication.

Girguis, PR, Lee RW, Desaulniers N, Childress JJ, Pospesel M, Felbeck H, Zal F.  2000.  Fate of nitrate acquired by the tubeworm Riftia pachyptila. Applied and Environmental Microbiology. 66:2783-2790.   10.1128/aem.66.7.2783-2790.2000   AbstractWebsite

The hydrothermal vent tubeworm Riftia pachyptila lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K, Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723-1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338-340, 1993), Here we report that at an in situ concentration of 40 mu M, nitrate is acquired by R, pachyptila at a rate of 3.54 mu mol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0.017 and 0.21 mu mol g(-1) h(-1), respectively), We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When R. pachyptila tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen "sink" Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen, Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts, Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.

Gros, O, Frenkiel L, Felbeck H.  2000.  Sulfur-oxidizing endosymbiosis in Divaricella quadrisulcata (Bivalvia : Lucinidae): Morphological, ultrastructural, and phylogenetic analysis. Symbiosis. 29:293-317. AbstractWebsite

Based on light and electron microscopy, the cellular organization of the gill filament of Divaricella quadrisulcata is described and compared with other gill filaments of lucinids examined to date. TEM observations revealed a dense population of Gram-negative bacteria located within bacteriocytes in the lateral zone of the gill filament which looks similar to that of Codakia orbicularis with typical "granule cells". The digestive tract of this shallow-water lucinid species is less modified than in other lucinid species. The stomach has a well developed gastric shield, a cristalline style protruding in the stomach from a typical style sac, and active digestive diverticula. The mid gut is coiled through the visceral mass. Therefore, D. quadrisulcata appears to be at least partially dependent on filter-feeding for nutrition. Only one type of bacterial 16S rRNA gene was FCR-amplified from symbiont-containing gill tissue of two specimens, indicating a symbiont population composed of a single species. Phylogenetic analysis showed that sequences of D. quadrisulcata- and C. orbicularis-symbiont were 100% identical at all nucleotide positions determined, suggesting that this other tropical lucinid species harbors the same bacterial symbiont species as the previously analyzed C. orbicularis. Thus, D. quadrisulcata appears as the fifth tropical bivalve colonized by the same symbiont species even though it lives in a different habitat as the four other ones. The symbiont transmission mode was investigated by PCR amplifications from mature ovaries and testes. The C. orbicularis-specific 16S rDNA primer set was unsuccesful in amplifying DNA target for all individuals investigated suggesting that the gill-endosymbionts are environmentally transmitted to the new host generation as for all lucinids examined to date.

Gros, O, Duplessis MR, Felbeck H.  1999.  Embryonic development and endosymbiont transmission mode in the symbiotic clam Lucinoma aequizonata (Bivalvia : Lucinidae). Invertebrate Reproduction & Development. 36:93-103.   10.1080/07924259.1999.9652683   AbstractWebsite

Lucinoma aequizonata is a large lucinid clam which lives in reducing mud around 500 m deep. Adults harbor intracellular chemoautotrophic sulfur-oxidizing bacteria in specialized gill cells called bacteriocytes. The embryonic and early larval development of L. aequizonata is described by using light and scanning electron microscopy. Gametes were obtained by injection of 0.2 ml of 4 mM serotonin solution in seawater into the posterior adductor muscle. The oocytes, 200 mu m in diameter, are surrounded by a glycoprotein capsule which gives to the egg a total diameter of 500 mu m. The development which occurs at 10 degrees C is slow. The first polar body is detected 2.5 h after contact between sperm and oocytes (T-0 + 2.5 h), and the first cleavage begins 10 h later (T-0 +12.5 h). The following successive cleavages produce a nonciliated morula, then a ciliated gastrula which begins to rotate within the egg-capsule at T-0 + 4.5 days. At this stage, the first shell pellicle appears on the dorsal side of the embryo. At T-0 + 8 days, the trochophore larvae develop discrete ciliary bands which constitute the prototroch. Typical straight-hinge veligers, D-shaped larvae, hatch from the egg-capsule 12 days after fertilization. The newly hatched larvae are 240 mu m in length and 200 mu m in height, and the straight hinge 150 mu m long. To elucidate the symbiont transmission mode, two symbiont-specific primers were designed and used in amplifications by PCR. This primer set was unsuccessful in amplifying symbiont DNA targets from mature gonads, spawned oocytes, eggs, and veligers whereas successful amplifications were obtained from symbiont-containing gill tissues. These data rule out the vertical transmission mode and strongly suggest that the symbionts are environmentally transmitted to the new host generation in L. aequizonata as for all tropical lucinids examined to date.