Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a yeast two-hybrid system.

The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.

Regulation of innate immunity in marine mussel Mytilus coruscus: MicroRNA Mc-novel_miR_196 targets McTLR-like1 molecule to inhibit inflammatory response and apoptosis.

Toll-like receptors (TLRs) are crucial players in immune recognition and regulation, with aberrant activation leading to autoimmune, chronic inflammatory, and infectious diseases. MicroRNAs (miRNAs) have been shown to regulate gene expression at transcriptional and post-transcriptional levels. While miRNA-mediated regulation of TLR signaling has been studied in mammals, the underlying mechanisms of TLR-miRNA interactions in molluscs remain unclear. In a previous study, one of the TLR genes potentially targeted by miRNAs was identified and named McTLR-like1. McTLR-like1 was later found to be targeted by miRNA Mc-novel_miR_196 through bioinformatic prediction. In this study, we aim to experimentally determine the interaction between McTLR-like1 and Mc-novel_miR_196, as well as their functional role in the innate immune response of molluscs. The results showed that the expression of Mc-novel_miR_196 was suppressed, while the expression of McTLR-like1 was enhanced in M. coruscus hemocytes treated with lipopolysaccharide (LPS). Moreover, in vitro assays demonstrated that Mc-novel_miR_196 directly targets the 5' UTR of McTLR-like1 and leads to the down-regulation of proinflammatory cytokines in hemocytes. In addition, co-transfection experiments confirmed that Mc-novel_miR_196 inhibits McTLR-like1 and inhibits the expression of proinflammatory cytokines. The Tunel assay also showed that Mc-novel_miR_196 inhibited apoptosis in hemocytes induced by LPS. Our findings suggest that microRNA Mc-novel_miR_196 acts as a regulator of innate immunity in M. coruscus by targeting McTLR-like1 and inhibiting inflammatory response and apoptosis. These results provide further insights into the complex molecular mechanisms underlying TLR signaling in molluscs.

Insights into the Deep Phylogeny and Novel Gene Rearrangement of Mytiloidea from Complete Mitochondrial Genome.

The extant marine mussels which belong to the Mytiloidea are widespread species inhabiting mostly coastal waters, with some distributed in the deep sea. To clarify the classification systems and phylogenetic relationships range from genus to family level within Mytiloidea, new sequence was used in a phylogenetic analysis including all the available Mytiloidea mitochondrial genomes. In this study, the complete mitochondrial genome of Vignadula atrata is 15,624 bp in length and contains 12 protein-coding genes (PCGs, atp8 is absent), two ribosomal RNA genes, and 22 transfer RNA genes. Phylogenetic analysis based on 12 PCGs showed that it has a close relationship to Bathymodiolus. The analysis of gene rearrangements in the Pteriomorphia showed that the arrangements are highly variable across species, novel gene rearrangements were found within Mytiloidea. The V. atrata mitogenome was provided in detail, with notes on the sequence and a key to the species of Vignadula. This study provides a perspective on the taxonomic histories of the marine mussels and refines the unclear relationship between the origin and evolution of species in Mytiloidea within Bivalvia.

Exploring the Role of a Novel Interleukin-17 Homolog from Invertebrate Marine Mussel Mytilus coruscus in Innate Immune Response: Is Negative Regulation by Mc-Novel_miR_145 the Key?

Interleukin-17 (IL-17) represents a class of proinflammatory cytokines involved in chronic inflammatory and degenerative disorders. Prior to this study, it was predicted that an IL-17 homolog could be targeted by Mc-novel_miR_145 to participate in the immune response of Mytilus coruscus. This study employed a variety of molecular and cell biology research methods to explore the association between Mc-novel_miR_145 and IL-17 homolog and their immunomodulatory effects. The bioinformatics prediction confirmed the affiliation of the IL-17 homolog with the mussel IL-17 family, followed by quantitative real-time PCR assays (qPCR) to demonstrate that McIL-17-3 was highly expressed in immune-associated tissues and responded to bacterial challenges. Results from luciferase reporter assays confirmed the potential of McIL-17-3 to activate downstream NF-κb and its targeting by Mc-novel_miR_145 in HEK293 cells. The study also produced McIL-17-3 antiserum and found that Mc-novel_miR_145 negatively regulates McIL-17-3 via western blotting and qPCR assays. Furthermore, flow cytometry analysis indicated that Mc-novel_miR_145 negatively regulated McIL-17-3 to alleviate LPS-induced apoptosis. Collectively, the current results showed that McIL-17-3 played an important role in molluscan immune defense against bacterial attack. Furthermore, McIL-17-3 was negatively regulated by Mc-novel_miR_145 to participate in LPS-induced apoptosis. Our findings provide new insights into noncoding RNA regulation in invertebrate models.

The Functional Significance of McMafF_G_K in Molluscs: Implications for Nrf2-Mediated Oxidative Stress Response.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of antioxidant gene expression in mammals, forming heterodimer complexes with small Maf proteins through its BZip domain. However, the underlying mechanism of Nrf2 action in molluscs remains poorly understood. The thick shell mussel, Mytilus coruscus, represents a model organism for the marine environment and molluscs interaction research. In this study, we used in silico cloning to obtain a small Maf homologue called McMafF_G_K from M. coruscus. McMafF_G_K possesses a typical BZip domain, suggesting its affiliation with the traditional small Maf family and its potential involvement in the Nrf2 signaling pathway. Transcriptional analysis revealed that McMafF_G_K exhibited a robust response to benzo[a]pyrene (Bap) in the digestive glands. However, this response was down-regulated upon interference with McMafF_G_K-siRNA. Interestingly, the expression levels of Nrf2, NAD(P)H: quinone oxidoreductase (NQO-1), and Glutathione Peroxidase (GPx), which are key players in oxidative stress response, showed a positive correlation with McMafF_G_K in digested adenocytes of M. coruscus. Furthermore, in vitro analysis of antioxidant capacity in digestive gland cells demonstrated that Bap exposure led to an increase in reactive oxygen species (ROS) levels, accompanied by an elevation in total antioxidant capacity (T-AOC), potentially counterbalancing the excessive ROS. Strikingly, transfection of McMafF_G_K siRNA resulted in a significant rise in ROS level and a down-regulation of T-AOC level. To validate the functional relevance of McMafF_G_K, a glutathione S-transferase (GST) pull-down assay confirmed its interaction with McNrf2, providing compelling evidence of their protein interaction. This study significantly contributes to our understanding of the functional role of McMafF_G_K in the Nrf2 signaling pathway and sheds light on its potential as a target for further research in oxidative stress response.

Genetic Diversity, Population Structure, and Environmental Adaptation Signatures of Chinese Coastal Hard-Shell Mussel Mytilus coruscus Revealed by Whole-Genome Sequencing.

The hard-shell mussel (Mytilus coruscus) is widespread in the temperate coastal areas of the northwest Pacific and holds a significant position in the shellfish aquaculture market in China. However, the natural resources of this species have been declining, and population genetic studies of M. coruscus are also lacking. In this study, we conducted whole-genome resequencing (WGR) of M. coruscus from eight different latitudes along the Chinese coast and identified a total of 25,859,986 single nucleotide polymorphism (SNP) markers. Our findings indicated that the genetic diversity of M. coruscus from the Zhoushan region was lower compared with populations from other regions. Furthermore, we observed that the evolutionary tree clustered into two primary branches, and the Zhangzhou (ZZ) population was in a separate branch. The ZZ population was partly isolated from populations in other regions, but the distribution of branches was not geographically homogeneous, and a nested pattern emerged, consistent with the population differentiation index (FST) results. To investigate the selection characteristics, we utilized the northern M. coruscus populations (Dalian and Qingdao) and the central populations (Zhoushan and Xiangshan) as reference populations and the southern ZZ population as the target population. Our selection scan analysis identified several genes associated with thermal responses, including Hsp70 and CYP450. These genes may play important roles in the adaptation of M. coruscus to different living environments. Overall, our study provides a comprehensive understanding of the genomic diversity of coastal M. coruscus in China and is a valuable resource for future studies on genetic breeding and the evolutionary adaptation of this species.

Molecular characterization of peptidoglycan recognition proteins from Mytilus coruscus.

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus.

Characterization of the complete mitochondrial genomes of two species of Penaeidae (Decapoda: Dendrobranchiata) and the phylogenetic implications for Penaeoidea.

In the present study, mitogenomes of the species Trachypenaeus curvirostris and Parapenaeus fissuroides (Decapoda: Dendrobranchiata: Penaeidae) were sequenced. The total lengths of the two species were 15,956 bp and 15,937 bp in length with A + T biases of 67.08% and 67.69%, respectively. Both two species showed positive AT skews (0.016, 0.058) and negative GC skews (-0.254, -0.310). Both mitogenomes contained 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Results of phylogenetic analyses support close relationships among Aristeidae, Benthesicymidae and Solenoceridae. The family Sicyoniidae was observed to be deeply nested within Penaeidae. Within Penaeidae, T. curvirostris and P. fissuroides were most closely related to the genus Parapenaeopsis and Metapenaeopsis, respectively, indicated that these two species belong to Penaeidae. These results will help to better understand the evolutionary position of Penaeidae and provide reference for further phylogenetic research on Penaeoidea species.

Sequence comparison of the mitochondrial genomes in two species of the genus Nerita (Gastropoda: Neritimorpha: Neritidae): phylogenetic implications and divergence time estimation for Neritimorpha.

Many Nerita species live in warm-water environments, and they are some of the few organisms from the intertidal zone that can live in both freshwater and seawater. Previous comparative studies of the mitogenomes of Nerita species suggest that the genome rearrangements are very conservative. Generally, the species possess a set of similar mitochondrial gene arrangements, but nucleotide sequences can be used to elucidate phylogenetic relationships at various levels of divergence. Here, the mitogenomes of Nerita undata and Nerita balteata were sequenced and found to be 15,583 bp and 15,571 bp, respectively. The mitogenomes of both species contain 13 protein-coding genes, 22 tRNA genes, and two rRNA genes. The nucleotides of the two mitogenomes are highly similar, with the same gene composition and genomic organization as those present in other Nerita species. The tRNA secondary structures were different from those of other gastropods: trnS2 is not folded into typical secondary structures, and the dihydrouridine (DHU) arm simply forms a loop. The phylogenetic analysis showed that Neritimorpha is a sister group of Vetigastropoda and Caenogastropoda. Nerita balteata is a sister group of Nerita versicolor and Nerita undata, and all three species belong to Neritimorpha. This study contributes towards the comparative mitogenomic analysis of Neritidae and phylogenetic considerations among Neritimorpha species. The estimation of divergence time revealed that the two Nerita species were differentiated in the late Paleogene of the Cenozoic Era, and their evolution may be related to environmental changes.

Strong genetic differentiation of the clam Meretrix lamarckii in the China Sea revealed by mitochondrial DNA marker.

The hard clam Meretrix lamarckii is ecologically and economically important in the coastal regions of China. In this study, we evaluated the genetic diversity and population structure of six M. lamarckii populations in the East China Sea and the South China Sea using mitochondrial cytochrome c oxidase subunit 1 (COI) and cytochrome b (Cytb) genes. We obtained 582 bp of partly sequences comprising 28 novel haplotypes of COI gene from 138 specimens and 1168 bp of partly sequences comprising 22 novel haplotypes of Cytb gene from 125 specimens. The haplotype diversity of COI and Cytb genes ranged from 0.606 to 0.862 and 0.562 to 0.863, respectively. The nucleotide diversity ranged from 0.0015 to 0.0038 in COI gene and ranged from 0.0007 to 0.0032 in Cytb gene. Thus, there is moderate-level genetic diversity in M. lamarckii in the China Sea. The F-statistics showed that the Zhoushan (ZS) and Xiangshan (XS) populations were significantly (P < 0.01) differed from the populations of Wenzhou (WZ), Zhangpu (ZP), Shantou (ST), and Zhanjiang (ZJ) in both COI and Cytb genes. Both haplotypes network and plot of STRUCTURE analysis suggested obviously genetic divergence between East China Sea and South China Sea regions. Knowledge on genetic variation and population structure of M. lamarckii populations along the Southeast China Sea obtained from this study will support the aquaculture management and conservation of M. lamarckii in China.

A novel invertebrate toll-like receptor with broad recognition spectrum from thick shell mussel Mytilus coruscus.

Toll-like receptors (TLRs) are a category of most well recognized pattern recognition molecules that act on a vital role in both innate and adaptive immunity. In the present study, a novel toll-like receptor (McTLRw) was identified and characterized in thick shell mussel Mytilus coruscus. McTLRw possesses one intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain, one transmembrane region (TM), one leucine rich repeat N-terminal domain (LRR_NT) and a few of leucine-rich repeats (LRRs), which all are common in typical TLRs. McTLRw transcripts were constitutively expressed in all examined tissues with higher expression levels in immune related tissues, and were significantly induced in haemocytes with the challenges of live Vibrio alginolyticus, lipopolysaccharide (LPS), peptidoglycans (PGN) and ß-glucan (GLU), but not induced by polyinosinic-polycytidylic acid (poly I:C). rMcTLRw exhibited affinity to LPS, PGN and GLU while no affinity to poly I:C. Further, the downstream of TLR signaling pathway myeloid differentiation factor 88a (MyD88a), interleukin-1 receptor-associated kinase-4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were significantly repressed in McTLRw silenced mussels while challenged with LPS. These results collectively indicated that McTLRw is one member of TLR family and involved in immune response to against invaders by taking participate in TLR mediated signaling pathway.

A novel interleukin-1 receptor-associated kinase-4 from thick shell mussel Mytilus coruscus is involved in inflammatory response.

Interleukin-1 receptor-associated kinase-4 (IRAK4) is considered as the most upstream kinase of IRAKs and plays a vital role in Toll-like receptor/Interleukin-1 receptor (TLR/IL-1R) signal transduction. In the present study, IRAK4 from thick shell mussel Mytilus coruscus (McIRAK4) was identified and characterized. McIRAK4 showed the most similarity to its counterparts in bivalves. The conserved death domain (DD) and catalytic domain of serine/threonine kinases (STKc) were predicted in all examined IRAK4s. McIRAK4 transcripts were constitutively expressed in all examined tissues with the higher expression level in immune related tissues, and were significantly induced in haemocytes upon lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further, the expression of McIRAK4 was obviously repressed by dsRNA mediated RNA interference (RNAi), meanwhile the proinflammatory cytokines TNF-alpha and IL17 were down-regulated while the antiinflammatory cytokine TGF-ß was up-regulated. Additionally, McIRAK4 showed a global cytoplasmic localization in HEK293T cells through fluorescence microscopy. These results collectively indicated that McIRAK4 is one member of IRAK4 subfamily and might play the potential signal transducer role in inflammatory response. The present study provides supplement for TLR-mediated signaling pathway triggered by pathogenic invasions in thick shell mussel, and contributes to the clarification of the innate immune response in molluscs.

Identification and functional characterization of three myeloid differentiation factor 88 (MyD88) isoforms from thick shell mussel Mytilus coruscus.

Myeloid differentiation factor 88 (MyD88) is a pivotal adapter protein that involved in interleukin-1 receptor/toll-like receptor (IL-1R/TLR) signal transduction, which could spur downstream cascades and eventually drawn into innate immune response. MyD88 has been extensively studied in vertebrates, however, the information ascribe to MyD88 in invertebrates is still very scarce especially its function annotation remains extremely obscure. At here, three novel MyD88 isoforms termed McMyD88a, McMyD88b and McMyD88c were firstly cloned from thick shell mussel Mytilus coruscus. McMyD88a, McMyD88b and McMyD88c shared domain topology containing the Death domain (DD) and TIR domain (TIR) with its counterparts in mammals. All three McMyD88s were ubiquitously expressed in examined tissues in thick shell mussel, with the higher expression levels in immune-related tissues such as haemocytes, gills and digestive glands. Upon Vibrio alginolyticus, polyinosine-polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) challenge, McMyD88a, McMyD88b and McMyD88c transcripts were significantly induced in haemocytes despite of differential expression levels and responsive time points. Overexpression of McMyD88a, McMyD88b and McMyD88c showed a dose-dependent induction to NF-κB or ISRE in mammalian cell lines. Taken together, these results suggested that McMyD88a, McMyD88b and McMyD88c are members of MyD88 family and play potential roles in innate immune response to pathogenic invasions in thick shell mussel. Moreover, these results suggested indirectly the existence of a MyD88-dependent signaling pathway in thick shell mussel, and provide insight into the immunoregulatory effect in molluscs.

Characterisation and expression analysis of two terminal complement components: C7 and C9 from large yellow croaker, Larimichthys crocea.

The large yellow croaker Larimichthys crocea, as one of the most economically important marine fish in China and East Asian countries, are facing the fatal attraction of various pathogens in recent years. Elucidation of the organism immunomodulatory mechanism of croaker response to pathogen infection is essential for the disease control. In present study, we reported for the first time the molecular characterization and expression analysis of two terminal complement components (TCCs) of croaker, Lc-C7 and Lc-C9. These two structural conserved TCCs were detected in many tissues in adult healthy fish, with highest levels detected in liver. The transcriptional expression analysis of Lc-C7 and Lc-C9 at different developmental stages showed a continuous increase towards hatch, however the two TCCs mRNA were not detected at the unfertilized stage, hinting the origination of these two TCCs after fertilization. Rapid and drastic responses to Vibrio alginolyticus challenge were observed for Lc-C7 and Lc-C9, suggesting the involvement of component C7 and C9 in innate immune responses to pathogenic invasion in teleost fish. These findings could deepen our understanding about immunomodulatory mechanisms of croaker and shed a new light to the role of component system in teleostean immunomodulation.

Molecular characterization and expression analysis of a complement component C3 in large yellow croaker (Larimichthys crocea).

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. Complement component 3 is a key molecule in the complement system, whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the large yellow croaker (Larimichthys crocea), which was high similarity to other complement C3. We reported the primary sequence, tissue expression profile, polypeptide domain architecture and phylogenetic analysis of L. crocea complement component C3 (L.c-C3) gene. Its open reading frame (ORF) is 4962 bp and encodes for 1653 amino acids with a putative signal peptide of 23 amino acid residues. The deduced amino acid sequence showed that L.c-C3 has conserved residues and domains known to be crucial for C3 function. Phylogenetic analysis showed that L. crocea was closely related to Miichthys miiuy. The mRNA expressions of L.c-C3 was detectable at different tissues. L.c-C3 was expressed in a wide range of adult tissues, it showed highest expression in the liver. But the different developmental stages from fertilized egg to newborn larvae of the large yellow croaker the highest expression levels of L.c-C3 gene were not found. Bacterial challenge experiments showed that the levels of L.c-C3 mRNA expression were up-regulated in the liver, spleen and brain of adult large yellow croaker respectively. The results showed that L.c-C3 mRNA expression in the large yellow croaker is influenced by bacterial stress and L.c-C3 might play an important role in immunity mechanisms. This study will further increase our understanding of the function of L.c-C3 and molecular mechanism of innate immunity in teleosts.

Identification and comparative analysis of the Pseudosciaena crocea microRNA transcriptome response to poly(I:C) infection using a deep sequencing approach.

Two sRNA libraries with or without poly(I:C) infection of large yellow croaker Pseudosciaena crocea were constructed and sequenced using the high-throughput Illumina/Solexa deep sequencing technology. The high-throughput sequencing pipeline yielded 163,79,272 and 217,07,070 raw reads corresponding to 132,27,594 and 206,86,409 clean reads for the normal and infected libraries, respectively. Bioinfromatic analysis identified 534 miRNAs, of which, 158 miRNAs were known in miRBase 20.0 and the remaining 376 were not found homology to any known metazoan miRNAs, suggesting a possible species-specificity. We analyzed the significance of differently expressed miRNAs between two libraries using pairwise comparison. There was significant differential expression of 112 miRNAs (p < 0.001) between two libraries. Thereinto, a number of known miRNAs were identified immune-related. Real-time quantitative PCR experiments (RT-qPCR) were preformed for 6 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first comprehensive study of miRNAs in P. crocea and of expression analysis of P. crocea miRNAs in response to poly(I:C) infection, and many miRNAs were differentially regulated under normal and infection conditions. These findings deepened our understanding of the role of miRNAs in the intricate host's immune system, and should be useful to develop new control strategies for host immune defense against various foreign infection in P. crocea.

Establishment of Nested PCR for the Detection of Pseudomonas plecoglossicida and Epidemiological Survey of Larimichthys crocea in the Southeast Coastal Region.

The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea.

Raising the Asymmetric Catalytic Efficiency of Chiral Covalent Organic Frameworks by Tuning the Pore Environment.

Chiral covalent organic frameworks (COFs) hold considerable promise in the realm of heterogeneous asymmetric catalysis. However, fine-tuning the pore environment to enhance both the activity and stereoselectivity of chiral COFs in such applications remains a formidable challenge. In this study, we have successfully designed and synthesized a series of clover-shaped, hydrazone-linked chiral COFs, each with a varying number of accessible chiral pyrrolidine catalytic sites. Remarkably, the catalytic efficiencies of these COFs in the asymmetric aldol reaction between cyclohexanone and 4-nitrobenzaldehyde correlate well with the number of accessible pyrrolidine sites within the frameworks. The COF featuring nearly one pyrrolidine moiety at each nodal point demonstrated excellent reaction yields and enantiomeric excess (ee) values, reaching up to 97 and 83%, respectively. The findings not only underscore the profound impact of a deliberately controlled chiral pore environment on the catalytic efficiencies of COFs but also offer a new perspective for the design and synthesis of advanced chiral COFs for efficient asymmetric catalysis.