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Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).

Animals↗

Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome.

To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no cbbM-specific signals. The RuBisCO form I gene (cbbL) was neither amplified by PCR nor detected by ISH. This is the first demonstration of chemoautotrophic symbiosis in the deep-sea gutless tubeworm, based on sequence data and in situ localization of both the 16S-rRNA- and RuBisCO-encoding genes.

Amino Acid Sequence↗

Sulfurimonas paralvinellae sp. nov., a novel mesophilic, hydrogen- and sulfur-oxidizing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent polychaete nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. and emended description of the genus Sulfurimonas.

A novel mesophilic bacterium, strain GO25(T), was isolated from a nest of hydrothermal vent polychaetes, Paralvinella sp., at the Iheya North field in the Mid-Okinawa Trough. Cells were motile short rods with a single polar flagellum. Growth was observed between 4 and 35 degrees C (optimum 30 degrees C; 13-16 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.1). The isolate was a facultatively anaerobic chemolithoautotroph capable of growth using molecular hydrogen, elemental sulfur or thiosulfate as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source and elemental sulfur, thiosulfate or yeast extract as the sole sulfur source. Strain GO25(T) represents the first deep-sea epsilonproteobacterium capable of growth by both hydrogen and sulfur oxidation. Nitrate or molecular oxygen (up to 10 % partial pressure) could serve as the sole electron acceptor to support growth. Metabolic products of nitrate reduction shifted in response to the electron donor provided. The G+C content of genomic DNA was 37.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurimonas and was most closely related to Sulfurimonas autotrophica OK10(T) (96.3 % sequence similarity). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurimonas autotrophica OK10(T). On the basis of the physiological and molecular properties of the novel isolate, the name Sulfurimonas paralvinellae sp. nov. is proposed, with strain GO25(T) (=JCM 13212(T)=DSM 17229(T)) as the type strain. Thiomicrospira denitrificans DSM 1251(T) (=ATCC 33889(T)) is phylogenetically associated with Sulfurimonas autotrophica OK10(T) and Sulfurimonas paralvinellae GO25(T). Based on the phylogenetic relationship between Thiomicrospira denitrificans DSM 1251(T), Sulfurimonas autotrophica OK10(T) and Sulfurimonas paralvinellae GO25(T), we propose the reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. (type strain DSM 1251(T)=ATCC 33889(T)). In addition, an emended description of the genus Sulfurimonas is proposed.

Anaerobiosis↗

Oxygen-binding characteristics of Potamilla chlorocruorin.

Accurate oxygen equilibrium curves of chlorocruorin of a marine polychaete annelid, Potamilla leptochaeta, were determined under a variety of experimental conditions. Like chlorocruorins from other species Potamilla chlorocruorin exhibited a low oxygen affinity, a large Bohr effect, and high cooperativity compared to those of human hemoglobin. However, in contrast to chlorocruorins from other species, the shape of the oxygen equilibrium curve for Potamilla chlorocruorin varied dramatically upon changes of pH or temperature. As observed in hemocyanins and annelid hemoglobins, cations, especially divalent ones such as Mg2+ and Ca2+, caused marked increase in oxygen affinity and cooperativity of Potamilla chlorocruorin. This finding together with the determination of cations in Potamilla blood has made clear the physiological role of chlorocruorin as an oxygen carrier. A graphical analysis based on the Monod-Wyman-Changeux allosteric model indicated that the number of sites for oxygen binding involved in heme-heme interactions is six, defining the functional unit of chlorocruorin molecule.

Allosteric Regulation↗

Evolution of poecilogony and the biogeography of North American populations of the polychaete Streblospio.

Invertebrate interspecific developmental patterns can be highly variable and, taxonomically, are considered only weakly constrained. Intraspecifically, some invertebrate species possess multiple developmental modes-a condition known as poecilogony. Closer examination of most putative poecilogenous species, however, has not supported poecilogony, but rather has uncovered hidden or cryptic species. The polychaete Streblospio benedicti is a well-known, poecilogenous species found along the coast of North America. We collected mitochondrial cytochrome subunit I DNA sequence data from 88 individuals taken from 11 locations along the Atlantic, Gulf, and Pacific Coasts of the United States to provide a phylogenetic framework from which to interpret intraspecific variation in larval life history and brooding structure morphology in this species. Our results are consistent with a recent revision of the species into two separate species: S. benedicti, a pouched brooding form distributed along the Atlantic and Pacific Coasts, and S. gynobranchiata, a branchiate brooding form in the Gulf of Mexico. Contrary to the redescription, S. benedicti is paraphyletic because the pouched brooding population in Vero Beach, Florida shows strong genetic affinity with Gulf of Mexico populations (S. gynobranchiata). However, S. benedicti is a true poecilogenous species, with both lecithotrophic and planktotrophic individuals possessing identical mitochondrial DNA haplotypes. Crossbreeding experiments further support the molecular phylogeny with reproductive isolation demonstrated between, but not within, the major phylogenetic clades consistent with the previously described species. The genetic break near Vero Beach, Florida, corresponds to a well-known phylogeographic boundary, but the estimated time of separation for the Streblospio spp., approximately 10 million years before present, predates all other known phylogeographic subdivisions in this area. This suggests that biogeographic sundering in this region is a recurrent event. Divergence times within the major Streblospio spp. clades are recent and indicate that changes in larval life history as well as brooding structure morphology are highly plastic and can evolve rapidly.

Animals↗

Foundations of gregariousness: a dispersal polymorphism among the planktonic larvae of a marine invertebrate.

Theory predicts that selection should favor genotypes that can vary their tendency to disperse in habitats that are spatially or temporally variable or those that remain near their carrying capacity. Although many marine habitats appear to fit these criteria, confirmed examples of dispersal polymorphism among marine invertebrates are exceedingly rare. Competent larvae of the gregarious tubeworm, Hydroides dianthus, settle specifically in response to living conspecific worms, but a small proportion of each spawn settle nonspecifically on uninhabited substrata concurrently with their gregarious siblings. Here, using a parental half-sib analysis, we show that the proportion of a spawn settling in response to uninhabited biofilm is highly heritable. When estimated as a continuous trait based on a one-way ANOVA, heritability is estimated to be 0.83 +/- 0.31. When founder production was analyzed as a threshold trait, heritability was estimated to be 0.68 +/- 0.10 based on the breeding design experiment and 0.65 +/- 0.09 based on the artificial selection experiments. Realized heritability based on the selection experiments was considerably lower, however (0.17 per generation and 0.02 cumulative). Artificial selection was ineffectual at sequentially increasing the proportion of founder larvae among inbred family lines, but after three generations of selection, the proportion of larvae settling in response to biofilm was significantly higher among inbred lines than among the field-collected parents. The obligate planktonic larval stage common among so many marine invertebrates is thought to preclude the evolution of dispersal polymorphisms in these animals. Theoretical expectations of variable dispersal may instead be realized through individual behavioral differences resulting in differential transport or settlement preference, but this possibility remains largely unexplored among marine invertebrates.

Animals↗

Heterochrony and the evolution of poecilogony: generating larval diversity.

Poecilogony is the production of more than one type of young within a single species of marine invertebrate. We chose a poecilogonous polychaete to investigate potential differences in morphogenesis among offspring that are polymorphic in dispersal potentials (planktonic, benthic) and trophic modes (planktotrophy, adelphophagy). Differences in morphogenesis occur and are strongly influenced by maternal type. Females that provide extra-embryonic nutrition (as nurse eggs; type III females) also produce offspring with an accelerated onset of juvenile traits, relative to planktotrophic offspring of females that do not provide extra-embryonic nutrition (type I females). Thus, progeny of some females appear morphologically preadapted for a benthic lifestyle. Surprisingly, differences in phenotype among offspring do not parallel offspring ecotype, as offspring with early onset of juvenile traits (III) are ecologically bimodal. Some Type III offspring eat the nurse eggs (adelphophagy), have accelerated development, and hatch as benthic juveniles. In contrast, their siblings hatch as small, planktotrophic, dispersive larvae that are morphologically similar to their type III siblings, but ecologically similar to Type I planktotrophic larvae. We propose that poecilogony evolved through sequence heterochrony in morphogenesis with accelerated onset of juvenile traits in type III offspring. In addition, we suggest that heterochrony in life-history events (hatching, metamorphosis) also occurs, thereby generating offspring that are dimorphic in both phenotype and ecotype. Over time, selection acting on different levels of ontogeny (morphogenesis vs. dispersal) may balance this polymorphism and allow poecilogony to persist.

Animal Nutritional Physiological Phenomena↗

Distinct patterns of genetic differentiation among annelids of eastern Pacific hydrothermal vents.

Population genetic and phylogenetic analyses of mitochondrial COI from five deep-sea hydrothermal vent annelids provided insights into their dispersal modes and barriers to gene flow. These polychaetes inhabit vent fields located along the East Pacific Rise (EPR) and Galapagos Rift (GAR), where hundreds to thousands of kilometers can separate island-like populations. Long-distance dispersal occurs via larval stages, but larval life histories differ among these taxa. Mitochondrial gene flow between populations of Riftia pachyptila, a siboglinid worm with neutrally buoyant lecithothrophic larvae, is diminished across the Easter Microplate region, which lies at the boundary of Indo-Pacific and Antarctic deep-sea provinces. Populations of the siboglinid Tevnia jerichonana are similarly subdivided. Oasisia alvinae is not found on the southern EPR, but northern EPR populations of this siboglinid are subdivided across the Rivera Fracture Zone. Mitochondrial gene flow of Alvinella pompejana, an alvinellid with large negatively buoyant lecithotrophic eggs and arrested embryonic development, is unimpeded across the Easter Microplate region. Gene flow in the polynoid Branchipolynoe symmytilida also is unimpeded across the Easter Microplate region. However, A. pompejana populations are subdivided across the equator, whereas B. symmitilida populations are subdivided between the EPR and GAR axes. The present findings are compared with similar evidence from codistributed species of annelids, molluscs and crustaceans to identify potential dispersal filters in these eastern Pacific ridge systems.

Animals↗

Comparative phylogeography of two coastal polychaete tubeworms in the Northeast Atlantic supports shared history and vicariant events.

The historic processes which have led to the present-day patterns of genetic structure in the marine coastal fauna of the Northeast Atlantic are still poorly understood. While tectonic uplifts and changes in sea level may have caused large-scale vicariance, warmer conditions during glacial maxima may have allowed pockets of diversity to persist to a much wider extent than in the Northwestern Atlantic. The large-scale geographic distribution of deeply divergent lineages of the coastal polychaete tubeworms Pectinaria koreni (two clades) and Owenia fusiformis (three clades) were compared using a fragment of the mitochondrial cytochrome oxidase I gene (mtCOI). All lineages were present along the biogeographic transition zone on the north coast of Brittany (France) and we found evidence pointing towards congruence in the timing of cladogenic events between Pectinaria sp. (P. auricoma/P. belgica and P. koreni) and Owenia sp., suggesting a shared history of vicariant events. More conserved 16SrRNA sequences obtained from four species of Pectinariidae together with mtCOI sequences of P. koreni seem consistent with an initial establishment of pectinariids in the north, and a southward colonization of the Northeast Atlantic. Phylogeographic patterns in O. fusiformis were also consistent with a north/south pattern of lineage splitting and congruent levels of divergence were detected between lineages of both species. We observed signatures of both persistence in small northern glacial refugia, and of northwards range expansion from regions situated closer to the Mediterranean. However, whether the recolonization of the Northeast Atlantic by both species actually reflects separate interglacial periods is unclear with regards to the lack of molecular clock calibration in coastal polychaete species.

Animals↗

The benefits of polyandry in the free-spawning polychaete Galeolaria caespitosa.

In many species, females are thought to benefit from polyandry due to the reduced risks of fertilization by genetically incompatible sperm. However, few studies that have reported such benefits have directly attributed variation in female reproductive success to the interacting effects of males and females at fertilization. In this paper, we determine whether male x female interactions influence fertilization in vitro in the free-spawning, sessile polychaete Galeolaria caespitosa. Furthermore, we determined whether polyandry results in direct fertilization benefits for females by experimentally manipulating the number of males contributing towards staged spawning events. To test for male x female interaction effects we performed an initial experiment that crossed seven males with six females (in all 42 combinations), enabling us to assess fertilization rates for each specific male-female pairing and attribute variation in fertilization success to males, females and their interaction. This initial experiment revealed a strong interaction between males and females at fertilization, confirming that certain male-female combinations were more compatible than others. A second experiment tested the hypothesis that polyandry enhances female reproductive success by exposing each female's eggs to either a single male's sperm (monandry) or the sperm from three males simultaneously (polyandry). We performed this second experiment at two ecologically relevant sperm concentrations. This latter experiment revealed a strong fertilization benefit of polyandry, independent of the effects of sperm concentration (which were also significant). We suggest that these direct fertilization gains arising from polyandry will constitute an important source of selection on females to mate multiply in nature.

Analysis of Variance↗

Outcrossing hermaphroditic polychaete worms adjust their sex allocation to social conditions.

Sex allocation theory predicts that simultaneous hermaphrodites shift sex allocation facultatively in response to variation in local group size. This study was performed to evaluate the relative investment in each sex function by the simultaneously hermaphroditic polychaete worm Ophryotrocha diadema and to test whether allocation to each sex depends on the number of reproductive competitors. Four experimental groups were set up (in a 2 x 2 factorial design) with small or large group size and with small or large enclosures to control for potential confounding effects of density. We measured the proportion of female and male investment in focal individuals. Results revealed that individuals regulated their reproductive output so that when reproductive competitors were present, the number of female gametes was strongly reduced and the male function increased. In contrast, under monogamy, individuals in small groups produced lower numbers of sperm but had a higher egg output than worms in large groups. Density did not affect sex allocation in our experiment. Our findings provide qualitative support for Local Mate Competition theory, but also show that the pattern of sex allocation specific to this species is more complex than expected by current theory.

Analysis of Variance↗

Does egg competition occur in marine broadcast-spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.

Animals↗

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis.

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.

Animals↗

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin.

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.

Animals↗

The effects of cytoskeleton inhibitors on cytoplasmic localization in Chaetopterus pergamentaceus.

We have treated fertilized and KC1-activated eggs of Chaetopterus pergamentaceus with microfilament and microtubule inhibitors to test the relationship of these cytoskeletal components to cytoplasmic localization. Low doses of cytochalasin B inhibited cleavage in fertilized egges. Such embryos underwent differentiation without cleavage, a process characterized by relocalization of the yolky endoplasm to the center of the uncleaved egg and by the formation of cilia. Similar treatment of KC1-activated eggs inhibited ciliation, but not endoplasmic relocalization. Reversible inhibition of the first cleavage resulted in equal cleavage and differentiation of a larva lacking an apical organ. Inhibition of the first two cleavages resulted in differentiation without cleavage. At all concentrations high enough to block mitosis, colchicine prevented ciliation and endoplasmic relocalization. Thus microtubule organization, but not microfilament organization, is required for ooplasmic reorganization and differentiation without cleavage.

Animals↗

Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.).

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G-100 gel filtration, benzamidine-cellulose and SBTI-Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM-Sepharose and DEAE-Sepharose ion-exchange chromatography. Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p-chloromercuribenzoate. They do not hydrolyse exopeptidase substrates. From their properties, they can be divided into five classes. 1. A trypsin-like protease, which hydrolyses only trypsin substrates and is inhibited by N-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35 000). 2. A chymotrypsin-like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z-PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31 000. 3. Two minor chymotrypsin-like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27 000). 4. Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z-PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27 000) but isoelectric points ranging from 4.0 to above 9.1. 5. The last one is very similar but has lower esterolytic activities. These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.

Animals↗