First transcriptome profiling in gill and hepatopancrease tissues of Metapenaeus ensis in response to acute ammonia-N stress.

The greasyback shrimp, Metapenaeus ensis, suffers from ammonia-N stress during intensive factory aquaculture. Optimizing ammonia-N stress tolerance has become an important issue in M. ensis breeding. The metabolic and adaptive mechanisms of ammonia-N toxicity in M. ensis have not been comprehensively understood yet. In this study, a large number of potential simple sequence repeats (SSRs) in the transcriptome of M. ensis were identified. Differentially expressed genes (DEGs) in the gill and hepatopancreas at 24 h post-challenges under high concentrations of ammonia-N treatment were detected. We obtained 20,108,851-27,681,918 clean reads from the control and high groups, assembled and clustered a total of 103,174 unigenes with an average of 876 bp and an N50 of 1189 bp. Comparative transcriptome analyses identified 2000 different expressed genes in the gill and 2010 different expressed genes in the hepatopancreas, a large number of which were related to immune function, oxidative stress, metabolic regulation, and apoptosis. The results suggest that M. ensis may counteract ammonia-N toxicity at the transcriptome level by increasing the expression of genes related to immune stress and detoxification metabolism, and that selected genes may serve as molecular indicators of ammonia-N. By exploring the genetic basis of M. ensis' ammonia-N stress adaptation, we constructed the genetic networks for ammonia-N adaptation. These findings will accelerate the understanding of M. ensis' ammonia-N adaptation, contribute to the research of future breeding, and promote the level of factory aquaculture of M. ensis.

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 transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

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.

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.

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.

Comprehensive expression analysis of the beta integrin from Penaeus monodon indicating its participation in innate immunity and ammonia nitrogen stress response.

The aim of the present study was to investigate the function of the beta integrin (PmItgb) in Penaeus monodon. The 3011 bp cDNA sequence of PmItgb was cloned from P. monodon using rapid amplification of cDNA ends (RACE) PCR. Phylogenetic tree analyses indicated that the amino acid sequence of PmItgb should be merged into Fenneropenaeus chinensis (93%). Quantitative real-time PCR (q RT-PCR) revealed that PmItgb mRNA was highly expressed in the hemocytes. In addition, with regard to developmental stages, PmItgb showed significantly higher expression in oosperm, nauplius IV, zoea I and III, and post larval stages than that in other development stages. PmItgb expression in the shrimp epidermis was higher in the postmolt (B) stage, and lower in other molting stages. We also found that Vibrio harveyi and V. anguillarum challenge enhanced PmItgb expression in the hepatopancreas and gills. When PmItgb was inhibited, innate immunity-related genes such as ALF, crustin 1, crustin 7, penaeidin 3, and penaeidin 5 were significantly down-regulated. Furthermore, we demonstrated that PmItgb knock-down by specific dsRNA reduced bacterial clearance. In high ammonia nitrogen concentrations, PmItgb was significantly up-regulated in the hepatopancreas and gills. After PmItgb was silenced, the rate of mortality owing to high ammonia nitrogen concentrations decreased; the expression of related anti-apoptotic genes was up-regulated, and that of the apoptotic genes was slightly down-regulated. These results suggested that PmItgb may be involved in shrimp innate immunity and mediate apoptosis of hepatopancreatic cells induced by high ammonia nitrogen environments.

Ecdysone inducible gene E75 from black tiger shrimp Penaeus monodon: Characterization and elucidation of its role in molting.

Molting is controlled by ecdysteroids, which are synthesized and secreted by the Y-organ in crustaceans. Ecdysone inducible gene, E75, is an early-response gene in the 20-hydroxyecdysone (20E) signaling pathway, with crucial roles in arthropod development. Complementary DNA (cDNA) encoding Penaeus monodon E75 (PmE75) was cloned using RT-PCR and RACE. PmE75 cDNA was 3526 bp long and encoded a 799-amino acid protein. Tissue distribution analysis showed that PmE75 was expressed ubiquitously in selected tissues, and was relatively abundant in the epidermis, muscle, and hepatopancreas. Developmental expression revealed that PmE75 was expressed throughout its life cycle. Silencing PmE75 significantly decreased PmE75 expression. Shrimps injected with PBS and dsGFP started molting on Day 7 and had almost completed molting on Day 9, whereas dsPmE75-injected shrimp presented no signs of molting. These results suggested that PmE75 might be involved in molting. In situ hybridization results support this hypothesis. To explore the role of 20E and eyestalks in the regulation of molting in P. monodon, exogenous 20E injection and eyestalk ablation (ESA) were performed, and showed that 20E can induce the transcription and expression of PmE75 in the hepatopancreas, epidermis, and muscle, which were significantly elevated after ESA. These results provide further insights into our understanding of molting.

C-type lectin response to bacterial infection and ammonia nitrogen stress in tiger shrimp (Penaeus monodon).

C-type lectins (CTLs) are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many shrimp, C-type lectins subfamily contain a large number of members with different functions that need to research in deep. In this present study, a new type of CTL, PmCL1 with 861 bp long full-length cDNA, that encodes a protein with 164-amino acid from a 495-bp open reading frame, was isolated and characterized from tiger shrimp (Penaeus monodon). The mRNA transcript of PmCL1 showed the highest expression in the hepatopancreas, whereas it was barely detected in the ovary. After the shrimp were stimulated by Vibrio harveyi and Vibrio anguillarum, PmCL1 expression in the hepatopancreas and gill was significantly upregulated. A carbohydrate-binding assay revealed the specificity of PmCL1 for pathogen-associated molecular patterns (PAMPs) that included peptidoglycan (PGN) and lipopolysaccharide (LPS), and saccharides that included d-glucose, galactosamine, α-lactose, treholose, and d-mannose. Recombinant PmCL1 agglutinated gram-positive (Staphylococcus aureus) and gram-negative bacteria (V. harveyi, V. anguillarum, Vibrio alginolyticus, Vibrio parahemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) in the presence of calcium ions and enhanced the efficiency of clearing the invading bacteria. Collectively, our results suggested that PmCL1 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, as well as the response towards ammonia nitrogen stress.

The comprehensive expression analysis of the G protein-coupled receptor from Penaeus monodon indicating it participates in innate immunity and anti-ammonia nitrogen stress.

The G protein-coupled receptors (GPCRs) composed a superfamily that played an important role in physiological processes of crustaceans, with multiple functions such as growth and development, acting as a defense against stimulations from external factors. In this paper, one kind of GPCRs were identified from Penaeus monodon, called PmGPCR, included an open reading frame (ORF) of 1113 bp. Bioinformatic analysis showed that PmGPCR protein had the typical structure of seven transmembrane domains (7TM), especially the special Asp-Arg-Try motif (DRY motif) between the third transmembrane structures (TM3) and the second intracellular loops (IL-2) which can prove that PmGPCR belongs to the rhodopsin-like family. The analyses of phylogenetic tree indicated that the amino acid sequence of PmGPCR should be merged into Procambarus clarkiic with high identity (98%). Quantitative real-time PCR (q RT-PCR) revealed that PmGPCR mRNA was highly expressed in hepatopancreas, abdominal ganglia and lymph, in which it was significantly higher than that of other tissues (P < 0.05). In addition, the expression of PmGPCR was analyzed during three days post-stimulation with the gram-positive/negative bacteria, the mRNA expression level increased after challenged with gram - positive bacteria in hepatopancreas, lymph and intestines. During the development stages, PmGPCR showed significantly higher expression in nauplius, zoea III, mysis III and post larvae stages than that in other development stages. Meanwhile, the highest transcripts expression of PmGPCR in abdominal ganglia, hepatopancreas, lymph and intestines respectively appeared at D0, D1, D2 and D3/D4 stages of molting. High or low concentration of ammonia nitrogen up-regulated the expression level of PmGPCR at the initial stage in hepatopancreas and gill, and then down-regulated at 48 h. These results indicated PmGPCR may mediate the pathways that involved in growth and development process, survival in the adversity, in addition, provided the useful data to research GPCR-mediated physiological and biological process and explain the mechanisms to defense pathogens and anti-stress in shrimp.

Transcriptome reveals involvement of immune defense, oxidative imbalance, and apoptosis in ammonia-stress response of the black tiger shrimp (Penaeus monodon).

Ammonia is a major aquatic environmental pollutant that negatively impacts shrimp health and commercial productivity. However, we currently do not fully understand the underlying molecular mechanisms of ammonia stress in shrimp. We therefore performed transcriptomic analysis of hepatopancreas from black tiger shrimp (Penaeus monodon) treated with ammonia-stress. We obtained 146,410,174 and 115,241,048 clean reads for the control and treatment groups, respectively. A total of 64,475 unigenes with an average length of 1275 bp and a N50 value of 2158 bp were assembled. A comparative transcriptome analysis identified 3462 differentially expressed genes, 177 of which are highly homologous with known proteins in aquatic species. Most of these genes showing the expression changes were related to immune function. Some significantly down-regulated genes are involved in purine metabolism and other metabolic pathways, which suggests that purineolytic capacity is an ammonia detoxification process in P. monodon, and metabolic depression is a strategy to reduce shrimp exposure to ammonia. Additionally, ammonia stress altered the expression patterns of key apoptosis genes (Bcl-xL, PERK, caspase 7, and caspase 10), confirmed that ammonia-stress induce oxidative stress and eventually even apoptosis. We also found evidence for the involvement of antioxidant defense in response to oxidative imbalance, given the regulation of peroxiredoxin 1, SOD, and CAT under ammonia stress. In conclusion, our study clarifies shrimp defensive response to ammonia toxicity and should benefit efforts to breed more ammonia-tolerant varieties.

Characterization and expression analysis of a chitinase gene (PmChi-5) from black tiger shrimp (Penaeus monodon) under pathogens infection and ambient ammonia-N stress.

Chitinases are crucial enzymes for crustaceans. Previous researches had already revealed that chitinases play important roles in digestion, molting and defense against viruses. In the present study, a chitinase cDNA was identified from black tiger shrimp (Penaeus monodon) and designated as PmChi-5. The full-length PmChi-5 cDNA was 2860 bp in size, containing an open reading frame (ORF) of 1731 bp that encoded a protein of 576 amino acids with a deduced molecular weight of 64.8 kDa. Expression of the PmChi-5 mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill and hepatopancreas. PmChi-5 was expressed throughout the whole larvae stages, and the highest level at Mysis3 stage, which indicated that PmChi-5 may be involved in larval metamorphosis. After challenged with Streptococcus agalactiae and Vibrio harveyi, the transcripts of PmChi-5 were found to be up-regulated significantly both in hepatopancreas and gill. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-5 transcripts were significantly changed in hepatopancreas and gill. The results showed that PmChi-5 may be involved in molting, larval metamorphosis, the immune defenses to pathogens infection and ammonia-N stress.

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%-88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

Characterization, expression and silencing by RNAi of p53 from Penaeus monodon.

The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.

Characterization of Argonaute2 gene from black tiger shrimp (Penaeus monodon) and its responses to immune challenges.

Argonaute2 binds to a short guide RNA (microRNA or short interfering RNA) and guides RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. Here we identified and characterized Argonaute2 from black tiger shrimp Penaeus monodon (designated as PmAgo2). The full-length cDNA of PmAgo2 contained a 5' untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 2616 bp and a 3' UTR of 123 bp. The predicted PmAgo2 protein is 99.4 KDa with the theoretical isoelectric point of 9.54. PmAgo2 shared the highest similarity of amino acid with Marsupenaeus japonicus Argonaute2 and Litopenaeus vannamei Argonaute2, at 69.0% and 68.5%, respectively. Phylogenic analysis showed PmAgo2 clustered with shrimp Argonaute2, and closed to the group of insects. Real-time quantitative PCR showed that PmAgo2 was widely expressed in almost all examined tissues except eyestalk, with high expression in lymph and haemocyte. mRNA expression also revealed that PmAgo2 was significantly up-regulated by Staphylococcus aureus and White Spot Syndrome Virus (WSSV) in hepatopancreas. Furthermore, our study also confirmed that dsRNA and ssRNA homologous poly (I:C) and R848 activated the expression of PmAgo2. The result indicated that PmAgo2 responded to both bacterial infection and viral infection, especially, it may induce an ssRNA-mediated RNAi with other core members of siRNA pathway in black tiger shrimp.

Evaluation of Genetic Parameters and Comparison of Stress Tolerance Traits in Different Strains of Litopenaeus vannamei.

Litopenaeus vannamei stands out globally in aquaculture for its fast growth, broad salt tolerance, disease resistance, and high protein levels. Selective breeding requires the precise estimation of the variance components and genetic parameters for important traits. This study formed lineages from 20 full sibling families of L. vannamei, with progenitors from Thailand and the USA. We then assessed the genetic resilience traits of juvenile shrimp from these families to high ammonia-N, high pH, and low salinity by performing a 96 h acute toxicity test. Mortality rates for the families under 96 h exposure to high ammonia-N, high pH, and low salinity were 19.52-92.22%, 23.33-92.22%, and 19.33-80.00%, respectively, showing significant variance in stress tolerance among families (p < 0.05). Survival heritability estimates, using threshold male and female models, were 0.44 ± 0.12 in high ammonia-N, 0.41 ± 0.12 in high pH, and 0.27 ± 0.08 in low salinity, respectively. Genetic correlations between growth and stress resistance traits varied from 0.0137 ± 0.2406 to 0.8327 ± 0.0781, and phenotypic correlations ranged from 0.0019 ± 0.0590 to 0.6959 ± 0.0107, indicating a low-to-high positive correlation significant at (p < 0.05). It was found that the survival rate of families No. 2 and No. 9 was higher under high ammonia-N and high pH stresses, while the survival rate of family No. 10 was higher under low salinity stress after comparing two selection criteria, the breeding values and phenotypic values. Thus, these three families are identified as potential breeding program candidates. Through the creation of a genetic parameter estimation model, the genetic variances across mating combinations for stress resistance traits were obtained and families with heightened stress resistance were identified, laying the groundwork for enhanced genetic selection of L. vannamei.

Effects of Enterococcus faecium (R8a) on nonspecific immune gene expression, immunity and intestinal flora of giant tiger shrimp (Penaeus monodon).

In this study, Penaeus monodon were gave basic feed supplemented with three levels of Enterococcus faecium. Then, the expression of non-specific immunity-related genes, and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (AKP), phenol oxidase (PO) were evaluated. Meanwhile, the disease resistance test and intestinal flora determination were conducted. The results showed that the MDA levels of 2% and 5% E. faecium groups were significantly lower than that of the control group (P < 0.05). While the SOD and T-AOC and ACP and AKP of experimental groups were significantly higher (P < 0.05), the PO of experimental groups were significantly lower than that of the control group (P < 0.05). In addition, the expressions of immunity-related genes (tlr22, dorsal, lysozyme, crustin, imd, and relish) in the 2% and 5% E. faecalis groups were significantly greater than those in the control group (P < 0.05). After P. monodon was challenged with Vibrio parahaemolyticus for 7 days, the average cumulative mortality of P. monodon in the 2% and 5% groups were significantly lower than that in the 0% group (P < 0.05). With the increase of feeding time, the number of effective OTUs in each group showed a downward trend. At the 14th d, Proteobacteria, Bacteroidetes and Firmicutes, the dominant flora in the intestinal tract of P. monodon. In summary, supplied with E. faecium could increase the expression of non-specific immunity-related genes, enhance the immune capacity of P. monodon.

Isolation and characterization of homologous TRBP cDNA for RNA interference in Penaeus monodon.

The transactivation response RNA-binding protein (TRBP) interacts with Dicer and binds to double-stranded RNA as a critical component of the RNA-induced silencing complex, which is a key complex in the RNA interference pathway. The full-length cDNA of TRBP from the tiger prawn, Penaeus monodon, (PmTRBP; 1548 bp long with a 1029 bp coding region) was isolated. The encoded polypeptide of 343 amino acids had a predicted molecular mass of 36.8 kDa. Sequence homology and phylogenetic analysis indicated that PmTRBP was evolutionarily closest to TRBP1 from Litopenaeus vannamei, with the three double-stranded RNA-binding motifs that were typical of the TRBP family. Tissue expression profile analysis by quantitative real-time reverse transcription polymerase chain reaction showed that PmTRBP1 was constitutively expressed in all the examined tissues, with a predominant expression in the lymphatic organs and with the weakest expression in the ovaries. Significantly upregulated PmTRBP1 expression was elicited by systemic injections of Staphylococcus aureus, Vibrio vulnificus, and white spot syndrome virus, thereby revealing its pathogen inducibility. Furthermore, exogenous viral nucleoside analogs (high-molecular-weight poly(I:C) dsRNAs as well as R484 single-stranded RNA) were remarkably induced PmTRBP1 transcription at 48 h and 9 h post-injection, respectively, which suggested that PmTRBP1 might function in tiger prawn antibacterial and antiviral response.

Molecular cloning and mRNA expression of M-phase phosphoprotein 6 gene in black tiger shrimp (Penaeus monodon).

It is widely accepted that protein phosphorylation is a major control event in regulating cell cycle. In the present study, a novel M-phase phosphoprotein 6 (MPP6) was identified from black tiger shrimp Penaeus monodon (designated as PmMPP6) by cDNA library and RACE approaches. The full-length cDNA of PmMPP6 was of 690 bp, including a 5'-terminal un-translated region (5'UTR) of 68 bp, a 3'UTR of 172 bp with a poly (A) tail, and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a predicted molecular weight of 17.01 kDa. Blastx and phylogenetic analysis together supported that PmMPP6 was a novel member of shrimp MPP6. The mRNA expression of PmMPP6 in thirteen tissues was examined by real-time PCR, and mRNA transcript of PmMPP6 was predominantly detectable in tissues of lymphoid and muscle, to a lesser degree in the tissues of gill, ovary and hepatopancreas, and mainly detected in haemocytes, heart and gonad. The temporal expression of PmMPP6 in different developmental stages of ovary was investigated by real-time PCR. During the six stages of ovary development, two peaks expression of PmMPP6 was detected in stage II with 3.78-fold increase and stage V with 3.48-fold increase compared to that in stage I. All these results indicated that PmMPP6 might be involved in regulating shrimp cell cycle and ovary development.

Effect of Air Exposure and Re-Submersion on the Histological Structure, Antioxidant Response, and Gene Expression of Procambarus Clarkii.

Air exposure is an important environmental stressor during the transportation and cultivation of Procambarus clarkii. We evaluated the effect of re-submersion for 24 h after dry transportation for 24 h on the histological structure, antioxidant activity, and gene expression of crayfish. The antioxidant parameters of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and the relative expression of CAT, SOD, HSP70, and ferritin genes were subsequently measured in the hepatopancreas and gills at both stages. Histopathology found that air exposure led to vacuolation of the hepatopancreas and disorderly arrangement of respiratory epithelial cells (REC) in the gills. The activities of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactic dehydrogenase (LDH) in the hepatopancreas and gills increased with short-term air exposure. The relative expression of genes (CAT, SOD, HSP70, and Ferritin) were induced after short-term air exposure. During re-submersion, MDA content and CAT and SOD activities in the hepatopancreas and gills were restored after 24 h, however, LDH activity and hepatopancreatic tissue damage were not repaired. Our results indicate that air exposure can cause oxidative damage to P. clarkii, and CAT and SOD can be used to determine the response of crayfish exposed to air, in addition to some damage that can be eliminated after re-submersion to a limited degree. This study provides foundational data that re-submersion can improve crayfish performance under hypoxic stress to a certain extent and will lead to the development of more effective transportation strategies and decrease economic losses in the future.