Search

Hydrothermal vents and hydrocarbon seeps in the deep ocean are rare oases fueled by chemosynthesis. Biological communities inhabiting these ecosystems are often distributed in widely separated habitats, raising intriguing questions on how these organisms achieve connectivity and whether habitat types facilitate intraspecific divergence. The deep-sea patellogastropod limpet Bathyacmaea nipponica that colonizes both vents and seeps across ∼2,400 km in the Northwest Pacific is a feasible model to answer these questions. We analyzed 123 individuals from four vents and three seeps using a comprehensive method incorporating population genomics and physical ocean modeling. Genome survey sequencing and genotyping-by-sequencing resulted in 9,838 single-nucleotide polymorphisms for population genomic analyses. Genetic divergence and demographic analyses revealed four habitat-linked (i.e., three seep and one vent) genetic groups, with the vent genetic group established via the opportunistic invasion of a few limpet larvae from a nearby seep genetic group. TreeMix analysis uncovered three historical seep-to-vent migration events. ADMIXTURE and divMigrate analyses elucidated weak contemporary gene flow from a seep genetic group to the vent genetic group. Physical ocean modeling underlined the potential roles of seafloor topography and ocean currents in shaping the genetic connectivity, contemporary migration, and local hybridization of these deep-sea limpets. Our study highlighted the power of integrating genomic and oceanographic approaches in deciphering the demography and diversification of deep-sea organisms. Given the increasing anthropogenic activities (e.g., mining and gas hydrate extraction) affecting the deep ocean, our results have implications for the conservation of deep-sea biodiversity and establishment of marine protected areas.

Objectives Pembrolizumab is a standard treatment for non-small cell lung cancer (NSCLC) with high-PD-L1 expression; however, its effect is dismal in patients with poor physical condition. Additionally, the effect of immunotherapy is generally limited in NSCLC harboring driver mutations such asEGFR, ALK, or RET gene aberrations. Results We report the beneficial effect of pembrolizumab in a patient with poor performance status and PD-L1+ lung adenocarcinoma with theCCDC6–RET fusion gene and co-occurring NF1/TP53 mutations, complicated by multiple cancer-associated thrombi and respiratory failure. Conclusions Further studies are warranted to establish the role of co-occurring NF1/TP53 mutations as a positive predictive biomarker for pembrolizumab in NSCLC harboring RET fusion genes.

A new pardaliscid amphipod, Princaxelia marianaensissp. nov., is described from a single female captured at the Shinkai Seep Field, Mariana Trench, from a depth of 5,689–5,683 m. A key to species of Princaxelia is provided. This is the first species of Princaxelia to be described from the Mariana Trench, and the second report of this genus from this region.

Two species of dirivultid copepod (Siphonostomatoida), Stygiopontius senckenbergi Ivanenko & Ferrari, 2013 and Dirivultus kaiko sp. nov., were discovered from hydrothermal vent fields in the Okinawa Trough, the western North Pacific. Since S. senckenbergi was originally described based on two adult males from the New Ireland Fore-Arc system, Papua New Guinea, the discovery here represents the second record. This species was attached on ventral setae covered with filamentous bacteria of the deep-sea squat lobster Shinkaia crosnieri (Decapoda: Munidopsidae). The female of S. senckenbergi is described for the first time as well as the nauplius I and the copepodid IV. Sexual dimorphism is shown on several appendages (i.e. antennule, leg 2 and leg 5). The nauplius I shows typical lethithotrophic characteristics. Dirivultus kaiko sp. nov., which was found on the tentacular crown of the siboglinid tubeworm Lamellibrachia columna, differs from its two congeners by the antennule, maxilliped and leg 5 of both sexes. These are the second and third dirivultid species to be reported from Japan.

Genetic connectivity provides a basis for evaluating the resilience of animal communities by elucidating gene flow and serves as a proxy for realized dispersal through planktonic larval dispersal, settlement, and reproductive success. Gandalfus yunohana (Takeda, Hashimoto & Ohta, 2000) is a brachyuran crab endemic to deep-sea hydrothermal vents in the northwestern Pacific. Although an iconic and often dominant species, the connectivity among its populations is yet to be examined. We obtained barcoding sequences of the mitochondrial COI gene of G. yunohana from four vent fields including two on the Izu Arc, one on the northern Mariana Arc, and one in the Okinawa Trough. Genetic diversity of populations on the Izu and northern Mariana arcs were similar and shared the dominant haplotypes, showing no genetic subdivision regardless of the habitat depth. The Okinawa Trough population, for which only one specimen was available, was not genetically different from specimens from the Izu/Mariana arc populations. Estimation of the number of immigrants among populations suggests that the migration from north to south is higher than in the reverse direction. Our results resonate with previous laboratory-culture experiments, suggesting a high dispersal capability for G. yunohana. Visual observations and sampling, however, suggest that G. yunohana is exceedingly rare in the Okinawa Trough. This perhaps results from a lack of sufficient larval supply from the Izu-Bonin-Mariana Arc vents, and there may not be a reproductive population in the Okinawa Trough.

Stygiopontius copepods (Dirivultidae, Siphonostomatoida, Crustacea) are among the most successful meiobenthic organisms at deep-sea hydrothermal vents. Most of their ecology is not yet known, including the spatiotemporal differences in their sex ratios and their controlling factors. We investigated spatial variation in the sex ratio of adult Stygiopontius senokuchiae and its association with environmental parameters, including food quality at hydrothermal vent chimney structures in the calderas of three neighboring sea knolls (Bayonnaise Knoll, Myojin Knoll, and Myojin-sho Caldera) in the western North Pacific Ocean. Their sex ratio was significantly biased to males from 1:1, which was different from some of the other Stygiopontius species. The ratios did not show a significant correlation with the density of total adults. While previous studies have shown that the abundance of S. senokuchiae is positively associated with δ13C values of detritus on active chimneys, multivariate analyses in this study did not detect any significant association between their sex ratio and any investigated parameters including δ13C. These findings suggest that neither population density nor chemoautotrophic food availability drives the sexual difference in the spatial distribution around vents among the adults of the species.

The Scaly-foot Snail, Chrysomallon squamiferum, presents a combination of biomineralised features, reminiscent of enigmatic early fossil taxa with complex shells and sclerites such as sachtids, but in a recently-diverged living species which even has iron-infused hard parts. Thus the Scaly-foot Snail is an ideal model to study the genomic mechanisms underlying the evolutionary diversification of biomineralised armour. Here, we present a high-quality whole-genome assembly and tissue-specific transcriptomic data, and show that scale and shell formation in the Scaly-foot Snail employ independent subsets of 25 highly-expressed transcription factors. Comparisons with other lophotrochozoan genomes imply that this biomineralisation toolkit is ancient, though expression patterns differ across major lineages. We suggest that the ability of lophotrochozoan lineages to generate a wide range of hard parts, exemplified by the remarkable morphological disparity in Mollusca, draws on a capacity for dynamic modification of the expression and positioning of toolkit elements across the genome., The Scaly-foot Snail, Chrysomallon squamiferum, is a model for understanding the evolution of biomineralised armour. Here, the authors present a chromosome-level reference genome assembly and tissue-specific transcriptomic data for this enigmatic organism.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated as INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription dataset of INp10 from biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help better understand ecological strategies of this possibly widespread marine-specific methanotrophic clade. Importance The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus spp. suggests that the hydrothermal field is a promising locale to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus.

Variations in offspring production according to feeding strategies or food supply have been recognized in many animals from various ecosystems. Despite an unusual trophic structure based on non-photosynthetic primary production, these relationships remain largely under-studied in chemosynthetic ecosystems. Here, we useRimicarisshrimps from deep-sea hydrothermal vents as a study case to explore relations between reproduction, diets and food supply in these environments. For that, we compared reproductive outputs of three congeneric shrimps differing by their diets. They inhabit vents located under oligotrophic waters of tropical gyres with opposed latitudes, allowing us to also examine the prevalence of phylogenetic vs environmental drivers in their reproductive rhythms. For this we used both our original data and a compilation of published observations on the presence of ovigerous females covering various seasons over the past 35 years. We report distinct egg production trends betweenRimicarisspecies relying solely on chemosymbiosis –R. exoculataandR. kairei– and those relying on mixotrophy –R. chacei– whereR. chaceiproduces broods with higher numbers of smaller eggs. Besides, our data and historical records suggest a reproductive period with substantial proportions of brooding females mostly between January and early April for all examined species whatever the region. Intriguingly, this periodicity does not correspond to seasonal variations in surface production with presence of brooding females during either boreal winter or austral summer. These observations contrast with the long-standing paradigm in deep-sea species for which periodic reproductive patterns have always been attributed to seasonal variations of photosynthetic production sinking from surface. Our results suggest the presence of intrinsic basis for biological rhythms in the deep sea, and bring to light the importance of having year-round observations in order to understand life history of vent animals.

Anti-programmed cell death 1 (PD-1) antibodies have poor efficacy in epidermal growth factor receptor (EGFR)-mutated lung cancer. We herein report a 72-year-old man with programmed cell death-ligand 1 (PD-L1)-negative lung adenocarcinoma harboring an EGFR mutation that responded to nivolumab for more than 2 years. A pathological examination revealed infiltration of CD8-positive lymphocytes and macrophages expressing CD68, CD206, and PD-L1 into the PD-L1-negative tumor; CD206 expression is a marker of immunosuppressive tumor-associated macrophages (TAMs). The presence of PD-L1-positive TAMs in the tumor environment might be a predictor of a positive response to anti-PD-1 antibodies.

Osimertinib, a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is the standard treatment for patients with lung cancer harboring EGFR T790M; however, acquired resistance is inevitable due to genetic and epigenetic changes in cancer cells. In addition, a recent randomized clinical trial revealed that the combination of osimertinib and bevacizumab failed to exhibit superior progression-free survival compared with osimertinib alone. The present study aimed to investigate the effect of triple therapy with osimertinib, bevacizumab and cetuximab in xenograft tumors with different initial tumor volumes (conventional model, 200 mm3 and large model, 500 mm3). The results demonstrated that osimertinib significantly inhibited tumor growth in both the conventional and large models; however, maximum tumor regression was attenuated in the large model in which hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-α (TGF-α) expression levels increased. Although the combination of osimertinib and bevacizumab exerted a greater inhibitory effect on tumor growth compared with osimertinib in the conventional model, the effect of this combination therapy was attenuated in the large model. TGF-α attenuated sensitivity to osimertinib in vitro; however, this negative effect was counteracted by the combination of osimertinib and cetuximab, but not osimertinib and bevacizumab. In the large xenograft tumor model, the triple therapy induced the greatest inhibitory effect on tumor growth compared with osimertinib alone and its combination with bevacizumab. Clinical trials of the triple therapy are required for patients with lung cancer with EGFR mutations and HIF-1α/TGF-α.

A Correction to this paper has been published: 10.1007/s12526-021-01192-x

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.

Molecular agents targeting the epidermal growth factor receptor (EGFR)‐, anaplastic lymphoma kinase (ALK)‐ or c‐ros oncogene 1 (ROS1) alterations have revolutionized the treatment of oncogene‐driven non‐small‐cell lung cancer (NSCLC). However, the emergence of acquired resistance remains a significant challenge, limiting the wider clinical success of these molecular targeted therapies. In this study, we investigated the efficacy of various molecular targeted agents, including erlotinib, alectinib, and crizotinib, combined with anti‐vascular endothelial growth factor receptor (VEGFR) 2 therapy. The combination of VEGFR2 blockade with molecular targeted agents enhanced the anti‐tumor effects of these agents in xenograft mouse models of EGFR‐, ALK‐, or ROS1‐altered NSCLC. The numbers of CD31‐positive blood vessels were significantly lower in the tumors of mice treated with an anti‐VEGFR2 antibody combined with molecular targeted agents compared with in those of mice treated with molecular targeted agents alone, implying the antiangiogenic effects of VEGFR2 blockade. Additionally, the combination therapies exerted more potent antiproliferative effects in vitro in EGFR‐, ALK‐, or ROS1‐altered NSCLC cells, implying that VEGFR2 inhibition also has direct anti‐tumor effects on cancer cells. Furthermore, VEGFR2 expression was induced following exposure to molecular targeted agents, implying the importance of VEGFR2 signaling in NSCLC patients undergoing molecular targeted therapy. In conclusion, VEGFR2 inhibition enhanced the anti‐tumor effects of molecular targeted agents in various oncogene‐driven NSCLC models, not only by inhibiting tumor angiogenesis but also by exerting direct antiproliferative effects on cancer cells. Hence, combination therapy with anti‐VEGFR2 antibodies and molecular targeted agents could serve as a promising treatment strategy for oncogene‐driven NSCLC., We found that VEGFR2 blockade augmented the anti‐tumor effects of molecular targeted agents in oncogene‐driven NSCLC, particularly in EGFR/ALK/ROS1‐driven NSCLC. We also identified 2 mechanisms underlying the synergistic effects of anti‐VEGFR2 therapy with molecular targeted agents. VEGFR2 blockade not only inhibited tumor angiogenesis but also exerted direct antiproliferative effects on cancer cells.

Having a noncoiled, limpet-like shell is a characteristic shared by numerous gastropod molluscs, including many lineages outside the true limpets (Patellogastropoda) where it has evolved convergently. The shell shape of limpet-formed gastropods has often been used as a key taxonomic character, and although studies have shown that it can vary depending on the substrate morphology, these have mostly been examples from true limpets. Over a dozen origins of limpet-form are known in Vetigastropoda, and these limpets are still generally assumed to have rather stable shell forms that are useful for taxonomy and species identification. Here, we show that the vetigastropod limpet Lepetodrilus nux (Okutani, Fujikura & Sasaki, 1993) from a deep-sea hot vent in the Okinawa Trough develop distinct shell forms when living on different substrate types. Sequences of the barcoding region of the mitochondrial cytochrome c oxidase subunit I gene among the three forms only differed by 0.31–0.63% (K2P distance) in a 637 bp alignment, in line with the differences in shell morphology being intraspecific. The extent of shell form shift seen in this species is likely the largest reported for a vetigastropod limpet and provide further evidence that such plasticity is not phylogenetically constrained but is an intrinsic part of having a limpet-like shell.

Pectinodontid limpets of the genus Bathyacmaea are endemic to hot vents and cold seeps and exhibit greatly variable shell and radular macro-morphologies, rendering reliable species-level identification challenging. Here, we analyzed shell microstructures of western Pacific vent/seep Bathyacmaea limpets using scanning electron microscopy and Raman spectrophotometry to test its usefulness in providing phylogenetic signals. Bathyacmaea shells comprised of two forms of calcitic microstructure including irregular spherulitic prismatic type-A (ISP type-A) and semi-foliated (SF), as well as the aragonitic crossed lamellar (CL) microstructure. Despite marked differences in macroscopic shell morphologies once leading them to be classified into different species or even genera, six morphotypes of Bathyacmaea nipponica from different chemosynthetic localities and substrates shared an outermost ISP-A layer and alternating layers of SF and CL structures in their outer and inner shell layers. A genetically divergent lineage recovered from the South Chamorro Seamount, however, differed in having a simple three-layered shell composition consisting of ISP-A, SF, and CL structures, in that order, from the outside, and an unusually thin inner shell layer consisting of only CL structure. Moreover, the ratio of aragonite and calcite varied with habitat conditions, with calcite dominating in vents and aragonite dominating in seeps. These results suggest that the shell microstructure of pectinodontids is under phylogenetic constraints and provides useful taxonomic signals, while the mineral polymorphism in aragonite/calcite ratio varies according to environmental factors. Furthermore, microstructures of two ‘species’ from Cretaceous seeps confirmed the same patterns in fossil lineages.

Studying population genetics of deep-sea animals helps us understand their history of habitat colonization and population divergence. Here, we report a population genetic study of the deep-sea mussel Bathymodiolus platifrons (Bivalvia: Mytilidae) widely distributed in chemosynthesis-based ecosystems in the Northwest Pacific. Three mitochondrial genes (i.e., atp6, cox1, and nad4) and 6,398 genomewide single nucleotide polymorphisms (SNPs) were obtained from 110 individuals from four hydrothermal vents and two methane seeps. When using the three mitochondrial genes, nearly no genetic differentiation was detected for B. platifrons in the Northwest Pacific. Nevertheless, when using SNP datasets, all individuals in the South China Sea (SCS) and three individuals in Sagami Bay (SB) together formed one genetic cluster that was distinct from the remaining individuals. Such genetic divergence indicated a genetic barrier to gene flow between the SCS and the open Northwest Pacific, resulting in the co-occurrence of two cryptic semi-isolated lineages. When using 125 outlier SNPs identified focusing on individuals in the Okinawa Trough (OT) and SB, a minor genetic subdivision was detected between individuals in the southern OT (S-OT) and those in the middle OT (M-OT) and SB. This result indicated that, although under the influence of the Kuroshio Current and the North Pacific Intermediate Water, subtle geographic barriers may exist between the S-OT and the M-OT. Introgression analyses based on these outlier SNPs revealed that Hatoma Knoll in the S-OT represents a possible contact zone for individuals in the OT-SB region. Furthermore, migration dynamic analyses uncovered stronger gene flow from Dai-yon Yonaguni Knoll in the S-OT to the other local populations, compared to the reverse directions. Taken together, the present study offered novel perspectives on the genetic connectivity of B. platifrons mussels, revealing the potential interaction of ocean currents and geographic barriers with adaption and reproductive isolation in shaping their migration patterns and genetic differentiation in the Northwest Pacific.

The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has shown dramatic effects in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusion genes. However, patients inevitably develop resistance to this agent. Therefore, a new treatment strategy is required for lung tumors with ROS1 fusion genes. In the present study, lung cancer cell lines, HCC78 harboring SLC34A2-ROS1 and ABC-20 harboring CD74-ROS1, were used as cell line-based resistance models. Crizotinib-resistant HCC78R cells were established from HCC78. We comprehensively screened the resistant cells using a phosphor-receptor tyrosine kinase array and RNA sequence analysis by next-generation sequencing. HCC78R cells showed upregulation of HB-EGF and activation of epidermal growth factor receptor (EGFR) phosphorylation and the EGFR signaling pathway. Recombinant HB-EGF or EGF rendered HCC78 cells or ABC-20 cells resistant to crizotinib. RNA sequence analysis by next-generation sequencing revealed the upregulation of AXL in HCC78R cells. HCC78R cells showed marked sensitivity to EGFR-TKI or anti-EGFR antibody treatment in vitro. Combinations of an AXL inhibitor, cabozantinib or gilteritinib, and an EGFR-TKI were more effective against HCC78R cells than monotherapy with an EGFR-TKI or AXL inhibitor. The combination of cabozantinib and gefitinib effectively inhibited the growth of HCC78R tumors in an in vivo xenograft model of NOG mice. The results of this study indicated that HB-EGF/EGFR and AXL play roles in crizotinib resistance in lung cancers harboring ROS1 fusions. The combination of cabozantinib and EGFR-TKI may represent a useful alternative treatment strategy for patients with advanced NSCLC harboring ROS1 fusion genes.

Due to increasing anthropogenic impacts on deep-sea hydrothermal vent ecosystems, it is essential to understand population structure and maintenance through larval recruitment and recovery of vent faunas after disturbances. In this study, we quantified vent animal recruitment in the Okinawa Trough, in the western Pacific Ocean. This is the first study to investigate recruitment patterns at a man-made hydrothermal vent. Colonization plates were deployed at three sites. Site 1 manifested new hydrothermal shimmering with small chimneys, white bacterial mats, and some alvinocaridid shrimp that arrived after drilling. Site 2 showed no evidence of newly arrived foundation species after drilling, and Site 3 had pre-existing animal communities in the vicinity of the new vent. Twenty-two months after deployment, colonization plates were retrieved and recruited animals were inventoried. Species composition and abundance differed among sites, but relatively high similarity in species composition was observed at Sites 1 and 3, though not at Site 2. Newly established communities on the plates at Sites 1 and 2 (no pre-existing fauna) showed lower species richness and abundance than at Site 3. Differences in abundance and size-frequency distributions of major recruits on the plates (i.e. Lepetodrilus nux , Bathymodiolus spp.) suggest the importance of reproductive and early life-history characteristics in spatial variability of recruitment. Lepetodrilus nux populations established on the plates at Site 1 showed high genetic connectivity. These results illustrate the importance of localized recruitment, which may have a significant impact on sustainability of vent faunal populations, despite the existence of regional metapopulations.

Recent genetic research has begun to reveal population structures of deep-sea, hydrothermal vent species, but detailed assessments of genetic diversity and connectivity in hydrothermal vent populations, based on multiple genetic loci, are still scarce, especially in the Northwest Pacific. Accordingly, we isolated 38 novel polymorphic microsatellite loci from the limpet, Lepetodrilus nux, and 14 from the squat lobster, Shinkaia crosnieri, two dominant hydrothermal vent species, using next-generation sequencing. These loci revealed polymorphism levels of 5–20 alleles per locus in L. nux and 5–25 in S. crosnieri. Observed and expected heterozygosities ranged from 0.240 to 0.960 and 0.283 to 0.938 in L. nux and from 0.450 to 0.950 and 0.620 to 0.941 in S. crosnieri, respectively. Twelve loci in L. nux and four loci in S. crosnieri showed significant deviation from Hardy–Weinberg equilibrium (p

The present study aimed to generate genome-wide single nucleotide polymorphisms (SNPs) for the deep-sea mussel Bathymodiolus platifrons via a combination of genome survey sequencing and the type IIB endonuclease restriction-site associated DNA (2b-RAD) sequencing, assess the potential use of SNPs in detecting fine-sale population genetic structure and signatures of divergent selection, as well as their cross-species application in other bathymodioline mussels. Genome survey sequencing was conducted for one individual of B. platifrons. De novo assembly resulted in 781,720 sequences with a scaffold N50 of 2.9 kb. Using these sequences as a reference, 9307 genome-wide SNPs were identified by 2b-RAD for 28 B. platifrons individuals collected from a seep and a vent population. Among these SNPs, nine outliers showed significant evidence for divergent selection, and their positions in the genes or scaffolds were identified. The F ST estimated based on the putative neutral SNPs was low (0.0126) indicating the two B. platifrons populations having a high genetic connectivity. However, the permutation test detected significant differences ( P Bathymodiolus japonicus , Bathymodiolus aduloides and Idas sp. with different phylogenetic distances from B. platifrons. Overall, our study has demonstrated the feasibility and effectiveness of combining genome survey sequencing and 2b-RAD to rapidly generate genomic resources for use in fine-scale population genetic studies, and various cross-species applications.

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

The ‘Shinkai Seep Field’ is a serpentinite-hosted chemosynthetic ecosystem in the Southern Mariana Forearc. In June 2015 the site was revisited and a number of rissoiform gastropods were collected. Taxonomic investigations revealed that these specimens represent a hitherto undescribed species ofProvanna(Gastropoda: Abyssochrysoidea), described herein asProvanna cingulatan. sp. This new species is characterized by numerous spiral keels, lack of significant axial sculpture, rounded and inflated whorls, and large size for the genus. With the shell height exceeding 16.5 mm (may reach 20 mm), it is the largestProvannaspecies known thus far. Phylogenetic analysis using 411 bp of the cytochrome oxidase c subunit I (COI) gene confirmed its systematic placement within the genusProvanna. This is the only gastropod from a family endemic to chemosynthetic ecosystems thus far known from the ‘Shinkai Seep Field’. Furthermore, with a collection depth of 5687 m, it represents the deepest known bathymetric range for the superfamily Abyssochrysoidea as a whole.

Temperature is an important factor affecting the distributions and life-history traits of marine animals. Deep-sea hydrothermal vents are suitable environments to examine ecologic differences related to temperature, due to the steep temperature gradients around the vents. Rearing experiments under various temperature conditions (5–30 °C) at atmospheric pressure demonstrated a difference in thermal effects on egg hatching and larva in two co-occurring, vent-associated alvinocaridid shrimps – the peripherally distributed Alvinocaris longirostris and the centrally distributed Shinkaicaris leurokolos. The duration before hatching became shorter as temperature increased, while the maximum hatching rate occurred at higher temperatures in S. leurokolos (10–20 °C) than in A. longirostris (10 °C). Hatched larvae of both species were negatively buoyant, and larva with normal abdominal length could actively swim and stay suspended in the mid- or surface water layers of the culture plates under our experimental conditions. However, no larvae settled or metamorphosed into juveniles under the rearing conditions used in this study. Larvae with shortened abdomens occurred under most of the experimental conditions, although they were less frequent at 10 °C in A. longirostris and 20 °C in S. leurokolos. The maximum survival periods at these temperatures were 88 days in A. longirostris and 30 days in S. leurokolos. These characteristics may cause differences in the distributional ranges of the two species. The present results indicate that temperature is an important factor controlling life-history traits of vent shrimps.

The South Chamorro Seamount (13°47'N, 146°00'E) on the southeastern Mariana Forearc is one of a series of serpentine mud volcanoes in the area, and hosts an active fluid seepage area on its...

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep-sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution.

Background: Non-small cell lung cancers (NSCLCs) harboring driver oncogene such as epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement are sensitive to the corresponding molecular targeted tyrosine kinase inhibitors (TKIs). However, acquired resistance inevitably develops, and more effective treatment strategies are needed. Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab significantly prolongs progression free survival when added to first-generation EGFR TKIs in EGFR mutant NSCLC. However, it remains unknown whether anti-VEGF receptor (VEGFR) antibody enhances the efficacy of molecular targeted agents as well. In this study, we investigated combinations with an anti-VEGFR antibody and EGFR or ALK TKIs in preclinical models Materials and Methods: We treated mice bearing subcutaneous tumor cell lines harboring EGFR mutation (PC-9, H3255) or ALK rearrangement (ABC-11, H3122) using combinations of anti-mouse VEGFR antibody DC101 and erlotinib (EGFR TKI) /alectinib (ALK TKI). Neovascularization of the tumors were determined by CD31 immunostaining. To determine the effects of VEGFR antibody on cancer cell itself, we also examined the efficacy of the combinations in vitro. Results: Combination therapy demonstrated significantly better tumor inhibition compared to erlotinib or alectinib alone in all the examined xenograft tumors. CD31 positive blood vessel staining was significantly reduced in the tumors treated with combination therapy. Mutant EGFR transfection in HEK293T cells increased VEGF expression in the cell culture supernatant. Furthermore, MTT assay showed significantly enhanced cell growth inhibition when anti-human VEGFR antibody ramucirumab combined with erlotinib (against PC-9 or H3255 cells) or alectinib (against ABC-11 or H3122 cells) suggesting that anti-VEGFR antibody affects not only on tumor vasculature but cancer cell itself. Consistent with this finding, combination with siVEGFR and erlotinib also inhibited the cell proliferation significantly better than erlotinib alone in PC-9 cells. Conclusion: A significant combination benefit was observed with anti-VEGFR2 antibody and EGFR or ALK TKI both in vivo and in vitro, suggesting anti-VEGFR antibody inhibits not only the blood vessels but may also exert direct anticancer effects. Combination therapy of erlotinib or alectinib and anti-VEGFR2 antibody could be a promising treatment strategy for oncogene addicted NSCLC. Citation Format: Hiromi Watanabe, Eiki Ichihara, Hiroe Kayatani, Hisao Higo, Go Makimoto, Hirohisa Kano, Kazuya Nishii, Naofumi Hara, Kiichiro Ninomiya, Toshio Kubo, Kadoaki Ohashi, Kammei Rai, Katsuyuki Hotta, Masahiro Tabata, Yoshinobu Maeda, Katsuyuki Kiura. Significant combination benefit of anti-VEGFR antibody and oncogene-targeted agents in EGFR or ALK mutant NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2131.

Although many deep-sea species are considered to have transoceanic distribution, this assumption has rarely been critically tested. Using Hesiolyra (Hesionidae) as a case study, we showed that careful molecular and morphological analyses are required for refuting/accepting claims about such wide distribution. Hesiolyra is a genus of polychaetes commonly found in hydrothermal vents of the Eastern Pacific, Atlantic and Indian Ocean. Nevertheless, H. bergi Blake, 1985, a species originally described from the Eastern Pacific Rise (EPR), is the only recognized species in this genus. Phylogenetic analyses based on the COI and 16 S rRNA genes revealed two monophyletic clades that were distinct from a clade consisting of H. bergi sequences. With the average K2P distances from 7.69% to 14.17% for the COI gene, we erected two new species of Hesiolyra, herein named as H. longqiensis n. Sp. from the Southwest Indian Ridge (SWIR) and H. heteropoda n. Sp. from the Central Indian Ridge (CIR) and the Carlsberg Ridge (CR). Among them, H. longqiensis n. Sp. from SWIR is more closely related to H. bergi from EPR than to H. heteropoda n. Sp. from CIR and CR, which called for alternative hypotheses on how historical oceanographic conditions might have driven the dispersal and speciation of this genus along the global mid-ocean ridges. Morphologically, the two newly described species can be easily distinguished from H. bergi. The notopodia and neuropodia of H. bergi and H. longqiensis n. Sp., are of similar sizes, but the morphology of their lyrate chaetae and their dorsal cirri alternation patterns are different. In H. heteropoda n. Sp., the notopodia are remarkably slimmer than the neuropodia, and it does not possess lyrate chaetae. Overall, in this study we have described two new species of Hesiolyra from the Indian Ocean ridges and show that the distribution of H. bergi is restricted to the Eastern Pacific Rise. Future exploration of other mid-ocean ridges is required to discover the true diversity of this genus of polychaetes inhabiting hydrothermal vent fields.

The detection of certain oncogenic driver mutations, including those of epidermal growth factor receptor (EGFR), is essential for determining treatment strategies for advanced non‑small cell lung cancer (NSCLC). The current study assessed the feasibility of testing exhaled breath condensate (EBC) for EGFR mutations by droplet digital PCR (ddPCR). Samples were collected from 12 patients with NSCLC harboring EGFR mutations that were admitted to Okayama University Hospital between June 1, 2014 and December 31, 2017. A total of 21 EBC samples were collected using the RTube™ method and EGFR mutations (L858R, exon 19 deletions or T790M) were assessed through ddPCR analysis (EBC‑ddPCR). A total of 3 healthy volunteer samples were also tested to determine a threshold value for each mutation. Various patient characteristics were determined, including sex (3 males and 9 females), age (range 54‑81 years; median, 66 years), smoking history (10 had never smoked; 2 were former smokers), histology (12 patients exhibited adenocarcinoma), clinical stage (9 patients were stage IV; 3 exhibited post‑operative recurrence) and EGFR mutation type (4 had L858R; 8 had exon 19 deletions; 8 had T790M). EBC‑ddPCR demonstrated positive droplets in 8 of the 12 patients. The sensitivity and specificity of each mutation was as follows: 27.3 and 80.0% for EGFR L858R, 30.0 and 90.9% for EGFR Ex19del, and 22.2 and 100% for EGFR T790M. EBC‑ddPCR analysis of EGFR mutations exhibited modest sensitivity and acceptable specificity. EBC‑ddPCR is a minimally invasive and replicable procedure and may be a complementary method for EGFR testing in patients where blood or tissue sampling proves difficult.

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

Pulmonary pleomorphic carcinoma (PPC) is a rare very aggressive subtype of non-small cell lung cancer. We herein report a case of PPC that showed a rapid response to nivolumab. The patient, whose multiple tumors had progressed very aggressively, was treated with nivolumab, an anti-programmed cell death-1 (PD-1) antibody. The tumors dramatically shrank after one cycle of nivolumab. The tumors were positive for programmed cell death ligand 1 (PD-L1). An immunohistochemical analysis revealed numerous PD-1+, CD68+ and CD206+ macrophages. This PD-1 antibody may be a good treatment option, especially in tumors that express PD-L1 and which show PD-1+ macrophage infiltration.

BackgroundGastropods of the genusProvannaare abundant and widely distributed in deep-sea chemosynthetic environments with seven extant species described in the northwestern Pacific.MethodsWe investigated the population history and connectivity of fiveProvannaspecies in the northwestern Pacific through population genetic analyses using partial sequences of the cytochromecoxidase subunit I gene.ResultsWe found thatP. subglabra, the most abundant and genetically diverse species, is genetically segregated by depth. Among the five species, the three comparatively shallower species (P. lucida, P. kuroshimensis, P. glabra) had a more constant demographic history compared to the deeper species (P. subglabra,P. clathrata).DiscussionEnvironmental differences, especially depth, appears to have a role in the segregation ofProvannasnails. The population ofP. clathratain the Irabu Knoll appears to have expanded afterP. subglabrapopulation. The remaining three species,P. lucida,P. kuroshimensis, andP. glabra, are only known from a single site each, all of which were shallower than 1,000 m. These data indicate thatProvannagastropods are vertically segregated, and that their population characteristics likely depend on hydrothermal activities.

A new species of dirivultid copepod (Siphonostomatoida) is described from hydrothermal vents in a volcanic seamount in Izu-Bonin Arc, western part of North Pacific Ocean. The copepod was collected during the research cruise NT13-09 using the R/V Natsushima with the ROV Hyper-Dolphin in April 2013. The type series of the new species was collected from the populations of Paralvinella spp. (Annelida: Alvinellidae) on an active vent chimney at the depth of 795 m. Stygiopontius senokuchiae n. sp. is most closely related to S. teres Humes, 1996 but clearly distinguished from the latter species by the possession of the following characters: the basis of leg 1 with an attenuated inner process; the genital double somite with a conical process lateral to the genital opening; and caudal rami without distal process. The findings of the copepod in the present study represents the first record of nominal species of the Dirivultidae from Japanese waters and a record of the shallowest depth of the genus. A key to species of the genus Stygiopontius from Western Pacific is provided.

Two new species of the crangonid shrimp genus Metacrangon Zarenkov, 1965, are described and illustrated on the basis of materials collected from the Okinawa Trough, Ryukyu Islands, southern Japan, during diving operations of remotely operated vehicles (ROVs): M. ryukyu n. sp. from off Iheya Island, at depth of 986 m; and Metacrangon kaiko n. sp. from NE of Yonaguni Island, at depth of 2205 m. The two new species resemble members of the M. munita (Dana, 1852) species group, but are both characteristic in having setose dactyli on pereopods 4 and 5. Some minor differences in morphology and genetic analysis using partial sequences of the barcoding mitochondrial COI gene support the recognition of the two new species. Holotypes of the two new species were collected from hydrothermally influenced areas, representing a previously unknown habitat for species of Metacrangon.

Understanding the formative factors of connectivity among deep-sea chemosynthetic communities may clarify the biogeographic mechanisms that facilitate the establishment and development of vent/seep fauna. This study identified interspecific differences in the geographical distribution and genetic population structure of 4 alvinocaridid shrimp species inhabiting hydrothermal vents in the Okinawa Trough, Northwest Pacific. Shinkaicaris leurokolos populations were widely distributed at 580 to 1651 m depth, with partial nucleotide sequences of the mitochondrial COI gene revealing extremely high genetic diversity (haplotype diversity: 0.98 to 1.00). In contrast, local populations of Alvinocaris dissimilis and A. longirostris, both of which have narrower bathymetric distributions, were characterised by low haplotype diversity (0.29 to 0.52). In the Okinawa Trough, Alvinocaris sp. only occurs at the Irabu Knoll, but this species also inhabits the Suiyo Seamount on the volcanic front of the Izu-Bonin Arc (Northwest Pacific). This undescribed species had relatively high haplotype diversity (0.70 to 0.82), with no genetic differentiation between the 2 sites. Certain factors may allow this species to occur at both sites (e.g. a chemical environment derived from similar substratum), despite geological differences in hydrothermal circulation (i.e. a back-arc basin versus an arc volcanic front, respectively). The findings of this study indicate that (1) the wide geographical and bathymetrical distributional ranges of the vent shrimp species cause high genetic diversity associated with stable population connectivity and (2) certain environmental features (e.g. depth and substratum) and life-history traits (e.g. feeding habitat and larval characteristics) represent important formative factors in the connectivity of alvinocaridids in the Northwest Pacific.

We herein report a case of congestive heart failure which developed during osimertinib treatment. A 78-year-old woman presented with mild exertional dyspnea three weeks after starting osimertinib for the treatment of epidermal growth factor receptor (EGFR) T790M-positive non-small cell lung cancer. She was diagnosed with congestive heart failure caused by the osimertinib. In contrast to trastuzumab, a human epidermal growth factor receptor 2 (HER2) monoclonal antibody that often causes cardiac dysfunction, the causal relationship between osimertinib and cardiotoxicity has so far received little attention and thus remains unclear. However, it inhibits HER2 in addition to mutant EGFR, thereby potentially causing cardiotoxicity.

The characterization of evolutionary and biogeographical patterns is of fundamental importance to identify factors driving biodiversity. Due to their widespread but discontinuous distribution, deep‐sea hydrothermal vent barnacles represent an excellent model for testing biogeographical hypotheses regarding the origin, dispersal and diversity of modern vent fauna. Here, we characterize the global genetic diversity of vent barnacles to infer their time of radiation, place of origin, mode of dispersal and diversification. Our approach was to target a suite of multiple loci in samples representing seven of the eight described genera. We also performed restriction‐site associated DNA sequencing on individuals from each species. Phylogenetic inferences and topology hypothesis tests indicate that vent barnacles have colonized deep‐sea hydrothermal vents at least twice in history. Consistent with preliminary estimates, we find a likely radiation of barnacles in vent ecosystems during the Cenozoic. Our analyses suggest that the western Pacific was the place of origin of the major vent barnacle lineage, followed by circumglobal colonization eastwards through the Southern Hemisphere during the Neogene. The inferred time of radiation rejects the classic hypotheses of antiquity of vent taxa. The timing and the mode of origin, radiation and dispersal are consistent with recent inferences made for other deep‐sea taxa, including nonvent species, and are correlated with the occurrence of major geological events and mass extinctions. Thus, we suggest that the geological processes and dispersal mechanisms discussed here can explain the current distribution patterns of many other marine taxa and have played an important role shaping deep‐sea faunal diversity. These results also constitute the critical baseline data with which to assess potential effects of anthropogenic disturbances on deep‐sea ecosystems.

After the adoption of the Aichi Target, data accumulation and evaluation regarding biodiversity have progressed rapidly. The use of ecologically and biologically significant areas (EBSAs) criteria to evaluate important areas enables the identification of effective and prioritized areas for ecosystem management. This includes strategic environmental assessment and discussions aimed at establishing protected marine areas based on scientific data. This paper reviews previous and current ideas as well as the methods used, for the identification of EBSAs. In particular, the following issues are addressed: problems associated with different types of marine ecosystems in the Japanese Archipelago, such as seagrass and seaweed beds, coral reefs, offshore pelagic plankton, and deep-sea benthic ecosystems; and problems associated with the integration of multiple criteria that are not totally exclusive. Several candidate variables accounting for each of the 7 criteria used to identify ecologically important areas are presented. Data availability is the most important criterion that allowed for the comprehensive evaluation of different types of ecosystems in the same localities. In particular, for coastal ecosystems such as seagrass, seaweed beds, and coral reefs, it is possible to carry out broad spatial comparisons using variables representing most of these 7 criteria. Regarding methods for the quantitative evaluation of each criterion and their integration, application of these methods to kelp forest ecosystems in Hokkaido, Northern Japan is presented as a case study.

BackgroundThe deep-sea hydrothermal vent is one of the most ‘extreme’ environments in the marine realm. Few species are capable of inhabiting such ecosystems, despite extremely high productivity there supported by microbial chemosynthesis, leading to high biomass and low species richness. Although gastropod molluscs are one of the main constituents of megafaunal communities at vent ecosystems, most species belong to several typical families (e.g., Provannidae, Peltospiridae, Lepetodrilidae) specialised and adapted to life at vents.MethodsDuring recent surveys of Okinawa Trough hydrothermal vent systems, two snails atypical of vent ecosystems were unexpectedly found in newly discovered hydrothermally influenced areas. Shell and radular characteristics were used to identify the gastropods morphologically.ResultsOne species was a vetigastropod, the calliostomatidTristichotrochus ikukoae(Sakurai, 1994); and the other was a caenogastropod, the muricidAbyssotrophon soyoae(Okutani, 1959). Both gastropods were previously only known from regular non-chemosynthetic deep-sea and very rare—only two definitive published records exist forT. ikukoaeand three forA. soyoae. The radula formula ofTristichotrochus ikukoaeis accurately reported for the first time and based on that it is returned to genusOtukaia. For both species, barcode sequences of the cytochromecoxidase I (COI) gene were obtained and deposited for future references.DiscussionThese new records represent the second record of calliostomatids from vents (third from chemosynthetic ecosystems) and the third record of muricids from vents (tenth from chemosynthetic ecosystems), and extend the distribution of both species to the southwest. Neither family has been recorded at chemosynthetic ecosystems in the western Pacific. Both were from weakly diffuse flow areas not subject to high temperature venting but were nevertheless associated with typical vent-reliant taxa such asLamellibrachiatubeworms andBathymodiolusmussels. These new records show that these species are capable of tolerating environmental stress associated with weak hydrothermally influenced areas, despite not being vent endemic species, adding to the list of known vent/non-vent species intersections. This signifies that such weakly influenced areas may provide key habitats for them, and that such areas may play a role in the evolution of biological adaptations to ‘extreme’ chemosynthetic ecosystems.

This paper considers the arrangement method of the peach on the handling unit that is a part of a peach moth inspection system that our group is developing using X-Ray. The peach fruit moth does not live in Taiwan. And the larva of the moth enters the peach, eats the pulp near the seed and damages to the peach. Therefore, transportation of the peach that the larva of the moth enters is prohibited. The developed system tries to detect whether the larva enters the peach or not. The arrangement direction is important, since effectiveness of detection of the larva and stability of the peach on the unit are influenced. First, the influence of the seed on the projection of the peach is considered. Secondly, the difficulty of the rotation of the peach on the handling unit is addressed so as to evaluate the stability. As a result, it is clear that there is trade off. And, the peach moth inspection system is demonstrated in a field experiment. It is confirmed that the arrangement direction of the peach for detection has to take priority, even if the stability is lower.

Next-generation sequencing (NGS) has become a powerful tool in phylogenetic and evolutionary studies. Here we applied NGS to recover two ribosomal RNA genes (18S and 28S) from 16 species and 15 mitochondrial genomes from 16 species of scale worms representing six families in the suborder Aphroditiformia (Phyllodocida, Annelida), a complex group of polychaetes characterized by the presence of dorsal elytra or scales. The phylogenetic relationship of the several groups of scale worms remains unresolved due to insufficient taxon sampling and low resolution of individual gene markers. Phylogenetic tree topology based on mitochondrial genomes is comparable with that based on concatenated sequences from two mitochondrial genes (cox1 and 16S) and two ribosomal genes (18S and 28S) genes, but has higher statistical support for several clades. Our analyses show that Aphroditiformia is monophyletic, indicating the presence of elytra is an apomorphic trait. Eulepethidae and Aphroditidae together form the sister group to all other families in this suborder, whereas Acoetidae is sister to Iphionidae. Polynoidae is monophyletic, but within this family the deep-sea subfamilies Branchinotogluminae and Macellicephalinae are paraphyletic. Mitochondrial genomes in most scale-worm families have a conserved gene order, but within Polynoidae there are two novel arrangement patterns in the deep-sea clade. Mitochondrial protein-coding genes in polynoids as a whole have evolved under strong purifying selection, but substitution rates in deep-sea species are much higher than those in shallow-water species, indicating that purifying selection is relaxed in deep-sea polynoids. There are positive selected amino acids for some mitochondrial genes of the deep-sea clade, indicating they may involve in the adaption of deep-sea polynoids. Overall, our study (1) provided more evidence for reconstruction of the phylogeny of Aphroditiformia, (2) provided evidence to refute the assumption that mitochondrial gene order in Errantia is conserved, and (3) indicated that the deep-sea extreme environment may have affected the mitochondrial genome evolution rate and gene order arrangement in Polynoidae.

The present study investigated spatiotemporal variations in meiofaunal abundance and composition at high taxonomic levels, and their associations with certain measures of food availability around hydrothermal vents on chimney structures and in adjacent non-vent fields in the calderas of three neighboring seamounts (Bayonnaise Knoll, Myojin Knoll, and Myojin-sho Caldera), in the Izu-Ogasawara Arc, western North Pacific Ocean. Total meiofaunal abundance in seafloor sediment at the bases of vent chimneys appeared to be greater than that in other non-vent habitats outside or inside the calderas of all seamounts, which was partly explained by temporal variations at the bases of the chimneys. There was no significant difference in the mean meiofaunal abundances among those habitats. A typical deep-sea meiofaunal composition (nematodes as the most abundant taxon, harpacticoid copepods as the second) was observed in the seafloor sediments in the non-vent fields, and even in the sediments at chimney bases. This was significantly different from the meiofaunal composition observed on the surfaces of vent chimneys, where copepods and their nauplii were most abundant. This spatial difference was significantly correlated with a difference in stable carbon isotope ratios (δ13C) of organic matter in sediment, suggesting that the availability of chemosynthetic food controls the spatial differences in meiofaunal composition around these hydrothermal vents, even at a high taxonomic level.

Polynoid scale worms (Polynoidae, Annelida) invaded deep-sea chemosynthesis-based ecosystems approximately 60 million years ago, but little is known about their genetic adaptation to the extreme deep-sea environment. In this study, we reported the first two transcriptomes of deep-sea polynoids (Branchipolynoe pettiboneae, Lepidonotopodium sp.) and compared them with the transcriptome of a shallow-water polynoid (Harmothoe imbricata). We determined codon and amino acid usage, positive selected genes, highly expressed genes and putative duplicated genes. Transcriptome assembly produced 98,806 to 225,709 contigs in the three species. There were more positively charged amino acids (i.e., histidine and arginine) and less negatively charged amino acids (i.e., aspartic acid and glutamic acid) in the deep-sea species. There were 120 genes showing clear evidence of positive selection. Among the 10% most highly expressed genes, there were more hemoglobin genes with high expression levels in both deep-sea species. The duplicated genes related to DNA recombination and metabolism, and gene expression were only enriched in deep-sea species. Deep-sea scale worms adopted two strategies of adaptation to hypoxia in the chemosynthesis-based habitats (i.e., rapid evolution of tetra-domain hemoglobin in Branchipolynoe or high expression of single-domain hemoglobin in Lepidonotopodium sp.).

Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with greater than or equal to98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

Barnacles of the genus Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2–19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise FST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.