RNA-seq is a recently developed approach widely used for transcriptome profiling in biological analyses that use next-generation sequencing technologies. Accurate estimation of gene expression levels ...is critical for answering biological questions. Here, we show that the commonly used measure of gene expression levels, fragments per kilobase of transcript per million mapped reads (FPKM), is biased in transcript length, GC content, and dinucleotide frequencies in the RNA-seq analysis of marine species. We used a generalized linear model to correct the observed biases of FPKM. We used RNA-seq data sets from eight species obtained by different sequencing methods to evaluate the correction methods. Our work contributes to the understanding of potential technical artifacts in RNA-seq experiments for marine species, and presents a means by which more accurate gene expression measures can be obtained.
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.
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)62+ 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)62+ 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.
Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about ...the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length caspase-3-like gene named Cgcaspase-3 was cloned from C.gigas cDNA, encoding a predicted protein containing caspase family p20 and p10 domain profiles and a conserved caspase active site motif. Phylogenetic analysis demonstrated that both Cgcaspase-3 and Cgcaspase-1 may function as effector caspases clustered in the invertebrate branch. Although the sequence identities between the two caspases was low, both enzymes possessed executioner caspase activity and were capable of inducing cell death. These results suggested that Cgcaspase-3 and Cgcaspase-1 were two effector caspases in C. gigas. We also observed that nucleus-localized Cgcaspase-3, may function as a caspase-3-like protein and cytoplasm-localized Cgcaspase-1 may function as a caspase-7-like protein. Both Cgcaspase-3 and Cgcaspase-1 mRNA expression increased after larvae settled on the substratum, suggesting that both caspases acted in several tissues or organs that degenerated after oyster larvae settlement. The highest caspase expression levels were observed in the gills indicating that both effector caspases were likely involved in immune or metabolic processes in C. gigas.
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
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.
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 F
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
) 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
and
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.
The northern bay scallop Argopecten irradians irradians (Lamarck) and the southern bay scallop Argopecten irradians concentricus (Say) were introduced into China in the 1980s and 1990s, and are now ...major aquaculture molluscs in China. Here, we report the transcriptome sequencing of the two subspecies and the subsequent association analysis on candidate gene on the trait of heat tolerance. In total, RNA from six tissues of 67 and 42 individuals of northern and southern bay scallops, respectively, were used and 55.5 and 34.9 million raw reads were generated, respectively. There were 82,267 unigenes produced in total, of which 32,595 were annotated. Altogether, 32,206 and 23,312 high-quality SNPs were identified for northern and southern bay scallops, respectively. For case-control analysis, two intercrossed populations were heat stress treated, and both heat-susceptible and heat-resistant individuals were collected. According to annotation and SNP allele frequency analysis, 476 unigenes were selected, and 399 pairs of primers were designed. Genotyping was conducted using the high-resolution melting method, and Fisher's exact test was performed for allele frequency comparison between the heat-susceptible and heat-resistant groups. SNP all-53308-760 T/C showed a significant difference in allele frequency between the heat-susceptible and heat-resistant groups. Notably, considerable difference in allele frequency at this locus was also observed between the sequenced natural populations. These results suggest that SNP all-53308-760 T/C may be related to the heat tolerance of the bay scallop. Moreover, quantitative expression analysis revealed that the expression level of all-53308 was negatively correlated with heat tolerance of the bay scallop.
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.
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.
In this study, the effect of sodium pyrophosphate (NaPP) on the separation of apatite from dolomite by flotation was systematically investigated. Flotation results revealed that NaPP could ...selectively depress the flotation of apatite, thus realizing the separation of apatite from dolomite. Further, the selective depression mechanism of NaPP was studied through zeta potential measurements, contact angle measurements, and X-ray photoelectron spectroscopy (XPS) analysis. The results demonstrated that the adsorption of sodium oleate (NaOL) onto apatite surface was depressed by the preferential interaction of NaPP with active Ca sites. For dolomite, while the presence of NaPP hindered the interaction of NaOL with active Ca sites, it appeared to enhance the reactivity with active Mg sites. Thus, the adsorption of NaOL onto dolomite surface was hardly influenced. In this way, the separation of apatite from dolomite was achieved.
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