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Abstract Helicobacter pylori, particularly cytotoxin-associated gene A (CagA)-positive strains, plays a key role in the progression of gastric cancer (GC). Ferroptosis, associated with lethal lipid peroxidation, has emerged to play an important role in malignant and infectious diseases, but the role of CagA in ferroptosis in cancer cells has not been determined. Here, we report that CagA confers GC cells sensitivity to ferroptosis both in vitro and in vivo. Mechanistically, CagA promotes the synthesis of polyunsaturated ether phospholipids (PUFA-ePLs), which is mediated by increased expression of alkylglycerone phosphate synthase (AGPS) and 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), leading to susceptibility to ferroptosis. This susceptibility is mediated by activation of the MEK/ERK/SRF pathway. SRF is a crucial transcription factor that increases AGPS transcription by binding to the AGPS promoter region. Moreover, the results demonstrated that CagA-positive cells are more sensitive to apatinib than are CagA-negative cells, suggesting that detecting the H. pylori CagA status may aid patient stratification for treatment with apatinib.

Summary: Pyruvate kinase (PK), as a key rate-limiting enzyme in glycolysis, has been widely used to assess the stress tolerance and sensitivity of organisms. However, its phosphorylation regulatory mechanisms mainly focused on human cancer research, with no reports in marine organisms. In this study, we firstly reported a conserved PK Ser11 phosphorylation site in mollusks, which enhanced enzyme activity by promoting substrate binding, thereby regulating divergent thermal metabolism of two allopatric congeneric oyster species with differential habitat temperature. It was phosphorylated by ERK kinase, and regulated by the classical MAPK pathway. The MAPK/ERK-PK signaling cascade responded to increased environmental temperature and exhibited stronger activation pattern in the relatively thermotolerant species (Crassostrea angulata), indicating its involvement in shaping temperature adaptation. These findings highlight the presence of complex and unique phosphorylation-mediated signaling transduction mechanisms in marine organisms, and provide new insights into the evolution and function of the crosstalk between classical pathways.

The Pacific abalone (Haliotis discus hannai) is widely cultivated in East Asian countries. Aquaculture of this species has become a thriving industry in China, with adequate seed production being a key contributing factor. However, maintaining sufficient diatom abundance during the nursery phase remains challenging. In this study, we assessed a transfer nursery culture method that involves prolonged cultivation with a sufficient diatom supply. This method involved treating postlarvae on diatom-depleted plates with a garlic juice (GJ) solution to facilitate detachment, followed by transfer to newly diatom-precoated plates for further growth in the nursery. We investigated the effects of garlic juice concentration (GJC) and animal size on detachment and mortality rates using a two-factor design. Our negative binomial regression analysis uncovered significant impacts of both factors and their interactions on the target traits. We noticed that the optimal GJC for smaller groups of abalone postlarvae (2.2 ± 1.0 mm) was 0.7 %, while for larger groups (3.8 ± 2.2 mm), it increased to 1.0 %. Notably, suitable GJC concentrations during post-treatment recovery resulted in no apparent mortality in Pacific abalone postlarvae. Our study demonstrated the effectiveness of the transfer nursery method, showing an improvement of over ∼1.1 mm in shell length and a 43.1 % increase in survival during a 30-day experiment. Our study presents a significant advancement, as it likely introduces the first reported method of large-scale postlarval removal of H. discus hannai for prolonged nursery periods with sufficient diatom provision. The integration of GJ treatment and the transfer nursery method exhibited promising results, highlighting the benefits of its application for enhancing husbandry procedures, nursery management, and the overall industrial productivity of Pacific abalone.

The low-grade zinc oxide ore was sulfidized to increase the efficiency of flotation, but the effect of pre-sulfidization on the adsorption mechanism of octadecyl amine (ODA) on the smithsonite surface is currently unclear. In this study, the effect of pre-sulfidization on the adsorption mechanism of ODA and the flotation behavior was studied using smithsonite and pre-sulfidized smithsonite as the samples by zeta potential, contact angle measurement, total organic carbon analyzer (TOC), quartz microcrystalline balance (QCM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and micro-flotation tests. Micro-flotation tests showed that the pretreatment of sulfidization could improve the floatability of smithsonite. Zeta potential and contact angle measurements demonstrated that pre-sulfidization could favor the adsorption of ODA, which is further confirmed by the adsorption tests of ODA using TOC and QCM. Furthermore, FTIR and XPS analysis showed that pre-sulfidization changes the adsorption mode of ODA, changing it from physical adsorption to chemical adsorption. These results suggested that the favorable effect of pre-sulfidization on the adsorption of ODA and the flotation of smithsonite might provide important guidance for industrial application.

This is an essay in the field of mineral processing engineering. The useful mineral of a pegmatite lithium deposit in Africa is spodumene, and the raw ore contains 0.87% Li2O. It belongs to low-grade lithium ore, which is relatively difficult to develop and use. In this study, a spodumene concentrate with a yield of 10.15%, containing 6.03% Li2O and lithium recovery of 70.14% was obtained through detailed flotation tests and the flotation process of "one roughing, two scavengings and three cleanings". The magnetic separation test results show that chemical grade -1 spodumene concentrate can be obtained by 0.2 T magnetic separation of lithium flotation concentrate, and ceramic grade spodumene concentrate can be obtained by 0.6 T magnetic separation of lithium flotation concentrate, which realizes the high-quality utilization of this low-grade lithium ore.

Abstract Comparing the responses of closely related species to environmental changes is an efficient method to explore adaptive divergence, for a better understanding of the adaptive evolution of marine species under rapidly changing climates. Oysters are keystone species thrive in intertidal and estuarine areas where frequent environmental disturbance occurs including fluctuant salinity. The evolutionary divergence of two sister species of sympatric estuarine oysters, Crassostrea hongkongensis and Crassostrea ariakensis, in response to euryhaline habitats on phenotypes and gene expression, and the relative contribution of species effect, environment effect, and their interaction to the divergence were explored. After a 2‐month outplanting at high‐ and low‐salinity locations in the same estuary, the high growth rate, percent survival, and high tolerance indicated by physiological parameters suggested that the fitness of C. ariakensis was higher under high‐salinity conditions and that of C. hongkongensis was higher under low‐salinity conditions. Moreover, a transcriptomic analysis showed the two species exhibited differentiated transcriptional expression in high‐ and low‐salinity habitats, largely caused by the species effect. Several of the important pathways enriched in divergent genes between species were also salinity‐responsive pathways. Specifically, the pyruvate and taurine metabolism pathway and several solute carriers may contribute to the hyperosmotic adaptation of C. ariakensis, and some solute carriers may contribute to the hypoosmotic adaptation of C. hongkongensis. Our findings provide insights into the phenotypic and molecular mechanisms underlying salinity adaptation in marine mollusks, which will facilitate the assessment of the adaptive capacity of marine species in the context of climate change and will also provide practical information for marine resource conservation and aquaculture.

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

High-temperature stress caused by global climate change poses a significant threat to marine ectotherms. This study investigated the role of protein phosphorylation modifications in the molecular regulation network under heat stress in oysters, which are representative intertidal organisms that experience considerable temperature changes. Firstly, the study compared the extent of thermal damage between two congeneric oyster species, the relative heat-tolerant Crassostrea angulata (C. angulata) and heat-sensitive Crassostrea gigas (C. gigas), under sublethal temperature (37 °C) for 12 h, using various physiological and biochemical methods. Subsequently, the comparative proteomic and phosphoproteomic analyses revealed that high-temperature considerably regulated signal transduction, energy metabolism, protein synthesis, cell survival and apoptosis, and cytoskeleton remodeling through phosphorylation modifications of related receptors and kinases. Furthermore, the protein kinase A, mitogen-activated protein kinase 1, tyrosine-protein kinase Src, and serine/threonine kinase AKT, exhibiting differential phosphorylation modification patterns, were identified as hub regulators that may enhance glycolysis and TCA cycle to increase the energy supply, distribute protein synthesis, inhibit Caspase-dependent apoptosis activated by endogenous mitochondrial cytochrome release and maintain cytoskeletal stability, ultimately shaping the higher thermal resistance of C. angulata. This study represents the first investigation of protein phosphorylation dynamics in marine invertebrates under heat stress, reveals the molecular mechanisms underlying the differential thermal responses between two Crassostrea oysters at the phosphorylation level, and provides new insights into understanding phosphorylation-mediated molecular responses in marine organisms during environmental changes and predicting the adaptive potential in the context of global warming.

Abstract Anti‐angiogenic therapy has long been considered a promising strategy for solid cancers. Intrinsic resistance to hypoxia is a major cause for the failure of anti‐angiogenic therapy, but the underlying mechanism remains unclear. Here, it is revealed that N4‐acetylcytidine (ac4C), a newly identified mRNA modification, enhances hypoxia tolerance in gastric cancer (GC) cells by promoting glycolysis addiction. Specifically, acetyltransferase NAT10 transcription is regulated by HIF‐1α, a key transcription factor of the cellular response to hypoxia. Further, acRIP‐sequencing, Ribosome profiling sequencing, RNA‐sequencing, and functional studies confirm that NAT10 in turn activates the HIF‐1 pathway and subsequent glucose metabolism reprogramming by mediating SEPT9 mRNA ac4C modification. The formation of the NAT10/SEPT9/HIF‐1α positive feedback loop leads to excessive activation of the HIF‐1 pathway and induces glycolysis addiction. Combined anti‐angiogenesis and ac4C inhibition attenuate hypoxia tolerance and inhibit tumor progression in vivo. This study highlights the critical roles of ac4C in the regulation of glycolysis addiction and proposes a promising strategy to overcome resistance to anti‐angiogenic therapy by combining apatinib with ac4C inhibition.

The fine scheelite flotation using garnet and glass beads as agitation medium was investigated through slurry rheology and froth property analysis. The apparent viscosity of fine scheelite, calcite and quartz (−20 μm) slurry were measured and the corresponding scheelite flotation behaviours, froth water recovery and froth height were studied. It was found that calcite slurry could aggregate into network structures and exhibit much higher apparent viscosity at the vicinity of pH 8.5–9.0 than scheelite and quartz (scheelite, calcite, and quartz slurry at 20.34, 38.66, and 14.58 mPa·s, respectively). Agitation medium containing garnet and glass beads could reduce the slurry apparent viscosity by effectively dispersing the calcite network structures in flotation slurry, and improve the fine scheelite flotation selectivity. As the apparent viscosity was reduced, the froth water recovery decreased from 24.33% to 6.37% and initial froth height of the flotation froth decreased from 69.6 to 8.7 mm, and finally results in increase in separation efficiency from 23.10% to 31.96%. The results could be potentially used in flotation of fine minerals by applying agitation medium to improve the process selectivity.

Transcriptional plasticity interacts with natural selection in complex ways and is crucial for the survival of species under rapid climate change. How 3D genome architecture affects transcriptional plasticity and its interaction with genetic adaptation are unclear. We transplanted estuarine oysters to a new environment and found that genes located in active chromatin regions exhibited greater transcriptional plasticity, and changes in these regions were negatively correlated with selective signals. This indicates a trade-off between 3D active regions and selective signals in shaping plastic responses to a new environment. Specifically, a mutation, lincRNA, and changes in the accessibility of a distal enhancer potentially affect its interaction with the ManⅡa gene, which regulates the muscle function and survival of oysters. Our findings reveal that 3D genome architecture compensates for the role of genetic adaptation in environmental response to new environments and provide insights into synergetic genetic and epigenetic interactions critical for fitness-related trait and survival in a model marine species.

As the world’s largest farmed marine animal, oysters have enormous economic and ecological value. However, mass summer mortality caused by high temperature poses a significant threat to the oyster industry. To investigate the molecular mechanisms underlying heat adaptation and improve the heat tolerance ability in the oyster, we conducted genome-wide association analysis (GWAS) analysis on the F2 generation derived from the hybridization of relatively heat-tolerant Crassostrea angulata ♀ and heat-sensitive Crassostrea gigas ♂, which are the dominant cultured species in southern and northern China, respectively. Acute heat stress experiment (semi-lethal temperature 42 °C) demonstrated that the F2 population showed differentiation in heat tolerance, leading to extremely differentiated individuals (approximately 20% of individuals die within the first four days with 10% survival after 14 days). Genome resequencing and GWAS of the two divergent groups had identified 18 significant SNPs associated with heat tolerance, with 26 candidate genes located near these SNPs. Eleven candidate genes that may associate with the thermal resistance were identified, which were classified into five categories: temperature sensor (Trpm2), transcriptional factor (Gata3), protein ubiquitination (Ube2h, Usp50, Uchl3), heat shock subfamily (Dnajc17, Dnaja1), and transporters (Slc16a9, Slc16a14, Slc16a9, Slc16a2). The expressional differentiation of the above genes between C. gigas and C. angulata under sublethal temperature (37 °C) further supports their crucial role in coping with high temperature. Our results will contribute to understanding the molecular mechanisms underlying heat tolerance, and provide genetic markers for heat-resistance breeding in the oyster industry.

The utilization of Ca ion as assistant depressant of CMC on talc has been widely reported. Thus, the study on the adsorption mechanism of Ca ion on talc surface is very crucial for understanding the performance of CMC on talc depression. In this paper, mechanism insights into hydrated Ca ion adsorption on talc (001) basal surface were creatively provided using DFT calculation. [Ca(H2O)6]2+ and [Ca(OH)(H2O)3]+ were determined as the effective hydrate components for Ca ion adsorption, and the top O site was the most favorable position for their adsorptions on talc surface. Furthermore, the adsorption mechanisms of [Ca(H2O)6]2+ and [Ca(OH)(H2O)3]+ on talc surface were found to be not the Ca—O chemical bond, but the hydrogen bonding formed by the H atom of the H2O ligand and the surface O atom. H2O acted like a bridge to connect them to the talc surface. Moreover, the hydrogen bonding was formed due to the hybridization of H 1s orbital with the O 2s, O 2p orbitals. Simultaneously, electrons transferred between the H atom and the surface O atom. This work provides theoretical insights into the Ca ion adsorption on talc surface, which can help deeply understand the talc flotation using CMC as depression.

Association of tumor microenvironment and immune checkpoint (e.g., PD-L1) is important for immune escape, impacting chemotherapy and immunotherapy efficacy. We aimed to investigate biomarkers and therapeutic targets against treatment resistance in gastric cancer. Abundances of tumor-infiltrating immune cells were estimated in multiple datasets. Three patient subgroups (A, B, and C) were identified based on seven types of PD-L1- and IFN-γ-associated immune cells. Patients yielded increased prognosis from subgroup A to C (p = 0.027). Subgroup A was characterized by high activated CD4+ memory T cell infiltration, while more resting CD4+ memory T cells were in subgroup C. Further, a risk score was developed for prognostication. Lipoma preferred partner (LPP), as the hub gene in subgroup-related regulatory network, was upregulated (p < 0.01) and was associated with high risk score (p < 0.001) and poor survival (p < 0.05). Bioinformatics analyses and experiments found that LPP expressed restrictively in fibroblasts and associated with activated CD4+ memory T cell infiltration and tumor growth. High-LPP patients yielded fewer benefits from chemotherapy or immunotherapy, compared with the low-LPP group. We finally identified 28 compounds as sensitive drugs for high-LPP patients. Our findings suggested LPP might be a biomarker for treatment response and therapeutic target in gastric cancer.

Ao Li, He Dai, and Ximing Guo et al. present a high-quality genome from the estuarine oyster, Crassostrea ariakensis. They highlight the climate impact on population divergence, a suite of genes likely important for adaptation to environments of different temperatures and salinities, and provide further insight into the evolution of this species.

Summary: Background: Exitron is a new type of non-canonical alternative splicing. Accumulating evidence implies exitron may have pathological function and contribute to another source of anti-tumor immunogenicity in various cancers. Its role in gastric cancer remains poorly understood. Large-scale, multi-omics analysis could comprehensively characterize the landscape of exitrons in gastric cancer, reveal undiscovered mechanism and hopefully identify molecular biomarkers for predicting immunotherapy response. Methods: We collected datasets from five studies for analysis. RNA sequencing was used for exitron identification. Somatic mutations were detected by whole exome sequencing. Neopeptides were confirmed by proteome mass spectrometry. Findings: 42174 gastric cancer-specific exitrons (GCSEs) were identified in 632 patients. GCSEs were clinically relevant to gender, age, Lauren type, tumor stage and prognosis. Tissue specificity test and pathogenic exitron prediction revealed their unique functional impact. GCSEs were mutually exclusive with mutations and demonstrated both unique and complementary function against TP53 mutation in gastric cancer. We further established splicing regulatory network to reveal upstream regulation of exitron splicing. We also evaluated the immunogenicity and diagnostic potential of GCSEs. Evidence of GCSEs-derived neopeptide expression was validated by whole proteome mass spectrometry. PD-1 and Siglecs were significantly increased in high neoantigen load patients. But exitron-related biomarkers failed to predict immunotherapy response, possibly due to small sample size and insufficient sequencing depth. Interpretation: The present study provided a comprehensive multidimensional landscape of gastric cancer exitrons and underscores insights into underexplored mechanism in gastric cancer pathology. Funding: The Guangdong Provincial Key Laboratory of Precision Medicine for Gastroinstestinal Cancer (2020B121201004), the Guangdong Provincial Major Talents Project (No. 2019JC05Y361) and National Natural Science Foundation of China (grant number:82172960 and 81872013).

The Ostreid herpesvirus 1 (OsHV-1) is a lethal pathogen of the Pacific oyster (Crassostrea gigas), an important aquaculture species. To understand the genetic architecture of the defense against the pathogen, we studied genomic variations associated with herpesvirus-caused mortalities by pooled whole-genome resequencing of before and after-mortality larval samples as well as dead and surviving adults from a viral challenge. Analysis of the resequencing data identified 5,271 SNPs and 1,883 genomic regions covering 3,111 genes in larvae, and 18,692 SNPs and 28,314 regions covering 4,863 genes in adults that were significantly associated with herpesvirus-caused mortalities. Only 1,653 of the implicated genes were shared by larvae and adults, suggesting that the antiviral response or resistance in larvae and adults involves different sets of genes or differentiated members of expanded gene families. Combined analyses with previous transcriptomic data from challenge experiments revealed that transcription of many mortality-associated genes was also significantly upregulated by herpesvirus infection confirming their importance in antiviral response. Key immune response genes especially those encoding antiviral receptors such as TLRs and RLRs displayed strong association between variation in regulatory region and herpesvirus-caused mortality, suggesting they may confer resistance through transcriptional modulation. These results point to previously undescribed genetic mechanisms for disease resistance at different developmental stages and provide candidate polymorphisms and genes that are valuable for understanding antiviral immune responses and breeding for herpesvirus resistance.

In vertebrates insulin is a direct transcriptional target of Pdx: the same is true in Pacific oysters and the authors show insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue, showing this gene interaction dates back to the origin of Bilateria.

Abstract Lophotrochozoan species exhibit wide morphological diversity; however, the molecular basis underlying this diversity remains unclear. Here, we explored the evolution of Notch pathway genes across 37 metazoan species via phylogenetic and molecular evolutionary studies with emphasis on the lophotrochozoans. We displayed the components of Notch pathway in metazoans and found that Delta and Hes/Hey-related genes, as well as their functional domains, are duplicated in lophotrochozoans. Comparative transcriptomics analyses allow us to pinpoint sequence divergence of multigene families in the Notch signalling pathway. We identified the duplication mechanism of a mollusc-specific gene, Delta2, and found it displayed complementary expression throughout development. Furthermore, we found the functional diversification not only in expanded genes in the Notch pathway (Delta and Hes/Hey-related genes), but also in evolutionary conservative genes (Notch, Presenilin, and Su(H)). Together, this comprehensive study demonstrates conservation and divergence within the Notch pathway, reveals evolutionary relationships among metazoans, and provides evidence for the occurrence of developmental diversity in lophotrochozoans, as well as a basis for future gene function studies.

Worldwide, the bivalve aquaculture industry has realized or recognized the potential gains from selective breeding programs using phenotypic and pedigree data. Yesso scallop Patinopecten yessoensis are among the most important commercial shellfish in China. A family-based breeding program to investigate the genetic variations for growth and survival-related traits at suspended and bottom environments of sea-based culture systems was reported in this study. We proposed and conducted a novel phenotyping technique to longitudinally evaluate the shell heights in the yesso scallop. At harvest after rearing for 20 months, the individual shell heights at 6, 10, 16, and 20 months were simultaneously obtained by the growth rings in the outer shells of the animals. Meanwhile, the body weight (BW) of the survived individuals at harvest was also recorded. Variance components and genetic parameters for growth and survival-related traits were estimated using an animal and threshold model, respectively. In the suspended environment, the heritability estimates for BW and shell heights at specific ages ranged from moderate to high (0.328–0.853). The estimated correlations between shell heights at contiguous ages were consistently high, ranging from 0.890 to 0.958 but decreased with increasing intervals between ages (0.496–0.828). The estimated correlations between shell heights at contiguous ages and BW at harvest were similar, ranging from 0.535 to 0.983. The heritability estimates for individual survival at harvest were at a low level of 0.128 by the probit-threshold model. While at the bottom environment, estimates of heritability for growth and survival-related traits were similar but slightly lower than those at the suspended environment. Furthermore, the genetic correlation for BW between the two environments was very small, which probably indicates genotype-by-environment interaction effects for growth in the yesso scallop. The study can provide prior information, which might develop a new idea for selection in this species. The results are discussed concerning selection work with yesso scallop, and solutions for accurate estimation of genetic parameters and increasing genetic gain are also outlined.

Infectious disease outbreaks are causing widespread declines of marine invertebrates including corals, sea stars, shrimps, and molluscs. Dermo is a lethal infectious disease of the eastern oyster Crassostrea virginica caused by the protist Perkinsus marinus. The Pacific oyster Crassostrea gigas is resistant to Dermo due to differences in the host-parasite interaction that is not well understood. We compared transcriptomic responses to P. marinus challenge in the two oysters at early and late infection stages. Dynamic and orchestrated regulation of large sets of innate immune response genes were observed in both species with remarkably similar patterns for most orthologs, although responses in C. virginica were stronger, suggesting strong or over-reacting immune response could be a cause of host mortality. Between the two species, several key immune response gene families differed in their expansion, sequence variation and/or transcriptional response to P. marinus, reflecting evolutionary divergence in host-parasite interaction. Of note, significant upregulation of inhibitors of apoptosis (IAPs) was observed in resistant C. gigas but not in susceptible C. virginica, suggesting upregulation of IAPs is an active defense mechanism, not a passive response orchestrated by P. marinus. Compared with C. gigas, C. virginica exhibited greater expansion of toll-like receptors (TLRs) and positive selection in P. marinus responsive TLRs. The C1q domain containing proteins (C1qDCs) with the galactose-binding lectin domain that is involved in P. marinus recognition, were only present and significantly upregulated in C. virginica. These results point to previously undescribed differences in host defense genes between the two oyster species that may account for the difference in susceptibility, providing an expanded portrait of the evolutionary dynamics of host-parasite interaction in lophotrochozoans that lack adaptive immunity. Our findings suggest that C. virginica and P. marinus have a history of coevolution and the recent outbreaks may be due to increased virulence of the parasite.

Understanding the genetic basis of trait variations and their coordination between relative species or populations distributing in different environmental conditions is important in evolutionary biology. In marine ectotherms, growth-defense trade-offs are a common ecological and evolutionary phenomenon. However, the biochemical and molecular mechanisms that govern these trade-offs in marine ectotherms in the evolutionary perspective remain poorly investigated. Oysters are among the most important species in global aquaculture. Crassostrea gigas (C. gigas) and Crassostrea angulata (C. angulata) are two allopatric congeneric dominant oyster species that inhabit the northern and southern intertidal areas of China. Wild C. gigas and C. angulata were spawned, and their F1 progeny were cultured in the same sites to reduce the environmental effects. Untargeted metabolomics and transcriptomics, together with phenotypic parameters including morphological traits (growth performance), nutritional content (glycogen, crude fat, and fatty acid content), physiology (normalized oxygen consumption rate and total antioxidant capacity) were applied to assess metabolic and transcript divergences between C. gigas and C. angulata. Integrated analyses of metabolites and transcriptomes showed that C. gigas allocated more energy to storage and defense by suppressing glycolysis, fatty acid oxidation and by upregulating fatty acid synthesis, antioxidant gene expression, and related metabolites. The metabolic and transcript results were further confirmed by the phenotypic data that C. gigas has higher glycogen and crude fat content and fatty acid unsaturation and stronger antioxidant capacity than C. angulata. In contrast, C. angulata exhibited better growth performance and a higher oxygen consumption rate. These findings suggest that C. angulata allocates more energy to growth, which is embodied in its stronger aerobic capacity and higher levels of protein synthesis genes, metabolites, and growth-related biomarkers. This study will help to enlighten the evolutionary patterns and genetic basis of growth-defense trade-offs in marine ectotherms and the biochemical and molecular mechanisms underlying energy allocation. Also, the key genes and metabolites of glycogen and fatty acids pathway identified in this study will be applied for meat quality improvement in the oyster industry.

Abstract Background The Pacific oyster Crassostrea gigas is an important fishery resource that is sensitive to temperature fluctuations. Thus, it has evolved a protection mechanism against heat stress by increasing the expression of the gene coding for heat shock protein (HSP) 70 under elevated temperatures. In other animals, heat shock response is a transcriptional response driven by the heat shock transcription factor 1 (HSF1) and thermal stress can trigger HSP70 expression to protect the organism via HSF1. However, the regulatory relationship between HSF1 and HSP remains unclear in Pacific oyster. Therefore, in the present study, we examined the transcriptomic response of several to thermal stress following HSF1 interference. Results We identified 150 genes responsive to heat shock including seven HSP genes, six of which belonging to the group of 17 HSP genes enriched in response to heat shock, according to weighted gene co-expression network analysis (WGCNA). The other gene was enriched in the module correlated with HSF1 interference. In addition, we found 48 and 47 genes that were upregulated and downregulated by HSF1 in response to heat shock, respectively. In the upregulated genes, we identified one HSP70 potentially regulated by HSF1 in response to heat shock. Furthermore, based on differentially expressed genes and WGCNA analyses, we found that the hypoxia signaling pathway was enriched under heat shock conditions. Five genes were then selected to detect dynamic changes through time. The results suggested that gene expression was correlated with HSF1 expression. The regulation of HSP70 by HSF1 was preliminarily confirmed by binding site predictions and by a dual luciferase assay. Conclusions Our results revealed that the expression of HSP70 and HSP20 was initially triggered after 2 h of heat shock, and one of the HSP70 genes was potentially regulated by HSF1. From these results, it is evident that not all heat-inducible genes were triggered simultaneously in response to heat shock stress. Overall, the results revealed a possible HSF1–HSP regulatory relationship in Pacific oyster, providing valuable information on the mechanisms of thermal tolerance in this commercially important oyster.

Abstract Background Oyster is rich in glycogen and free amino acids and is called “the milk of sea”. To understand the main genetic effects of these traits and the genetic networks underlying their correlation, we have conducted the whole genome resequencing with 427 oysters collected from the world-wide scale. Results After association analysis, 168 clustered significant single nucleotide polymorphism (SNP) loci were identified for glycogen content and 17 SNPs were verified with 288 oyster individuals in another wide populations. These were the most important candidate loci for oyster breeding. Among 24 genes in the 100-kb regions of the leading SNP loci, cytochrome P450 17A1 (CYP17A1) contained a non-synonymous SNP and displayed higher expressions in high glycogen content individuals. This might enhance the gluconeogenesis process by the transcriptionally regulating the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Also, for amino acids content, 417 clustered significant SNPs were identified. After genetic network analysis, three node SNP regions were identified to be associated with glycogen, protein, and Asp content, which might explain their significant correlation. Conclusion Overall, this study provides insights into the genetic correlation among complex traits, which will facilitate future oyster functional studies and breeding through molecular design.

Triosteum himalayanum, Triosteum pinnatifidum (Triosteum L., Caprifoliaceae, Dipsacales) are widely distributed in China while Triosteum sinuatum mainly occurrs in northeast China. Few reports have been determined on the genus Triosteum. In the present research, we sequenced 2 chloroplast genomes of Triosteum and analyzed 18 chloroplast genomes, trying to explore the sequence variations and phylogeny of genus Triosteum in the order Dipsacales. The chloroplast genomes of the genus Triosteum ranged from 154,579 bp to 157,178 bp, consisting of 132 genes (86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes). Comparative analyses and phylogenetic analysis supported the division of Dipsacales into two clades, Adoxaceae and six other families. Among the six families, a clade of Valerianaceae+Dipsacaceae was recovered as a sister to a clade of Morinaceae+Linnaeaceae. A closer relationship of T. himalayanum and T. pinnatifidum among three species was revealed. Our research supported that Loniceraferdinandi and Triosteum was closely related. Zabelia had a closer relationship with Linnaea borealis and Dipelta than Morinaceae. The divergence between T. sinuatum and two other species in Triosteum was dated to 13.4 mya.

The Pacific oyster is a globally important aquaculture species inhabiting the intertidal environment, which experiences great temperature variation. Mass deaths in the summer pose a major challenge for the oyster industry. We initiated an artificial selection breeding program in 2017 using acute heat shock treatments of the parents to select for thermotolerance in oysters. In this study, we compared the respiration rate, summer survival rate, gene expression, and gene structure of F2 selected oysters and non-selected wild oysters. A transcriptional analysis revealed global divergence between the selected and control groups at the larval stage, including 4764 differentially expressed genes, among which 79 genes were heat-responsive genes. Five heat shock proteins were enriched, and four of the six genes (five heat stock genes in the enriched GO terms and KEGG pathways and BAG4) were differentially expressed in 1-year-old oysters. Integration of the transcriptomic and re-sequencing data of the selected and the control groups revealed 1090 genes that differentiated in both gene structure and expression. Two SNPs (single nucleotide polymorphism) that may mediate the expression of CGI_10022585 and CGI_10024709 were validated. In addition, the respiration rate of 1-year-old oysters varied significantly between the selected group and the control group at room temperature (20°C). And the summer survival rate of the selected population was significantly improved. This study not only shows that artificial selection has a significant effect on the gene structure and expression of oysters, but it also helps reveal the mechanism underlying their tolerance of high temperature as well as the ability of oysters to adapt to climate change.

Natural selection and isolation are both important for understanding the geographic distribution of marine species and environmental responses to changing climate. In this study, we revealed distinct genetic variation in Crassostrea ariakensis by comparing the COI gene segment sequence in northern and southern oysters partitioned by the Yangtze River estuary. Reciprocal hybridization and intrapopulation crosses clarified their taxonomic status as the same species. There was no heterosis in the survival and growth of the hybrids, while the maternal effect was observed in larvae from eggs in the native habitat that showed higher fitness. Both the northern and southern F1 progenies exhibited positive performance in fitness traits, including survivorship, respiration rate, and growth, in their native habitats compared to that in their non-native habitats, indicating a strong signature of local adaptation. The oysters dwelling in the warm/southern habitats evolved a higher thermotolerance of LT50, while the oysters inhabiting the high-salinity/northern habitats had a 2.43‰ higher LS50 than that of their southern counterparts. After strong natural selection in the northern environments, the higher survival of the F1 progenies from the southern oysters under heat shock indicates an evolved genetic basis for its higher thermal tolerance. Strong environmental gradients, especially for temperature and salinity, and geographic isolation by the interaction between coastal currents and the Yangtze River estuary potentially contribute to shaping the distribution pattern and adaptive divergence of C. ariakensis in China.

The most common beneficiation method for feldspar is flotation with a cationic (amine) collector under acidic conditions. However, there are several disadvantages to this, such as environmental pollution and equipment corrosion. In order to resolve such problems, it is important to study the flotation of feldspar using anionic collectors under natural pH conditions. The purpose of this paper is to study the effects and mechanism of Fe3+ on flotation separation of feldspar and epidote using sodium oleate (NaOL) at a natural pH. Through flotation experiments, adsorption measurements, zeta potential testing, FTIR analysis and X-ray photoelectron spectroscopy (XPS), the mechanism of Fe3+ on the surface of feldspar and epidote is revealed, and the reason behind the difference in flotation of the two minerals is discussed. The flotation test results show that Fe3+ can significantly improve the flotation behavior of minerals when NaOL is used as a collector under natural pH, and the highest recovery rates of feldspar and epidote are 90% and 43%, respectively. Analysis of the solution and adsorption measurement results show that Fe3+ is adsorbed on the minerals′ surface in the form of Fe(OH)3, which promotes the adsorption of NaOL on the minerals’ surface through Fe(OH)3, activating the flotation of feldspar and epidote. The difference in adsorption of Fe3+ between feldspar and epidote is the reason for this difference in flotation behavior. The results of the zeta potentials show that after being treated with Fe3+, the electrostatic adsorption of NaOL displays a significant negative shift on the surface of feldspar, while there is almost no electrostatic adsorption of NaOL on the surface of Fe3+-treated epidote. FTIR analysis confirmed that the difference in the adsorption of Fe3+ and NaOL on the surface of feldspar and epidote is due to the fact that there are more active particles (metal bonds) on the surface of feldspar than on the surface of epidote, and the properties of these metal bonds can be changed by Fe3+, which allows NaOL to be more easily adsorbed on the mineral surface through –COO−. The possible adsorption form is “mineral-Fe3+–COO−“. Compared with the infrared spectrum of epidote, there is a new absorption peak at 1713.68 cm−1, which can be attributed to the C=O characteristic peak of NaOL in the infrared spectrum of Fe3+–NaOL-treated feldspar, which is why the floatability of feldspar is better than epidote. XPS confirmed that the Fe on the surface of feldspar is Fe3+ in the form of Fe(OH)3, while Fe on the surface of epidote is mainly Fe2O3 (Fe–O) contained in mineral crystals. Furthermore, there is less adsorption of Fe3+ on the surface of epidote, and this discrepancy leads to the difference in the adsorption of NaOL on the minerals’ surface, which itself leads to the difference in flotation behavior between feldspar and epidote. These findings indicate that the flotation separation of feldspar and epidote can be achieved using Fe3+ and NaOL under natural pH. This study may provide a reference for the flotation mechanism of feldspar and epidote under natural pH.

Heat shock transcription factor 1 (HSF1) plays an important role in regulating heat shock, which can activate heat shock proteins (HSPs). HSPs can protect organisms from thermal stress. Oysters in the intertidal zone can tolerate thermal stress. The Pacific oyster (Crassostrea gigas gigas) and Fujian oyster (C. gigas angulata)—allopatric subspecies with distinct thermal tolerances—make good study specimens for analyzing and comparing thermal stress regulation. We cloned and compared HSF1 isoforms, which is highly expressed under heat shock conditions in the two subspecies. The results revealed that two isoforms (HSF1a and HSF1d) respond to heat shock in both Pacific and Fujian oysters, and different heat shock conditions led to various combinations of isoforms. Subcellular localization showed that isoforms gathered in the nucleus when exposed to heat shock. The co-immunoprecipitation revealed that HSF1d can be a dimer. In addition, we selected HSPs that are expressed under the heat shock response, according to the RNA-seq and proteomic analyses. For the HSPs, we analyzed the coding part and the promoter sequences. The result showed that the domains of HSPs are conserved in two subspecies, but the promoters are significantly different. The Dual-Luciferase assay showed that the induced expression isoform HSF1d had the highest activity in C. gigas gigas, while the constitutively-expressed HSF1a was most active in C. gigas angulata. In addition, variation in the level of HSP promoters appeared to be correlated with gene expression. We argue that this gene is regulated based on the different expression levels between the two subspecies’ responses to heat shock. In summary, various stress conditions can yield different HSF1 isoforms and respond to heat shock in both oyster subspecies. Differences in how the isoforms and promoter are activated may contribute to their differential expressions. Overall, the results comparing C. gigas gigas and C. gigas angulata suggest that these isoforms have a regulatory relationship under heat shock, providing valuable information on the thermal tolerance mechanism in these commercially important oyster species.

Ostreid herpesvirus-1 microvariant (OsHV-1 μVar) is considered a major infectious microbe that can reduce the survival of natural or cultured oysters in summer. Because they lack an adaptive immune system, oysters are dependent on their innate immune systems to fight pathogens. The duplication and functional divergence of innate immune genes in the oyster have been studied, but the contribution of molecular mechanisms underlying innate immunity remains to be defined. Here, we identified the interacting proteins associated with Crassostrea gigas Toll-like receptors (CgTLR) using a yeast two-hybrid (Y2H) screening system. A total of eight proteins were identified that could interact with CgTLR. Three of these appeared at least four times in the screening and were related to MyD88. Two genes encoding these MyD88-like proteins, CgMyD88-1 and CgMyD88-2, possessed typical death and TIR domains. The third gene encoding an MyD88-like protein possessed only a TIR domain, and we named it CgMyD88s. CgMyD88s interacted only with CgTLR, but not CgMyD88-1 or CgMyD88-2. Both CgMyD88-1 and CgMyD88-2 mRNAs were upregulated after OsHV-1 μVar infection, whereas the expression of CgMyD88s decreased. When overexpressed in HEK293T cells, CgMyD88-1 and CgMyD88-2 activated an NF-κB reporter, whereas CgMyD88s impaired activation induced by CgMyD88-1 or CgMyD88-2. Intriguingly, the silencing of CgMyD88s using double-stranded RNA (dsRNA)-mediated RNA interference increased the expression of CgMyD88-1 and CgMyD88-2. Taken together, our results revealed that CgMyD88-1, CgMyD88-2, and CgMyD88s may all participate in the TLR-mediated innate immune pathway and that CgMyD88s served as a plug to avoid oysters from excessive inflammatory response during OsHV-1 μVar infections.

Investigating responses of organisms to stressful or new environments with selection pressure is one of the crucial problems in evolutionary biology, and it is of importance to understand the phenotypic and molecular mechanism underlying thermal tolerance under the context of the climate change. The Pacific oyster, Crassostrea gigas, inhabiting the environment with high variation in temperature, is a worldwide aquaculture species. However, summer mortality relevant to the high temperature is one of the problems challenging the oyster industry. An artificial selective breeding program was initiated to select for the thermal tolerance of oysters in an attempt to increase the summer survival rates since 2017 in our study. Parents of thermotolerance oyster selection is are based on acute thermal tolerance under controlled heat stress to strengthen the selection intensity. Furthermore, the phenotypic and the genotypic response to extreme high temperature were investigated based on the comparison of the F1 progeny of the selected and natural populations in growth, physiology, mortality rate post heat stress, genetic structure, and gene expression. The parameter of growth showed no significant change between the selected and natural populations for the 6-month-old oysters. The selected population exhibited a higher survival rate after exposure to heat stress in the laboratory, which is in line with result of the filed experience that summer mortality of selected population was significantly lower than that of natural population. Further, the respiration rate of the selected population increased at 38°C, while it increased at 35°C in the natural population. Simultaneously, metabolism-related enzymes (PK, SOD) showed higher activity levels in the selected population. Furthermore, phylogenetic analysis, population structure, and principal component analysis (PCA) suggested that the selected and natural populations exhibited genetic divergence, with eight genes (IF4A2, IF6, EIF3A, MANBA, DDX43, RECS, CAT2, and BAG4) in the selected regions showing differential expression patterns in response to heat stress in the two populations. This study suggests that artificial selection has a significant effect on phenotype and genome structure for the oyster, our study providing an alternative way to reveal the mechanism underlying thermotolerance that plays an important role to predict the potential adaptation to the climate change.

Flexible electrochromic energy storage devices (FECESDs) for powering flexible electronics have attracted considerable attention. Silver nanowires (AgNWs) are one kind of the most promising flexible transparent electrodes (FTEs) materials for the emerging flexible devices. Currently, fabricating FECESD based on AgNWs FTEs is still hindered by their intrinsic poor electrochemical stability. To address this issue, a hybrid AgNWs/Co(OH) _2 /PEDOT:PSS electrode is proposed. The PEDOT:PSS could not only improve the resistance against electrochemical corrosion of AgNWs, but also work as functional layer to realize the color-changing and energy storage properties. Moreover, the Co(OH) _2 interlayer further improved the color-changing and energy storage performance. Based on the improvement, we assembled the symmetrical FECESDs. Under the same condition, the areal capacitance (0.8 mF cm ^−2 ) and coloration efficiency (269.80 cm ^2 C ^−1 ) of AgNWs/Co(OH) _2 /PEDOT:PSS FECESDs were obviously higher than AgNWs/PEDOT:PSS FECESDs. Furthermore, the obtained FECESDs exhibited excellent stability against the mechanical deformation. The areal capacitance remained stable during 1000 times cyclic bending with a 25 mm curvature radius. These results demonstrated the broad application potential of the AgNWs/Co(OH) _2 /PEDOT:PSS FECESD for the emerging portable and multifunctional electronics.

Abstract Background Both growth and nutritional traits are important economic traits of Crassostrea gigas (C. gigas) in industry. But few work has been done to study the genetic architecture of nutritional traits of the oyster. In this study, we constructed a high-density genetic map of C. gigas to help assemble the genome sequence onto chromosomes, meanwhile explore the genetic basis for nutritional traits via quantitative trait loci (QTL) mapping. Results The constructed genetic map contained 5024 evenly distributed markers, with an average marker interval of 0.68 cM, thus representing the densest genetic map produced for the oyster. According to the high collinearity between the consensus map and the oyster genome, 1574 scaffold (about 70%) of the genome sequence of C. gigas were successfully anchored to 10 linkage groups (LGs) of the consensus map. Using this high-qualified genetic map, we then conducted QTL analysis for growth and nutritional traits, the latter of which includes glycogen, amino acid (AA), and fatty acid (FA). Overall, 41 QTLs were detected for 17 traits. In addition, six candidate genes identified in the QTL interval showed significant correlation with the traits on transcriptional levels. These genes include growth-related genes AMY and BMP1, AA metabolism related genes PLSCR and GR, and FA metabolism regulation genes DYRK and ADAMTS. Conclusion Using the constructed high-qualified linkage map, this study not only assembled nearly 70% of the oyster genome sequence onto chromosomes, but also identified valuable markers and candidate genes for growth and nutritional traits, especially for AA and FA that undergone few studies before. These findings will facilitate genome assembly and molecular breeding of important economic traits in C. gigas.

Many marine ectotherms, especially those inhabiting highly variable intertidal zones, develop high phenotypic plasticity in response to rapid climate change by modulating gene expression levels. Herein, we examined the regulatory architecture of heat-responsive gene expression plasticity in oysters using expression quantitative trait loci (eQTL) analysis. Using a backcross family of Crassostrea gigas and its sister species Crassostrea angulata under acute stress, 56 distant regulatory regions accounting for 6–26.6% of the gene expression variation were identified for 19 heat-responsive genes. In total, 831 genes and 164 single nucleotide polymorphisms (SNPs) that could potentially regulate expression of the target genes were screened in the eQTL region. The association between three SNPs and the corresponding target genes was verified in an independent family. Specifically, Marker13973 was identified for heat shock protein (HSP) family A member 9 (HspA9). Ribosomal protein L10a (RPL10A) was detected approximately 2 kb downstream of the distant regulatory SNP. Further, Marker14346-48 and Marker14346-85 were in complete linkage disequilibrium and identified for autophagy-related gene 7 (ATG7). Nuclear respiratory factor 1 (NRF1) was detected approximately 3 kb upstream of the two SNPs. These results suggested regulatory relationships between RPL10A and HSPA9 and between NRF1 and ATG7. Our findings indicate that distant regulatory mutations play an important role in the regulation of gene expression plasticity by altering upstream regulatory factors in response to heat stress. The identified eQTLs provide candidate biomarkers for predicting the persistence of oysters under future climate change scenarios.

Abstract Background The Pacific oyster Crassostrea gigas is an important marine fishery resource, which contains high levels of glycogen that contributes to the flavor and the quality of the oyster. However, little is known about the molecular and chemical mechanisms underlying glycogen content differences in Pacific oysters. Using a homogeneous cultured Pacific oyster family, we explored these regulatory networks at the level of the metabolome and the transcriptome. Results Oysters with the highest and lowest natural glycogen content were selected for differential transcriptome and metabolome analysis. We identified 1888 differentially-expressed genes, seventy-five differentially-abundant metabolites, which are part of twenty-seven signaling pathways that were enriched using an integrated analysis of the interaction between the differentially-expressed genes and the differentially-abundant metabolites. Based on these results, we found that a high expression of carnitine O-palmitoyltransferase 2 (CPT2), indicative of increased fatty acid degradation, is associated with a lower glycogen content. Together, a high level of expression of phosphoenolpyruvate carboxykinase (PEPCK), and high levels of glucogenic amino acids likely underlie the increased glycogen production in high-glycogen oysters. In addition, the higher levels of the glycolytic enzymes hexokinase (HK) and pyruvate kinase (PK), as well as of the TCA cycle enzymes malate dehydrogenase (MDH) and pyruvate carboxylase (PYC), imply that there is a concomitant up-regulation of energy metabolism in high-glycogen oysters. High-glycogen oysters also appeared to have an increased ability to cope with stress, since the levels of the antioxidant glutathione peroxidase enzyme 5 (GPX5) gene were also increased. Conclusion Our results suggest that amino acids and free fatty acids are closely related to glycogen content in oysters. In addition, oysters with a high glycogen content have a greater energy production capacity and a greater ability to cope with stress. These findings will not only provide insights into the molecular mechanisms underlying oyster quality, but also promote research into the molecular breeding of oysters.

Abstract Despite the mitochondrial antiviral signalling protein (MAVS)-dependent RIG-I-like receptor (RLR) signalling pathway in the cytosol plays an indispensable role in the antiviral immunity of the host, surprising little is known in invertebrates. Here we characterized the major members of RLR pathway and investigated their signal transduction a Molluscs. We show that genes involved in RLR pathway were significantly induced during virus challenge, including CgRIG-I-1, CgMAVS, CgTRAF6 (TNF receptor-associated factor 6), and CgIRFs (interferon regulatory factors. Similar to human RIG-I, oyster RIG-I-1 could bind poly(I:C) directly in vitro and interact with oyster MAVS via its caspase activation and recruitment domains. We also show that transmembrane domain-dependent self-association of CgMAVS may be crucial for its signalling and that CgMAVS can recruit the downstream signalling molecule, TRAF6, which can subsequently activate NF-κB signal pathway. Moreover, oyster IRFs appeared to function downstream of CgMAVS and were able to activate the interferon β promoter and interferon stimulated response elements in mammalian cells. These results establish invertebrate MAVS-dependent RLR signalling for the first time and would be helpful for deciphering the antiviral mechanisms of invertebrates and understanding the development of the vertebrate RLR network.

Investigating the physiological mechanisms of closely related species that exhibit distinct geographic distributions and thermal niches is essential for understanding their thermal tolerance capacities and local adaptations in view of climate warming. The variations in upper thermal limits (LT50) under acute heat shock and cardiac activity, standard metabolic rate (SMR), anaerobic metabolite production and molecular responses (expression of molecular chaperones and glycolysis metabolism genes) under increasing temperatures in two oyster subspecies were studied. The populations of two oyster subspecies, Crassostrea gigas gigas and C. gigas angulata, exhibit different latitudinal distributions along the northern and southern coastlines of China, respectively, which experience different environmental conditions. The LT50 was significantly higher, by ∼1°C, in the southern than in the northern oysters. In both subspecies, temperature increases had powerful effects on heart rate, SMR and gene expression. The southern oysters had the highest Arrhenius breakpoint temperatures for heart rate (31.4 ± 0.17°C) and SMR (33.09°C), whereas the heart rate (28.86 ± 0.3°C) and SMR (29.22°C) of the northern oysters were lower. The same patterns were observed for the Q10 coefficients. More thermal sensitivity was observed in the northern oysters than in their southern counterparts, as the heat-shock proteins (HSPs) in the northern oysters were expressed first and had a higher induction at a lower temperature than those of southern oysters. Furthermore, different expression patterns of energetic metabolism genes (HK, PK, and PEPCK) were observed. In the northern oysters, increasing anaerobic glycolysis genes (PEPCK) and end products (succinate) were found at 36–43°C, indicating a transition from aerobic to anaerobic metabolism and a lower aerobic scope compared with the southern oysters. These two subspecies experience different environmental conditions, and their physiological performances suggested species-specific thermal tolerance windows in which the southern oysters, with mild physiological flexibility, had a higher potential capability to withstand heat stress. Overall, our results indicate that comparing and unifying physiological and molecular mechanisms can provide a framework for understanding the likely effects of global warming on marine ectotherms in intertidal regions.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKKα/β-2 gene. We found that oyster IKKα/β-2, a homolog of human IKKα/IKKβ, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/β-2 activated the promoters of NF-κB, TNFα, and IFNβ, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/β-1, we investigated the signal transduction pathways mediated by these two IKKα/β proteins. Our results showed that IKKα/β-1 and IKKα/β-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/β-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/βs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/β-1 and IKKα/β-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/β proteins interacted with NEMO protein, indicates that oyster IKKα/βs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/βs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster “interferon (IFN)-like” antiviral pathway. Moreover, the expression of oyster IKKα/β-1 and IKKα/β-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/β-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/β proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

The Pacific oyster (Crassostrea gigas) is one of the most important aquaculture species worldwide. Glycogen contributes greatly to the special taste and creamy white color of oysters. Previous genome-wide association studies (GWAS) identified several single nucleotide polymorphism (SNP) sites that were strongly related to glycogen content. Genes within 100 kb upstream and downstream of the associated SNPs were screened. One gene annotated as protein phosphatase 1 regulatory subunit 3B (PPP1R3B), which can promote glycogen synthesis together with protein phosphatase 1 catalytic subunit (PPP1C) in mammals, was selected as a candidate gene in this study. First, full-length CgPPP1R3B was cloned and its function was characterized. The gene expression profiles of CgPPP1R3B in different tissues and seasons showed a close relationship to glycogen content. RNA interference (RNAi) experiments of this gene in vivo showed that decreased CgPPP1R3B levels resulted in lower glycogen contents in the experimental group than in the control group. Co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) assays indicated that CgPPP1R3B can interact with CgPPP1C, glycogen synthase (CgGS) and glycogen phosphorylase (CgGP), thus participating in glycogen metabolism. Co-sedimentation analysis in vitro demonstrated that the CgPPP1R3B protein can bind to glycogen molecules directly, and these results indicated the conserved function of the CgPPP1R3B protein compared to that of mammals. In addition, thirteen SNPs were precisely mapped in this gene. Ten of the thirteen SNPs were confirmed to be significantly (p < 0.05) related to glycogen content in an independent wild population (n = 288). The CgPPP1R3B levels in oysters with high glycogen content were significantly higher than those of oysters with low glycogen content, and gene expression levels were significantly associated with various genotypes of four associated SNPs (p < 0.05). The data indicated that the associated SNPs may control glycogen content by regulating CgPPP1R3B expression. These results suggest that CgPPP1R3B is an important gene for glycogen metabolic regulation and that the associated SNPs of this gene are potential markers for oyster molecular breeding for increased glycogen content.

Oysters are among the most important species in global aquaculture. Crassostrea gigas, and its subspecies C. angulata, are the major cultured species. To determine the genetic basis of growth-related traits in oysters, we constructed a second-generation linkage map from 3367 single-nucleotide polymorphisms (SNPs) based on genotyping-by-sequencing, genotyped from a C. gigas × C. angulata hybrid family. These 3367 SNPs were distributed on 1695 markers, which were assigned to 10 linkage groups. The genetic linkage map had a total length of 1084.3 cM, with an average of 0.8 cM between markers; it thus represents the densest genetic map constructed for oysters to date. Twenty-seven quantitative trait loci (QTL) for five growth-related traits were detected. These QTL could explain 4.2–7.7% (mean = 5.4%) of the phenotypic variation. In total, 50.8% of phenotypic variance for shell width, 7.7% for mass weight, and 34.1% for soft tissue weight were explained. The detected QTL were distributed among eight linkage groups, and more than half (16) were concentrated within narrow regions in their respective linkage groups. Thirty-eight annotated genes were identified within the QTL regions, two of which are key genes for carbohydrate metabolism. Other genes were found to participate in assembly and regulation of the actin cytoskeleton, signal transduction, and regulation of cell differentiation and development. The newly developed high-density genetic map, and the QTL and candidate genes identified provide a valuable genetic resource and a basis for marker-assisted selection for C. gigas and C. angulata.

Background: Reduced-representation sequencing technology is widely used in genotyping for its economical and efficient features. A popular way to construct the reduced-representation sequencing libraries is to digest the genomic DNA with restriction enzymes. A key factor of this method is to determine the restriction enzyme(s). But there are few computer programs which can evaluate the usability of restriction enzymes in reduced-representation sequencing. SimRAD is an R package which can simulate the digestion of DNA sequence by restriction enzymes and return enzyme loci number as well as fragment number. But for linkage mapping analysis, enzyme loci distribution is also an important factor to evaluate the enzyme. For phylogenetic studies, comparison of the enzyme performance across multiple genomes is important. It is strongly needed to develop a simulation tool to implement these functions. Results: Here, we introduce a Perl module named RestrictionDigest with more functions and improved performance. It can analyze multiple genomes at one run and generate concise comparison of enzyme performance across the genomes. It can simulate single-enzyme digestion, double-enzyme digestion and size selection process and generate comprehensive information of the simulation including enzyme loci number, fragment number, sequences of the fragments, positions of restriction sites on the genome, the coverage of digested fragments on different genome regions and detailed fragment length distribution. Conclusions: RestrictionDigest is an easy-to-use Perl module with flexible parameter settings. With the help of the information produced by the module, researchers can easily determine the most appropriate enzymes to construct the reduced-representation libraries to meet their experimental requirements.

Fine-scale adaptive evolution is always constrained by strong gene flow at vertical level in marine organisms. Rapid environmental fluctuations and phenotypic plasticity through optimization of fitness-related traits in organisms play important roles in shaping intraspecific divergence. The coastal systems experience strong variations in multiple abiotic environmental factors, especially the temperature. We used a typical intertidal species, Pacific oyster (Crassostrea gigas), to investigate the interaction between plasticity and adaptive evolution. We collected intertidal and subtidal oysters from two ecological niches and carried out common garden experiments for one generation. We identified fine-scale vertical adaptive divergence between intertidal and subtidal F1 progeny at both sites, based on different hierarchical phenotypes, including morphological, physiological, and molecular traits. We further quantified the global plasticity to thermal stress through transcriptomic analysis. The intertidal oysters exhibited slow growth rate. However, they showed high survival and metabolic rates under heat stress, indicating vertically fine-scale phenotypic adaptive mechanisms and evolutionary trade-offs between growth and thermal tolerance. Transcriptomic analysis confirmed that the intertidal oysters have evolved high plasticity. The genes were classified into three types: evolutionarily divergent, concordantly plastic, and adaptive plastic genes. The evolved divergence between intertidal and subtidal oysters for these gene sets showed a significant positive correlation with plastic changes of subtidal populations in response to high temperature. Furthermore, the intertidal oysters exhibited delayed large-scale increase in expressional plasticity than that in subtidal counterparts. The same direction between plasticity and selection suggests that the oysters have evolved adaptive plasticity. This implies that adaptive plasticity facilitates the oyster to adapt to severe intertidal zones. The oysters exposed to strong environmental variability are thermal tolerant and have high adaptive potential to face the current global warming. Our findings will not only provide new insights into the significant role of plasticity in adaptive evolution that can be extended to other marine invertebrates, but also provide basic information for oyster resources conservation and reef reestablishment.

Single nucleotide polymorphisms (SNPs) are widely used in genetics and genomics research. The Pacific oyster (Crassostrea gigas) is an economically and ecologically important marine bivalve, and it possesses one of the highest levels of genomic DNA variation among animal species. Pacific oyster SNPs have been extensively investigated; however, the mechanisms by which these SNPs may be used in a high-throughput, transferable, and economical manner remain to be elucidated. Here, we constructed an oyster 190K SNP array using Affymetrix Axiom genotyping technology. We designed 190,420 SNPs on the chip; these SNPs were selected from 54 million SNPs identified through re-sequencing of 472 Pacific oysters collected in China, Japan, Korea, and Canada. Our genotyping results indicated that 133,984 (70.4%) SNPs were polymorphic and successfully converted on the chip. The SNPs were distributed evenly throughout the oyster genome, located in 3,595 scaffolds with a length of ~509.4 million; the average interval spacing was 4,210 bp. In addition, 111,158 SNPs were distributed in 21,050 coding genes, with an average of 5.3 SNPs per gene. In comparison with genotypes obtained through re-sequencing, ~69% of the converted SNPs had a concordance rate of >0.971; the mean concordance rate was 0.966. Evaluation based on genotypes of full-sib family individuals revealed that the average genotyping accuracy rate was 0.975. Carrying 133 K polymorphic SNPs, our oyster 190K SNP array is the first commercially available high-density SNP chip for mollusks, with the highest throughput. It represents a valuable tool for oyster genome-wide association studies, fine linkage mapping, and population genetics.

DNA methylation is one of the most important epigenetic modifications of eukaryotic genomes and is believed to play integral roles in diverse biological processes. Although DNA methylation has been well studied in mammals, data are limited in invertebrates, particularly Mollusca. The Pacific oyster Crassostrea gigas is an emerging genetic model for functional analysis of DNA methylation in Mollusca. Recent studies have shown that there is a positive association between methylation status and gene expression in C. gigas; however, whether this association exists at the exon level remains to be determined.In this study, we characterized the genome-wide methylation pattern across two different tissues of C. gigas and found that methylated genes are expressed in more tissues and development stages than unmethylated genes. Furthermore, we found that different types of exons had different methylation levels, with the lowest methylation levels in the first exons, followed by the last exons, and the internal exons. We found that the exons included in the gene transcript contained significantly higher DNA methylation levels than skipped exons. We observed that the DNA methylation levels increased slowly after the start sites and end sites of exons seperately, and then decreased quickly towards the middle sites of exons. We also found that methylated exons were significantly longer than unmethylated exons.This study constitutes the first genome-wide analysis to show an association between exon-level DNA methylation and mRNA expression in the oyster. Our findings suggest that exon-level DNA methylation may play a role in the construction of alternative splicing by positively influencing exon inclusion during transcription.

Chlorite and serpentine are common magnesium-containing gangue minerals in copper sulfide flotation. In this study, sodium alginate, a natural hydrophilic polysaccharide, was introduced as a selective depressant for these gangue minerals. Micro-flotation tests were conducted on both single minerals and synthetic mixtures. The flotation results showed that sodium alginate could simultaneously depress the flotation of chlorite and serpentine effectively, but seldom influenced the floatability of chalcopyrite at pH 9. In the ternary mixture flotation, a concentrate with a Cu grade of 31% could be achieved at Cu recovery of 90%. The selective depression of chlorite and serpentine was also validated by the real ore flotation experiments. The selective depression mechanism was investigated through adsorption tests, zeta potential measurements, and FTIR analyses. The adsorption density results implied that sodium alginate selectively adsorbed on the surface of phyllosilicates, but no adsorption on the chalcopyrite surface was observed. The zeta potential results showed that the sodium alginate could selectively decrease the surface charge of chlorite and serpentine. The FTIR results revealed the chemical adsorption of sodium alginate on the chlorite and serpentine surface and no form of adsorption on chalcopyrite, agreeing well with the adsorption density results. On the basis of these results, a selective adsorption model of sodium alginate on the mineral surface was proposed.

A novel method to limit the adverse effect of fine serpentine on the flotation of pyrite was investigated in this paper. The flotation results showed that coarser serpentine possessed a weaker depression effect on the pyrite flotation process, and the use of KAl(SO4)2·12H2O could efficiently limit the detrimental effect of fine serpentine on pyrite with a maximum increase of pyrite recovery from 14% to 86% at pH 9.0. The results of particle size measurements and rheological measurements exhibited that the addition of KAl(SO4)2·12H2O increased the particle size of serpentine buta hrdly affected the particle size of pyrite, then limited the formation of serpentine-pyrite aggregates. Adsorption test results showed that the adsorption density of potassium butyl xanthate (PBX) onto pyrite regained with the addition of KAl(SO4)2·12H2O, thereby achieving good flotation improvement. It can be concluded that KAl(SO4)2·12H2O is likely to be an effective pyrite flotation reagent, especially in the presence of fine serpentine.

Hypoxia-inducible factor (HIF), a critical member of the basic-helix-loop-helix (bHLH)-containing Per-Arnt-Sim (PAS) protein family, is a master transcription factor involved in maintaining oxygen homeostasis. In the present study, we isolated and characterized a novel bHLH-PAS family member, CgHIFα-like gene, from the Pacific oyster Crassostrea gigas, and determined its importance during hypoxia stress. The 3020-bp CgHIFα-like cDNA encoded a protein of 888 amino acids. The predicted CgHIFα-like amino acid sequence was conserved in the N-terminal bHLH, PAS, and PAC domains (but not in the C-terminal domain) and was most closely related to the HIF family in the bHLH-PAS protein phylogenic tree. Similar to the mammalian HIF-1α, CgHIFα-like could be expressed as four mRNA isoforms containing alternative 5'-untranslated regions and different translation initiation codons. At the mRNA level, these isoforms were expressed in a tissue-specific manner and showed increased transcription to varying degrees under hypoxic conditions. Additionally, the western blot analysis demonstrated that CgHIFα-like was induced by hypoxia. Electrophoretic mobility shift assay indicated that CgHIFα-like could bind to the hypoxia responsive element (HRE), whereas dual-luciferase reporter analysis demonstrated that CgHIFα-like could transactivate the reporter gene containing the HREs. In addition to CgHIFα-like, we identified CgARNT from the C. gigas, analyzed its expression pattern, and confirmed its interaction with CgHIFα-like using a yeast two-hybrid assay. In conclusion, this is the first report on the cloning and characterization of a novel hypoxia transcription factor in mollusks, which could accumulate under hypoxia and regulate hypoxia related gene expression by binding to HRE and dimerizing with CgARNT. As only one member of HIF has been identified in invertebrates to date, our results provide new insights into the unique mechanisms of hypoxia tolerance in mollusks.

Octopamine (OA) and its precursor, tyramine (TA), participate in invertebrate development such as growth, maturation, and reproduction by activating their corresponding G protein-coupled receptors (GPCRs). Although OA was first discovered in mollusks (octopus), subsequent studies on OA, TA and related receptors have primarily been conducted in Ecdysozoa, especially in insects. Accordingly, only limited reports on OA/TA receptors in mollusks are available and their physiological roles remain unclear. Here, a full-length cDNA encoding a putative 524 amino acid OA/TA receptor (CgGPR1) was isolated from the Pacific oyster Crassostrea gigas. CgGPR1 was most closely related to the Lymnaea stagnalis OA receptor OAR2 in sequence. Phylogenetic analysis showed that CgGPR1 belongs to a poorly studied subfamily of invertebrate OA/TA receptors. The spatio-temporal expression of CgGPR1 in C. gigas larvae was examined by quantitative real-time PCR and Western blot analysis. CgGPR1 was expressed during all developmental stages of C. gigas with higher levels at mid-developmental stages, indicating its potential role in embryogenesis and tissue differentiation. Immunoreactive fluorescence of CgGPR1 was mainly observed in the velum, foot, gill and mantle of C. gigas larvae. CgGPR1 transcripts were detected in all the tested organs of adult C. gigas, with highest level in the mantle. Pharmacological analysis showed that cAMP and Ca2+ concentrations remained unchanged in HEK293 cells expressing CgGPR1 upon addition of OA, TA or related amines, suggesting that CgGPR1 modulates other unknown molecules rather than cAMP and Ca2+. Our study sheds light on CgGPR1 function in oysters.

Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks.