Cardiac performance and heart gene network provide dynamic responses of bay scallop Argopecten irradians irradians exposure to marine heatwaves.

The increased frequency of marine heat waves (MHWs) caused by global climate change is predicted to threaten the survival of economic bivalves, therefore having severely adverse effects on local ecological communities and aquaculture production. However, the study of scallops facing MHWs is still scarce, particularly in the scallop Argopecten irradians irradians, which has a significant share of "blue foods" in northern China. In the present study, bay scallop heart was selected to detect its cardiac performance, oxidative impairment and dynamic molecular responses, accompanied by assessing survival variations of individuals in the simulated scenario of MWHs (32 °C) with different time points (0 h, 6 h, 12 h, 24 h, 3 d, 6 d and 10 d). Notably, cardiac indices heart rate (HR), heart amplitude (HA), rate-amplitude product (RAP) and antioxidant enzyme activities superoxide dismutase (SOD) and catalase (CAT) all peaked at 24 h but sharply dropped on 3 d, coinciding with mortality. Transcriptome analysis revealed that the heart actively defended against heat stress at the acute stage (<24 h) via energy supply, misfolded proteins correction and enhanced signal transduction, whereas regulation of the defense response and apoptotic process combined with twice transcription initiation were the dominant responses at the chronic stage (3-10 d). In particular, HSP70 (heat shock protein 70), HSP90 and CALR (calreticulin) in the endoplasmic reticulum were identified as the hub genes (top 5 %) in the HR-associated module via WGCNA (weighted gene co-expression network analysis) trait-module analysis, followed by characterization of their family members and diverse expression patterns under heat exposure. Furthermore, RNAi-mediated knockdown of CALR expression (after 24 h) significantly weakened the thermotolerance of scallops, as evidenced by a drop of 1.31 °C in ABT (Arrhenius break temperature) between the siRNA-injected group and the control group. Our findings elucidated the dynamic molecular responses at the transcriptome level and verified the cardiac functions of CALR in bay scallops confronted with stimulated MHWs.

Tissue-Specific and Time-Dependent Expressions of PC4s in Bay Scallop (Argopecten irradians irradians) Reveal Function Allocation in Thermal Response.

Transcriptional coactivator p15 (PC4) encodes a structurally conserved but functionally diverse protein that plays crucial roles in RNAP-II-mediated transcription, DNA replication and damage repair. Although structures and functions of PC4 have been reported in most vertebrates and some invertebrates, the PC4 genes were less systematically identified and characterized in the bay scallop Argopecten irradians irradians. In this study, five PC4 genes (AiPC4s) were successfully identified in bay scallops via whole-genome scanning through in silico analysis. Protein structure and phylogenetic analyses of AiPC4s were conducted to determine the identities and evolutionary relationships of these genes. Expression levels of AiPC4s were assessed in embryos/larvae at all developmental stages, in healthy adult tissues and in different tissues (mantles, gills, hemocytes and hearts) being processed under 32 °C stress with different time durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d and 10 d). Spatiotemporal expression profiles of AiPC4s suggested the functional roles of the genes in embryos/larvae at all developmental stages and in healthy adult tissues in bay scallop. Expression regulations (up- and down-) of AiPC4s under high-temperature stress displayed both tissue-specific and time-dependent patterns with function allocations, revealing that AiPC4s performed differentiated functions in response to thermal stress. This work provides clues of molecular function allocation of PC4 in scallops in response to thermal stress and helps in illustrating how marine bivalves resist elevated seawater temperature.

Identification, characterization and expression analyses of PC4 genes in Yesso scallop (Patinopecten yessoensis) reveal functional differentiations in response to ocean acidification.

Transcriptional coactivator p15 (PC4), considered a multifunctional chromosome associated protein, is actively involved in transcription regulation, DNA replication, damage repair and chromosome formation. Although studies have reported significant effects of PC4 in most vertebrates and some invertebrates, the complete PC4 gene members are less systematically identified and characterized in scallops. In this study, seven PC4 genes (PyPC4s) were identified in the Yesso scallop Patinopecten yessoensis using whole-genome scanning via bioinformatic analyses. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the seven genes. Expression profiles of PyPC4s were further investigated in embryos/larvae at all developmental stages, healthy adult tissues, and mantles that were exposed to low pH stress (pH 6.5 and 7.5) with different time durations (3, 6, 12 and 24 h). Spatiotemporal expression patterns indicated the functional roles of PyPC4s at all development stages and in healthy adult tissues, with PY-3235.33 demonstrating remarkably high constitutive expressions. Expression regulations (up- and down-regulation) of PyPC4s under low pH stress levels demonstrated a time-dependent pattern with functional complementation and/or enhancement, revealing that PyPC4s exhibited differentiated functions in response to ocean acidification (OA). Collectively, our data offer a novel perspective stating that low pH is a potential inducer leading to functional differentiation of PyPC4s in scallops. The results provide preliminary information on the versatile roles of PC4(s) in bivalves in response to OA.

Molecular allocation of PC4s provides implications for deciphering thermal response in Zhikong scallop (Chlamys farreri).

The increasing sea temperature caused by global warming has led to serious death of Zhikong scallop (Chlamys farreri) and improving its thermal tolerance has become an active research area in scallop aquaculture industry. Gene transcriptional coactivator p15 (PC4) plays pivotally multi-faced roles in most vertebrates and some invertebrates, but the systematic identification and characterization of PC4 genes have less been reported in scallops. In this study, 15 PC4 genes (CfPC4s) were identified in Zhikong scallop through whole-genome scanning, including two pairs of tandem duplicate genes located in the same scaffold (CF-19495.9 and CF-19495.10, CF-6819.1 and CF-6819.2). Protein structural and phylogenetic analyses were performed to verify identities and evolutionary relationships of these genes. Spatiotemporal expression patterns were determined at different development stages and in healthy adult tissues, as well as expression regulations in selected tissues (mantles, gills, hemocytes and hearts) after high temperatures challenge (27 °C) with different durations (3 h, 6 h, 12 h, 24 h, 3 d, 6 d, 15 d and 30 d). Spatiotemporal expressions of CfPC4s were ubiquitous but exhibited different patterns, suggesting the functional roles of CfPC4s in all stages of growth and development of the scallop. Expression regulations of CfPC4s and their functional related factors (TFIIA, TFIID, TFIIH and RNAPII) in pre-initiation complex (PIC) in various tissues displayed up- and/or down-regulated responses at different time points, showing time- and/or tissue-dependent expression patterns with function allocation upon different thermal durations. Collectively, this study demonstrated that gene allocation of CfPC4s provided implications for deciphering thermal response in Zhikong scallop and potentially helped in developing strategies for long-term healthy sustainable Zhikong scallop culture.

Fine-mapping and association analysis of candidate genes for papilla number in sea cucumber, Apostichopus japonicus.

The papilla number is one of the most economically important traits of sea cucumber in the China marketing trade. However, the genetic basis for papilla number diversity in holothurians is still scarce. In the present study, we conducted genome-wide association studies (GWAS) for the trait papilla number of sea cucumbers utilizing a set of 400,186 high-quality SNPs derived from 200 sea cucumbers. Two significant trait-associated SNPs that passed Bonferroni correction (P < 1.25E-7) were located in the intergenic region near PATS1 and the genic region of EIF4G, which were reported to play a pivotal role in cell growth and proliferation. The fine-mapping regions around the top two lead SNPs provided precise causative loci/genes related to papilla formation and cellular activity, including PPP2R3C, GBP1, and BCAS3. Potential SNPs with P < 1E-4 were acquired for the following GO and KEGG enrichment analysis. Moreover, the two lead SNPs were verified in another population of sea cucumber, and the expressive detection of three potential candidate genes PATS1, PPP2R3C, and EIF4G that near or cover the two lead SNPs was conducted in papilla tissue of TG (Top papilla number group) and BG (Bottom papilla number group) by qRT-PCR. We found the significantly higher expression profile of PATS1 (3.34-fold), PPP2R3C (4.90-fold), and EIF4G (4.23-fold) in TG, implying their potential function in papilla polymorphism. The present results provide valuable information to decipher the phenotype differences of the papilla trait and will provide a scientific basis for selective breeding in sea cucumbers. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00139-w.

Genome-Wide Association Study Reveals PC4 as the Candidate Gene for Thermal Tolerance in Bay Scallop (Argopecten irradians irradians).

The increasing sea temperature caused by global warming has resulted in severe mortalities in maricultural scallops. Therefore, improving thermal tolerance has become an active research area in the scallop farming industry. Bay scallop (Argopecten irradians irradians) was introduced into China in 1982 and has developed into a vast aquaculture industry in northern China. To date, genetic studies on thermal tolerance in bay scallops are limited, and no systematic screening of thermal tolerance-related loci or genes has been conducted in this species. In the present study, we conducted a genome-wide association study (GWAS) for thermal tolerance using the Arrhenius break temperature (ABT) indicators of 435 bay scallops and 38,011 single nucleotide polymorphism (SNP) markers. The GWAS identified 1,906 significant thermal tolerance-associated SNPs located in 16 chromosomes of bay scallop. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that 638 genes were enriched in 42 GO terms, while 549 annotated genes were enriched in aggregation pathways. Additionally, the SNP (15-5091-20379557-1) with the lowest P value was located in the transcriptional coactivator p15 (PC4) gene, which is involved in regulating DNA damage repair and stabilizing genome functions. Further analysis in another population identified two new thermal tolerance-associated SNPs in the first coding sequence of PC4 in bay scallops (AiPC4). Moreover, AiPC4 expression levels were significantly correlated (r = 0.675-0.962; P < 0.05) with the ABT values of the examined bay scallops. Our data suggest that AiPC4 might be a positive regulator of thermal tolerance and a potential candidate gene for molecular breeding in bay scallop aiming at thermal tolerance improvement.

RNA m6A reader YTHDF2 facilitates lung adenocarcinoma cell proliferation and metastasis by targeting the AXIN1/Wnt/ß-catenin signaling.

Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related deaths worldwide. YTHDF2 is a reader of N6-methyladenosine (m6A) on RNA and plays a critical role in the initiation and propagation of myeloid leukemia; however, whether YTHDF2 controls the development of LUAD remains to be explored. Here, we found that YTHDF2 was significantly upregulated in LUAD compared with paracancerous normal tissues, and YTHDF2 knockdown drastically inhibited, while its overexpression promoted, cell growth, colony formation and migration of LUAD cells in vitro. In addition, YTHDF2 knockdown significantly inhibited tumorigenesis in a murine tumor xenograft model. Through the integrative analysis of RNA-seq, m6A-seq, CLIP-seq, and RIP-seq datasets, we identified a set of potential direct targets of YTHDF2 in LUAD, among which we confirmed AXIN1, which encodes a negative regulator of the Wnt/ß-catenin signaling, as a direct target of YTHDF2. YTHDF2 promoted AXIN1 mRNA decay and subsequently activated the Wnt/ß-catenin signaling. Knockout of AXIN1 sufficiently rescued the inhibitory effect of YTHDF2 depletion on lung cancer cell proliferation, colony-formation, and migration. These results revealed YTHDF2 to be a contributor of LUAD development acting through the upregulation of the AXIN1/Wnt/ß-catenin signaling, which can be a potential therapeutic target for LUAD.

Genome-wide identification, characterization of RLR genes in Yesso scallop (Patinopecten yessoensis) and functional regulations in responses to ocean acidification.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), are crucial sensors with a conserved structure in cytoplasm, inducing the production of cytokines, chemokines and host restriction factors which mediate a variety of intracellular activities to interfere with distinct PAMPs (pathogen-associated molecular patterns) for eliminating pathogens in innate immune system. Although RLR genes have been investigated in most vertebrates and some invertebrates, the systematic identification and characterization of RLR genes have not been reported in scallops. In this study, four RLR genes (PY-10413.4, PY-10413.5, PY-443.7 and PY-443.8, designated PyRLRs) were identified in Yesso scallop (Patinopecten yessoensis) through whole-genome scanning through in silico analysis, including two pairs of tandem duplicate genes located on the same scaffold (PY-10413.4 and PY-10413.5, PY-443.7 and PY-443.8, respectively). Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyRLRs were determined in all developmental stages, in healthy adult tissues, and in mantles that simulated ocean acidification (OA) exposure (pH = 6.5 and 7.5) at different time points (3, 6, 12 and 24 h). Spatiotemporal expression patterns suggested the functional roles of PyRLRs in all stages of development and growth of the scallop. Regulation expressions revealed PY-10413.4 and PY-10413.5 with one or two CARD(s) (caspase activation and recruitment domain) were up-regulated expressed at most time points, whereas PY-443.8 and PY-10413.4 without CARD were significantly down-regulated at each time points, suggesting functional differentiations in the two pairs of PyRLRs based on the structural differences in response to OA. Collectively, this study demonstrated gene duplication of RLR family genes and provide primary analysis for versatile roles in the response of the bivalve innate immune system to OA challenge.

Development of Novel Cardiac Indices and Assessment of Factors Affecting Cardiac Activity in a Bivalve Mollusc Chlamys farreri.

Cardiac activity has been widely used in marine molluscs as an indicator for their physiological status in response to environmental changes, which is, however, largely less studied in scallops. Here, we monitored cardiac performance of Zhikong scallop Chlamys farreri using an infrared-based method, and evaluated the effects of several biotic (shell height, total weight, and age) and environmental factors (circadian rhythm and temperature) on scallop heart rate (HR), amplitude (HA), and rate-amplitude product (RAP). Results revealed that size has a significant effect on both HR (negative) and HA (positive), but RAP values are similar in different sized scallops. Age also affects scallop cardiac performance, significantly for HR, but not for HA or RAP. Circadian rhythm affects cardiac activity, with significant elevation of HR, HA and RAP during 1:00-8:00 and 17:00-19:00. With seawater temperature elevation, HR peaks at 30.03 ± 0.23°C, HA at 15.08 ± 0.02°C, and RAP at 15.10 ± 0.19 and 30.12 ± 0.28°C. This suggests HR is a good indicator for thermal limit, whereas HA may indicate optimal growth temperature, and RAP could be an index of myocardial oxygen consumption to indicate myocardium stress. Our study provides basic information on the factors that may affect scallop cardiac performance. It also elucidates the feasibility of HA and RAP as cardiac indices in marine molluscs.

Identification and characterization of TEP family genes in Yesso scallop (Patinopecten yessoensis) and their diverse expression patterns in response to bacterial infection.

Thioester-containing protein (TEP) family members are characterized by their unique intrachain ß-cysteinyl-γ-glutamyl thioesters, and they play important roles in innate immune responses. Although significant effects of TEP members on immunity have been reported in most vertebrates, as well as certain invertebrates, the complete TEP family has not been systematically characterized in scallops. In this study, five TEP family genes (PyC3, PyA2M, PyTEP1, PyTEP2 and PyCD109) were identified from Yesso scallop (Patinopecten yessoensis) through whole-genome scanning, including one pair of tandem duplications located on the same scaffold. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the five genes (PyTEPs). The vast distribution of PyTEPs in TEP subfamilies confirmed that the Yesso scallop contains relatively comprehensive types of TEP members in evolution. The expression profiles of PyTEPs were determined in hemocytes after bacterial infection with gram-positive (Micrococcus luteus) and gram-negative (Vibrio anguillarum) using quantitative real-time PCR (qRT-PCR). Expression analysis revealed that the PyTEP genes exhibited disparate expression patterns in response to the infection by gram bacteria. A majority of PyTEP genes were overexpressed after bacterial stimulation at most time points, especially the notable elevation displayed by duplicated genes after V. anguillarum challenge. Interestingly, at different infection times, PyTEP1 and PyTEP2 shared analogous expression patterns, as did PyC3 and PyCD109. Taken together, these results help to characterize gene duplication and the evolutionary origin of PyTEPs and supplied valuable resources for elucidating their versatile roles in bivalve innate immune responses to bacterial pathogen challenges.