Integrated analysis of intestinal microbiota and transcriptome reveals that a coordinated interaction of the endocrine, immune system and gut microbiota response to heat stress in Litopenaeus vannamei.

Due to the ongoing global warming, the risk of heatwaves in the oceans is continuously increasing while our understanding of the physiological response of Litopenaeus vannamei under extreme temperature conditions remains limited. Therefore, this study aimed to evaluate the physiological responses of L. vannamei under heat stress. Our results indicated that as temperature rose, the structure of intestinal and hepatopancreatic tissues was damaged sequentially. Activity of immune-related enzymes (acid phosphatase/alkaline phosphatase) initially increased before decreased, while antioxidant enzymes (superoxide dismutase and glutathione-S transferase) activity and malondialdehyde content increased with rising temperature. In addition, the total antioxidant capacity decreased with rising temperature. With the rising temperature, there was a significant increase in the expression of caspase-3, heat shock protein 70, lipopolysaccharide-induced tumor necrosis factor-α, transcriptional enhanced associate domain and yorkie in intestinal and hepatopancreatic tissues. Following heat stress, the number of potentially beneficial bacteria (Rhodobacteraceae and Gemmonbacter) increased which maintain balance and promote vitamin synthesis. Intestinal transcriptome analysis revealed 852 differentially expressed genes in the heat stress group compared with the control group. KEGG functional annotation results showed that the endocrine system was the most abundant in Organismal systems followed by the immune system. These results indicated that heat stress leads to tissue damage in shrimp, however the shrimp may respond to stress through a coordinated interaction strategy of the endocrine system, immune system and gut microbiota. This study revealed the response mechanism of L. vannamei to acute heat stress and potentially provided a theoretical foundation for future research on shrimp environmental adaptations.

Mechanisms of neurocentral-eyestalk-intestinal immunotoxicity in whiteleg shrimp Litopenaeus vannamei under ammonia nitrogen exposure.

Ammonia-N, as the most toxic nitrogenous waste, has high toxicity to marine animals. However, the interplay between ammonia-induced neuroendocrine toxicity and intestinal immune homeostasis has been largely overlooked. Here, a significant concordance of metabolome and transcriptome-based "cholinergic synapse" supports that plasma metabolites acetylcholine (ACh) plays an important role during NH4Cl exposure. After blocking the ACh signal transduction, the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the cerebral ganglia increased, while the release of NPF in the thoracic ganglia and NE in the abdominal ganglia, and crustacean hyperglycemic hormone (CHH) and neuropeptide F (NPF) in the eyestalk decreased, finally the intestinal immunity was enhanced. After bilateral eyestalk ablation, the neuroendocrine system of shrimp was disturbed, more neuroendocrine factors, such as corticotropin releasing hormone (CRH), adrenocorticotropic-hormone (ACTH), ACh, DA, 5-HT, and norepinephrine (NE) were released into the plasma, and further decreased intestinal immunity. Subsequently, these neuroendocrine factors reach the intestine through endocrine or neural pathways and bind to their receptors to affect downstream signaling pathway factors to regulate intestinal immune homeostasis. Combined with different doses of ammonia-N exposure experiment, these findings suggest that NH4Cl may exert intestinal toxicity on shrimp by disrupting the cerebral ganglion-eyestalk axis and the cerebral ganglion-thoracic ganglion-abdominal ganglion axis, thereby damaging intestinal barrier function and inducing inflammatory response.

Safety evaluation and effects of dietary phlorotannins on the growth, health, and intestinal microbiota of Litopenaeus vannamei.

Phlorotannins are phenolic compounds with diverse biological activities, yet their efficacy in aquatic animals currently remains unclear. This investigation scrutinized the influence of phlorotannins on the growth, immunity, antioxidant capacity, and intestinal microbiota in Litopenaeus vannamei, concurrently evaluating the potential adverse effects of phlorotannins on L. vannamei. A base diet without phlorotannins supplementation was used as a control, and 4 groups of diets with different concentrations (0, 0.5, 1.0, 2.0 g kg-1) of phlorotannins were formulated and fed to juvenile shrimp (0.25 ± 0.01 g) for 60 days followed by a 24-hour challenge with Vibrio parahaemolyticus with triplicate in each group. Compared with the control, dietary 2.0 g kg-1 phlorotannins significantly improved the growth of the shrimp. The activities of enzymes related to cellular immunity, humoral immunity, and antioxidants, along with a notable upregulation in the expression of related genes, significantly increased. After V. parahaemolyticus challenge, the cumulative survival rates of the shrimp demonstrated a positive correlation with elevated concentrations of phlorotannins. In addition, the abundance of Bacteroidetes and functional genes associated with metabolism increased in phlorotannins supplementation groups. Phlorotannins did not elicit any detrimental effects on the biological macromolecules or histological integrity of the hepatopancreas or intestines. Simultaneously, it led to a significant reduction in malondialdehyde content. All results indicated that phlorotannins at concentrations of 2.0 g kg-1 can be used as safe feed additives to promote the growth, stimulate the immune response, improve the antioxidant capacity and intestinal health of L. vannamei, and an protect shrimp from damage caused by oxidative stress.

Effectiveness of a formulation based on Ocimum gratissimum essential oil and cashew gum as inhibitors of quality loss and melanosis in shrimp.

In this study, two nanoemulsions were formulated with essential oil (EO) of Ocimum gratissimum with (EON) or without (EOE) cashew gum (CG). Subsequently, inhibition of melanosis and preservation of the quality of shrimp stored for 16 days at 4 ± 0.5 °C were evaluated. A computational approach was performed to predict the system interactions. Dynamic light scattering (DLS) and atomic force microscopy (AFM) were used for nanoparticle analysis. Gas chromatography and flame ionization detector (GC-FID) determined the chemical composition of the EO constituents. Shrimps were evaluated according to melanosis's appearance, psychrotrophic bacteria's count, pH, total volatile basic nitrogen, and thiobarbituric acid reactive substances. EON exhibited a particle size three times smaller than EOE. The shrimp treated with EON showed a more pronounced sensory inhibition of melanosis, which was considered mild by the 16th day. Meanwhile, in the other groups, melanosis was moderate (EOE) or severe (untreated group). Both EON and EOE treatments exhibited inhibition of psychrotrophic bacteria and demonstrated the potential to prevent lipid oxidation, thus extending the shelf life compared to untreated fresh shrimp. EON with cashew gum, seems more promising due to its physicochemical characteristics and superior sensory performance in inhibiting melanosis during shrimp preservation.

Bactericidal efficacy of plasma-activated water against Vibrio parahaemolyticus on Litopenaeus vannamei.

In this study, the antimicrobial mechanism of plasma-activated water (PAW) against Vibrio parahaemolyticus and the effectiveness of PAW in artificially contaminated Litopenaeus vannamei were investigated. The results demonstrated a significant reduction (p < 0.05) in viable counts of V. parahaemolyticus with increasing plasma discharge time (5, 10, 20, and 30 min) and PAW immersion time (3, 5, 10, 20, and 30 s). Specifically, the count of V. parahaemolyticus decreased by 2.1, 2.7, 3.3, and 4.4 log CFU/mL after exposed to PAW 5, PAW 10, PAW 20, and PAW 30 for 30 s, respectively. Significant cell surface wrinkling, accompanied by notable nucleic acid and protein leakage were observed after treatment with PAW. The permeability of the inner and outer cell membranes was significantly increased (p < 0.05), along with an increase in electrical conductivity (p < 0.05). The reactive oxygen species (ROS) within V. parahaemolyticus cells were significantly increased (p < 0.05), while superoxide dismutase (SOD) activity, and the relative expression of the ompW, emrD, and luxS genes were significantly decreased (p < 0.05). A reduction number of 1.3, 1.8, 2.1, and 2.2 log CFU/g of V. parahaemolyticus in artificially contaminated L. vannamei was obtained with PAW for 5 min. The study elucidated that PAW could destroy cell membranes, leading to cell death. The findings would strengthen strategies for V. parahaemolyticus control and provide a potential application of PAW for preserving aquatic products.

PvGeneExpDB: An integrative gene expression database for in-depth understanding on the Pacific white shrimp (Litopenaeus vannamei).

The Pacific white shrimp (Litopenaeus vannamei) is a high-valued economic farming species. With the development of high-throughput sequencing technology, cumulative large-scale transcriptomic studies have been revealing molecular landscape of various biological conditions including genetic selection, breeding, evolution, disease landscape, etc. However, no single experiment or databases allow thorough investigations of transcriptomic dynamics for these progressions. Meanwhile, the available datasets are often scattered and lack management. Here, we have established PvGeneExpDB, the first gene expression database for L. vannamei (www.bio-marine-scau.com/pv_ex/), which encompasses gene expression profiles, differential expression, and co-expression analyses under various biological conditions. Based on the analyses of 7 datasets, which include 53 samples with accurate and detailed records, PvGeneExpDB identifies 20,599 novel transcripts, shows expression profiles of a total of 20,817 genes, and implements Gene Ontology (GO) reconstruction of 76.7 % of these genes. Besides, 26 co-expressed groups were first identified by large-scale, cross-sample Weighted Gene Co-expression Network Analysis (WGCNA). By integrating the gene expression data in the database, our goal is to deepen the biological understanding of L. vannamei.

Shrimp Cultured in Low-Salt Saline-Alkali Water has a Better Amino Acid Nutrition and Umami─Comparison of Flavors between Saline-Alkali Water- and Seawater-Cultured Litopenaeus vannamei.

The advantages of Litopenaeus vannamei farming in saline-alkali water have gradually attracted attention, but few studies have focused on its flavor. In this study, L. vannamei cultured in saline-alkali water (SS) and ordinary seawater (CS) (both have a breeding time of 120 days) were selected for analysis (n = 5). High-performance liquid chromatography (HPLC) was used to measure free amino acids and flavoring nucleotides in the muscles of L. vannamei, while the taste activity value (TAV) and equivalent umami concentration (EUC) were used to analyze the degree of umami. The total essential amino acids (TEAA) in the SS group were 238.41 ± 46.24 mg/mL, significantly higher than that in the CS group (107.06 ± 15.65 mg/mL). The total amount of flavor nucleotides in the SS group was 2948.51 ± 233.66 µg/mL, significantly higher than those in the CS group (2530.37 ± 114.67 µg/mL). The content and TAV of some free amino acids (Glu, Cys-s) in the SS group were significantly higher. Meanwhile, due to the significant increase in IMP, the synergistic effect of free amino acids and flavored nucleotides leads to higher EUC. The significant separation of SS and CS samples in principal component analysis (PCA) indicates a significant difference between the two groups. Our results indicate that shrimp cultured in saline-alkali water has a stronger umami. This study enriches the basic theories related to the flavor of salt-alkali water crustaceans.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection.

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

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.

Optimized swarming motility assay to identify anti-virulence products against Vibrio parahaemolyticus, a pathogen of farmed shrimp.

Swarming motility is a type of movement used by pathogenic flagellated bacteria as virulence factor to colonize surfaces and cause damage to the host. Vibrio parahaemolyticus is a pathogenic flagellated bacterium that increases its virulence by switching from swimmer to swarming cells. The hosts of pathogenic V. parahaemolyticus include farmed shrimp. Therefore, methods to detect and quantify this movement are important to control shrimp diseases caused by pathogenic V. parahaemolyticus strains. We developed an optimized swarming motility assay by identifying the most optimal type of agar, and drying time of the culture medium, agar concentration and volume of the bacterial culture to achieve the fastest swarming motility during the migration of V. parahaemolyticus on Petri dishes during a 24-hour incubation period. The method includes data analysis that could be used as a tool to identify potential anti-virulence products by comparing the slopes of the linearized diameters of the swarming halos of bacteria treated with the products, as they migrate on Petri dishes over a 24-hour incubation period. Here we report:•A simple method for detection and quantification of swarming motility halos of V. parahaemolyticus bacteria.•A method that could be used as a tool to identify potential anti-virulence products.

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.

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.

Exposure to polystyrene nanoplastics induces apoptosis, autophagy, histopathological damage, and intestinal microbiota dysbiosis of the Pacific whiteleg shrimp (Litopenaeus vannamei).

Nanoplastics (NPs) are widely distributed environmental pollutants that can disrupt intestinal immunity of crustaceans. In this study, the effects of NPs on gut immune enzyme activities, cell morphology, apoptosis, and microbiota diversity of Litopenaeus vannamei were investigated. L. vannamei was exposed to five concentrations of NPs (0, 0.1, 1, 5, and 10 mg/L) for 28 days. The results showed that higher concentrations of NPs damaged the intestinal villi, promoted formation of autophagosomes, increased intestinal non-specific immunoenzyme activities, and significantly increased apoptosis at 10 mg/L. In response to exposure to NPs, the expression levels of ATG3, ATG4, ATG12, Caspase-3, p53, and TNF initially increased and then decreased. In addition, the concentration of NPs was negatively correlated to the expression levels of the genes of interest and intestinal enzyme activities, suggesting that exposure to NPs inhibited apoptosis and immune function. The five dominant phyla of the gut microbiota (Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria, and Actinomycetes) were similar among groups exposed to different concentrations of NPs, but the abundances tended to differ. Notably, exposure to NPs increased the abundance of pathogenic bacteria. These results confirm that exposure to NPs negatively impacted intestinal immune function of L. vannamei. These findings provide useful references for efficient breeding of L. vannamei.

The interactions between CpG oligodeoxynucleotides and Toll-like receptors in Pacific white shrimp Litopenaeus vannamei.

CpG oligodeoxynucleotides (ODNs), as a novel type of adjuvant with immunomodulatory effects, are recognized by Toll-like receptors (TLRs) in Litopenaeus vannamei. In the present study, eleven LvTLRs-pCMV recombinants (rLvTLRs) were constructed to investigate the relationships between various CpG ODNs and different LvTLRs in human embryonic kidney 293T (HEK293T) cells, which was further confirmed by bio-layer interferometry (BLI) technique. The results of dual luciferase reporter assay showed that every LvTLR could activate multiple downstream genes, mainly including NF-κB, CREB, ISRE, IL-6-promoter, TNF-α-promoter and Myc, thereby inducing main signaling pathways in shrimps. Most CpG ODNs possessed affinities to more than one LvTLR, while each LvTLR could recognize multiple CpG ODNs, and the widely recognized ligands within CpG ODNs are A-class and B-class. Moreover, BLI analysis showed that CpG 2216, Cpg 2006, CpG 2143 and CpG 21425 exhibited dose-dependent affinity to the expressed TLR protein, which were consistent with the results in HEK293T cells. It suggested that the interactions of CpG ODNs with LvTLRs were indispensable for the immune regulation triggered by CpG ODNs, and these findings would lay foundations for studying the activations of LvTLRs to immune signaling pathways and shedding lights on the immune functions and mechanisms of CpG ODNs.

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.

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.

Structure and function analyses of the SRC gene in Pacific white shrimp Litopenaeus vannamei.

SRC gene encodes scavenger receptor class C, a member of the scavenger receptor family, and has only been identified and investigated in invertebrates. Our previous studies have revealed that SRC is a novel candidate gene associated with body weight in Pacific white shrimp (Litopenaeus vannamei). In order to comprehend the underlying mechanism by which LvSRC affects shrimp growth, we analyzed the structure, phylogeny, expression profiles and RNA interference (RNAi) of this gene in L. vannamei. We found that LvSRC contains two CCP domains and one MAM domain, with the highest expression level in the heart and relatively low expression level in other tissues. Notably, LvSRC exhibited significantly higher expression levels in the fast-growing group among groups with different growth rates, suggesting its potential involvement as a gene contributing to the growth of L. vannamei. RNAi of LvSRC inhibited body length and body weight gain compared to the control groups. Moreover, through RNA-seq analysis, we identified 598 differentially expressed genes (DEGs), including genes associated with growth, immunity, protein processing and modification, signal transduction, lipid synthesis and metabolism. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significant changes in the signaling pathways related to growth, lipid metabolism and immune response, suggesting that LvSRC exhibits the potential to participate in diverse physiological processes and immune regulation. These findings deepen our understanding of the structure and function of the SRC in shrimp and lay the foundation for further research into the regulatory mechanism of LvSRC. Additionally, they provide potential applications in shrimp genetics and breeding.

Transcriptomics reveals different response mechanisms of Litopenaeus vannamei hemocytes to injection of Vibrio parahaemolyticus and WSSV.

As the most important cultural crustacean species worldwide, studies about Pacific white shrimp (Litopenaeus vannamei) have received more attention. It has been well-documented that various pathogens could infect L. vannamei, resulting in huge economic losses. The studies about the responding mechanism of L. vannamei to sole pathogens such as Vibrio parahaemolyticus and white spot virus (WSSV) have been extensively reported, while the studies about the differently responding mechanisms remain unclear. In the present study, we identified the differently expressed genes (DEGs) of L. vannamei hemocytes post V. parahaemolyticus and WSSV infection with RNA-seq technology and compared the DEGs between the two groups. The results showed 2672 DEGs post the V. parahaemolyticus challenge (1079 up-regulated and 1593 down-regulated genes), while 1146 DEGs post the WSSV challenge (1067 up-regulated and 513 down-regulated genes). In addition, we screened the genes that simultaneously respond to WSSV and V. parahaemolyticus (434), solely respond to WSSV (1146), and V. parahaemolyticus challenge (2238), respectively. Six DEGs involved in innate immunity were quantified to validate the RNA-seq results, and the results confirmed the high consistency of both methods. Furthermore, we found plenty of innate immunity-related genes that responded to V. parahaemolyticus and WSSV infection, including pattern recognition receptors (PRRs), the proPO activating system, antimicrobial peptides (AMPs), and other immunity-related proteins. The results revealed that they were differently expressed after different pathogen challenges, demonstrating the complex and specific recognition systems involved in defending against the invasion of different pathogens in the environment. The present study improved our understanding of the molecular response of hemocytes of L. vannamei to V. parahaemolyticus and WSSV stimulation.

Investigating the Impact of Varied Dietary Protein Levels on Litopenaeus vannamei: An Exploration of the Intestinal Microbiota and Transcriptome Responses.

This study explored the effects of dietary protein levels on Litopenaeus vannamei with its intestinal microbiota and transcriptome responses. Previous studies on the effects of dietary protein levels on L. vannamei have focused on growth performance, antioxidant indices, and digestive enzyme activity, but few studies have been conducted at the microbiological and molecular levels. In this study, five isolipid experimental diets with protein levels of 32% (P32), 36% (P36), 40% (P40), 44% (P44), and 48% (P48) were used in an L. vannamei (0.63 ± 0.02 g) feeding trial for 56 days. At the end of the feeding trial, the growth performance, immunity, intestinal health, and transcriptional responses of L. vannamei were determined. This study demonstrated that higher protein levels (P44) led to superior weight gain and growth rates for L. vannamei, with lower feed conversion ratios (FCR) observed in the P48 and P44 groups compared to the P32 and P36 groups (p ≤ 0.05). The P44 and P48 groups also showed a notably higher protein efficiency ratio (PER) compared to others (p ≤ 0.05), and there was no significant difference between them. Upon Vibrio parahaemolyticus infection, the P48 group exhibited a significantly lower survival rate (SR) within 48 h, while during 72 h of white spot syndrome virus (WSSV) infection, the P44 group had a notably higher survival rate than the P32 group (p ≤ 0.05). Digestive enzyme activity and antioxidant levels in L. vannamei initially increased and then decreased as protein levels increased, usually peaking in the P40 or P44 groups. Lower dietary protein levels significantly reduced the relative abundance of beneficial bacteria and increased the relative abundance of pathogenic bacteria in the intestines of L. vannamei. Transcriptome sequencing analysis revealed that most differentially expressed genes (DEGs) were up-regulated and then down-regulated as dietary protein levels increased. Furthermore, KEGG pathway enrichment analysis indicated that several immune and metabolic pathways, including metabolic pathways, glutathione metabolism, cytochrome P450, and lysosome and pancreatic secretion, were significantly enriched. In summary, the optimal feed protein level for L. vannamei shrimp was 40-44%. Inappropriate feed protein levels reduced antioxidant levels and digestive enzyme activity and promoted pathogen settlement, deceasing factors in various metabolic pathways that respond to microorganisms through transcriptional regulation. This could lead to stunted growth in L. vannamei and compromise their immune function.

Profile of the gut microbiota of Pacific white shrimp under industrial indoor farming system.

The gut microbial communities interact with the host immunity and physiological functions. In this study, we investigated the bacterial composition in Litopenaeus vannamei shrimp's gut and rearing water under different host (developmental stage: juvenile and adult; health status: healthy and diseased) and environmental factors (temperature 25 °C and 28 °C; and light intensity: low and high). The PCoA analysis showed that all water samples were clustered together in a quarter, whereas the gut samples spread among three quarters. In terms of functional bacteria, gut samples of adult shrimp, healthy adult shrimp, adult shrimp raised at 28 °C, and juvenile shrimp under high light intensity exhibited a higher abundance of Vibrionaceae compared to each other opposite group. Gut samples of juvenile shrimp, infected adult shrimp, juvenile shrimp with low light intensity, and adult shrimp with a water temperature of 25 °C showed a higher abundance of Pseudoaltromonadaceae bacteria compared to each other opposite group. Gut samples of juvenile shrimp, healthy adult shrimp, adult shrimp raised at a water temperature of 28 °C, and juvenile shrimp with high light intensity showed the higher abundance of Firmicutes/Bacteroidota ratio compared to each other opposite group. Our results showed that L. vannamei juveniles are more sensitive to bacterial infections; besides, water temperature of 28 °C and high light intensity groups were both important conditions improving the shrimp gut bacterial composition under industrial indoor farming systems. KEY POINTS: • Bacteria diversity was higher among shrimp intestinal microbiota compared to the rearing water. • Shrimp juveniles are more sensitive to bacterial infection compared to adults. • Water temperature of 28 °C and high light intensity are recommended conditions for white shrimp aquaculture.