Toxicity of three microcystin variants on the histology, physiological and metabolism of hepatopancreas and intestinal microbiota of Litopenaeus vannamei.

Microcystins (MCs) are prevalent harmful contaminants within shrimp aquaculture systems, exhibiting a diverse array of variants. Gut microbiota can engage in mutual interactions with the host through the gut-liver axis. In this study, the shrimp Litopenaeus vannamei were subjected to three different variants of MCs (LR, YR, RR) at a concentration of 1 µg/L each, and elucidated the alterations in both intestinal microbiota and hepatopancreas physiological homeostasis. The results showed that all three variants of MCs prompted histological alterations in the hepatopancreas, induced elevated levels of oxidative stress biomarkers (H2O2, T-SOD, and CAT), disturbed the transcription levels of immune-related genes (Crus, ALF, and Lys), along with an increase in apoptotic genes (Casp-3 and P53). Furthermore, the metabolic profiles of the hepatopancreas were perturbed, particularly in amino acid metabolism such as "lysine degradation" and "ß-alanine metabolism"; the mTOR and FoxO signaling were also influenced, encompassing alterations in the transcription levels of related genes. Additionally, the alterations were observed in the intestinal microbiota's diversity and composition, particularly potential beneficial bacteria (Alloprevotella, Bacteroides, Collinsella, Faecalibacterium, and Prevotellaceae UCG-001), which exhibited a positive correlation with the metabolite berberine. These findings reveal that the three MCs variants can impact the health of the shrimp by interfering with the homeostasis of intestinal microbial and hepatopancreas physiology.

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.

Dinotefuran exposure alters biochemical, metabolomic, gut microbiome, and growth responses in decapoda pacific white shrimp Penaeus vannamei.

Dinotefuran, a neonicotinoid insecticide, may impact nontarget organisms such as Decapoda P. vannamei shrimp with nervous systems similar to insects. Exposing shrimp to low dinotefuran concentrations (6, 60, and 600 µg/L) for 21 days affected growth, hepatosomatic index, and survival. Biomarkers erythromycin-N-demethylase, alanine aminotransferase, and catalase increased in all exposed groups, while glutathione S-transferase is the opposite; aminopyrin-N-demethylase, malondialdehyde, and aspartate aminotransferase increased at 60 and 600 µg/L. Concentration-dependent effects on gut microbiota altered the abundance of bacterial groups, increased potentially pathogenic and oxidative stress-resistant phenotypes, and decreased biofilm formation. Gram-positive/negative microbiota changed significantly. Metabolite differences between the exposed and control groups were identified using mass spectrometry and KEGG pathway enrichment. N-acetylcystathionine showed potential as a reliable dinotefuran metabolic marker. Weighted correlation network analysis (WGCNA) results indicated high connectivity of cruecdysone in the metabolite network and significant enrichment at 600 µg/L dinotefuran. The WGCNA results revealed a highly significant negative correlation between two key metabolites, caldine and indican, and the gut microbiota within co-expression modules. Overall, the risk of dinotefuran exposure to non-target organisms in aquatic environments still requires further attention.

Biochemical, histological and transcriptional response of intestines in Litopenaeus vannamei under chronic zinc exposure.

Zinc (Zn) is an essential trace element for the normal physiological function of aquatic organisms, but it could become toxic to organisms when the concentration increased in water. As the first line of defense, the shrimp intestines are the most susceptible organ to environmental stress. In this study, the chronic toxicity of 0 (control, IC), 0.01(IL), 0.1(IM) and 1 mg/L (IH) Zn in intestines of Litopenaeus vannamei was investigated from the perspectives of biochemical, histological and transcriptional changes after exposure for 30 days. The results showed that the intestinal tissue basement membrane is swollen in the IM and IH groups and detached in the IH group. The total antioxidant capacities (T-AOC) were reduced while the content of malondialdehyde (MDA) were increased significantly in IM and IH groups. The production of reactive oxygen species (ROS) was increased significantly in IH group. Many differentially expressed genes (DEGs) were identified in IL, IM and IH groups, respectively. GO and KEGG enrichment analyses were conducted on the DEGs to obtain the underlying biological processes and pathways. The gene modules related to the sample were identified by weighted gene co-expression network analysis (WGCNA), and genes in modules highly corelated with IH group were mainly enriched in immune related pathways. Nine DEGs were selected for validation by quantitative real time PCR (qRT-PCR) and the expression profiles of these DEGs kept a well consistent with the high-throughput data, which confirmed reliability of transcriptome results. Additionally, 10 DEGs were screened to detect the changes of expression level in different groups. All these results indicated that Zn exposure could damage the intestinal barrier, provoke oxidative stress, reduce the immune function, increase the susceptibility to bacterial infections of L. vannamei and cause inflammation, ultimately result in cell apoptosis. Our study provides more perspective on the stress response of crustacean under Zn exposure.

Acute Ammonia Causes Pathogenic Dysbiosis of Shrimp Gut Biofilms.

Acute ammonia exposure has detrimental effects on shrimp, but the underlying mechanisms remain to be fully explored. In the present study, we investigated the impact of acute ammonia exposure on the gut microbiota of the white shrimp Litopenaeus vannamei and its association with shrimp mortality. Exposure to a lethal concentration of ammonia for 48 h resulted in increased mortality in L. vannamei, with severe damage to the hepatopancreas. Ammonia exposure led to a significant decrease in gut microbial diversity, along with the loss of beneficial bacterial taxa and the proliferation of pathogenic Vibrio strains. A phenotypic analysis revealed a transition from the dominance of aerobic to facultative anaerobic strains due to ammonia exposure. A functional analysis revealed that ammonia exposure led to an enrichment of genes related to biofilm formation, host colonization, and virulence pathogenicity. A species-level analysis and experiments suggest the key role of a Vibrio harveyi strain in causing shrimp disease and specificity under distinct environments. These findings provide new information on the mechanism of shrimp disease under environmental changes.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

Single-cell RNA-seq revealed heterogeneous responses and functional differentiation of hemocytes against white spot syndrome virus infection in Litopenaeus vannamei.

Shrimp hemocytes are the vital immune cells participating in innate immune response to defend against viruses. However, the lack of specific molecular markers for shrimp hemocyte hindered the insightful understanding of their functional clusters and differential roles in combating microbial infections. In this study, we used single-cell RNA sequencing to map the transcriptomic landscape of hemocytes from the white spot syndrome virus (WSSV)-infected Litopenaeus vannamei and conjointly analyzed with our previous published single-cell RNA sequencing technology data from the healthy hemocytes. A total of 16 transcriptionally distinct cell clusters were identified, which occupied different proportions in healthy and WSSV-infected hemocytes and exerted differential roles in antiviral immune response. Following mapping of the sequencing data to the WSSV genome, we found that all types of hemocytes could be invaded by WSSV virions, especially the cluster 8, which showed the highest transcriptional levels of WSSV genes and exhibited a cell type-specific antiviral response to the viral infection. Further evaluation of the cell clusters revealed the delicate dynamic balance between hemocyte immune response and viral infestation. Unsupervised pseudo-time analysis of hemocytes showed that the hemocytes in immune-resting state could be significantly activated upon WSSV infection and then functionally differentiated to different hemocyte subsets. Collectively, our results revealed the differential responses of shrimp hemocytes and the process of immune-functional differentiation post-WSSV infection, providing essential resource for the systematic insight into the synergistic immune response mechanism against viral infection among hemocyte subtypes. IMPORTANCE: Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.

Microplastics weaken the exoskeletal mechanical properties of Pacific whiteleg shrimp Litopenaeus vannamei.

The ubiquitous presence of microplastics (MPs) in aquatic environments poses a significant threat to crustaceans. Although exoskeleton quality is critical for crustacean survival, the impact of MPs on crustacean exoskeletons remains elusive. Our study represents a pioneering effort to characterize the effects of MPs exposure on crustacean exoskeletons. In this study, the mechanical properties of whiteleg shrimp Litopenaeus vannamei exoskeletons were analyzed after exposure to environmentally realistic levels of MPs. Nanoindentation data demonstrated that MPs exposure significantly increased the hardness and modulus of both the carapace and abdominal segments of L. vannamei. Moreover, fractures and embedded MPs were detected on the exoskeleton surface using SEM-EDS analysis. Further analysis demonstrated that the degree of chitin acetylation (DA) in the shrimp exoskeleton, as indicated by FTIR peaks, was reduced by MPs exposure. In addition, exposure to MPs significantly inhibited the muscle Ca2+-ATPase activity and hemolymph calcium levels. Transcriptome and metabolome analyses revealed that the expression levels of genes encoding key enzymes and metabolites in the chitin biosynthetic pathway were significantly affected by MPs exposure. In conclusion, MPs at environmentally relevant concentrations may affect the exoskeletal mechanical properties of L. vannamei through a comprehensive mechanism involving the disruption of the crystalline structure of chitin, assimilation into the exoskeleton, and dysregulation of exoskeleton biosynthesis-related pathways.

Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.

Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes.

BACKGROUND: Acute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria. RESULTS: A subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a 'minimal' type I-F CRISPR mechanism. CONCLUSIONS: This study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

Transcriptome analysis of reproductive tract tissues of male river prawn Macrobrachium americanum.

BACKGROUND: The river prawn, Macrobrachium americanum (M. americanum), is one of the largest prawns of the genus in Latin America and is an amphidromous species distributed along the Pacific coast of America. This prawn has commercial value due to its size and taste, making it a good option for aquaculture production. Its culture has been attempted in ponds and concrete tanks, but no successful technique can still support commercial production. Understanding the mechanisms that regulate reproduction at the molecular level is very important. This knowledge can provide tools for manipulating transcripts, which could increase the number or size of animals in the culture. Our understanding of the mechanism that regulates the reproduction of M. americanum at the molecular level is limited. AIM: Perform and analyze the transcriptome assembly of the testes, vas deferens, and terminal ampulla of M. americanum. to provide new molecular information about its reproduction. METHODS AND RESULTS: The cDNA library was constructed and sequenced for each tissue to identify novel transcripts. A combined transcriptome with the three tissues was assembled using Trinity software. Unigenes were annotated using BLASTx and BLAST2GO. The transcriptome assembly generated 1,059,447 unigenes, of which 7222 genes had significant hits (e-value < 1 × 10-5) when compared against the Swiss-Prot database. Around 75 genes were related to sex determination, testis development, spermatogenesis, spermiogenesis, fertilization, maturation of testicular cells, neuropeptides, hormones, hormone receptors, and/or embryogenesis. CONCLUSIONS: These results provide new molecular information about M. americanum reproduction, representing a reference point for further genetic studies of this species.

Mechanisms of ammonotelism, epithelium damage, cellular apoptosis, and proliferation in gill of Litopenaeus vannamei under NH4Cl exposure.

Excessive ammonia-N in coastal environment and aquaculture threatens the health of marine organisms. To explore the mechanism of gill damage induced by ammonia-N, transcriptome of Litopenaeus vannamei 's gill was carried out under 20 mg/L NH4Cl for 0, 6, and 48 h. K-means clustering analysis suggested that ammonia excretion and metabolism-related genes were elevated. GO and KEGG enrichment analysis suggested that glycosyltransferase activity and amino acid metabolism were affected by ammonia. Moreover, histological observation via three staining methods gave clues on the changes of gill after ammonia-N exposure. Increased mucus, hemocyte infiltration, and lifting of the lamellar epithelium suggested that gill epithelium was suffering damage under ammonia-N stress. Meanwhile, the composition of extracellular matrix (ECM) in connective tissue changed. Based on the findings of transcriptomic and histological analysis, we further investigated the molecular mechanism of gill damage under multiple concentrations of NH4Cl (0, 2, 10, 20 mg/L) for multiple timepoints (0, 3, 6, 12, 24, 48, 72 h). First, ammonia excretion was elevated via ion channel, transporter, and exocytosis pathways, but hemolymph ammonia still kept at a high level under 20 mg/L NH4Cl exposure. Second, we focused on glycosaminoglycan metabolism which was related to the dynamics of ECM. It turned out that the degradation and biosynthesis of chondroitin sulfate (CS) were elevated, suggesting that the structure of CS might be destructed under ammonia-N stress and CS played an important role in maintaining gill structure. It was enlightening that the destructions occurred in extracellular regions were vital to gill damage. Third, ammonia-N stress induced a series of cellular responses including enhanced apoptosis, active inflammation, and inhibited proliferation which were closely linked and jointly led to the impairment of gill. Our results provided some insights into the physiological changes induced by ammonia-N and enriched the understandings of gill damage under environmental stress.

Integrated physiological, transcriptome, and metabolome analyses of the hepatopancreas of Litopenaeus vannamei under cold stress.

Temperature is a limiting factor in the growth of aquatic organisms and can directly affect many chemical and biological processes, including metabolic enzyme activity, aerobic respiration, and signal transduction. In this study, physiological, transcriptomic, and metabolomic analyses were performed to characterize the response of Litopenaeus vannamei to cold stress. We subjected L. vannamei to gradually decreasing temperatures (24 °C, 20 °C, 18 °C, 14 °C, and 12 °C) and studied the changes in the hepatopancreas. The results showed that extreme cold stress (12 °C) caused structural damage to the hepatopancreas of L. vannamei. However, shrimp exhibited response mechanisms to enhance cold tolerance, through regulating changes in key genes and metabolites in amino acid, lipid metabolism, and carbohydrate metabolism, including (a) increased level of methylation in cells to enhance cold tolerance; (b) increased content of critical amino acids, such as proline, alanine, glutamic acid and taurine, to ameliorate energy metabolism, protect cells from cold-induced osmotic imbalance, and promote ion transport and DNA repair; (c) accumulation of unsaturated fatty acids to improve cell membrane fluidity; and (d) regulation of the metabolic pattern shift to rely on anaerobic metabolism with a gradual decrease in aerobic metabolism and enhance glycolysis to produce enough ATP to maintain energy metabolic balance. When the temperature dropped further, cold stress impaired antioxidant and immune defense responses in shrimp. This study provides an integrated analysis of the physiology, transcriptome, and metabolome of L. vannamei in response to cold stress.

Investigating the transcriptomic variances in two phases Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei.

This study explores the transcriptomic differences in two distinct phases of Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei, a crucial aspect in shrimp health management. We employed high-throughput sequencing to categorize samples into two phases, 'Phase A' and 'Phase B', defined by the differential expression of PTP2 and TPS1 genes. Our analysis identified 2057 genes, with 78 exhibiting significant variances, including 62 upregulated and 16 downregulated genes. Enrichment analyses via GO and KEGG pathways highlighted these genes' roles in cellular metabolism, signal transduction, and immune responses. Notably, genes like IQGAP2, Rhob, Pim1, and PCM1 emerged as potentially crucial in EHP's infection process and lifecycle. We hypothesize that these genes may influence trehalose metabolism and glucose provision, impacting the biological activities within EHP during different phases. Interestingly, a lower transcript count in 'Phase A' EHP suggests a reduction in biological activities, likely preparing for host cell invasion. This research provides a foundational understanding of EHP infection mechanisms, offering vital insights for future studies and therapeutic interventions.

Temperature-Induced Sex Differentiation in River Prawn (Macrobrachium nipponense): Mechanisms and Effects.

Macrobrachium nipponense is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually results in higher yield. To investigate the impact of temperature on sexual differentiation in M. nipponense, two temperature treatments (26 °C and 31 °C) were conducted. The results showed that compared to the 31 °C treatment (3.20 ± 0.12), the 26 °C treatment displayed a lower female/male ratio (2.20 ± 0.11), which implied that a lower temperature could induce masculinization in M. nipponense. The temperature-sensitive sex differentiation phase was 25-35 days post hatching (DPH) at 26 °C while 15-20 DPH at 31 °C. Transcriptome and qPCR analysis revealed that a lower temperature up-regulated the expression of genes related to androgen secretion, and down-regulated the expressions of genes related to oogonia differentiation. Thirty-one temperature-regulated sex-differentiation genes were identified and the molecular mechanism of temperature-regulated sex differentiation was suggested. The finding of this study indicates that temperature regulation can be proposed as an innovative strategy for improving the culture yield of M. nipponense.

Smart nano generation of transgenic algae expressing white spot syndrome virus in shrimps for inner ear-oral infection treatments using the spotted hyena optimizer (SHO)-Long short-term memory algorithm.

Nanotechnology offers a promising avenue to amplify the effectiveness and precision of using transgenic algae in managing WSSV in shrimp by possibly crafting nano-carriers for targeted therapeutic agent delivery or modifying algae cells at a molecular level. Leveraging the capabilities of nano-scale interventions, this study could explore innovative means to manipulate cellular processes, control biological interactions, and enhance treatment efficacy while minimizing undesirable impacts in aquatic environments. The White Spot Syndrome Virus (WSSV) is a double-stranded DNA virus with a tail and rod form that belongs to theNimaviridaefamily. There is no workable way to manage this illness at the moment. This research proposes a new model based on the Long Short-Term Memory (LSTM) and Spotted Hyena Optimizer (SHO) method to control the inner ear-oral infection, utilizing transgenic algae (Chlamydomonas reinhardtii). It is pretty tricky to modify the weight matrix in LSTM. The output will be more accurate if the weight of the neurons is exact. Histological examinations and nested polymerase chain reaction (PCR) testing were performed on the challenged shrimp every 4 h to assess the degree of white spot disease. The SHO-LSTM has shown the highest accuracy and Roc value (98.12% and 0.93, respectively) and the lowest error values (MSE = 0.182 and MAE = 0.48). The hybrid optimized model improves the overall inner ear-oral linked neurological diseases detection ratio. Additionally, with the slightest technical complexity, it effectively controls the forecast factors required to anticipate the ENT. Algal cells were found to be particularly well-suited for inner ear-oral infections, and shrimps fed a transgenic line had the best survival ratio in WSSV infection studies, with 87% of the shrimp surviving. This shows that using this line would effectively stop the spread of WSSV in shrimp populations.

A native strain of Bacillus subtilis increases lipid accumulation and modulates expression of genes related to digestion and amino acid metabolism in Litopenaeus vannamei.

In the field of shrimp aquaculture, the utilization of probiotics represents a promising avenue, due to the well-documented benefits conferred by these microorganisms. In the current study, a Bacillus subtilis strain, referred to as strain E, was isolated from the gastrointestinal tract of the shrimp Litopenaeus vannamei and subsequently identified via molecular methods and phylogeny. The probiotic potential of strain E was characterized, and its application as a feed shrimp additive was evaluated in a 45-day experiment. Several parameters were assessed, including zootechnical performance, muscle tissue proximate composition, hepatopancreas lipid concentration, and the expression of genes associated with digestion, amino acid metabolism, and antioxidant defense mechanisms in various shrimp tissues. Although no significant impact on zootechnical performance was observed, supplementation with strain E led to an increase in lipid concentration within both muscle and hepatopancreas tissues. Furthermore, a marked decrease in the expression of genes linked to digestion and amino acid metabolism was noted. These findings suggest that the addition of the B. subtilis strain E to shrimp feed may enhance nutrient absorption and modulate the expression of genes related to digestion and amino acid metabolism.

Effectiveness of dietary heat-killed Bacillus subtilis harboring plasmid containing 60 copies of CpG-ODN 1668 against Vibrio harveyi in Penaeus vannamei.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

Comparative transcriptome analysis of hepatopancreas reveals the potential mechanism of shrimp resistant to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus carrying a pathogenic plasmid (VPAHPND) is one of the main causative agents of acute hepatopancreatic necrosis disease (AHPND) in shrimp aquaculture. Knowledge about the mechanism of shrimp resistant to VPAHPND is very helpful for developing efficient strategy for breeding AHPND resistant shrimp. In order to learn the mechanism of shrimp resistant to AHPND, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp from different families with different resistance to VPAHPND. Through comparative analysis on the hepatopancreas of shrimp from VPAHPND resistant family and susceptible family, we found that differentially expressed genes (DEGs) were mainly involved in immune and metabolic processes. Most of the immune-related genes among DEGs were highly expressed in the hepatopancreas of shrimp from resistant family, involved in recognition of pathogen-associated molecular patterns, phagocytosis and elimination of pathogens, maintenance of reactive oxygen species homeostasis and other immune processes etc. However, most metabolic-related genes were highly expressed in the hepatopancreas of shrimp from susceptible family, involved in metabolism of lipid, vitamin, cofactors, glucose, carbohydrate and serine. Interestingly, when we analyzed the expression of above DEGs in the shrimp after VPAHPND infection, we found that the most of identified immune-related genes remained at high expression levels in the hepatopancreas of shrimp from the VPAHPND resistant family, and most of the identified metabolic-related genes were still at high expression levels in the hepatopancreas of shrimp from the VPAHPND susceptible family. The data suggested that the differential expression of these immune-related and metabolic-related genes in hepatopancreas might contribute to the resistance variations of shrimp to VPAHPND. These results provided valuable information for understanding the resistant mechanism of shrimp to VPAHPND.

Shrimp SIRT4 promotes white spot syndrome virus replication.

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.