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.

Distribution of microplastics in bathyal- to hadal-depth sediments and transport process along the deep-sea canyon and the Kuroshio Extension in the Northwest Pacific.

Understanding microplastic (MP) behavior in oceans is crucial for reducing marine plastic pollution. However, the complex process underlying MP transportation to the deep seafloor remains unknown despite the deep sea being considered its major sink. We focused on MP distribution in Sagami Bay (adjacent to highly populated areas of Japan), the plate triple junction connected through the Sagami Trough, and the abyssal plain immediately below the Kuroshio Extension. We observed the highest number of MPs in the abyssal stations, more than previously reported. The polymer types and aspect ratio of MPs in the abyssal stations significantly differed from those in the bathyal/hadal stations. The study suggests that MPs accumulated in the open ocean surface layer sink to the abyssal plains immediately below it, while MPs from land sources accumulate in the bathyal depth and are transported to the hadal depth near the coast through turbidity currents along the submarine canyon.

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.

Plastic additives in deep-sea debris collected from the western North Pacific and estimation for their environmental loads.

Plastic waste has become a growing concern in terms of marine pollution, but little information is available on plastic debris and its possible risks of chemical additives exposure in the deep-sea. This study focused on identification of polymer type and additive concentrations in 21 plastic debris collected from deep-sea of Sagami Bay, Japan and West Pacific Ocean under the Kuroshio Extension and its recirculation gyre (KERG) zone (water depth: 1388-5819 m). Polyethylene (PE) was dominant polymer (57% of the total) in samples, followed by polyvinylchloride (PVC), epoxy resin, polyester (PES), and polypropylene. In plastic additives, bis (2-ethylhexyl) phthalate (DEHP) was detected to be contained in a PVC sheet at concentration of 48%. Butylated hydroxytoluene (BHT) was also detected in PE plastic debris with median concentration of 12,000 ng/g. PES clothes were detected to contain dyeing mixtures, 1,2,4-trichlorobenzene (1,2,4-TCB), up to 42,000 ng/g. Knowing the estimated number of plastic debris under KE current, the minimum burden of chemical additives were estimated that 720 kg of dibutyl phthalate, 570 kg of BHT, 230 kg of DEHP, and 160 kg of 1,2,4-TCB exist on the seabed of KERG zone. This result strongly suggests that enormous amount of hazardous additives lie within plastic debris on abyssal level of the ocean.

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.

Massive occurrence of benthic plastic debris at the abyssal seafloor beneath the Kuroshio Extension, the North West Pacific.

The abyss (3500-6500 m) covers the bulk of the deep ocean floor yet little is known about the extent of plastic debris on the abyssal seafloor. Using video imagery we undertook a quantitative assessment of the debris present on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific. This body of water is one of the major transit pathways for the massive amounts of debris that are entering the North Pacific Ocean from Asia. Shallower sites (1400-1500 m depth) were also investigated for comparison. The dominant type of debris was single-use plastics - mainly bags and food packaging. The density of the plastic debris (mean 4561 items/km2) in the abyssal zone was the highest recorded for an abyssal plain suggesting that the deep-sea basin in the Northwest Pacific is a significant reservoir of plastic debris.

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.

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.

A new small device made of glass for separating microplastics from marine and freshwater sediments.

Separating microplastics from marine and freshwater sediments is challenging, but necessary to determine their distribution, mass, and ecological impacts in benthic environments. Density separation is commonly used to extract microplastics from sediments by using heavy salt solutions, such as zinc chloride and sodium iodide. However, current devices/apparatus used for density separation, including glass beakers, funnels, upside-down funnel-shaped separators with a shut-off valve, etc., possess various shortcomings in terms of recovery rate, time consumption, and/or usability. In evaluating existing microplastic extraction methods using density separation, we identified the need for a device that allows rapid, simple, and efficient extraction of microplastics from a range of sediment types. We have developed a small glass separator, without a valve, taking a hint from an Utermöhl chamber. This new device is easy to clean and portable, yet enables rapid separation of microplastics from sediments. With this simple device, we recovered 94-98% of <1,000 µm microplastics (polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, and polystyrene). Overall, the device is efficient for various sizes, polymer types, and sediment types. Also, microplastics collected with this glass-made device remain chemically uncontaminated, and can, therefore, be used for further analysis of adsorbing contaminants and additives on/to microplastics.

A small, stainless-steel sieve optimized for laboratory beaker-based extraction of microplastics from environmental samples.

Removing non-plastic materials is a mandatory process for studying microplastics in environmental samples, and non-plastic materials, both inorganic and organic matter, are often removed chemically through sequential processes. In the multiple chemical treatment processes, the samples need to be collected and the reagent removed at the end of each chemical treatment before the samples are again exposed to a different reagent in a separate container. This leads to a loss of microplastics to some extent. Here, we developed a new, yet simple, small sieve made of stainless-steel that can fit in a laboratory beaker (e.g. 200 ml volume), allowing it to be transferred as-is between chemical treatments of environmental samples, even being soakable in a beaker of acid solution. The collection rates of microplastics were significantly higher in the small stainless-steel sieve than the commonly used filter method for different size of microplastic particles. The use of the new sieve means the processes of rinsing off and filtering samples can be abbreviated throughout the entire process of non-plastic matter removal from environmental samples, contributing to a lower chance of microplastic loss. The time consumed in the sieve method was also significantly lower than for the filtering method due to the elimination of the collection and rinsing steps, thus the use of this sieve can reduce processing time for the samples. The new method is innovative in terms of reducing both the microplastic loss and processing time during chemical treatment processes. •The method developed allows the lower chance of microplastic loss during chemical digestion process•The method reduces the time of sequential processes during chemical digestion.

A new method for acquiring images of meiobenthic images using the FlowCAM.

The purpose of this study was to develop a new method for investigating sediment-inhabiting meiobenthos using the Flow Cytometer And Microscope (FlowCAM). Meiobenthos are widely recognized as a useful indicator for assessing the effects of anthropogenic and natural disturbances in both shallow and deep ocean ecosystems. These small benthic invertebrates are traditionally investigated by individually counting and identifying specimens under a microscope, which is labor intensive and time consuming. However, FlowCAM, which was originally developed to semiautomatically analyze microplankton, has the potential to resolve these challenges. Meiobenthic specimens were extracted from sediment using the centrifugal separation method and were then pipetted into the FlowCAM system and imaged. The images were then used to classify and count the specimens at high taxonomic levels to verify the effectiveness of this method compared with traditional methods. We found that FlowCAM system: •Enabled sufficient meiobenthic images to be obtained to allow the identification and classification of specimens at high taxonomic levels.•Obtained comparable numbers of individuals to traditional methods.•Has the potential to rapidly process large the volumes of meiobenthos samples that are required when monitoring seasonal and spatial variation in ocean ecosystems and conducting long-term environmental impact assessments.

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.