Effects of Dietary Gracilaria lichenoides and Bacillus amyloliquefaciens on Growth Performance, Antioxidant Capacity, and Intestinal Health of Penaeus monodon.

This research sought to assess the effects of dietary supplements with Gracilaria lichenoides and Bacillus amyloliquefaciens, either individually or combined, on the growth performance, antioxidant capacity, and intestinal function of Penaeus monodon. A total of 840 shrimps were randomly assigned to 28 tanks with an average initial weight of (1.04 ± 0.03) g (30 shrimp per tank) with 7 different treatment groups and 4 replicates per treatment. The control treatment (C) consisted of a basal diet; in contrast, the experimental groups were complement with varying levels of G. lichenoides (3% or 8%), either alone (S3 and S8) or in combination with B.amyloliquefaciens at different concentrations (3% G. lichenoides and 109 CFU/g-S3B9; 8% G. lichenoides and 1011 CFU/g B. amyloliquefaciens-S8B11; 109 CFU/g B. amyloliquefaciens-S9; 1011 CFU/g B. amyloliquefaciens-B11). The results indicated that the maximum values of final body weight (FBW) (10.49 ± 0.90) g, weight gain rate (WGR) (908.94 ± 33.58) g, and specific growth rate (SGR) (4.20 ± 0.06) g were perceived in the 3% G. lichenoide diet treatment, and compared with the control group, the difference was significant (p < 0.05). The whole-body lipid content of shrimp in the B9 group was significantly higher than that in the B11 group (p < 0.05), but no significant difference was observed when compared with shrimp fed other diets (p > 0.05). The ash content of shrimp in the B9 group was found to be significantly higher than that in the S3B9 group (p < 0.05). Furthermore, the lipase activity in the stomach and intestines of the experimental groups exhibited a statistically significantly increase compared to the control (p < 0.05). In comparison to the control group, the hepatopancreas of the S3 group exhibited a significant increase in the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and antioxidant genes [SOD, catalase (CAT), GSH-Px, thioredoxin (Trx), Hippo, and NF-E2-related factor 2 (Nrf2)] expression levels (p < 0.05). Additionally, the activities of total antioxidant capacity (T-AOC), SOD, peroxidase (POD), and antioxidant genes (CAT, GSH-Px, Trx, and Hippo) in the S3B9 treatment of hepatopancreas showed significant improvement (p < 0.05). The inclusion of dietary G. lichenoides and B. amyloliquefaciens resulted in enhanced relative expression of intestinal lipid metabolism genes (fatty acid synthetase (FAS), lipophorin receptor (LR), fatty acid transport protein 1 (FATP1)) and suppressed the expression of the long-chain fatty acid-CoA ligase 4 (LCL4) gene. Analysis of microbiota sequencing indicated improvements in composition and structure, with notable increases in Firmicutes at the phylum level and Vibrio at the genus level in the S3 group, as well as an increase in Tenericutes at the genus level in the S8B11 group. Overall, the inclusion of dietary G. lichenoides and B. amyloliquefaciens positively impacted the growth, antioxidant capacity, and microbial composition of shrimp, with particular enhancement observed in shrimp fed a supplementary 3% G. lichenoides diet.

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.

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 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.

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.

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.

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.

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.

Automatic Penaeus Monodon Larvae Counting via Equal Keypoint Regression with Smartphones.

Today, large-scale Penaeus monodon farms no longer incubate eggs but instead purchase larvae from large-scale hatcheries for rearing. The accurate counting of tens of thousands of larvae in these transactions is a challenging task due to the small size of the larvae and the highly congested scenes. To address this issue, we present the Penaeus Larvae Counting Strategy (PLCS), a simple and efficient method for counting Penaeus monodon larvae that only requires a smartphone to capture images without the need for any additional equipment. Our approach treats two different types of keypoints as equip keypoints based on keypoint regression to determine the number of shrimp larvae in the image. We constructed a high-resolution image dataset named Penaeus_1k using images captured by five smartphones. This dataset contains 1420 images of Penaeus monodon larvae and includes general annotations for three keypoints, making it suitable for density map counting, keypoint regression, and other methods. The effectiveness of the proposed method was evaluated on a real Penaeus monodon larvae dataset. The average accuracy of 720 images with seven different density groups in the test dataset was 93.79%, outperforming the classical density map algorithm and demonstrating the efficacy of the PLCS.

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.

Genes expression in Penaeus monodon of Bangladesh; challenged with AHPND-causing Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the causative agent of Acute hepatopancreatic necrosis disease (AHPND), was discovered in 2013 as a unique isolate that produces toxins and kills penaeid shrimps in devasting nature in Bangladesh and causes severe economic losses. This research aimed to understand the expressions of immune genes in different stages of the host species, Penaeus monodon, against virulence and toxin genes upon being challenged with V. parahaemolyticus. Healthy post-larvae (PL) samples were collected from southwestern of Bangladesh from July 2021 to August 2022. The tryptic soy agar with 1.5% sodium chloride (NaCl) was used to inoculate the cells of V. parahaemolyticus, and the tryptic soy broth (TSB) with 1.5% NaCl was used to transfer the colonies. The spectrophotometry measured bacteria density. PCR, qPCR, SDS-PAGE, and Western blot measured gene expression and survivability after the immersion challenge. The 1 × 105CFU/mL of V. parahaemolyticus was used for 144 h.p.i (hours post-infection) challenge to six stages of post-larvae (PL) of P. monodon (PL20, PL25, PL30, PL35, PL40, and PL45), PL30 and PL35 showed 100% mortality by day 72 (h.p.i.) after exposure that indicated most vulnerable to V. parahaemolyticus. The expression of immune and toxic genes was confirmed by qPCR. The immune genes toll-like receptors (TLR), prophenoloxidase (ProPO), lysozyme (lyso), and penaeidin (PEN) of PL20 and PL25 of P. monodon were expressed robustly up-trends. PL30 and PL35 showed the lowest gene expression at the end of 72 (h.p.i.). At the end of the 144 (h.p.i.) exposure, the immune genes TLR, ProPO, lyso, and PEN expressed highest in PL45 than other post-larvae stages of P. monodon. The toxic genes (pirA, ToxR, ToxA, ToxB, tlh, tdh, and trh) in PL30 and PL35 of P. monodon after exposure of V. parahaemolyticus were expressed highest at the end of the 72 (h.p.i.). The lowest toxic genes expressions were revealed in PL20 and PL45 at the end of the 144 (h.p.i.). The SDS-PAGE analysis of proteins from the bacterium revealed identical protein profiles with toxic genes, and those toxins were further confirmed by Western blot. The 20 kDa, 78 kDa (ToxR), 20 kDa, 25 kDa (ToxA), 25 kDa (ToxB), 20 kDa, 27 kDa, 75 kDa (tdh), and 20 kDa, 27 kDa, 75 kDa, and 78 kDa (trh) proteins were strong responses in Western blot, indicating the crucial involvement of these immune-related genes in the defense and recovery of the first-line defense mechanisms during V. parahaemolyticus infection to shrimp. The all-toxic genes showed a unique homology and those derived from the common ancestor compared with V. parahaemolyticus (NCBI accession no. AP014859.1). All clades were derived with different traits with very low genetic distance, where the overall mean distance was 3.18 and showed a very uniform and homogenous pattern among the lineages. The V. parahaemolyticus infection process in different PL stages in P. monodon revealed novel insights into the immune responses. The responses may lead to the subsequent production of a DNA vaccine, enhancing shrimp health management to minimize the economic losses due to AHPND experiencing an outbreak of early mortality syndrome (EMS) toward sustainable production P. monodon (shrimp).

Transcriptome and molecular regulatory mechanisms analysis of gills in the black tiger shrimp Penaeus monodon under chronic low-salinity stress.

Background: Salinity is one of the main influencing factors in the culture environment and is extremely important for the survival, growth, development and reproduction of aquatic animals. Methods: In this study, a comparative transcriptome analysis (maintained for 45 days in three different salinities, 30 psu (HC group), 18 psu (MC group) and 3 psu (LC group)) was performed by high-throughput sequencing of economically cultured Penaeus monodon. P. monodon gill tissues from each treatment were collected for RNA-seq analysis to identify potential genes and pathways in response to low salinity stress. Results: A total of 64,475 unigenes were annotated in this study. There were 1,140 upregulated genes and 1,531 downregulated genes observed in the LC vs. HC group and 1,000 upregulated genes and 1,062 downregulated genes observed in the MC vs. HC group. In the LC vs. HC group, 583 DEGs significantly mapped to 37 signaling pathways, such as the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway; in the MC vs. HC group, 444 DEGs significantly mapped to 28 signaling pathways, such as the MAPK signaling pathway, Hippo signaling pathway and calcium signaling pathway. These pathways were significantly associated mainly with signal transduction, immunity and metabolism. Conclusions: These results suggest that low salinity stress may affect regulatory mechanisms such as metabolism, immunity, and signal transduction in addition to osmolarity in P. monodon. The greater the difference in salinity, the more significant the difference in genes. This study provides some guidance for understanding the low-salt domestication culture of P. monodon.

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.

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.

Expression Analysis of a Novel Oxidoreductase Glutaredoxin 2 in Black Tiger Shrimp, Penaeus monodon.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that is an important component of the redox system in organisms. However, there is a serious lack of sequence information and functional validation related to Grx in crustaceans. In this study, a novel Grx was identified in Penaeus monodon (PmGrx2). The full-length cDNA of PmGrx2 is 998 bp, with an open reading frame (ORF) of 441 bp, encoding 119 amino acids. Sequence alignment showed that PmGrx2 had the highest identity with Grx2 of Penaeus vannamei at 96.64% and clustered with Grx2 of other crustaceans. Quantitative real-time PCR (qRT-PCR) analysis showed that PmGrx2 was expressed in all examined tissues, with higher expression levels in the stomach and testis. PmGrx2 was continuously expressed during development and had the highest expression level in the zygote stage. Both ammonia-N stress and bacterial infection could differentially induce the expression of PmGrx2 in hepatopancreas and gills. When PmGrx2 was inhibited, the expression of antioxidant enzymes was suppressed, the degree of apoptosis increased, and the GSH content decreased with the prolongation of ammonia-N stress. Inhibition of PmGrx2 resulted in shrimp being exposed to a greater risk of oxidative damage. In addition, an SNP locus was screened on the exons of PmGrx2 that was significantly associated with an ammonia-N-stress-tolerance trait. This study suggests that PmGrx2 is involved in redox regulation and plays an important role in shrimps' resistance to marine environmental stresses.

Genetic parameters of color phenotypes of black tiger shrimp (Penaeus monodon).

Black tiger shrimp (Penaeus monodon) is the second most important aquaculture species of shrimp in the world. In addition to growth traits, uncooked and cooked body color of shrimp are traits of significance for profitability and consumer acceptance. This study investigated for the first time, the phenotypic and genetic variances and relationships for body weight and body color traits, obtained from image analyses of 838 shrimp, representing the progeny from 55 sires and 52 dams. The color of uncooked shrimp was subjectively scored on a scale from 1 to 4, with "1" being the lightest/pale color and "4" being the darkest color. For cooked shrimp color, shrimp were graded firstly by subjective scoring using a commercial grading score card, where the score ranged from 1 to 12 representing light to deep coloration which was subsequently found to not be sufficiently reliable with poor repeatability of measurement (r = 0.68-0.78) Therefore, all images of cooked color were regraded on a three-point scale from brightest and lightest colored cooked shrimp, to darkest and most color-intense, with a high repeatability (r = 0.80-0.92). Objective color of both cooked and uncooked color was obtained by measurement of RGB intensities (values range from 0 to 255) for each pixel from each shrimp. Using the "convertColor" function in "R", the RGB values were converted to L*a*b* (CIE Lab) systems of color properties. This system of color space was established in 1976, by the International Commission of Illumination (CIE) where "L*" represents the measure of degree of lightness, values range from 0 to 100, where 0 = pure black and 100 = pure white. The value "a*" represents red to green coloration, where a positive value represents the color progression towards red and a negative value towards green. The value "b*" represents blue to yellow coloration, where a positive value refers to more yellowish and negative towards the blue coloration. In total, eight color-related traits were investigated. An ordinal mixed (threshold) model was adopted for manually (subjectively) scored color phenotypes, whereas all other traits were analyzed by linear mixed models using ASReml software to derive variance components and estimated breeding values (EBVs). Moderate to low heritability estimates (0.05-0.35) were obtained for body color traits. For subjectively scored cooked and uncooked color, EBV-based selection would result in substantial genetic improvement in these traits. The genetic correlations among cooked, uncooked and body weight traits were high and ranged from -0.88 to 0.81. These suggest for the first time that 1) cooked color can be improved indirectly by genetic selection based on color of uncooked/live shrimp, and 2) intensity of coloration is positively correlated with body weight traits and hence selection for body weight will also improve color traits in this population.