Deciphering the impact of circRNA-mediated autophagy on tumor therapeutic resistance: a novel perspective.

Cancer therapeutic resistance remains a significant challenge in the pursuit of effective treatment strategies. Circular RNAs (circRNAs), a class of non-coding RNAs, have recently emerged as key regulators of various biological processes, including cancer progression and drug resistance. This review highlights the emerging role of circRNAs-mediated autophagy in cancer therapeutic resistance, a cellular process that plays a dual role in cancer by promoting both cell survival and death. Increasing evidence suggests that circRNAs can modulate autophagy pathways, thereby influencing the response of cancer cells to therapeutic agents. In this context, the intricate interplay between circRNAs, autophagy, and therapeutic resistance is explored. Various mechanisms are discussed through which circRNAs can impact autophagy, including direct interactions with autophagy-related genes, modulation of signaling pathways, and cross-talk with other non-coding RNAs. Furthermore, the review delves into specific examples of how circRNA-mediated autophagy regulation can contribute to resistance against chemotherapy and radiotherapy. Understanding these intricate molecular interactions provides valuable insights into potential strategies for overcoming therapeutic resistance in cancer. Exploiting circRNAs as therapeutic targets or utilizing them as diagnostic and predictive biomarkers opens new avenues for developing personalized treatment approaches. In summary, this review underscores the importance of circRNA-mediated autophagy in cancer therapeutic resistance and proposes future directions for research in this exciting and rapidly evolving field.

Toxic effects of cadmium exposure on intestinal histology, oxidative stress, microbial community, and transcriptome change in the mud crab (Scylla paramamosain).

Cadmium is one of hazardous pollutants that has a great threat to aquatic organisms and ecosystems. The intestine plays important roles in barrier function and immunity to defend against environmental stress. However, whether cadmium exposure caused the intestine injury is not well studied. Thus, the aim of this study was to explore the potential mechanisms of cadmium toxicity in the intestine of mud crab (Scylla paramamosain) via physiological, histological, microbial community, and transcriptional analyses. Mud crabs were exposed to 0, 0.01, and 0.125 mg/L cadmium. After a 21-day of cadmium exposure, 0.125 mg/L cadmium caused intestine damaged by decreasing superoxide dismutase and catalase activities, and increasing hydrogen peroxide and malondialdehyde levels. Integrated biological index analysis confirmed that the toxicity of cadmium exhibited a concentration-dependent manner. Comparative transcriptional analyses showed that the up-regulations of several genes associated with heat shock proteins, detoxification and anti-oxidant defense, and two key signaling pathways (PI3k-Akt and apoptosis) revealed an adaptive response mechanism against cadmium exposure. Transcriptomic analysis also suggested that cadmium exposure disturbed the expression of ion transport and immune-related genes, indicating that it has negative effects on the immune functions of the mud crab. Furthermore, the intestinal microbial diversity and composition were significantly influenced by cadmium exposure. The abundance of the dominant phyla Fusobacteria and Bacteroidetes significantly changed after cadmium exposure. KEGG pathway analysis demonstrated that cadmium exposure could change energy metabolism and environmental information processing. Overall, we concluded that excessive cadmium exposure could be potentially exerted adverse effects to the mud crab health by inducing oxidative damage, decreasing immune system, disrupting metabolic function, and altering intestinal microbial composition. These results provided a novel insight into the mechanism of cadmium toxicity on crustaceans.

Identification and functional characterization of glutaredoxin 5 from the mud crab (Scylla paramamosain) in response to cadmium and bacterial challenge.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which plays a key role in maintaining redox homeostasis and regulating cell survival pathways. However, the expression pattern and function of Grx remain unknown in the mud crab (Scylla paramamosain). In the present study, a novel full-length of Grx 5 from the mud crab (designated as Sp-Grx 5) was cloned and characterized. The open reading frame of Sp-Grx 5 was 441 bp, which encoded a putative protein of 146 amino acids. The amino acid sequence of Sp-Grx 5 contained a typical C-G-F-S redox active motif and several GSH binding sites. Sp-Grx 5 widely existed in all tested tissues with a high-level expression in hepatopancreas. Subcellular localization showed that Sp-Grx 5 was located in the cytoplasm and nucleus. The expression of Sp-Grx 5 was significantly up-regulated after Vibrio parahaemolyticus infection and cadmium exposure, suggesting that Sp-Grx 5 was involved in innate immunity and detoxification. Furthermore, overexpression of Sp-Grx 5 could improve cells viability after H2O2 exposure. All these results indicated that Sp-Grx 5 played important roles in the redox homeostasis and innate immune response in crustaceans.

Mechanism of Cadmium Exposure Induced Hepatotoxicity in the Mud Crab (Scylla paramamosain): Activation of Oxidative Stress and Nrf2 Signaling Pathway.

Cadmium, one of the most toxic heavy metals, can cause severe oxidative damage to aquatic animals. However, the mechanism whereby the mud crabs respond to cadmium exposure remains unclear. This study investigated the effects of cadmium exposure on oxidative stress and histopathology changes and evaluated the role of the Nrf2 signaling pathway in regulating responses to cadmium-induced hepatotoxicity were investigated in mud crabs. Mud crabs were exposed to 0, 0.01, 0.05, and 0.125 mg/L cadmium for 21 d. The present results indicated that cadmium exposure increased hydrogen peroxide (H2O2) production, lipid peroxidation and tissue damage, but decreased the activity of superoxide dismutase (SOD) and catalase (CAT), and caused lipid peroxidation and tissue damage. The results of an integrated biomarker index analysis suggested that the toxicity of cadmium was positively related to cadmium concentration. The expression levels of the Nrf2 signaling pathway (Nrf2, metallothionein, and cytochrome P450 enzymes) were up-regulated after cadmium exposure. Silencing of Nrf2 in vivo decreased antioxidant gene (SOD, CAT, and glutathione S-transferase) expression, suggesting that Nrf2 can regulate antioxidant genes. Knocking down Nrf2 in vivo also significantly decreased the activity of SOD and CAT after cadmium exposure. Moreover, silencing of Nrf2 in vivo enhanced H2O2 production and the mortality rates of mud crabs after cadmium exposure. The present study indicated that cadmium exposure induced hepatotoxicity in the mud crab by increasing H2O2 content, which decreased the antioxidant capacity, leading to cell injury. In addition, the Nrf2 is activated to bound with antioxidant response element, initiating the expression of antioxidant enzyme genes during cadmium induced hepatotoxicity in the mud crabs.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

The role of tumor suppressor protein p53 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

The tumor suppressor protein p53 plays important roles in DNA repair, cell cycle and genetic stability. In the present study, a p53 gene in the mud crab (Scylla paramamosain) (designated as Sp-53) was identified and characterized. The open reading frame of Sp-53 was comprised a 1383 bp, which encoded a putative protein of 460 amino acids. Sp-53 is expressed in all examined tissues, with the highest expression in hepatopancreas and hemocytes. Vibrio parahaemolyticus infection induced oxidative stress, and led to DNA damage. The Sp-53 transcriptions in hepatopancreas were significantly up-regulated after V. parahaemolyticus infection. RNA interference (RNAi) experiment was used to understand the roles of Sp-53 in response to V. parahaemolyticus infection. Knocking down Sp-53 in vivo significantly reduced the expression of the Mn-SOD, Gpx3 and caspase 3 after V. parahaemolyticus infection. Moreover, the mortality of mud crabs and DNA damage in Sp-53-silenced mud crab challenged with V. parahaemolyticus were significantly higher than those in the control group. All these results suggested that Sp-53 played an important role in responses to V. parahaemolyticus infection through its participation in regulation of antioxidant defense, DNA repair and apoptosis.

Oxidative stress, cell cycle arrest, DNA damage and apoptosis in the mud crab (Scylla paramamosain) induced by cadmium exposure.

Cadmium is one of the most common heavy metal pollutants in the aquatic environment. Mud crab (Scylla paramamosain) is considered a model organism to monitor the impact of heavy metals. However, knowledge about toxicological mechanism of cadmium in crustaceans still remains limited. In this study, mud crabs were exposed to different concentrations of cadmium (0, 1.25, 2.5, 5 and 10 mg/L) for 72 h. Cadmium exposure significantly decreased superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidative capacity (T-AOC), and significantly increased malondialdehyde (MDA) and H2O2 levels. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity significantly increased after cadmium exposure. Moreover, integrated biological responses version 2 (IBRv2) analysis suggested that cadmium exposure exerted stronger toxicity on mud crab. Furthermore, oxidative stress induced by cadmium exposure could decrease total hemocyte count (THC), interrupt Ca2+ homeostasis, and lead to cytological damage. Cadmium exposure induced DNA damage, which activated DNA damage response signaling ATR-CHK1-p53 pathway. Our results also showed that cadmium exposure significantly increased the apoptosis and caspase-3 mRNA levels, which implied that cadmium induced apoptosis through a caspase-3 pathway.

A double chitin catalytic domain-containing chitinase targeted by c-Jun is involved in immune responses in shrimp.

Chitinases are a group of chitin-degrading enzymes widely distributed in organisms. Chitinases containing two chitin catalytic domains have been widely found in arthropods but their functions remain unclear. In this study, a member of these chitinases from Litopenaeus vannamei (dChi) was identified and functionally studied in the context of immunity. The promoter of dChi contained activator protein 1 (AP-1) binding sites and could be regulated by c-Jun. The recombinant dChi protein showed no bacteriostatic activity in vitro but knockdown of dChi in vivo increased the mortality of shrimp and the bacterial load in tissues after Vibrio parahaemolyticus infection, suggesting that dChi could play a positive role in antibacterial responses. However, silencing of dChi expression significantly decreased the mortality of WSSV-infected shrimp and down-regulated the viral load in tissues, indicating that dChi could facilitate WSSV infection. We further demonstrated that dChi was involved in regulation of the bacterial phagocytosis of hemocytes and expression of a series of immune related transcription factors and antimicrobial peptides. These indicated that the roles of dChi in antibacterial responses and anti-WSSV responses in vivo could result from its regulatory effects on the immune system. Taken together, the current study suggests that double chitin catalytic domain-containing chitinases could be important players in immune regulation in crustaceans.

Immune and physiological responses of mud crab (Scylla paramamosain) under air exposure.

The immune and physiological responses of mud crab (Scylla paramamosain) under air exposure were studied. The results showed that air exposure increased plasma activities of AST, ALT, ALP. There was a significant increase in glucose (GLU) and malondialdehyde (MDA) levels after air exposure. The transcript levels of SOD, CAT, HSP90, HSP70, p53, and hypoxia-inducible factor-1 (HIF-1) were induced by air exposure. Furthermore, caspase-3 transcript significantly increased at 48 and 72 h, while it significantly decreased at 96 h and 120 h under air exposure. These results suggested that oxidative stress occurred in the prolonged period of air exposure. HIF-1 and p53 signaling pathways played an important role under air exposure.

Expression of lactate dehydrogenase is induced during hypoxia via HIF-1 in the mud crab Scylla paramamosain.

Lactate dehydrogenase (LDH) is a key enzyme involved in anaerobic metabolism in most organisms. In the present study, we determined the structure and function of LDH sequence in Scylla paramamosain (SpLDH) by gene cloning, expression and RNA interference techniques in order to explore the genetic characteristics of LDH and its relationship with HIF-1 during hypoxia. The full-length cDNA was 1453 bp with an open reading frame (ORF) of 996 bp, and encoded a polypeptide of 332 amino acids. Homology analysis showed that the SpLDH gene is highly similar to arthropods. The SpLDH transcript increased after hypoxia in all tested tissues. The silencing of HIF-1 blocked the increase in LDH mRNA and activity, which were induced by hypoxia in gill and muscle tissues. Our results indicated that SpLDH expression was regulated transcriptionally by HIF-1.

Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

Identification of a Novel Small RNA srvg23535 in Vibrio alginolyticus ZJ-T and Its Characterization With Phenotype MicroArray Technology.

Small non-coding RNAs (sRNAs) are important modulators of gene expression and are involved in the pathogenesis and survival of prokaryotes. However, few studies have been conducted with Vibrio alginolyticus, which limits our ability to probe the regulation of virulence and environmental adaptation by sRNAs in this opportunistic pathogen. In this study, the sRNA candidate srvg23535 was identified in V. alginolyticus ZJ-T. The precise transcript end, secondary structure, and sequence conservation were determined. A srvg23535 null mutant was constructed and characterized by using Phenotype MicroArray (PM) technology. In silico target prediction was conducted by IntaRNA and TargetRNA2. Subsequently, a 107 nt transcript was validated with a sigma70 promoter at the 5' end and a Rho-independent terminator at the 3' end. The sRNA srvg23535 had four stem-loop structures and was conserved among Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio splendidus. Deletion of srvg23535 in V. alginolyticus ZJ-T led to a weaker utilization of D-mannose, D-melibiose, lactulose, and inosine as carbon sources but stronger utilization of L-cysteine as nitrogen source. Moreover, the srvg2353 mutant showed stronger resistance to osmotic stress but weaker resistance to pH stress. Additionally, a total of 22 common targets were identified and several were related to the observed phenotype of the mutant. This study indicated that the novel sRNA, srvg23535, is conserved and restricted to Vibrio spp., affecting the utilization of several carbon and nitrogen sources and the response to osmotic and pH stress. These results extend our understanding of sRNA regulation in V. alginolyticus and provide a significant resource for the further study of the precise target mRNAs of srvg23535, which may provide targets for antibacterial therapeutic or attenuated vaccines against Vibrio spp.

Effect of dietary astaxanthin on the growth performance, non-specific immunity, and antioxidant capacity of pufferfish (Takifugu obscurus) under high temperature stress.

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320 mg kg-1 were fed to fish for 8 weeks. The results showed that the fish fed diet with 80, 160, and 320 mg kg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48 h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320 mg kg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress in pufferfish.

Molecular and immune response characterizations of a novel Bax inhibitor-1 gene in pufferfish, Takifugu obscurus.

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis, and immune responses. Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that protects cells from endoplasmic reticulum stress-induced apoptosis. In this study, a BI-1 gene from the pufferfish Takifugu obscurus (Pf-BI-1) was identified and characterized. The full length of Pf-BI-1 cDNA was 1387 bp, including a 5'-UTR of 82 bp, a 3'-UTR of 591 bp containing a poly-(A) tail, and an open reading frame (ORF) of 714 bp that encodes a polypeptide of 237 amino acids. Pf-BI-1 was ubiquitously expressed in various tissues, with the highest expression levels in the blood, brain, and gill. The expression of Pf-BI-1 was up-regulated in a time-dependent manner after heat shock stress, ammonia stress, and bacterial challenge. Intracellular localization revealed that Pf-BI-1 was primarily localized in the cell cytoplasm. Furthermore, over-expression of Pf-BI-1 could active NF-кB reporter genes in HeLa cells. These results indicated that Pf-BI-1 may be involved in the apoptosis and immunity process against ambient stressors in pufferfish.

Identification, characterization and expression analysis of tumor suppressor protein p53 from pufferfish (Takifugu obscurus) after the Vibrio alginolyticus challenge.

The tumor suppressor protein p53 plays a critical role in cell cycle, apoptosis and DNA repair. In this study, the full-length pufferfish p53 (Pf-p53) was obtained, containing an open reading frame of 1095 bp, a 5'UTR of 157 bp and a 3'UTR of 285 bp with a poly (A) tail. The Pf-p53 encoded a polypeptide of 364 amino acids with a theoretical isoelectric point of 8.03 and predicted molecular weight of 40.6 kDa. Pf-p53 was ubiquitously expressed in various tissues with a high-level expression in kidney, liver and gill. Vibrio alginolyticus challenge could induce ROS production and disrupt Ca2+ homeostasis, subsequently leading to the induction of DNA damage and apoptosis, while the Vibrio alginolyticus-induced oxidative stress can also increase the non-specific immunity. The pufferfish challenged with Vibrio alginolyticus showed a sharp increase of Pf-p53 transcript in liver. Subcellular localization analysis revealed that Pf-p53 was primarily localized in nucleus. Furthermore, overexpression of Pf-p53 in Hela cells could inhibit cell proliferation and the transcriptional activities of the NF-ĸB promoter. Taken together, our results indicated that Pf-p53 may play an important role in the immune response to Vibrio alginolyticus challenge.

Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification.

Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 µmol/L F3/B3, 1.6 µmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR.

Identification and expression analysis of a CC chemokine from cobia (Rachycentron canadum).

Chemokines are small, secreted cytokine peptides known principally for their ability to induce migration and activation of leukocyte populations and regulate the immune response mechanisms. The cobia (Rachycentron canadum), a marine finfish species, has a great potential for net cage aquaculture in the South China Sea. We isolated and characterized a CC chemokine cDNA from cobia-designated RcCC2. Its cDNA is 783 bp in length and encodes a putative protein of 110 amino acids. Homology and phylogenetic analysis revealed that the RcCC2 gene, which contains four conserved cysteine residues, shares a high degree of similarity with other known CC chemokine sequences and is closest to the CCL19/21 clade. The mRNA of RcCC2 is expressed constitutively in all tested tissues, including gill, liver, muscle, spleen, kidney, head kidney, skin, brain, stomach, intestine and heart, but not blood, with the highest level of expression in gill and liver. The reverse transcription quantitative polymerase chain reaction was used to examine the expression of the RcCC2 gene in immune-related tissues, including head kidney, spleen and liver, following intraperitoneal injection of the viral mimic polyriboinosinic polyribocytidylic acid, formalin-killed Vibrio carchariae (bacterial vaccine) and phosphate-buffered saline as a control. RcCC2 gene expression was up-regulated differentially in head kidney, spleen and liver during 12 h after challenge. These results indicate that the RcCC2 gene is inducible and is involved in immune responses, suggesting RcCC2 has an important role in the early stage of viral and bacterial infections.

Identification of a cobia (Rachycentron canadum) CC chemokine gene and its involvement in the inflammatory response.

The chemokines regulate immune cell migration under inflammatory and physiological conditions. We investigated a CC chemokine gene (RcCC1) from cobia (Rachycentron canadum). The full-length RcCC1 cDNA is comprised 673 nucleotides and encodes a four-cysteine arrangement 99-amino-acid protein typical of known CC chemokines. The genomic DNA of RcCC1 consists of three exons and two introns. Phylogenetic analysis showed that RcCC1 was closest to the MIP group of CC chemokines. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed RcCC1 was constitutively expressed in all tissues examined, with relative strong expression in gill, blood, kidney, spleen, and head kidney. The RcCC1 transcripts in the head kidney, spleen, and liver were quickly up-regulated after stimulation with formalin-inactivated Vibrio carchariae (bacterial vaccine) or polyriboinosinic polyribocytidylic acid (poly I:C). These results indicate RcCC1 not only plays a role in homeostasis, but also may be involved in inflammatory responses to bacterial and viral infection.

Functional annotation of an expressed sequence tag library from Haliotis diversicolor and analysis of its plant-like sequences.

The small abalone, Haliotis diversicolor, is a widely distributed and cultured species in the subtropical coastal area of China. To identify and classify functional genes of this important species, a normalized expressed sequence tag (EST) library, including 7069 high quality ESTs from the total body of H. diversicolor, was analyzed. A total of 4781 unigenes were assembled and 2991 novel abalone genes were identified. The GC content, codon and amino acid usage of the transcriptome were analyzed. For the accurate annotation of the abalone library, different influencing factors were evaluated. The gene ontology (GO) database provided a higher annotation rate (69.6%), and sequences longer than 800bp were easily subjected to a BLAST search. The taxonomy of the BLAST results showed that lancelet and invertebrates are most closely related to abalone. Sixty-seven identified plant-like genes were further examined by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, only seven of these were real transcripts in abalone. Phylogenic trees were also constructed to illustrate the positions of two Cystatin sequences and one Calmodulin protein sequence identified in abalone. To perform functional classification, three different databases (GO, KEGG and COG) were used and 60 immune or disease-related unigenes were determined. This work has greatly enlarged the known gene pool of H. diversicolor and will have important implications for future molecular and genetic analyses in this organism.

Identification of the structural proteins of VP1 and VP2 of a novel mud crab dicistrovirus.

Mud crab dicistrovirus (MCDV), a newly identified single-stranded positive RNA virus, is an important pathogen that causes serious economic losses to mud crab aquaculture. In this study, MCDV was purified, and three structural proteins of MCDV were separated by SDS-PAGE. The N-terminal 15 amino acids were sequenced and aligned with the main structural proteins of other dicistrovirus. The three structural proteins were named VP1, VP2 and VP3. Monoclonal antibodies (MAbs) against the two main structural proteins, VP1 and VP2, were prepared, and the two structural proteins were then identified using these MAbs. The results of Western blot analyses demonstrated that five MAbs recognised VP1 and two recognised VP2. The results of immunogold transmission electron microscopy (IEM) revealed that the epitopes of the two structural proteins recognised by the MAbs were located at the outer surface of the virions, which suggested that the two structural proteins are MCDV capsid proteins. The identification of the two structural proteins of MCDV is useful for studying their functions, as well as the mechanism of infection and the pathogenesis of MCDV.