Unique evolution of foraminiferal calcification to survive global changes.

Foraminifera, the most ancient known calcium carbonate-producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidification, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca2+) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.

The coral reef-dwelling Peneroplis spp. shows calcification recovery to ocean acidification conditions.

Large Benthic Foraminifera are a crucial component of coral-reef ecosystems, which are currently threatened by ocean acidification. We conducted culture experiments to evaluate the impact of low pH on survival and test dissolution of the symbiont-bearing species Peneroplis spp., and to observe potential calcification recovery when specimens are placed back under reference pH value (7.9). We found that Peneroplis spp. displayed living activity up to 3 days at pH 6.9 (Ωcal < 1) or up to 1 month at pH 7.4 (Ωcal > 1), despite the dark and unfed conditions. Dissolution features were observed under low Ωcal values, such as changes in test density, peeled extrados layers, and decalcified tests with exposed organic linings. A new calcification phase started when specimens were placed back at reference pH. This calcification's resumption was an addition of new chambers without reparation of the dissolved parts, which is consistent with the porcelaneous calcification pathway of Peneroplis spp. The most decalcified specimens displayed a strong survival response by adding up to 8 new chambers, and the contribution of food supply in this process was highlighted. These results suggest that porcelaneous LBF species have some recovery abilities to short exposure (e.g., 3 days to 1 month) to acidified conditions. However, the geochemical signature of trace elements in the new calcite was impacted, and the majority of the new chambers were distorted and resulted in abnormal tests, which might hinder the specimens' reproduction and thus their survival on the long term.

Micro- and nanometric characterization of the celestite skeleton of acantharian species (Radiolaria, Rhizaria).

We clarified the specific micrometric arrangement and nanometric structure of the radiolarian crystalline spines that are not a simple single crystal. A body of the celestite (SrSO4) skeleton of acantharian Acanthometra cf. multispina (Acanthometridae) composed of 20 radial spines having four blades was characterized using microfocus X-ray computed tomography. The regular arrangement of three types of spines was clarified with the connection of the blades around the root of each spine. The surface of the spines was covered with a chitin-based organic membrane to prevent from dissolution in seawater. In the nanometric scale, the mesocrystalline structure that consists of nanoscale grains having distorted single-crystal nature was revealed using scanning- and transmission electron microscopies, electron diffraction, and Raman spectroscopy. The acantharian skeletons have a crystallographically controlled architecture that is covered with a protective organic membrane. These facts are important for penetrating the nature of biogenic minerals.

Discovery of a colossal slickhead (Alepocephaliformes: Alepocephalidae): an active-swimming top predator in the deep waters of Suruga Bay, Japan.

A novel species of the family Alepocephalidae (slickheads), Narcetes shonanmaruae, is described based on four specimens collected at depths greater than 2171 m in Suruga Bay, Japan. Compared to other alepocephalids, this species is colossal (reaching ca. 140 cm in total length and 25 kg in body weight) and possesses a unique combination of morphological characters comprising anal fin entirely behind the dorsal fin, multiserial teeth on jaws, more scale rows than congeners, precaudal vertebrae less than 30, seven branchiostegal rays, two epurals, and head smaller than those of relatives. Mitogenomic analyses also support the novelty of this large deep-sea slickhead. Although most slickheads are benthopelagic or mesopelagic feeders of gelatinous zooplankton, behavioural observations and dietary analyses indicate that the new species is piscivorous. In addition, a stable nitrogen isotope analysis of specific amino acids showed that N. shonanmaruae occupies one of the highest trophic positions reported from marine environments to date. Video footage recorded using a baited camera deployed at a depth of 2572 m in Suruga Bay revealed the active swimming behaviour of this slickhead. The scavenging ability and broad gape of N. shonanmaruae might be correlated with its colossal body size and relatively high trophic position.

Guanine crystals regulated by chitin-based honeycomb frameworks for tunable structural colors of sapphirinid copepod, Sapphirina nigromaculata.

Sapphirinid copepods, which are marine zooplankton, exhibit tunable structural colors originating from a layered structure of guanine crystal plates. In the present study, the coloring portion of adult male of a sapphirinid copepod, Sapphirina nigromaculata, under the dorsal body surface was characterized to clarify the regulation and actuation mechanism of the layered guanine crystals for spectral control. The coloring portions are separated into small domains 70-100 µm wide consisting of an ordered array of stacked hexagonal plates ~1.5 µm wide and ~80 nm thick. We found the presence of chitin-based honeycomb frameworks that are composed of flat compartments regulating the guanine crystal plates. The structural color is deduced to be tuned from blue to achromatic via yellow and purple by changing the interplate distance according to vital observation and optical simulation using a photonic array model. The framework structures are essential for the organization and actuation of the particular photonic arrays for the exhibition of the tunable structural color.

Complex factors shape phenotypic variation in deep-sea limpets.

Pectinodontid limpets are important members of deep-sea hot vents and cold seeps as can be seen by their conspicuous presence in both extant and extinct systems. They have traditionally been classified into different genera and species based on shell and radula characteristics; the reliability of these characters has been questioned but not tested thoroughly. Here, for the first time in taxa endemic to deep-sea chemosynthetic ecosystems, we combine substrate translocation with molecular data to assess the plasticity and variability of key phenotypic characters. Molecular data revealed that several 'species' of extant vent/seep pectinodontids actually represent intergrading morphotypes of a single, highly plastic, evolutionary lineage, with each morphological trait being possibly influenced differently by environmental and genetic factors. Our results challenge previous interpretations of paleoecology at fossil chemosynthetic ecosystems and highlight the importance of modern analogues in understanding fossil systems.

Evidence of selective pressure in whale fall microbiome proteins and its potential application to industry.

The present study addresses the microbiome of the first whale fall (YOKO 16) that has been described in the deep sea in the southern Atlantic Ocean (São Paulo Plateau; 4204 m depth), in terms of its metabolic uniqueness. Sets of ten thousand protein sequences from YOKO 16 and 29 public domain metagenomes (SRA and GenBank databases) that represent various marine, terrestrial and gut-associated microbial communities were analyzed. The determination of protein functionality, based on the KAAS server, indicated that the YOKO 16 microbiome has industrially-relevant proteins, such as proteases and lipases, that have low similarity (~50%) with previously-described enzymes. The amino acid usage in the YOKO 16 protein sequences (based on blastp and Clustal analysis) revealed a pattern of preference similar to that of extremophiles, with an increased usage of polar, charged and acidic amino acids and a decreased usage of nonpolar residues. We concluded that the targeted microbiome is of potential biotechnological use, which justifies the allocation of resources for the discovery of enzymes in deep-sea whale fall communities.

Gut Microbial Divergence between Two Populations of the Hadal Amphipod Hirondellea gigas.

Hadal environments sustain diverse microorganisms. A few studies have investigated hadal microbial communities consisting of free-living or particle-associated bacteria and archaea. However, animal-associated microbial communities in hadal environments remain largely unexplored, and comparative analyses of animal gut microbiota between two isolated hadal environments have never been done so far. In the present study, 228 Gb of gut metagenomes of the giant amphipod Hirondellea gigas from two hadal trenches, the Mariana Trench and Japan Trench, were sequenced and analyzed. Taxonomic analysis identified 49 microbial genera commonly shared by the gut microbiota of the two H. gigas populations. However, the results of statistical analysis, in congruency with the alpha and beta diversity analyses, revealed significant differences in gut microbial composition across the two trenches. Abundance variation of Psychromonas, Propionibacterium, and Pseudoalteromonas species was observed. Microbial cooccurrence was demonstrated for microbes that were overrepresented in the Mariana trench. Comparison of functional potential showed that the percentage of carbohydrate metabolic genes among the total microbial genes was significantly higher in the guts of H. gigas specimens from the Mariana Trench. Integrating carbon input information and geological characters of the two hadal trenches, we propose that the differences in the community structure might be due to several selective factors, such as environmental variations and microbial interactions.IMPORTANCE The taxonomic composition and functional potential of animal gut microbiota in deep-sea environments remain largely unknown. Here, by performing comparative metagenomics, we suggest that the gut microbial compositions of two Hirondellea gigas populations from the Mariana Trench and the Japan Trench have undergone significant divergence. Through analyses of functional potentials and microbe-microbe correlations, our findings shed light on the contributions of animal gut microbiota to host adaptation to hadal environments.

Compositional and Functional Shifts in the Epibiotic Bacterial Community of Shinkaia crosnieri Baba & Williams (a Squat Lobster from Hydrothermal Vents) during Methane-Fed Rearing.

The hydrothermal vent squat lobster Shinkaia crosnieri Baba & Williams harbors an epibiotic bacterial community, which is numerically and functionally dominated by methanotrophs affiliated with Methylococcaceae and thioautotrophs affiliated with Sulfurovum and Thiotrichaceae. In the present study, shifts in the phylogenetic composition and metabolic function of the epibiont community were investigated using S. crosnieri individuals, which were reared for one year in a tank fed with methane as the energy and carbon source. The results obtained indicated that indigenous predominant thioautotrophic populations, such as Sulfurovum and Thiotrichaceae members, became absent, possibly due to the lack of an energy source, and epibiotic communities were dominated by indigenous Methylococcaceae and betaproteobacterial methylotrophic members that adapted to the conditions present during rearing for 12 months with a supply of methane. Furthermore, the overall phylogenetic composition of the epibiotic community markedly changed from a composition dominated by chemolithotrophs to one enriched with cross-feeding heterotrophs in addition to methanotrophs and methylotrophs. Thus, the composition and function of the S. crosnieri epibiotic bacterial community were strongly affected by the balance between the energy and carbon sources supplied for chemosynthetic production as well as that between the production and consumption of organic compounds.

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.

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

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.

Cytological Observations of the Large Symbiotic Foraminifer Amphisorus kudakajimensis Using Calcein Acetoxymethyl Ester.

Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. Amphisorus kudakajimensis was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living A. kudakajimensis. We showed that at least two types of reticulopodia exist in A. kudakajimensis: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of A. kudakajimensis performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera.

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.

Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments.

Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much (15)N or (34)S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200-500 nm in diameter and co-occurred with possible endobionts, were labeled with (15)N originated from (15)N-labeled nitrate under anoxia and were labeled with both (15)N and (34)S under dysoxia. The labeling with (15)N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions.

Deep-sea whale fall fauna from the Atlantic resembles that of the Pacific Ocean.

Whale carcasses create remarkable habitats in the deep-sea by producing concentrated sources of organic matter for a food-deprived biota as well as places of evolutionary novelty and biodiversity. Although many of the faunal patterns on whale falls have already been described, the biogeography of these communities is still poorly known especially from basins other than the NE Pacific Ocean. The present work describes the community composition of the deepest natural whale carcass described to date found at 4204 m depth on Southwest Atlantic Ocean with manned submersible Shinkai 6500. This is the first record of a natural whale fall in the deep Atlantic Ocean. The skeleton belonged to an Antarctic Minke whale composed of only nine caudal vertebrae, whose degradation state suggests it was on the bottom for 5-10 years. The fauna consisted mainly of galatheid crabs, a new species of the snail Rubyspira and polychaete worms, including a new Osedax species. Most of the 41 species found in the carcass are new to science, with several genera shared with NE Pacific whale falls and vent and seep ecosystems. This similarity suggests the whale-fall fauna is widespread and has dispersed in a stepping stone fashion, deeply influencing its evolutionary history.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Cytologic and Genetic Characteristics of Endobiotic Bacteria and Kleptoplasts of Virgulinella fragilis (Foraminifera).

The benthic foraminifer Virgulinella fragilis Grindell and Collen 1976 has multiple putative symbioses with both bacterial and kleptoplast endobionts, possibly aiding its survival in environments from dysoxia (5-45 µmol-O2 /L) to microxia (0-5 µmol-O2 /L) and in the dark. To clarify the origin and function of V. fragilis endobionts, we used genetic analyses and transmission electron microscope observations. Virgulinella fragilis retained δ-proteobacteria concentrated at its cell periphery just beneath the cell membranes. Unlike another foraminifer Stainforthia spp., which retains many bacterial species, V. fragilis has a less variable bacterial community. This suggests that V. fragilis maintains a specific intracellular bacterial flora. Unlike the endobiotic bacteria, V. fragilis klepto-plasts originated from various diatom species and are found in the interior cytoplasm. We found evidence of both retention and digestion of kleptoplasts, and of fragmentation of the kleptoplastid outer membrane that likely facilitates transport of kleptoplastid products to the host. Accumulations of mitochondria were observed encircling endobiotic bacteria. It is likely that the bacteria use host organic material for carbon oxidation. The mitochondria may use oxygen available around the δ-proteobacteria and synthesize adenosine triphosphate, perhaps for sulfide oxidation.

Unexpected biotic resilience on the Japanese seafloor caused by the 2011 Tohoku-Oki tsunami.

On March 11(th), 2011 the Mw 9.0 2011 Tohoku-Oki earthquake resulted in a tsunami which caused major devastation in coastal areas. Along the Japanese NE coast, tsunami waves reached maximum run-ups of 40 m, and travelled kilometers inland. Whereas devastation was clearly visible on land, underwater impact is much more difficult to assess. Here, we report unexpected results obtained during a research cruise targeting the seafloor off Shimokita (NE Japan), shortly (five months) after the disaster. The geography of the studied area is characterized by smooth coastline and a gradually descending shelf slope. Although high-energy tsunami waves caused major sediment reworking in shallow-water environments, investigated shelf ecosystems were characterized by surprisingly high benthic diversity and showed no evidence of mass mortality. Conversely, just beyond the shelf break, the benthic ecosystem was dominated by a low-diversity, opportunistic fauna indicating ongoing colonization of massive sand-bed deposits.

Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater.

BACKGROUND: Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. RESULTS: A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). CONCLUSIONS: A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.