Yukiko Nagai

Proton pumping accompanies calcification in foraminifera

Ongoing ocean acidification is widely reported to reduce the ability of calcifying marine organisms to produce their shells and skeletons. Whereas increased dissolution due to acidification is a largely inorganic process, strong organismal control over biomineralization influences calcification and hence complicates predicting the response of marine calcifyers. Here we show that calcification is driven by rapid transformation of bicarbonate into carbonate inside the cytoplasm, achieved by active outward proton pumping. Moreover, this proton flux is maintained over a wide range of pCO2 levels. We furthermore show that a V-type H+ ATPase is responsible for the proton flux and thereby calcification. External transformation of bicarbonate into CO2 due to the proton pumping implies that biomineralization does not rely on availability of carbonate ions, but total dissolved CO2 may not reduce calcification, thereby potentially maintaining the current global marine carbonate production.

Heterogeneous composition of key metabolic gene clusters in a vent mussel symbiont population.

Chemosynthetic symbiosis is one of the successful systems for adapting to a wide range of habitats including extreme environments, and the metabolic capabilities of symbionts enable host organisms to expand their habitat ranges. However, our understanding of the adaptive strategies that enable symbiotic organisms to expand their habitats is still fragmentary. Here, we report that a single-ribotype endosymbiont population in an individual of the host vent mussel, Bathymodiolus septemdierum has heterogeneous genomes with regard to the composition of key metabolic gene clusters for hydrogen oxidation and nitrate reduction. The host individual harbours heterogeneous symbiont subpopulations that either possess or lack the gene clusters encoding hydrogenase or nitrate reductase. The proportions of the different symbiont subpopulations in a host appeared to vary with the environment or with the host’s development. Furthermore, the symbiont subpopulations were distributed in patches to form a mosaic pattern in the gill. Genomic heterogeneity in an endosymbiont population may enable differential utilization of diverse substrates and confer metabolic flexibility. Our findings open a new chapter in our understanding of how symbiotic organisms alter their metabolic capabilities and expand their range of habitats.

Surfing the vegetal pole in a small population: extracellular vertical transmission of an 'intracellular' deep-sea clam symbiont

Symbiont transmission is a key event for understanding the processes underlying symbiotic associations and their evolution. However, our understanding of the mechanisms of symbiont transmission remains still fragmentary. The deep-sea clam Calyptogena okutanii harbours obligate sulfur-oxidizing intracellular symbiotic bacteria in the gill epithelial cells. In this study, we determined the localization of their symbiont associating with the spawned eggs, and the population size of the symbiont transmitted via the eggs. We show that the symbionts are located on the outer surface of the egg plasma membrane at the vegetal pole, and that each egg carries approximately 400 symbiont cells, each of which contains close to 10 genomic copies. The very small population size of the symbiont transmitted via the eggs might narrow the bottleneck and increase genetic drift, while polyploidy and its transient extracellular lifestyle might slow the rate of genome reduction. Additionally, the extracellular localization of the symbiont on the egg surface may increase the chance of symbiont exchange. This new type of extracellular transovarial transmission provides insights into complex interactions between the host and symbiont, development of both host and symbiont, as well as the population dynamics underlying genetic drift and genome evolution in microorganisms.

Mesoscopic crystallographic textures on shells of a hyaline radial foraminifer Ammonia beccarii

Most foraminifera, simple unicellular protists, create calcareous shells with precisely controlled architectures. We investigated the crystal fabric of porous shells of a hyaline radial foraminifer, Ammonia beccarii. The complex structures of the calcitic shell include micrometric single-crystalline domains around a straight pore ∼2 μm in diameter which are divided by strained intermediate regions. The c axes of calcite in the domains were almost perpendicular to the shell surface. The mesoscopic textures, such as periodic lateral lines with a ∼200–450 nm interval and vertical cracks with a ∼150 nm interval, were observed throughout the single-crystalline domains in the shell wall. Specific mechanical properties, such as a lower Youngs modulus and a higher hardness of the shells than those of Iceland spar, are ascribed to the presence of the specific mesoscopic textures.

Long-term Cultivation of the Deep-Sea Clam Calyptogena okutanii: Changes in the Abundance of Chemoautotrophic Symbiont, Elemental Sulfur, and Mucus.

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams’ gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

Decalcification and survival of benthic foraminifera under the combined impacts of varying pH and salinity

Coastal areas display natural large environmental variability such as frequent changes in salinity, pH, and carbonate chemistry. Anthropogenic impacts - especially ocean acidification - increase this variability, which may affect the living conditions of coastal species, particularly, calcifiers. We performed culture experiments on living benthic foraminifera to study the combined effects of lowered pH and salinity on the calcification abilities and survival of the coastal, calcitic species Ammonia sp. and Elphidium crispum. We found that in open ocean conditions (salinity ∼35) and lower pH than usual values for these species, the specimens displayed resistance to shell (test) dissolution for a longer time than in brackish conditions (salinity ∼5 to 20). However, the response was species specific as Ammonia sp. specimens survived longer than E. crispum specimens when placed in the same conditions of salinity and pH. Living, decalcified juveniles of Ammonia sp. were observed and we show that desalination is one cause for the decalcification. Finally, we highlight the ability of foraminifera to survive under Ωcalc < 1, and that high salinity and [Ca2+] as building blocks are crucial for the foraminiferal calcification process.

Effects of a long-term rearing system for deep-sea vesicomyid clams on host survival and endosymbiont retention

Deep-sea vesicomyid clams, including the genus Phreagena, harbor obligate sulfur-oxidizing symbiotic bacteria in gill epithelial cells. Difficulty in maintaining Phreagena clams in rearing tanks has been a major obstacle to achieving a better understanding of their unique biology. To improve the method of rearing Phreagena clams, here we reared them in an artificial chemosynthetic aquarium and evaluated the effects of the aquarium system on long-term clam rearing. We compared the survival of clams reared in the artificial chemosynthetic tank with the survival of those in the normal tank, and analyzed the symbiont abundance using semi-quantification of fluorescent in situ hybridization signals. Our results indicate that the artificial chemosynthetic aquarium system had specific effects on symbiont abundance and possibly on host survival. Furthermore, transmission electron microscopic observations of sulfur globules in the symbiont cells and expression analyses of the dsrA gene of the symbiont indicated that stocked elemental sulfur could be consumed as an energy source to reduce sulfide shortages. We discuss the importance of higher and more stable sulfide concentrations and the proportions of available O2 and CO2 in driving appropriate metabolic functions of the symbiont and improving the survival of the clams.