Rapid, contamination-less, and efficient environmental DNA filtration system.

Due to the sporadic distribution and trace amount of environmental DNA (eDNA) in deep-sea water, in the context of biodiversity monitoring, large volumes of filtration and multiple filtration replicates are required for eDNA metabarcoding. To address issues tied to the use of multiple filtration devices and large filtration volumes (e.g., contamination, time consumption, etc.), we have developed two systems for simple, rapid, and contamination-less filtration simultaneously that allow for the processing of multiple sample replicates from large volumes of water. First, the water from a Niskin bottle was filtered directly using a solenoid pump. Second, the pumped deep-sea water, using the siphon effect, was directly filtered by a filtration device driven by water pressure. This system can process 24 replicates simultaneously without the need for expensive equipment and active driving force. Compared with conventional filtering methods, e.g., peristaltic pumps, the proposed systems reduce filtration time, minimizing contamination, and enabling the simultaneous acquisition of multiple replicates. Overall, the systems presented here provide an effective approach for eDNA metabarcoding analysis, particularly for the filtration of large volumes of water containing small amounts of eDNA, such as deep-sea water. •The present systems reduce filtration time and contamination without water having to be transferred.•Simultaneous multiple replicates improve the efficiency and reliability of biodiversity assessments.

mTORC1 regulates phagosome digestion of symbiotic bacteria for intracellular nutritional symbiosis in a deep-sea mussel.

Phagocytosis is one of the methods used to acquire symbiotic bacteria to establish intracellular symbiosis. A deep-sea mussel, Bathymodiolus japonicus, acquires its symbiont from the environment by phagocytosis of gill epithelial cells and receives nutrients from them. However, the manner by which mussels retain the symbiont without phagosome digestion remains unknown. Here, we show that controlling the mechanistic target of rapamycin complex 1 (mTORC1) in mussels leads to retaining symbionts in gill cells. The symbiont is essential for the host mussel nutrition; however, depleting the symbiont's energy source triggers the phagosome digestion of symbionts. Meanwhile, the inhibition of mTORC1 by rapamycin prevented the digestion of the resident symbionts and of the engulfed exogenous dead symbionts in gill cells. This indicates that mTORC1 promotes phagosome digestion of symbionts under reduced nutrient supply from the symbiont. The regulation mechanism of phagosome digestion by mTORC1 through nutrient signaling with symbionts is key for maintaining animal-microbe intracellular nutritional symbiosis.

Complete Genomic Sequence of an Agarolytic Pseudoalteromonas Species Isolated from Deep Seawater.

We report the complete genomic sequence of the agarolytic bacterium Pseudoalteromonas sp. strain MM1, recovered from deep seawater. The genome has two circular chromosomes with sizes of 3,686,652 bp and 802,570 bp and GC contents of 40.8 and 40.0%, and it carries 3,967 protein-coding sequences, 24 rRNA genes, and 103 tRNA genes.

Investigation of the anti-tumor mechanism of tirabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, by phosphoproteomics and transcriptomics.

Tirabrutinib is a highly selective Bruton's tyrosine kinase (BTK) inhibitor used to treat hematological malignancies. We analyzed the anti-tumor mechanism of tirabrutinib using phosphoproteomic and transcriptomic methods. It is important to check the drug's selectivity against off-target proteins to understand the anti-tumor mechanism based on the on-target drug effect. Tirabrutinib's selectivity was evaluated by biochemical kinase profiling assays, peripheral blood mononuclear cell stimulation assays, and the BioMAP system. Next, in vitro and in vivo analyses of the anti-tumor mechanisms were conducted in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells followed by phosphoproteomic and transcriptomic analyses. In vitro kinase assays showed that, compared with ibrutinib, tirabrutinib and other second-generation BTK inhibitors demonstrated a highly selective kinase profile. Data from in vitro cellular systems showed that tirabrutinib selectively affected B-cells. Tirabrutinib inhibited the cell growth of both TMD8 and U-2932 cells in correlation with the inhibition of BTK autophosphorylation. Phosphoproteomic analysis revealed the downregulation of ERK and AKT pathways in TMD8. In the TMD8 subcutaneous xenograft model, tirabrutinib showed a dose-dependent anti-tumor effect. Transcriptomic analysis indicated that IRF4 gene expression signatures had decreased in the tirabrutinib groups. In conclusion, tirabrutinib exerted an anti-tumor effect by regulating multiple BTK downstream signaling proteins, such as NF-κB, AKT, and ERK, in ABC-DLBCL.

Buccal fat pad flap for transoral repair of radiation-induced necrosis in the lateral oropharyngeal wall.

Soft tissue necrosis (STN) is a late toxicity after radiotherapy. Extensive tissue defects due to STN near the carotid artery, such as in the lateral oropharyngeal wall, may lead to infectious pseudoaneurysms associated with fatal bleeding. Such defects are usually treated with transcervical reconstructive surgeries, which are highly invasive and technically difficult. We report a case in which a buccal fat pad (BFP) flap was used for minimally invasive transoral repair of tissue defects due to radiation-induced STN in the lateral oropharyngeal wall. The BFP flap covered the tissue defect, and the wound epithelialized completely. The patient had no dysfunctional mouth opening, speech, or swallowing. The BFP flap can be easily harvested via a minimally invasive transoral approach and is expected to be further utilized for radiation-induced STN in the lateral oropharyngeal wall.

Long-Term Outcomes of End-Flexible-Rigidscopic Transoral Surgery for Pharyngolaryngeal Cancer.

OBJECTIVE: End-flexible-rigidscopic transoral surgery (E-TOS) is a new and minimally invasive transoral surgery for resection of Tis-selected T3 pharyngolaryngeal cancers. We evaluated long-term oncological outcomes and whether postoperative voice and swallowing function were preserved following E-TOS. METHODS: In this retrospective single-center study, 154 patients treated with E-TOS using a curved retractor, flexible-tip rigid endoscope, and thin curved instruments were included. Their survival rate, larynx preservation rate, and disease control rate were estimated using the Kaplan-Meier method. Postoperative voice function was evaluated using both objective and subjective tests. Postoperative swallowing function was assessed using the Hyodo score and the functional outcome swallowing scale. RESULTS: The 3-year and 5-year overall survival, disease-specific survival, disease-free survival, laryngectomy-free survival, local control, and loco-regional control rates post E-TOS were 89.8% and 82.2%, 95.6% and 92.3%, 78.5% and 70.3%, 87.2% and 80.9%, 93.9% and 92.5%, and 87.2% and 85.7%, respectively. Both objective and subjective postoperative voice and swallowing function tests were within normal limits in more than 90% of the patients. CONCLUSION: E-TOS is an effective, safe, low-cost, and minimally invasive transoral surgery for Tis-selected T3 pharyngolaryngeal cancer; it also preserves postoperative voice, larynx, and swallowing function. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:1415-1424, 2023.

Heterologous expression and biochemical comparison of two homologous SoxX proteins of endosymbiotic Candidatus Vesicomyosocius okutanii and free-living Hydrogenovibrio crunogenus from deep-sea environments.

Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of Phreagena okutanii, a clam that inhabits deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli co-expressing cytochrome c maturation genes for comparative biochemical analysis. The VoSoxX recombinant had significantly lower thermal stability than HcSoxX, reflecting the difference in growth conditions of the source bacteria. The endosymbiont inhabits a mild intracellular environment, whereas the free-living bacterium dwells in a harsh environment. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, allowing further biochemical studies of the molecular mechanism of sulfur oxidation in deep-sea environments.

Phagocytosis of exogenous bacteria by gill epithelial cells in the deep-sea symbiotic mussel Bathymodiolus japonicus.

Animals that live in nutrient-poor environments, such as the deep sea, often establish intracellular symbiosis with beneficial bacteria that provide the host with nutrients that are usually inaccessible to them. The deep-sea mussel Bathymodiolus japonicus relies on nutrients from the methane-oxidizing bacteria harboured in epithelial gill cells called bacteriocytes. These symbionts are specific to the host and transmitted horizontally, being acquired from the environment by each generation. Morphological studies in mussels have reported that the host gill cells acquire the symbionts via phagocytosis, a process that facilitates the engulfment and digestion of exogenous microorganisms. However, gill cell phagocytosis has not been well studied, and whether mussels discriminate between the symbionts and other bacteria in the phagocytic process remains unknown. Herein, we aimed to investigate the phagocytic ability of gill cells involved in the acquisition of symbionts by exposing the mussel to several types of bacteria. The gill cells engulfed exogenous bacteria from the environment indiscriminately. These bacteria were preferentially eliminated through intracellular digestion using enzymes; however, most symbionts were retained in the bacteriocytes without digestion. Our findings suggest that regulation of the phagocytic process after engulfment is a key mechanism for the selection of symbionts for establishing intracellular symbiosis.

Molecular detection of a novel perkinsid associated with the deep-sea clam Phreagena okutanii.

Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

Recovery process of the muscle activities during walking with efficient early quadriceps training and gait exercises after total knee arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) by the trivector approach is less invasive to the knee extensor mechanism; early quadriceps training is possible and a good prognosis is expected after surgery. However, investigations regarding lower limb muscle activity during gait have not been reported after using the trivector approach. To determine an effective postoperative rehabilitation program, we analyzed the recovery processes of leg muscle activities during walking. METHODS: Fourteen subjects with severe knee osteoarthritis (OA) who underwent early exercises after TKA by trivector approach were included in the TKA group. The control group consisted of eight patients with mild knee OA. Surface electromyography of the vastus medialis (VM), vastus lateralis (VL), and rectus femoris (RF) muscles were recorded and gait speed and step length were measured. The TKA group was measured postoperatively at 3, 12, and 24 weeks. RESULTS: Gait speed of TKA group significantly increased at 12 weeks post-surgery and recovered equal to the control group at 24 weeks. Additionally, step length reached the level of control subjects at 24 weeks. Postoperative activity of VM returned to that of the control group at 12 weeks. VL continued decreasing until 12 weeks compared with the preoperative values, but gradually increased and became equal to the control group at 24 weeks. RF slightly increased at 3 weeks postoperation and remained stable. CONCLUSIONS: VM injury by the trivector approach might be small and temporary.Functional recovery of VL was important and early starting rehabilitation program up to 24 weeks is appropriate.

Massive digital gene expression analysis reveals different predictive profiles for immune checkpoint inhibitor therapy between adenocarcinoma and squamous cell carcinoma of advanced lung cancer.

BACKGROUND: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. METHODS: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test. RESULTS: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. CONCLUSIONS: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

Blocking EP4 down-regulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model.

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis and suppresses the anti-tumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient 3LL lung adenocarcinoma cell line was down-regulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs). On the basis of these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1 antibody was evaluated. EP4i, but not anti-PD-1 antibody, decreased tumor metabolism including glycolysis, fatty acid oxidation and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2-like to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 antibody treatment. Although CD8 T-cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 antibody treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicated a novel effect of EP4i for the metabolic reprogramming of tumors and revealed unique features of EP4i that can synergize with anti-PD-1 antibody to promote IFNγ production by NK cells, polarize TAMs into the M1 phenotype, and reduce hypoxia through normalization of the tumor vasculature.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish.

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.

Structural Comparison of Diplonemid Communities around the Izu Peninsula, Japan.

Diplonemea (diplonemids) is one of the most abundant and species-rich protist groups in marine environments; however, their community structures among local and seasonal samples have not yet been compared. In the present study, we analyzed four diplonemid community structures around the Izu Peninsula, Japan using barcode sequences amplified from environmental DNA. These sequences and the results of statistical analyses indicated that communities at the same site were more similar to each other than those in the same season. Environmental variables were also measured, and their influence on diplonemid community structures was examined. Salinity, electrical conductivity, and temperature, and their correlated variables, appeared to influence the structures of diplonemid communities, which was consistent with previous findings; however, since the results obtained did not reach statistical significance, further studies are required. A comparison of each diplonemid community indicated that some lineages were unique to specific samples, while others were consistently detected in all samples. Members of the latter type are cosmopolitan candidates and may be better adapted to the environments of the studied area. Future studies that focus on the more adaptive members will provide a more detailed understanding of the mechanisms by which diplonemids are widely distributed in marine environments and will facilitate their utilization as indicator organisms to monitor environmental changes.

Inhibition of mutant KRAS-driven overexpression of ARF6 and MYC by an eIF4A inhibitor drug improves the effects of anti-PD-1 immunotherapy for pancreatic cancer.

Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating ß1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations. Video abstract.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Symbiont Transmission onto the Cell Surface of Early Oocytes in the Deep-Sea Clam Phreagena okutanii.

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.

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.

Possible Roles of Hypotaurine and Thiotaurine in the Vesicomyid Clam Phreagena okutanii.

AbstractVesicomyid clams, which inhabit deep-sea hydrothermal vents and hydrocarbon seeps, are nutritionally dependent on symbiotic, chemoautotrophic bacteria that produce organic matter by using hydrogen sulfide. Vesicomyid clams absorb hydrogen sulfide from the foot and transport it in their hemolymph to symbionts in the gill. However, mechanisms to cope with hydrogen sulfide toxicity are not fully understood. Previous studies on vent-specific invertebrates, including bathymodiolin mussels, suggest that hypotaurine, a precursor of taurine, mitigates hydrogen sulfide toxicity by binding it to bisulfide ion, so as to synthesize thiotaurine. In this study, we cloned cDNAs from the vesicomyid clam Phreagena okutanii for the taurine transporter that transports hypotaurine into cells and for cysteine dioxygenase and cysteine-sulfinate decarboxylase, major enzymes involved in hypotaurine synthesis. Results of reverse-transcription polymerase chain reaction indicate that mRNAs of these three genes are most abundant in the foot, followed by the gill. However, hypotaurine and thiotaurine levels, measured by reverse-phase high-performance liquid chromatography, were low in the foot and high in the gill. In addition, thiotaurine was detected in hemolymph cells. Hypotaurine synthesized in the foot may be transported to the gill after binding to bisulfide ion, possibly by hemolymph cells.

Inside or out? Clonal thiotrophic symbiont populations occupy deep-sea mussel bacteriocytes with pathways connecting to the external environment.

Deep-sea Bathymodiolus mussels are generally thought to harbour chemosynthetic symbiotic bacteria in gill epithelial cells called bacteriocytes. However, previously observed openings at the apical surface of bacteriocytes have not been conclusively explained and investigated as to whether the Bathymodiolus symbiosis is intracellular or extracellular. In this study, we show that almost all the membranous chambers encompassing symbionts in a single bacteriocyte of Bathymodiolus septemdierum are interconnected and have pathways connecting to the external environment. Furthermore, the symbiont population colonising a single bacteriocyte is mostly clonal. This study hypothesises on a novel model of cellular localization at the interface between extra- and intracellular symbiosis, and the cellular-level process of symbiont acquisition in Bathymodiolus mussels.