Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

Fungal Abundance and Diversity in the Mariana Trench, the Deepest Ecosystem on Earth.

Hadal trenches host abundant and diversified benthic prokaryotic assemblages, but information on benthic fungi is still extremely limited. We investigated the fungal abundance and diversity in the Challenger Deep (at ca. 11,000 m depth) and the slope of the Mariana Trench in comparison with three sites of the adjacent abyssal plain. Our results indicate that trench sediments are a hotspot of fungal abundance in terms of the 18S rRNA gene copy number. The fungal diversity (as the number of amplicon sequence variants, ASVs) was relatively low at all sites (10-31 ASVs) but showed a high turnover diversity among stations due to the presence of exclusive fungal taxa belonging to Aspergillaceae, Trichosphaeriaceae, and Nectriaceae. Fungal abundance and diversity were closely linked to sediment organic matter content and composition (i.e., phytopigments and carbohydrates), suggesting a specialization of different fungal taxa for the exploitation of available resources. Overall, these findings provide new insights into the diversity of deep-sea fungi and the potential ecological role in trench sediments and pave the way for a better understanding of their relevance in one of the most extreme ecosystems on Earth.

Microbial decomposition of biodegradable plastics on the deep-sea floor.

Microbes can decompose biodegradable plastics on land, rivers and seashore. However, it is unclear whether deep-sea microbes can degrade biodegradable plastics in the extreme environmental conditions of the seafloor. Here, we report microbial decomposition of representative biodegradable plastics (polyhydroxyalkanoates, biodegradable polyesters, and polysaccharide esters) at diverse deep-sea floor locations ranging in depth from 757 to 5552 m. The degradation of samples was evaluated in terms of weight loss, reduction in material thickness, and surface morphological changes. Poly(L-lactic acid) did not degrade at either shore or deep-sea sites, while other biodegradable polyesters, polyhydroxyalkanoates, and polysaccharide esters were degraded. The rate of degradation slowed with water depth. We analysed the plastic-associated microbial communities by 16S rRNA gene amplicon sequencing and metagenomics. Several dominant microorganisms carried genes potentially encoding plastic-degrading enzymes such as polyhydroxyalkanoate depolymerases and cutinases/polyesterases. Analysis of available metagenomic datasets indicated that these microorganisms are present in other deep-sea locations. Our results confirm that biodegradable plastics can be degraded by the action of microorganisms on the deep-sea floor, although with much less efficiency than in coastal settings.

A deep-learning automated image recognition method for measuring pore patterns in closely related bolivinids and calibration for quantitative nitrate paleo-reconstructions.

Eutrophication is accelerating the recent expansion of oxygen-depleted coastal marine environments. Several bolivinid foraminifera are abundant in these oxygen-depleted settings, and take up nitrate through the pores in their shells for denitrification. This makes their pore density a possible nitrate proxy. This study documents three aspects related to the porosity of bolivinids. 1. A new automated image analysis technique to determine the number of pores in bolivinids is tested. 2. The pore patterns of Bolivina spissa from five different ocean settings are analysed. The relationship between porosity, pore density and mean pore size significantly differs between the studied locations. Their porosity is mainly controlled by the size of the pores at the Gulf of Guayaquil (Peru), but by the number of pores at other studied locations. This might be related to the presence of a different cryptic Bolivina species in the Gulf of Guayaquil. 3. The pore densities of closely related bolivinids in core-top samples are calibrated as a bottom-water nitrate proxy. Bolivina spissa and Bolivina subadvena showed the same correlation between pore density and bottom-water nitrate concentrations, while the pore density of Bolivina argentea and Bolivina subadvena accumeata is much higher.

Effectiveness of hierarchical Bayesian models for citizen science data with missing values: A case study on the factors influencing beach litter in Shimane Prefecture, Japan.

Citizen science can play an important role in addressing the issue of marine debris. However, citizen science data are often composed of inconsistent methods compared to data collected by experts. In this study, we applied beach cleanup data, collected in different survey years at different survey sites, to a hierarchical Bayesian model to elucidate the factors affecting the distribution of beach litter. The results showed the model accounting for differences between years had a smaller Watanabe-Akaike Information criterion than the model that did not account for it, indicating better accuracy of the model. The amount of beach litter was influenced by current velocity and bay openness, and these effects varied across years. The results indicate that citizen science data, which may contain missing values due to various constraints such as economic and human resources, can make an important contribution toward solving marine debris issues by flexible statistical analysis methods.

Foraminifera and plastic pollution: Knowledge gaps and research opportunities.

Plastic has become one of the most ubiquitous and environmentally threatening sources of pollution in the Anthropocene. Beyond the conspicuous visual impact and physical damages, plastics both carry and release a cocktail of harmful chemicals, such as monomers, additives and persistent organic pollutants. Here we show through a review of the scientific literature dealing with both plastic pollution and benthic foraminifera (Rhizaria), that despite their critical roles in the structure and function of benthic ecosystems, only 0.4% of studies have investigated the effects of micro- and nano-plastics on this group. Consequently, we urge to consider benthic foraminifera in plastic pollution studies via a tentative roadmap that includes (i) the use of their biological, physiological and behavioral responses that may unveil the effects of microplastics and nanoplastics and (ii) the evaluation of the indicative value of foraminiferal species to serve as proxies for the degree of pollution. This appears particularly timely in the context of the development of management strategies to restore coastal ecosystems.

Deep-sea infauna with calcified exoskeletons imaged in situ using a new 3D acoustic coring system (A-core-2000).

The deep ocean is Earth's largest habitable space inhabited by diverse benthic organisms. Infauna play crucial roles in shaping sedimentary structures, relocating organic matter, porewater chemistry, and hence biogeochemical cycles. However, the visualization and quantification of infauna in situ inside deep-sea sediment has been challenging, due to their sparse distribution and that deep-sea cameras do not visualize animals living below the sediment surface. Here, we newly developed a 3D acoustic "coring" system and applied it to visualize and detect burrowing bivalves in deep-sea sediments. The in situ acoustic observation was conducted at a dense colony of vesicomyid clams in a hydrocarbon seep in Sagami Bay, Japan, focusing on a patch of juvenile clams with a completely infaunal life style. We clearly observed strong backscatters from the top and lower edges of animals in our 3D acoustic data. At least 17 reflectors were identified in the survey area (625 cm2), interpreted to correspond to living clams. The estimated depths of the lower edge of clams ranged between 41 and 98 mm. The acoustic system presented here is effective for detecting and monitoring infauna with calcified exoskeletons. This novel tool will help us better assess and understand the distribution of deep-sea infauna, particularly those groups with hard exoskeletons, as well as biogeochemical cycles.

In situ experimental evidences for responses of abyssal benthic biota to shifts in phytodetritus compositions linked to global climate change.

Abyssal plains cover more than half of Earth's surface, and the main food source in these ecosystems is phytodetritus, mainly originating from primary producers in the euphotic zone of the ocean. Global climate change is influencing phytoplankton abundance, productivity, and distribution. Increasing importance of picoplankton over diatom as primary producers in surface oceans (especially projected for higher latitudes) is projected and hence altering the quantity of organic carbon supplied to the abyssal seafloor as phytodetritus, consequences of which remain largely unknown. Here, we investigated the in situ responses of abyssal biota from viruses to megafauna to different types of phytoplankton input (diatoms or cyanobacteria which were labeled with stable isotopes) at equatorial (oligotrophic) and temperate (eutrophic) benthic sites in the Pacific Ocean (1°N at 4277 m water depth and 39°N at 5260 m water depth, respectively). Our results show that meiofauna and macrofauna generally preferred diatoms as a food source and played a relatively larger role in the consumption of phytodetritus at higher latitudes (39°N). Contrarily, prokaryotes and viruses showed similar or even stronger responses to cyanobacterial than to diatom supply. Moreover, the response of prokaryotes and viruses was very rapid (within 1-2 days) at both 1°N and 39°N, with quickest responses reported in the case of cyanobacterial supply at higher latitudes. Overall, our results suggest that benthic deep-sea eukaryotes will be negatively affected by the predicted decrease in diatoms in surface oceans, especially at higher latitudes, where benthic prokaryotes and viruses will otherwise likely increase their quantitative role and organic carbon cycling rates. In turn, such changes can contribute to decrease carbon transfer from phytodetritus to higher trophic levels, with strong potential to affect oceanic food webs, their biodiversity and consequently carbon sequestration capacity at the global scale.

16S rRNA Gene Metabarcoding Indicates Species-Characteristic Microbiomes in Deep-Sea Benthic Foraminifera.

Foraminifera are unicellular eukaryotes that are an integral part of benthic fauna in many marine ecosystems, including the deep sea, with direct impacts on benthic biogeochemical cycles. In these systems, different foraminiferal species are known to have a distinct vertical distribution, i.e., microhabitat preference, which is tightly linked to the physico-chemical zonation of the sediment. Hence, foraminifera are well-adapted to thrive in various conditions, even under anoxia. However, despite the ecological and biogeochemical significance of foraminifera, their ecology remains poorly understood. This is especially true in terms of the composition and diversity of their microbiome, although foraminifera are known to harbor diverse endobionts, which may have a significant meaning to each species' survival strategy. In this study, we used 16S rRNA gene metabarcoding to investigate the microbiomes of five different deep-sea benthic foraminiferal species representing differing microhabitat preferences. The microbiomes of these species were compared intra- and inter-specifically, as well as with the surrounding sediment bacterial community. Our analysis indicated that each species was characterized with a distinct, statistically different microbiome that also differed from the surrounding sediment community in terms of diversity and dominant bacterial groups. We were also able to distinguish specific bacterial groups that seemed to be strongly associated with particular foraminiferal species, such as the family Marinilabiliaceae for Chilostomella ovoidea and the family Hyphomicrobiaceae for Bulimina subornata and Bulimina striata. The presence of bacterial groups that are tightly associated to a certain foraminiferal species implies that there may exist unique, potentially symbiotic relationships between foraminifera and bacteria that have been previously overlooked. Furthermore, the foraminifera contained chloroplast reads originating from different sources, likely reflecting trophic preferences and ecological characteristics of the different species. This study demonstrates the potential of 16S rRNA gene metabarcoding in resolving the microbiome composition and diversity of eukaryotic unicellular organisms, providing unique in situ insights into enigmatic deep-sea ecosystems.

Distribution and Genetic Divergence of Deep-Sea Hydrothermal Vent Copepods (Dirivultidae: Siphonostomatoida: Copepoda) in the Northwestern Pacific.

Copepods in the family Dirivultidae are one of the most successful meiofauna in deep-sea hydrothermal vent fields and are abundant near venting fluid. Although vents are spatially limited ocean habitats, they are distributed widely in the Atlantic, Pacific, and Indian Oceans. However, knowledge of dirivultid biogeography and phylogeography remains limited, especially in the northwestern Pacific. Here, we obtained partial mitochondrial COI gene sequences of three dirivultids from the northwestern Pacific-Stygiopontius senokuchiae and an unidentified Chasmatopontius species from vent fields in the Izu-Bonin Arc and Stygiopontius senckenbergi associated with the squat lobster Shinkaia crosnieri in the Okinawa Trough-and analyzed them in comparison with existing data. The among-species sequence diversity exceeded 80 out of 560 bp (14% or 0.166 in Kimura 2-parameter distance), whereas the within-species diversity was less than 10 bp (2% or 0.018 in Kimura 2-parameter distance), with no genetic saturation. Each species formed a monophyletic clade and the genetic region targeted is deemed reliable for identifying species and populations for these copepods. Among the three genera targeted, only Chasmatopontius formed a monophyletic cluster, while Aphotopontius and Stygiopontius did not. Species delimitation analyses suggested the existence of cryptic species in Chasmatopontius. Subdivision among local populations was observed in Aphotopontius, but not in Stygiopontius in the same distribution, implying potential differences in dispersal ability among different genera of dirivultids. Further sampling is required, to fill the spatial gaps to elucidate the biogeography and evolution of dirivultids in the global deep ocean.

Genomes of Thaumarchaeota from deep sea sediments reveal specific adaptations of three independently evolved lineages.

Marine sediments represent a vast habitat for complex microbiomes. Among these, ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are one of the most common, yet little explored, inhabitants, which seem extraordinarily well adapted to the harsh conditions of the subsurface biosphere. We present 11 metagenome-assembled genomes of the most abundant AOA clades from sediment cores obtained from the Atlantic Mid-Ocean ridge flanks and Pacific abyssal plains. Their phylogenomic placement reveals three independently evolved clades within the order Nitrosopumilales, of which no cultured representative is known yet. In addition to the gene sets for ammonia oxidation and carbon fixation known from other AOA, all genomes encode an extended capacity for the conversion of fermentation products that can be channeled into the central carbon metabolism, as well as uptake of amino acids probably for protein maintenance or as an ammonia source. Two lineages encode an additional (V-type) ATPase and a large repertoire of DNA repair systems that may allow to overcome the challenges of high hydrostatic pressure. We suggest that the adaptive radiation of AOA into marine sediments occurred more than once in evolution and resulted in three distinct lineages with particular adaptations to this extremely energy-limiting and high-pressure environment.

Development of robust models for rapid classification of microplastic polymer types based on near infrared hyperspectral images.

Hyperspectral data in the near infrared range were examined for nine common types of plastic particles of 1 mm and 100-500 µm sizes on dry and wet glass fiber filters. Weaker peak intensities were detected for small particles compared to large particles, and the reflectances were weaker at longer wavelengths when the particles were measured on a wet filter. These phenomena are explainable due to the effect of the correlation between the particle size and the absorption of infrared light by water. We constructed robust classification models that are capable of classifying polymer types, regardless of particle size or filter conditions (wet vs. dry), based on hyperspectral data for small particles measured on wet filters. Using the models, we also successfully classified the polymer type of polystyrene beads covered with microalgae, which simulates the natural conditions of microplastics in the ocean. This study suggests that hyperspectral imaging techniques with appropriate classification models allow the identification of microplastics without the time- and labor-consuming procedures of drying samples and removing biofilms, thus enabling more rapid analyses.

Abundant Chitinous Structures in Chilostomella (Foraminifera, Rhizaria) and Their Potential Functions.

Benthic foraminifera, members of Rhizaria, inhabit a broad range of marine environments and are particularly common in hypoxic sediments. The biology of benthic foraminifera is key to understanding benthic ecosystems and relevant biogeochemical cycles, especially in hypoxic environments. Chilostomella is a foraminiferal genus commonly found in hypoxic deep-sea sediments and has poorly understood ecological characteristics. For example, the carbon isotopic compositions of their lipids are substantially different from other co-occurring genera, probably reflecting unique features of its metabolism. Here, we investigated the cytoplasmic and ultrastructural features of Chilostomella ovoidea from bathyal sediments of Sagami Bay, Japan, based on serial semi-thin sections examined using an optical microscope followed by a three-dimensional reconstruction, combined with TEM observations of ultra-thin sections. Observations by TEM revealed the presence of abundant electron-dense structures dividing the cytoplasm. Based on histochemical staining, these structures are shown to be composed of chitin. Our 3D reconstruction revealed chitinous structures in the final seven chambers. These exhibited a plate-like morphology in the final chambers but became rolled up in earlier chambers (toward the proloculus). These chitinous, plate-like structures may function to partition the cytoplasm in a chamber to increase the surface/volume ratio and/or act as a reactive site for some metabolic functions.

Optimization of a hyperspectral imaging system for rapid detection of microplastics down to 100 µm.

Plastic pollution has become one of the most emergent issues threating aquatic and terrestrial ecosystems. However, it is still challenging to rapidly detect small microplastics. Here, we present a method to rapidly detect microplastics using hyperspectral imaging in which we optimized a commercially available hyperspectral imaging system (Pika NIR-640, Resonon Inc., USA). The optimizations included: (1) changing the four-lamp assembly to a symmetrical set of converged-light near-infrared lamps that are placed sideways instead of above the sample stage; (2) adopting a macro-photography technique by applying an extension tube between the camera and the lens, and moving the lens of the hyperspectral camera to the imaging target (working distance of ~3 cm); (3) adjusting the exposure and aspect ratio by tuning the frame rate and scan speed of the imaging system. After optimization, the detection resolution of each pixel improved from 250 µm to 14.8 µm. With the optimized system, microplastics down to 100 µm in size were rapidly detected. This result is promising for the application of our new method in the accelerated detection of microplastics and will contribute to a better understanding of the microplastic pollution situation.

Characterization of microplastics on filter substrates based on hyperspectral imaging: Laboratory assessments.

Microplastic pollution has become an urgent issue because it adversely affects ecosystems. However, efficient methods to detect and characterize microplastic particles are still in development. By conducting a series of laboratory assessments based on near-infrared hyperspectral imaging in the wavelength range of 900-1700 nm, we report the fundamental spectral features of (i) 11 authentic plastics and (ii) 11 filter substrate materials. We found that different plastic polymers showed distinct spectral features at 1150-1250 nm, 1350-1450 nm and 1600-1700 nm, enabling their automatic recognition and identification with spectral separation algorithms. Using an improved hyperspectral imaging system, we demonstrated the detection of three types of microplastic particles, polyethylene, polypropylene and polystyrene, down to 100 µm in diameter. As a filter substrate, a gold-coated polycarbonate filter (GPC0847-BA) showed constant reflectance over 900-1700 nm and a large radiative contrast against loaded plastic particles. Glass fiber filters (GF10 and GF/F) would also be suitable substrates due to their low cost and easy commercial availability. This study provides key parameters for applying hyperspectral imaging techniques for the detection of microplastics.

Microbial community and geochemical analyses of trans-trench sediments for understanding the roles of hadal environments.

Hadal trench bottom (>6000 m below sea level) sediments harbor higher microbial cell abundance compared with adjacent abyssal plain sediments. This is supported by the accumulation of sedimentary organic matter (OM), facilitated by trench topography. However, the distribution of benthic microbes in different trench systems has not been well explored yet. Here, we carried out small subunit ribosomal RNA gene tag sequencing for 92 sediment subsamples of seven abyssal and seven hadal sediment cores collected from three trench regions in the northwest Pacific Ocean: the Japan, Izu-Ogasawara, and Mariana Trenches. Tag-sequencing analyses showed specific distribution patterns of several phyla associated with oxygen and nitrate. The community structure was distinct between abyssal and hadal sediments, following geographic locations and factors represented by sediment depth. Co-occurrence network revealed six potential prokaryotic consortia that covaried across regions. Our results further support that the OM cycle is driven by hadal currents and/or rapid burial shapes microbial community structures at trench bottom sites, in addition to vertical deposition from the surface ocean. Our trans-trench analysis highlights intra- and inter-trench distributions of microbial assemblages and geochemistry in surface seafloor sediments, providing novel insights into ultradeep-sea microbial ecology, one of the last frontiers on our planet.

Drivers of Bacterial α- and ß-Diversity Patterns and Functioning in Subsurface Hadal Sediments.

Oceanic trenches at hadal (>6,000 m) depths are hot spots of organic matter deposition and mineralization and can host abundant and active bacterial assemblages. However, the factors able to shape their biodiversity and functioning remain largely unexplored, especially in subsurface sediments. Here, we investigated the patterns and drivers of benthic bacterial α- and ß-diversity (i.e., OTU richness and turnover diversity) along the vertical profile down to 1.5 m sediment depth in the Izu-Bonin Trench (at ~10,000 m water depth). The protease and glucosidase enzymatic activity rates were also determined, as a proxy of organic matter degradation potential in the different sediment layers. Molecular fingerprinting based on automated ribosomal intergenic spacer analysis (ARISA) indicated that the α-diversity of bacterial assemblages remained high throughout the vertical profile and that the turnover (ß-) diversity among sediment horizons reached values up to 90% of dissimilarity. Multivariate distance-based linear modeling (DISTLM) pointed out that the diversity and functioning of the hadal bacterial assemblages were influenced by the variability of environmental conditions (including the availability of organic resources and electron donors/acceptors) and of viral production rates along the sediment vertical profile. Based on our results, we can argue that the heterogeneity of physical-chemical features of the hadal sediments of the Izu-Bonin Trench contribute to increase the niches availability for different bacterial taxa, while viruses contribute to maintain high levels of bacterial turnover diversity and to enhance organic matter cycling in these extremely remote and isolated ecosystems.

Sediment sampling with a core sampler equipped with aluminum tubes and an onboard processing protocol to avoid plastic contamination.

Microplastics are abundant even on the deep-sea floor far from land and the ocean surface where human activities take place. To obtain samples of microplastics from the deep-sea floor, a research vessel and suitable sampling equipment, such as a multiple corer, a box corer, or a push corer manipulated by a remotely operated (ROV) or human occupied vehicle (HOV) are needed. Most such corers use sampling tubes made of plastic, such as polycarbonate, acrylic, or polyvinyl chloride. These plastic tubes are easily scratched by sediment particles, in particular during collection of coarse sandy sediments, and, consequently, the samples may become contaminated with plastic from the tube. Here, we report on the use of aluminum tubes with both a multiple corer and a push corer to prevent such plastic contamination. When compared with plastic tubes, aluminum tubes have the disadvantages of heavier weight and non-transparency. We suggest ways to overcome these problems, and we also present an onboard processing protocol to prevent plastic contamination during sediment core sampling when plastic tubes are used. •Use of a sediment corer with aluminum tubes reduces the risk of plastic contamination in the sediment samples•The proposed method allows undisturbed sediment cores to be retrieved with comparable efficiency to conventional transparent core tubes.

Viral Infections Boost Prokaryotic Biomass Production and Organic C Cycling in Hadal Trench Sediments.

Hadal trenches are among the most remote and least explored ecosystems on Earth and can support high benthic microbial standing stocks and activities. However, information on the role of viruses in such ecosystems and their interactions with prokaryotic hosts is very limited. Here, we investigated activities of benthic viruses and prokaryotes and their interactions in three hadal trenches (Japan, Izu-Ogasawara and Mariana trenches) and in their nearby abyssal sites. Our findings reveal that these hadal trenches, compared with the surrounding abyssal sites, support higher abundances and biomasses of prokaryotes. In addition, the high prokaryotic biomasses of hadal trenches could favor high rates of viral infection and cell lysis, especially in the Japan Trench. Hadal viruses can release large amounts of highly labile and promptly available organic material by inducing cell lysis, which could contribute to sustain benthic prokaryotes and decrease their dependency on the enzymatic digestion of the more refractory fraction of sediment organic matter. Our results suggest that this process can contribute to explain the discrepancy between high prokaryote biomass and apparent low efficiency in the utilization of the sedimentary organic matter in the hadal ecosystems. Concluding, hadal trenches may be characterized by a highly dynamic viral component, which can boost prokaryotic biomass production, thereby profoundly influencing the functioning of these remote and extreme ecosystems.

Compound-specific isotope analysis of benthic foraminifer amino acids suggests microhabitat variability in rocky-shore environments.

The abundance and biomass of benthic foraminifera are high in intertidal rocky-shore habitats. However, the availability of food to support their high biomass has been poorly studied in these habitats compared to those at seafloor covered by sediments. Previous field and laboratory observations have suggested that there is diversity in the food preferences and modes of life among rocky-shore benthic foraminifera. In this study, we used the stable nitrogen isotopic composition of amino acids to estimate the trophic position, trophic niche, and feeding strategy of individual foraminifera species. We also characterized the configuration and structure of the endobiotic microalgae in foraminifera using transmission electron microscopy, and we identified the origin of endobionts based on nucleotide sequences. Our results demonstrated a large variation in the trophic positions of different foraminifera from the same habitat, a reflection of endobiotic features and the different modes of life and food preferences of the foraminifera. Foraminifera did not rely solely on exogenous food sources. Some species effectively used organic matter derived from endobionts in the cell cytoplasm. The high biomass and species density of benthic foraminifera found in intertidal rocky-shore habitats are thus probably maintained by the use of multiple nitrogen resources and by microhabitat segregation among species as a consequence.