Study on the mechanism of miR-7562 regulating ATG5 and ATG12 genes in Penaeus monodon under Vibrio harveyi infection.

MicroRNAs (miRNAs) play a fundamental role in the post-transcriptional regulation of genes and are pivotal in modulating immune responses in marine species, particularly during pathogen assaults. This study focused on the function of miR-7562 and its regulatory effects on autophagy against Vibrio harveyi infection in the black tiger shrimp (Penaeus monodon), an economically important aquatic species. We successfully cloned and characterized two essential autophagy-related genes (ATGs) from P. monodon, PmATG5 and PmATG12, and then identified the miRNAs potentially involved in co-regulating these genes, which were notably miR-7562, miR-8485, and miR-278. Subsequent bacterial challenge experiments and dual-luciferase reporter assays identified miR-7562 as the principal regulator of both genes, particularly by targeting the 3'UTR of each gene. By manipulating the in vivo levels of miR-7562 using mimics and antagomirs, we found significant differences in the expression of PmATG5 and PmATG12, which corresponded to alterations in autophagic activity. Notably, miR-7562 overexpression resulted in the downregulation of PmATG5 and PmATG12, leading to a subdued autophagic response. Conversely, miR-7562 knockdown elevated the expression levels of these genes, thereby enhancing autophagic activity. Our findings further revealed that during V. harveyi infection, miR-7562 continued to influence the autophagic pathway by specifically targeting the ATG5-ATG12 complex. This research not only sheds light on the miRNA-dependent mechanisms governing autophagic immunity in shrimp but also proposes miR-7562 as a promising target for therapeutic strategies intended to strengthen disease resistance within the crustacean aquaculture industry.

Antiviral properties of Penaeus monodon cyclophilin A in response to white spot syndrome virus infection in the black tiger shrimp.

Cyclophilin A (CypA) or peptidylprolyl isomerase A, plays an important role in protein folding, trafficking, environmental stress, cell signaling and apoptosis etc. In shrimp, the mRNA expression level of PmCypA was stimulated by LPS. In this study, all three types of shrimp hemocytes: hyaline cell, granulocyte and semi-granulocyte expressed the PmCypA protein. The mRNA expression level of PmCypA was found to be up-regulate to four-fold in white spot syndrome virus (WSSV) infected hemocytes at 48 h. Interestingly, PmCypA protein was only detected extracellularly in shrimp plasma at 24 h post WSSV infection. To find out the function of extracellular PmCypA, the recombinant PmCypA (rPmCypA) was produced and administrated in shrimp primary hemocyte cell culture to observe the antiviral properties. In rPmCypA-administrated hemocyte cell culture, the mRNA transcripts of WSSV intermediate early gene, ie1 and early gene, wsv477 were significantly decreased but not that of late gene, vp28. To explore the antiviral mechanism of PmCypA, the expression of PmCypA in shrimp hemocytes was silenced and the expression of immune-related genes were investigated. Surprisingly, the suppression of PmCypA affected other gene expression, decreasing of penaeidin, PmHHAP and PmCaspase and increasing of C-type lectin. Our results suggested that the PmCypA might plays important role in anti-WSSV via apoptosis pathway. Further studies of PmCypA underlying antiviral mechanism are underway to show its biological function in shrimp immunity.

Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon.

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Reference genes for qPCR expression in black tiger shrimp, Penaeus monodon.

BACKGROUND: Gene expression profiling via qPCR is an essential tool for unraveling the intricate molecular mechanisms underlying growth and development. Identifying and validating the most appropriate reference genes is essential for qPCR experiments. Nevertheless, there exists a deficiency in a thorough assessment of reference genes concerning the expression of the genes in the research in the context of the growth and development of the Black Tiger Shrimp, P. monodon. This popular marine crustacean is extensively raised for human consumption. In this study, we assessed the expression stability of seven reference genes (ACTB, 18S, EF-1α, AK, PK, cox1, and CLTC) in adult tissues (hepatopancreas, gills, and stomach) of small and large polymorphs of P. monodon. METHODS AND RESULTS: The stability of gene expressions was assessed utilizing NormFinder, BestKeeper, and geNorm, and a comprehensive ranking of these genes was conducted through the online tool RefFinder. In the overall ranking, 18S and CLTC emerged as the most stable genes in the hepatopancreas and stomach, while CLTC and AK exhibited significant statistical reliability in the gills of adult P. monodon. The validation of these identified stable genes was carried out using a growth-associated gene, insr-1. CONCLUSION: The results indicated that 18S and CLTC stand out as the most versatile reference genes for conducting qPCR analysis focused on the growth of P. monodon. This study represents the first comprehensive exploration that identifies and assesses reference genes for qPCR analysis in P. monodon, providing valuable tools for research involving similar crustaceans.

Marine actinomycete Streptomyces variabilis S26 as a biocontrol agent for vibriosis in shrimp larval rearing systems.

Indiscriminate use of antibiotics has led to the emergence of antibiotic-resistant microbes and the loss of natural flora in aquaculture systems necessitating the ban of many of these chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine-derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16 S ribosomal RNA (rRNA) gene sequencing was then checked for the production of antibiotic in five different fermentation media and the one which showed maximum production was chosen for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was highly significant for the response concerned that is, antimicrobial activity as evident from the Fisher's F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38%; soy powder, 0.88%; ammonium sulfate, 0.16% and salinity, 27.76‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in vibrios and conferred better protection to P. monodon in culture system compared with control.

Characterization of a novel lytic bacteriophage VPMCC14 which efficiently controls Vibrio harveyi in Penaeus monodon culture.

Vibrio harveyi causes luminous vibriosis diseases in shrimp, which lead to shrimp mortalities. Considering the emergence of antibiotic-resistant bacteria, a Vibrio-infecting bacteriophage, VPMCC14, was characterized, and its lysis ability was evaluated on a laboratory scale. VPMCC14 was shown to infect V. harveyi S5A and V. harveyi ATCC 14126. VPMCC14 also exhibited a latent period of 30 min, with a burst size of 38 PFU/cell on its propagation strain. The bacteriophage was stable at a wide range of pHs (3-9), temperatures (0-45°C), and salinities (up to 40 ppt). VPMCC14 exhibited strict virulence properties as the bacteriophage entirely lysed V. harveyi S5A in liquid culture inhibition after 5 h and 4 h at very low MOIs such as MOI 0.1 and MOI 1, respectively. VPMCC14 could control V. harveyi infection in aquariums at MOI 1 and decrease the mortality of Penaeus monodon challenged by V. harveyi. VPMCC14 genome was 134,472 bp long with a 34.5 G+C% content, and 240 open reading frames. A unique characteristic of VPMCC14 was the presence of the HicB family antitoxin-coding open reading frame. Comparative genomic analyses suggested that VPMCC14 could be a representative of a new genus in the Caudoviricetes class. This novel bacteriophage, VPMCC14, could be applied as a biocontrol agent for controlling V. harveyi infection.

Molecular characterization and functional analysis of TRIM37 from black tiger shrimp (Penaeus monodon).

The family of TRIM proteins with E3 ubiquitin ligase activity served important roles in the regulation of innate immune processes, in particular antiviral and proinflammatory cytokine responses. In this study, a novel TRIM37 homolog was identified from Penaeus monodon (named PmTRIM37). The PmTRIM37 protein contained three conserved domains (one RING finger domain, a B-box, and one Coiled-coil region) at its N-terminal and one Meprin and MATH domain at its C-terminal. The MATH domain was the characteristic of TRIM37 family. PmTRIM37 has relatively high expression in immune-related tissues such as hepatopancreas, gills, lymphoid organs and hemocytes. The expression levels of PmTRIM37 in hepatopancreas and lymphoid organs were significantly up-regulated after white spot syndrome virus (WSSV) infection. Knock down of PmTRIM37 promoted WSSV replication and VP28 expression, suggesting that PmTRIM37 played a negative role in WSSV infection. Further studies revealed that PmTRIM37 positively regulated the NF-κB pathway and Antimicrobial peptides (AMP) expression during WSSV infection. These findings indicated that PmTRIM37 might restrict WSSV replication by positively regulating NF-κB pathway during WSSV infection in P. monodon.

Thymosin beta-4 participate in antibacterial immunity and wound healing in black tiger shrimp, Penaeus monodon.

Thymosin beta-4 (Tß4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates, which play fundamental roles in innate immune against pathogens and wound healing. In this study, the full-length cDNA of Tß4 was cloned from Penaeus monodon (designated as PmTß4), using the technology of rapid amplification of cDNA ends (RACE). The cDNA of PmTß4 was 1361 bp with an open reading frame (ORF) of 501 bp, which encoding a polypeptide of 166 amino acid. The Quantitative Real-time PCR (qRT-PCR) analysis results showed that PmTß4 was ubiquitously expressed in all the tested shrimp tissues, with the highest expression level was detected in the hemolymph, while the lowest expression level in the muscle. The expression level of PmTß4 was significantly up-regulated in hepatopancreas after challenged by Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In vitro antimicrobial test showed that the recombinant protein of PmTß4 (rPmTß4) had broad-spectrum of antimicrobial activity, which could inhibit both the growth of gram-negative bacteria and gram-positive bacteria, including Vibrio vulnificus, V. parahaemolyticus, Streptococcus agalactiae, S. aureus and Aeromonas hydrophila. Moreover, rPmTß4 had a certain binding ability to different bacteria, and this binding ability exhibits a strong dose-dependent effect. In vivo, PmTß4 could facilitate external bacterial clearance in shrimp, and have beneficial to shrimp survival post V. parahaemolyticus infection. Furthermore, wound-healing assay was carried out to study the role of PmTß4 in the process of wound healing. The results showed that the PmTß4 expression was significantly up-regulated by injury treatment, and exerted positive effects to promote wound healing. In addition, PmTß4 can significantly increase the expression level of superoxide dismutase (SOD) and Catalase (CAT) after injury treatment in shrimp, which would involve in scavenging reactive oxygen species (ROS) caused by the wound. In conclusion, these results indicated that PmTß4 may play important roles in antibacterial immunity and wound healing in Penaeus monodon.

Ambient copper modulates immunocompetence and induces physiological responses in Penaeus monodon against white spot syndrome virus infection.

Among trace metals, copper is essential for crustaceans' normal growth and metabolism. In the present study, an attempt was made to determine whether the addition of copper in rearing water influences the physiological and immunological responses of Penaeus monodon to white spot syndrome virus infection (WSSV). Adult P. monodon were distributed in experimental tanks and exposed to 0, 0.05, 0.1, 0.2 and 0.3 mg l-1 copper concentrations. After 14 days, the shrimps were challenged with WSSV and the biochemical/immune variables were determined on post-metal exposure day 14 and post-challenge days 2 and 5. Significant variations could be observed in the haemolymph (biochemical and immune) variables of P. monodon on exposure to copper and WSSV challenge. Shrimps exposed to copper at 0.1 mg l-1 showed higher total haemocyte count, phenol oxidase activity, nitro blue tetrazolium salt reduction, alkaline/acid phosphatase activity, total protein, carbohydrates, lipids, glucose and cholesterol besides maximum post-challenge survival. However, exposure to copper at 0.2 and 0.3 mgl-1 increased the susceptibility to WSSV infection, showing a decrease in the biochemical/immune variables. Therefore, the present study concludes that copper in ambient water induces immunomodulation and evokes physiological responses in P. monodon at sub-lethal doses. Immunostimulatory effects elicited by copper at 0.1 mg l-1 enhanced the immunocompetence and reduced the susceptibility of P. monodon to WSSV infection, conferring protection to the animals and resulting in higher survival.

Pathogenicity and transcriptomic exploration of Vibrio fortis in Penaeus monodon.

In this study, a strain (recorded as Y6) was isolated from the biofloc pool, its DNA was extracted for 16S rDNA sequencing and compared in the NCBI database, and it was identified as Vibrio fortis. The V. fortis was activated, cultured, and artificially injected into Penaeus monodon to observe the symptoms and calculate the semi-lethal concentration (LC50). It was found that the symptoms of the red leg, an empty stomach, and enlarged hepatopancreas of P. monodon after infection with V. fortis. The LC50 was 4.00 × 107, 2.24 × 107, 1.82 × 107, 1.41 × 107, 7.52 × 106 and 3.31 × 106 CFU/mL at 16, 24, 32, 48, 128, and 144 hpi, respectively. The K-B disk method was used to detect the sensitivity of V. fortis to various antibiotic drugs. V. fortis resisted Ampicillin, Piperacillin, Cefazolin, Cephalothin and Cefoxitin. Highly sensitive to Polymyxin B, Tobramycin, Gentamicin, Cefepime, Cefoperazone and Streptomycin. To explore the molecular response mechanism of V. fortis infection in P. monodon, the hepatopancreas of P. monodon infected with V. fortis at 24 and 48 hpi by transcriptome sequencing, and a total of 347 DEGs were obtained (214 up-regulated DEGs and 133 down-regulated DEGs). In the KEGG pathway enrichment analysis of DEGs, significant changes were found in genes and signaling pathways related to immune system and substance metabolism, including NOD-like receptor signaling pathways, Toll and Imd signaling pathways, C-type lectin receptor signaling pathways and pyruvate metabolism. This study initially revealed the immune response of P. monodon to V. fortis infection from the molecular level and provided a reference for further understanding of the study and control of the vibriosis of shrimp.

PmKuSPI is regulated by pmo-miR-bantam and contributes to hemocyte homeostasis and viral propagation in shrimp.

The Kunitz-type serine protease inhibitor (KuSPI) is a low molecular weight protein that plays a role in modulating a range of biological processes. In Penaeus monodon, the PmKuSPI gene has been found to be highly expressed in the white spot syndrome virus (WSSV)-infected shrimp and is predicted to be regulated by a conserved microRNA, pmo-miR-bantam. We reported that, despite being upregulated at the transcriptional level, the PmKuSPI protein was also upregulated after WSSV infection. Silencing the PmKuSPI gene in healthy shrimp had no effect on phenoloxidase activity or apoptosis but resulted in a delay in the mortality of WSSV-infected shrimp as well as a reduction in the total hemocyte number and WSSV copies. According to an in vitro luciferase reporter assay, the pmo-miR-bantam bound to the 3'UTR of the PmKuSPI gene as predicted. In accordance with the loss of function studies using dsRNA-mediated RNA interference, the administration of the pmo-miR-bantam mimic into WSSV-infected shrimp lowered the expression of the PmKuSPI transcript and the PmKuSPI protein, as well as the WSSV copy number. According to these results, the protease inhibitor PmKuSPI is posttranscriptionally controlled by pmo-miR-bantam and plays a role in hemocyte homeostasis, which in turn affects shrimp susceptibility to WSSV infection.

Molecular cloning and characterization of a cyclin B gene on the ovarian maturation stage of black tiger shrimp (Penaeus monodon).

The techniques of homology cloning and anchored PCR were used to clone the cyclin B gene from black tiger shrimp. The full length cDNA of black tiger shrimp cyclin B (btscyclin B) contained a 5' untranslated region (UTR) of 102 bp, an ORF of 1,206 bp encoding a polypeptide of 401 amino acids with an estimated molecular mass of 45 kDa and a 3' UTR of 396 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btscyclin B was homological to the cyclin B of other species and even the mammalians. Two conserved signature sequences of cyclin B gene family were found in the btscyclin B deduced amino acid sequence. The temporal expressions of cyclin B gene in the different tissues, including liver, ovary, muscle, brain stomach, heart and intestine, were measured by RT-PCR. mRNA expression of cyclin B could be detected in liver, ovary, muscle, brain, stomach, heart and strongest in the ovary, but almost not be detected in the intestine. In ovarian maturation stages, the expression of btscyclin B was different. The result indicated that btscyclin B was constitutive expressed and played an important role in the cell division stage.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 µg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 µg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 µg/body weight) was administered than the group fed with a lower concentration of LPS (4 µg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV.

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Ultrastructure of cells constituting lymphoid tubules and circulating hemocytes in Penaeus monodon.

The two main groups of cells in the lymphoid tubule wall of Penaeus monodon are fixed cells and migrating hemocytes. Fixed cells include endothelial, stromal, and capsular cells. Together, they form the scaffold that defines the structure of the lymphoid tubule and provide physical support as well as a niche for transmigrating hemocytes. The luminal surface of lymphoid tubule was lined by elongated, spindle-shaped endothelial cells with a centrally located nucleus and rather thick plasma membrane. Stromal cells were the smallest type of fixed cell. They are stellate cells located between the inner endothelial and outer capsular cells. These cells formed a cyto-reticular network for migrating hemocytes. Capsular cells have a flattened and irregular shape with a ruffled border with long filamentous microvilli. The nucleus is centrally located within a small mass of cytoplasm. Together they form the outermost layer of the lymphoid tubular wall. Transmigrating hemocytes within the lymphoid tubules, as opposed to circulating hemocytes, were classified into hyaline (HH), small granular (SGH) and large granular (LGH) hemocytes. The HH have very few granules and a few cytoplasmic organelles, reflecting low synthetic activity. The granular hemocytes (SGH and LGH), despite being different in size, have similar ultrastructural characteristics. They contain high amounts of rough endoplasmic reticulum, ribosomes, mitochondria, and three types of granules. These characteristics implicate their higher synthetic as well as immunologic activities. Based on these characteristics we believe that all the hemocytes belong to a single line of cell differentiation.

Transcriptome analysis of hepatopancreas in penaeus monodon under acute low pH stress.

The decrease of seawater pH can affect the metabolism, acid-base balance, immune response and immunoprotease activity of aquatic animals, leading to aquatic animal stress, impairing the immune system of aquatic animals and weakening disease resistance, etc. In this study, we performed high-throughput sequencing analysis of the hepatopancreas transcriptome library of low pH stress penaeus monodon, and after sequencing quality control, a total of 43488612-56271828 Clean Reads were obtained, and GO annotation and KEGG pathway enrichment analysis were performed on the obtained Clean Reads, and a total of 395 DEGs were identified. we mined 10 differentially expressed and found that they were significantly enriched in the Metabolic pathways (ko01100), Biosynthesis of secondary metabolites (ko01110), Nitrogen metabolism (ko00910) pathways, such as PIGA, DGAT1, DGAT2, UBE2E on Metabolic pathways; UGT, GLT1, TIM genes on Biosynthesis of secondary metabolites; CA, CA2, CA4 genes on Nitrogen metabolism, are involved in lipid metabolism, induction of oxidative stress and inflammation in the muscular body of spot prawns. These genes play an important role in lipid metabolism, induction of oxidative stress and inflammatory response in the muscle of the shrimp. In summary, these genes provide valuable reference information for future breeding of low pH-tolerant shrimp.

Shrimp pmo-miR-750 regulates the expression of sarcoplasmic calcium-binding protein facilitating virus infection in Penaeus monodon.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and play crucial roles in antiviral responses. Penaeus monodon miR-750 (pmo-miR-750) was found to be strongly up-regulated in the late phase of white spot syndrome virus (WSSV) infection, but its function remains uncharacterized. Herein, the targets that were translationally down-regulated in the shrimp stomach following a pmo-miR-750 mimic injection were identified using two-dimensional gel electrophoresis. Sarcoplasmic calcium-binding protein (Scp) and actin1 (Act1) were revealed to be down-regulated protein spots. The genuine binding of pmo-miR-750 mimic to Scp but not Act1 mRNA was validated in vitro. In addition, a negative correlation between the Scp transcript and pmo-miR-750 expression level in WSSV-infected P. monodon stomach implies that pmo-miR-750 regulates Scp expression in vivo. When injected into WSSV-infected shrimp, the pmo-miR-750 mimic suppressed Scp expression but significantly increased the WSSV copy number. Consistent with the miRNA mimic-mediated Scp suppression, the loss of function assay of Scp in WSSV-challenged shrimp by RNA interference revealed a decreased survival rate with a dramatic increase in viral copy number. Besides that, apoptosis was activated in the hemocytes of the Scp knockdown shrimp upon WSSV infection. Collectively, our findings reveal that up-regulated pmo-miR-750 suppresses Scp expression at both the transcript and protein levels in the late stage of WSSV infection, which contributes to modulating apoptosis and eventually enabling viral propagation.

Identification of multifunctionality of the PmE74 gene and development of SNPs associated with low salt tolerance in Penaeus monodon.

Members of the E74-like factor (ELF) subfamily are involved in the immune stress process of organisms by regulating immune responses and the development of immune-related cells. PmE74 of Penaeus monodon was characterized and functionally analyzed in this study. The full length of PmE74 was 3106 bp, with a 5'-UTR of 297 bp, and a 3'-UTR of 460 bp. The ORF (Open reading frame) was 2349 bp and encoded 782 amino acids. Domain analysis showed that PmE74 contains a typical Ets domain. Multiple sequence alignment and phylogenetic tree analysis showed that PmE74 clustered with Litopenaeus vannamei E74 and displayed significant similarity (98.98%). PmE74 was expressed in all tissues tested in P. monodon, with the highest levels of expression observed in the testis, intestine, and epidermis. Different pathogen stimulation studies have revealed that PmE74 expression varies in response to different pathogen stimuli. A 96-h acute low salt stress study revealed that PmE74 in the hepatopancreas was upregulated and downregulated in the salinity 17 group and considerably downregulated in the salinity 3 group, whereas PmE74 in gill tissue was considerably downregulated in both groups. Further, by knocking down PmE74 and learning the trends of its linkage genes PmAQP1, PmNKA, PmE75, PmFtz-f1, PmEcR, and PmRXR in response to low salt stress, it was further indicated that PmE74 could have a vital role in the regulation of low salt stress. The SNP test revealed that PmE74-In1-53 was significantly associated with low salt tolerance traits in P. monodon (P < 0.05). The findings of this study can aid in the advancement of molecular marker-assisted breeding in P. monodon, as well as provide fundamental data and methodologies for further investigation of its low salt tolerance strains in P. monodon.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.