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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.

The 2011 off the Pacific coast of Tohoku Earthquake occurred off southern Sanriku, Japan, on 11 March and generated strong shaking and a huge tsunami along the entire eastern coast of Tohoku. The mainshock and numerous aftershocks caused mass-wasting sedimentation events on the continental shelf, slope, and trench bottom. To investigate the impacts of the 2011 Tohoku Earthquake on the deep-sea environment off Sanriku, we conducted shipboard surveys up to ~2000 dbar during 2011–2018 and long-term monitoring of the seafloor on the continental slope using a deep-sea station (~1000 dbar) off Otsuchi Bay during 2014–2018. The turbidity calculated from light transmissometer anomaly data for the bottom layer deeper than 500 dbar on the continental slope was ~6% and higher than the values usually observed there. This high turbidity during 2011–2018 was caused by sporadic sediment resuspension induced by frequent aftershocks in the area off Sanriku. We found that dissolved oxygen concentrations between 1000 dbar and 1500 dbar dropped significantly by about 10% after the earthquake, whereas nutrients and dissolved inorganic carbon showed no significant difference and displayed a wide variation. The correlation between light transmissometer anomaly values and chemical parameters at 1000–1500 dbar suggests that turbidity increased the concentrations of phosphate, silicate, dissolved inorganic carbon, and methane, and decreased dissolved oxygen and nitrate. The deep-sea chemical environment was changed by remineralization resulting from the respiration and denitrification of microbial communities after the earthquake. The environmental change in the deep sea was maintained by sediment resuspension caused by continued aftershocks and was likely caused by variations in dissolved inorganic carbon and nutrients on the slope after the mainshock and aftershocks. The concentration and carbon isotope ratio of methane between 1000 and 2000 dbar near the hypocenter shows two peaks with high light transmissometer anomaly values near the isopycnal surfaces of 27.38σθ (1000 dbar) and 27.56σθ (1500 dbar). These sources of chemical input contributed by sediment resuspension induced by the mainshock and aftershocks might have been derived from shallow sediment on the continental slope and deep sub-seafloor reservoirs.

In this study, sediments from whale-fall chemosynthetic ecosystems (two different sites, one naturally occurring at 4200 m water depth in South Atlantic Ocean and one artificially immersed at 100 m water depth in Kagoshima Bay, Japan) were investigated by Ion Torrent PGM sequencing of the ITS region of ribosomal RNA to reveal fungal communities in these unique marine environments. As a result, a total of 107 (897 including singletons) Operational Taxonomic Units (OTUs) were obtained from the samples explored. Composition of the 107 OTUs at the phylum level among the five samples from two different whale-fall sites was assigned to Ascomycota (46%), Basidiomycota (7%), unidentified fungi (21%), non-fungi (10%), and sequences with no affiliation to any organisms in the public database (No-match) (16%). The high detection of the unidentified fungi and unassigned fungi was revealed in the whale-fall environments in this study. Some of these unidentified fungi are allied to early diverging fungi and they were more abundant in the sediments not directly in contact with whalebone. This study suggests that a cryptic fungal community exists in unique whale-fall ecosystems.

Marine ecosystems are continuously subjected to anthropogenic environmental pollution. Understanding the spread of pollution and the potential risks it poses to deep-sea ecosystems is important for developing better conservation measures. Here, we identified non-negligible levels of persistent organic pollutants in deep-sea chemosynthetic bivalves with limited or no filter feeding. The bivalves were collected from two sites: one located near a highly populated region and the other located relatively far from human activity. Analyses of samples collected nearly every decade in a period of 30 years suggested that environmental policy restrictions might be effective in reducing chemical pollution. However, the detection of contamination in deep-sea chemosynthetic animals suggests that the pollution could be spreading globally to chemosynthetic organisms with limited or no feeding. To protect these highly endemic and vulnerable deep-sea chemosynthetic ecosystems, our findings indicate that further research on chemical contamination and its effects on these ecosystems is required.

Few studies have investigated the prevalence of polybrominated diphenyl ethers (PBDEs) in deep-sea sharks. In this study, the levels and profiles of PBDEs were determined in liver samples of eight different species of deep-sea sharks collected in Suruga Bay, Japan. Widespread contamination of PBDEs in the deep-sea environment was reconfirmed in this study as these persistent organic pollutants (POPs) were detected in all specimens analyzed. Mean ΣPBDE levels in the deep-sea sharks ranged from 7 to 517 ng/g of lipid weight. The distribution patterns of BDE homologues were similar in all species where tetra-BDEs provided the dominant contribution to total PBDEs (46%). PBDEs levels were similar to, or higher than, those seen in other deep-sea sharks from different regions. The levels of PBDEs were likely to reflect their feeding preferences as higher PBDE levels were seen in species with higher trophic positions.

Oomycete infection of various freshwater animals, including salmonid eggs, causes significant economic damage to aquaculture worldwide. In this study, we detected oomycetes in infected chum salmon Oncorhynchus keta eggs at two hatcheries in northern Japan, in the source water used for egg incubation, and in the air at the hatcheries to clarify the source(s) of oomycete transmission using a DNA molecular marker. Seven oomycete taxa, belonging to Saprolegniaceae and Pythiaceae, were detected from the infected eggs. From the source water used for egg incubation and the air at the hatcheries, nine oomycete taxa, including those found on infected eggs, were detected, suggesting that both water and air are potential sources of oomycete transmission. There is no report of airborne transmission of these oomycetes detected in this study so far. Regarding protection and sterilization against oomycete infection in aquaculture hatcheries, not only water used at hatcheries but also the air in hatcheries may need to be considered hereafter.

We report the discovery of the deep-sea fungus, Alisea longicolla on a wood log artificially immersed at 495 m depths in deep sea, off the Nansei Islands, Japan. Three different species of wood logs, whale bones and coconuts were deployed at approximate depths of 250, 500, 1000, 2000, 3000 and 5000 m. Densely colonised A. longicolla was found on the Ubame oak (Quercus phillyraeoides) wood log from the 500 m deployment site, which was collected 1302 d (about 3 y and 7 mo) after the deployment. Alisea longicolla was originally described as a new genus of deep-sea ascomycete within the family Halosphaeriaceae, associated with sunken wood collected in 630–791 m water depths off the Vanuatu Islands. Our results provide further evidence to support that A. longicolla may be an endemic deep-sea fungus, and it grows slowly but is active and reproductive in deep-sea environments. The occurrence of obligate deep-sea fungi appears to be very rare in the environments and few data is available. Further investigation on A. longicolla will extend our understanding of the ecology, physiology and evolution of deep-sea fungi.

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 (Sao 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.

Graphical abstract, 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

Graphical abstract, 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

Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii.

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing., Graphical abstract Image, graphical abstract

The deep-sea is considered to be an ultimate sink for marine plastic debris. The abyssal (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. We undertook a quantitative assessment of debris presents on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific, which is one of the major transit points for massive amounts of debris sourced from Asia that are entering the North Pacific Ocean. The dominant type of debris was single-use plastics-mainly bags and food packaging. The density of plastic debris (average 4561 items km−2) in the abyssal zone was the highest recorded for an abyssal plain, suggesting that the deep-sea basin of the Northwest Pacific is a significant reservoir of plastic debris.

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., Graphical abstract Image, graphical abstract

To elucidate the localization of oomycetes (water molds) and bacteria in a single egg of chum salmon, Oncorhynchus keta, eggs infected with oomycetes were examined by bright-field microscopy, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH) using oomycete universal and bacterial species-specific probes. Furthermore, DNA barcoding for oomycetes and bacteria was performed on the chorion and cytoplasm of the eggs using genomic DNA extracted from the sections of eggs used for FISH analyses. Using bright-field microscopy and SEM, it was shown that oomycete hyphae penetrated the chorion and invaded the cytoplasm of the eggs. Based on DNA barcoding, two oomycetes (Pythium monospermum and Pythium sp. SE-OP1), and three bacterial species (Flavobacterium plurextorum, Flavobacterium sp. SE-BF1 and Undibacterium pigrum) were predominantly detected in the infected eggs. FISH analyses indicated that localization of the oomycetes was not noticeable different between the chorion and cytoplasm of the eggs. In contrast, FISH analyses using bacterial species-specific probes demonstrated clear differences in their localization. Flavobacterium sp. SE-BF1 and U. pigrum were predominantly found inside the chorion and around the oomycete hyphae invading the cytoplasm, respectively. These results are the first to indicate the possibility that U. pigrum has a specific relationship with oomycetes and coordinately invades eggs with oomycetes. However, it seems that Flavobacterium sp. SE-BF1 invades by a mechanism different from that of U. pigrum. Although this study is preliminary and the interaction between oomycetes and U. pigrum associated with chum salmon egg infection needs to be elucidated in detail, this finding may provide new insights into the infection mechanism of bacteria in salmon eggs, which is a longstanding problem in salmon hatcheries.

We report a novel nanoflagellate, Ophirina amphinema n. gen. n. sp., isolated from a lagoon of the Solomon Islands. The flagellate displays ‘typical excavate’ morphological characteristics, such as the presence of a ventral feeding groove with vanes on the posterior flagellum. The cell is ca. 4 µm in length, bears two flagella, and has a single mitochondrion with flat to discoid cristae. The flagellate exists in two morphotypes: a suspension-feeder, which bears flagella that are about the length of the cell, and a swimmer, which has longer flagella. In a tree based on the analysis of 156 proteins, Ophirina is sister to jakobids, with moderate bootstrap support. Ophirina has some ultrastructural (e.g. B-fibre associated with the posterior basal body) and mtDNA (e.g. rpoA–D) features in common with jakobids. Yet, other morphological features, including the crista morphology and presence of two flagellar vanes, rather connect Ophirina to non-jakobid or non-discobid excavates. Ophirina amphinema has some unique features, such as an unusual segmented core structure within the basal bodies and a rightward-oriented dorsal fan. Thus, Ophirina represents a new deeply-branching member of Discoba, and its mosaic morphological characteristics may illuminate aspects of the ancestral eukaryotic cellular body plan.

This study reports plastic debris pollution in the deep-sea based on the information from a recently developed database. The Global Oceanographic Data Center (GODAC) of the Japan Agency for Marine-Earth Science and Technology (JAMSTEC) launched the Deep-sea Debris Database for public use in March 2017. The database archives photographs and videos of debris that have been collected since 1983 by deep-sea submersibles and remotely operated vehicles. From the 5010 dives in the database, 3425 man-made debris items were counted. More than 33% of the debris was macro-plastic, of which 89% was single-use products, and these ratios increased to 52% and 92%, respectively, in areas deeper than 6000 m. The deepest record was a plastic bag at 10898 m in the Mariana Trench. Deep-sea organisms were observed in the 17% of plastic debris images, which include entanglement of plastic bags on chemosynthetic cold seep communities. Quantitative density analysis for the subset data in the western North Pacific showed plastic density ranging from 17 to 335 items km−2 at depths of 1092–5977 m. The data show that, in addition to resource exploitation and industrial development, the influence of land-based human activities has reached the deepest parts of the ocean in areas more than 1000 km from the mainland. Establishment of international frameworks on monitoring of deep-sea plastic pollution as an Essential Ocean Variable and a data sharing protocol are the keys to delivering scientific outcomes that are useful for the effective management of plastic pollution and the conservation of deep-sea ecosystems.

An initial multiple biogeochemical dataset was acquired from the first discovered asphalt seeps in the Brazil margin during deep-sea dive surveys in 2013 using a manned submersible. These surveys were conducted on the outer escarpment of the North São Paulo Plateau. Sediment cores taken from the submersible were processed for pore water and sediment biogeochemistry. The silica concentration, as a chemical geothermometer, showed a steep gradient in the pore water, which indicates the possibility of an active brine system operating in the seepage area. Rare earth elements were used as powerful tracers of chemical processes. Low rare earth element concentrations in both asphalt and Fe-Mn oxyhydroxide-phase sediments suggests that rare earth elements were released during the oil fractionation and biodegradation processes and further depleted under the reducing environment. The main bacterial communities of the sediment were Proteobacteria in the asphalt sites, while at non-asphalt sites, the main bacterial communities of sediment were Firmicutes. Stable carbon and nitrogen isotopes were used to determine the food sources of the heterotrophs, and results suggest that asphalt probably provides a carbon source for these benthic animals. This study may provide useful information to clarify the impact of heavy hydrocarbon seepage on the marine ecosystem.

Diplonemids were recently found to be the most species-rich group of marine planktonic protists. Based on phylogenetic analysis of 18S rRNA gene sequences and morphological observations, we report the description of new members of the genus Rhynchopus - R. humris sp. n. and R. serpens sp. n., and the establishment of two new genera - Lacrimia gen. n. and Sulcionema gen. n., represented by L. lanifica sp. n. and S. specki sp. n., respectively. In addition, we describe the organism formerly designated as Diplonema sp. 2 (ATCC 50224) as Flectonema neradi gen. n., sp. n. The newly described diplonemids share a common set of traits. Cells are sac-like but variable in shape and size, highly metabolic, and surrounded by a naked cell membrane, which is supported by a tightly packed corset of microtubules. They carry a single highly reticulated peripheral mitochondrion containing a large amount of mitochondrial DNA, with lamellar cristae. The cytopharyngeal complex and flagellar pocket are contiguous and have separate openings. Two parallel flagella are inserted sub-apically into a pronounced flagellar pocket. Rhynchopus species have their flagella concealed in trophic stages and fully developed in swimming stages, while they permanently protrude in all other known diplonemid species.

We investigated the fungal diversity in a total of 20 deep-sea sediment samples (of which 14 samples were associated with natural asphalt seeps and 6 samples were not associated) collected from two different sites at the Sao Paulo Plateau off Brazil by Ion Torrent PGM targeting ITS region of ribosomal RNA. Our results suggest that diverse fungi (113 operational taxonomic units (OTUs) based on clustering at 97% sequence similarity assigned into 9 classes and 31 genus) are present in deep-sea sediment samples collected at the Sao Paulo Plateau, dominated by Ascomycota (74.3%), followed by Basidiomycota (11.5%), unidentified fungi (7.1%), and sequences with no affiliation to any organisms in the public database (7.1%). However, it was revealed that only three species, namely Penicillium sp., Cadophora malorum and Rhodosporidium diobovatum, were dominant, with the majority of OTUs remaining a minor community. Unexpectedly, there was no significant difference in major fungal community structure between the asphalt seep and non-asphalt seep sites, despite the presence of mass hydrocarbon deposits and the high amount of macro organisms surrounding the asphalt seeps. However, there were some differences in the minor fungal communities, with possible asphalt degrading fungi present specifically in the asphalt seep sites. In contrast, some differences were found between the two different sampling sites. Classification of OTUs revealed that only 47 (41.6%) fungal OTUs exhibited >97% sequence similarity, in comparison with pre-existing ITS sequences in public databases, indicating that a majority of deep-sea inhabiting fungal taxa still remain undescribed. Although our knowledge on fungi and their role in deep-sea environments is still limited and scarce, this study increases our understanding of fungal diversity and community structure in deep-sea environments.

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.

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper-Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. T. hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. While Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans. This article is protected by copyright. All rights reserved.

In a previous report, we analyzed the stomach contents of juvenile chum salmon Oncorhynchus keta by morphological observation and also by molecular identification using the mitochondrial cytochrome c oxidase subunit I (COI) region. However, one of the most frequently detected COI sequences could not be assigned to any specific taxon, even at the phylum level. In the present study, we conducted in situ hybridization (ISH) on the stomach contents of juvenile chum salmon using the COI sequence and polymerase chain reaction amplification of a 18S ribosomal RNA gene from the tissue sections where ISH signals were detected. As a result, the organism that was enigmatic at the phylum level was found to be an appendicularian. Moreover, Oikopleura longicauda collected from the bay where the juvenile chum salmon samples were obtained was shown to have the same COI sequences as this taxonomic “orphan” COI sequence from the stomach contents. The present results suggest that the COI sequences previously deposited in public databases for “Oikopleura” are actually derived from taxonomic groups other than appendicularians, and that this may have hampered our understanding of prey richness in the stomach or gut of certain marine animals based on DNA barcoding.

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen-dependent multi-enzyme pathways. However, there are known exceptions―ciliated protozoans, such as Tetrahymena, along with diverse low-oxygen adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol-like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol-like lipids in eukaryotes. This article is protected by copyright. All rights reserved.