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.
Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better ...understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality.
► Describing the evaluation of C. gigas housekeeping genes in developmental stages. ► RL7 and RS18 are the most suitable internal controls for qRT-PCR in C. gigas larvae. ► EF-1α, 18S, 28S, GAPDH and ACT are not suitable internal controls in C. gigas larvae.
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 (
), 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 F
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.
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.
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.
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.
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•Diverged phosphorylation mediated differential heat tolerance between two Crassostrea oysters.•Heat considerably regulated signal transduction by protein phosphorylation.•Heat influenced energy supply, protein synthesis, apoptosis and cytoskeleton.
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.
Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes. However, the endogenous PUFA biosynthesis pathway is poorly understood in marine bivalves. ...Previously, a fatty acyl desaturase (Fad) with Δ5 activity was functionally characterized and an elongase termed Elovl2/5 was reported to efficiently elongate 18:2n–6 and 18:3n–3 to 20:2n–6 and 20:3n–3 respectively in Chlamys nobilis. In this study, another elongase and another Fad were identified. Functional characterization in recombinant yeast showed that the newly cloned elongase can elongate 20:4n–6 and 20:5n–3 to C22 and C24, while the newly cloned scallop Fad exhibited a Δ8-desaturation activity, and could desaturate exogenously added PUFA 20:3n–3 and 20:2n–6 to 20:4n–3 and 20:3n–6 respectively, providing the first compelling evidence that noble scallop could de novo biosynthesize 20:5n–3 and 20:4n–6 from PUFA precursors though the “Δ8 pathway”. No Δ6 or Δ4 activity was detected for this Fad. Searching against our scallop transcriptome database failed to find any other Fad-like genes, indicating that noble scallop might have limited ability to biosynthesize 22:6n–3. Interestingly, like previously characterized Elovl2/5, the two newly cloned genes showed less efficient activity toward n–3 PUFA substrates than their homologous n–6 substrates, resulting in a relatively low efficiency to biosynthesize n–3 PUFA, implying an adaption to marine environment.
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.
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.
Overall, this study provides insights into the genetic correlation among complex traits, which will facilitate future oyster functional studies and breeding through molecular design.
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.
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