Impact of dietary administration of Arthrospira platensis free-lipid biomass on growth performance, body composition, redox status, immune responses, and some related genes of pacific whiteleg shrimp, Litopenaeus vannamei.

The current study aimed to assess the influence of dietary inclusion of cyanobacterium Arthrospira platensis NIOF17/003 as a dry material and as a free-lipid biomass (FL) on the growth performance, body composition, redox status, immune responses, and gene expression of whiteleg shrimp, Litopenaeus vannamei postlarvae. L. vannamei were fed five different supplemented diets; the first group was fed on an un-supplemented diet as a negative control group (C-N), the second group was fed on a commercial diet supplemented with 2% of A. platensis complete biomass as a positive control group (C-P20), whereas, the three remaining groups were fed on a commercial diet supplemented with graded amounts of FL at 1%, 2%, and 3% (FL10, FL20, and FL30, respectively). The obtained results indicated that the diet containing 1% FL significantly increased the growth performance, efficiency of consumed feed, and survival percentage of L. vannamei compared to both C-N and C-P20 groups. As for the carcass analysis, diets containing A. platensis or its FL at higher levels significantly increased the protein, lipid, and ash content compared to the C-N group. Moreover, the shrimp group fed on C-P20 and FL10 gave significantly stimulated higher digestive enzyme activities compared with C-N. The shrimp fed C-P20 or FL exhibited higher innate immune responses and promoted their redox status profile. Also, the shrimp fed a low FL levels significantly upregulated the expression of both the peroxiredoxin (Prx) and prophenoloxidase (PPO1) genes than those receiving C-N. The current results recommended that dietary supplementation with 1% FL is the most effective treatment in promoting the performance and immunity of whiteleg shrimp.

Characterization of DmToll and DmToll7 homologue in Litopenaeus vannamei based on structure analysis.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that recognize invading pathogens and activate downstream signaling pathways. The number of 10 Tolls is found in Litopenaeus vannamei but have not yet been identified as the corresponding Toll homologue of model animal. In this study, we predicted the three-dimensional (3D) structures of 10 LvTolls (LvToll1-10) with AlphaFold2 program. The per-residue local distance difference test (pLDDT) scores of LvTolls showed the predicted structure of LvTolls had high accuracy (pLDDT>70). By structural analysis, 3D structures of LvToll2 and LvToll3 had high similarity with Drosophila melanogaster Toll and Toll7, respectively. 3D structure of LvToll7 and LvToll10 were not similar to that of other LvTolls. Moreover, we also predicted that LvSpätzle4 had high structural similarity to DmSpätzle. There were 9 potential hydrogen bonds in LvToll2-LvSpätzle4 complex. Importantly, co-immunoprecipitation assay showed that LvToll2 could bind with LvSpätzle4. Collectively, this study provides new insight for researching invertebrate immunity by identifying the protein of model animal homologue.

Assessment of fish protein hydrolysate as a substitute for fish meal in white shrimp diets: Impact on growth, immune response, and resistance against Vibrio parahaemolyticus infection.

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo, four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin, Midline-fas, and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp.

Nucleic Acid Sensing by STING Induces an IFN-like Antiviral Response in a Marine Invertebrate.

The cytosolic detection of pathogen-derived nucleic acids has evolved as an essential strategy for host innate immune defense in mammals. One crucial component in this process is the stimulator of IFN genes (STING), which acts as a vital signaling adaptor, connecting the cytosolic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) to the downstream type I IFN signaling pathway. However, this process remains elusive in invertebrates. In this study, we present evidence demonstrating that STING, an ortholog found in a marine invertebrate (shrimp) called Litopenaeus vannamei, can directly detect DNA and initiate an IFN-like antiviral response. Unlike its homologs in other eukaryotic organisms, which exclusively function as sensors for cyclic dinucleotides, shrimp STING has the ability to bind to both double-stranded DNA and cyclic dinucleotides, including 2'3'-cGAMP. In vivo, shrimp STING can directly sense DNA nucleic acids from an infected virus, accelerate IFN regulatory factor dimerization and nuclear translocation, induce the expression of an IFN functional analog protein (Vago4), and finally establish an antiviral state. Taken together, our findings unveil a novel double-stranded DNA-STING-IKKε-IRF-Vago antiviral axis in an arthropod, providing valuable insights into the functional origins of DNA-sensing pathways in evolution.

Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis.

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight ß-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Expression of octopamine/tyramine receptors and immune regulation in Litopenaeus vannamei under acute and chronic thermal stress.

This study investigated the impact of hyperthermal (34 °C) and hypothermal (14 °C) stress on the expression of the octopamine/tyramine receptor (LvOA/TA-R) and immune parameters in Litopenaeus vannamei, which is a species critical to the aquaculture industry. Given the sensitivity of aquatic organisms to climate change, understanding the physiological and immune responses of L. vannamei to temperature variations is essential for developing strategies to mitigate adverse effects. This research focuses on the immune response and expression changes of LvOA/TA-R under acute (0.5, 1, and 2 h) and chronic (24, 72, and 168 h) thermal stress conditions. Our findings reveal that thermal stress induces changes in LvOA/TA-R expression and impacts immune responses. Immune parameters such as total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, lysozyme activity, clearance efficiency, and phagocytosis exhibited a general trend of significant decline under the stress conditions. LvOA/TA-R had a higher expression in haemocyte under hyperthermal stress. The study elucidated that thermal stress modifies the expression of the LvOA/TA-R and diminishes immune functionality in L. vannamei, underscoring the potential influence of climate change on industry.

A Negative Regulatory Feedback Loop within the JAK-STAT Pathway Mediated by the Protein Tyrosine Phosphatase DUSP14 in Shrimp.

The JAK-STAT pathway is a central communication node for various biological processes. Its activation is characterized by phosphorylation and nuclear translocation of the transcription factor STAT. The regulatory balance of JAK-STAT signaling is important for maintenance of immune homeostasis. Protein tyrosine phosphatases (PTPs) induce dephosphorylation of tyrosine residues in intracellular proteins and generally function as negative regulators in cell signaling. However, the roles of PTPs in JAK-STAT signaling, especially in invertebrates, remain largely unknown. Pacific white shrimp Penaeus vannamei is currently an important model for studying invertebrate immunity. This study identified a novel member of the dual-specificity phosphatase (DUSP) subclass of the PTP superfamily in P. vannamei, named PvDUSP14. By interacting with and dephosphorylating STAT, PvDUSP14 inhibits the excessive activation of the JAK-STAT pathway, and silencing of PvDUSP14 significantly enhances humoral and cellular immunity in shrimp. The promoter of PvDUSP14 contains a STAT-binding motif and can be directly activated by STAT, suggesting that PvDUSP14 is a regulatory target gene of the JAK-STAT pathway and mediates a negative feedback regulatory loop. This feedback loop plays a role in maintaining homeostasis of JAK-STAT signaling and is involved in antibacterial and antiviral immune responses in shrimp. Therefore, the current study revealed a novel inhibitory mechanism of JAK-STAT signaling, which is of significance for studying the regulatory mechanisms of immune homeostasis in invertebrates.

Dietary Bacillus velezensis T23 fermented products supplementation improves growth, hepatopancreas and intestine health of Litopenaeus vannamei.

This study aimed to elucidate the effects of dietary fermented products of Bacillus velezensis T23 on the growth, immune response and gut microbiota in Pacific white shrimp (Litopenaeus vannamei). Shrimp were fed with diets containing fermentation products of B. velezensis T23 at levels of (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 g/kg) for 4 weeks, to assess the influence on shrimp growth. The results showed that 0.3 and 0.4 g/kg T23 supplementation improved shrimp growth and feed utilization. Based on these results we selected these three diets (Control, 0.3T23 and 0.4T23) to assess the effect on immune response and gut microbiota of shrimp. Compared with the control, the 0.3T23 and 0.4T23 groups enhanced lipase and α-amylase activities in the gut significantly. Moreover, the 0.4T23 group decreased TAG and MDA levels in hepatopancreas, ALT and AST levels of serum significantly (P < 0.05). In hepatopancreas, CAT and SOD activities were improved observably and the MDA content was reduced markedly in both T23 groups. The expressions of antimicrobial related genes, Cru and peroxinectin in the 0.3T23 group, and proPO and peroxinectin in the 0.4T23 group were up-regulated remarkably (P < 0.05). Moreover, hepatopancreas of shrimp fed with a diet amended with T23 showed a significant down-regulated expression of nf-kb and tnf-α genes, while expressions of tgf-ß was considerably up-regulated. Furthermore, serum LPS and LBP contents were reduced markedly in T23 groups. Intestinal SOD and CAT were noteworthy higher in T23 groups (P < 0.05). In the intestine of shrimp fed on the diet enriched with T23 the expression of nf-κb and tnf-α exhibited markedly down-regulated, whereas hif1α was up-regulated (P < 0.05). Besides, in the intestine of shrimp grouped under T23, Cru and peroxinectin genes were markedly up-regulated (P < 0.05). Dietary 0.3 g/kg T23 also upregulated the ratio of Rhodobacteraceae to Vibrionaceae in the gut of the shrimp. Taken together, the inclusion of B. velezensis T23 in the diet of shrimp enhanced the growth and feed utilization, enhanced hepatopancreas and intestine health.

Influence of stocking density on the growth, immune and physiological responses, and cultivation environment of white-leg shrimp (Litopenaeus vannamei) in biofloc systems.

Biofloc (BF) stands out as a promising system for sustainable shrimp farming. Optimizing various culture conditions, such as stocking density, carbohydrate source, and feeding management, is crucial for the widespread adoption of the BF system. This study compares the growth performance of white-leg shrimp (Litopenaeus vannamei) in culture ponds at low density (LD) with 50 organisms/m2 and high density (HD) with 200 organisms/m2. Post-larvae of white-leg shrimp were stocked for 16 weeks in both LD and HD groups. The LD group exhibited a superior survival rate, growth rate, and feed consumption compared to the HD group. The BF from the LD system recorded a significantly higher protein content (16.63 ± 0.21%) than the HD group (15.21 ± 0.34%). Heterotrophic bacterial counts in water did not significantly differ with stocking density. However, Vibrio count in water samples was higher in the HD group (3.59 ± 0.35 log CFU/mL) compared to the LD group (2.45 ± 0.43 log CFU/mL). The whole shrimp body analysis revealed significantly higher protein and lipid content in the LD group. In contrast, the total aerobic bacterial count in shrimp from the HD group was high, with the identification of Salmonella enterica ssp. arizonae. Additionally, Vibrio counts in shrimp samples were significantly higher in the HD group (4.63 ± 0.32 log CFU/g) compared to the LD group (3.57 ± 0.22 log CFU/g). The expression levels of immune-associated genes, including prophenoloxidase, transglutaminase, penaiedin 3, superoxide dismutase, lysozyme, serine proteinase, and the growth-related gene ras-related protein (rap-2a), were significantly enhanced in the LD group. Conversely, stress-related gene expression increased significantly in the HD group. Hepatopancreases amylase, lipase, and protease were higher in the LD group, while trypsin activity did not differ significantly. Antioxidant enzyme activity (catalase, glutathione, glutathione peroxidase, and superoxide dismutase) significantly increased in the LD group. The histological structure of hepatopancreas, musculature, and female gonads remained similar in both densities. However, negative effects were observed in the gills' histology of the HD group. These results suggest that increasing stocking density is associated with significantly negative biological, microbial, and physiological effects on white-leg shrimp under the BF system.

Comparative Transcriptomic Analysis of WSSV-Challenged Penaeus vannamei with Variable Resistance Levels.

The Pacific white shrimp, Penaeus vannamei, is highly susceptible to white spot syndrome virus (WSSV). Our study explored the transcriptomic responses of P. vannamei from resistant and susceptible families, uncovering distinct expression patterns after WSSV infection. The analysis revealed a higher number of differentially expressed genes (DEGs) in the susceptible family following WSSV infection compared to the resistant family, when both were evaluated against their respective control groups, indicating that the host resistance of the family line influences the transcriptome. The results also showed that subsequent to an identical duration following WSSV infection, there were more DEGs in P. vannamei with a high viral load than in those with a low viral load. To identify common transcriptomic responses, we profiled DEGs across families at 96 and 228 h post-infection (hpi). The analysis yielded 64 up-regulated and 37 down-regulated DEGs at 96 hpi, with 33 up-regulated and 34 down-regulated DEGs at 228 hpi, showcasing the dynamics of the transcriptomic response over time. Real-time RT-PCR assays confirmed significant DEG expression changes post-infection. Our results offer new insights into shrimp's molecular defense mechanisms against WSSV.

Immune signaling of Litopenaeus vannamei c-type lysozyme and its role during microsporidian Enterocytozoon hepatopenaei (EHP) infection.

The microsporidian Enterocytozoon hepatopenaei (EHP) is a fungi-related, spore-forming parasite. EHP infection causes growth retardation and size variation in shrimp, resulting in severe economic losses. Studies on shrimp immune response have shown that several antimicrobial peptides (AMPs) were upregulated upon EHP infection. Among those highly upregulated AMPs is c-type lysozyme (LvLyz-c). However, the immune signaling pathway responsible for LvLyz-c production in shrimp as well as its function against the EHP infection are still poorly understood. Here, we characterized major shrimp immune signaling pathways and found that Toll and JAK/STAT pathways were up-regulated upon EHP infection. Knocking down of a Domeless (DOME) receptor in the JAK/STAT pathways resulted in a significant reduction of the LvLyz-c and the elevation of EHP copy number. We further elucidated the function of LvLyz-c by heterologously expressing a recombinant LvLyz-c (rLvLyz-c) in an Escherichia coli. rLvLyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus. Interestingly, we found an antifungal activity of rLvLyz-c against Candida albican, which led us to further investigate the effects of rLvLyz-c on EHP spores. Incubation of the EHP spores with rLvLyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that rLvLyz-c possibly digest a chitin coat on the EHP spores. Transmission electron microscopy analysis revealed that an endospore layer, which is composed mainly of chitin, was digested by rLvLyz-c. Lastly, we observed that EHP spores that were treated with rLvLyz-c showed a significant reduction of the spore germination rate. We hypothesize that thinning of the endospore of EHP would result in altered permeability, hence affecting spore germination. This work provides insights into shrimp immune signaling pathways responsible for LvLyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.

Dietary Poly-ß-Hydroxybutyrate Improved the Growth, Non-specific Immunity, Digestive Enzyme Activity, Intestinal Morphology, Phagocytic Activity, and Disease Resistance Against Vibrio parahaemolyticus of Pacific White Shrimp, Penaeus vannamei.

This study assessed the effects of dietary supplementation of poly-ß-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology, and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00% (Con, P0.25, P0.5, P1.0, P2.0, and P4.0, respectively). Triplicate groups of 90 shrimps (initial body weight 0.25 ± 0.01 g) per treatment were randomly assigned and fed an experimental diet for 56 days. The growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin, and pepsin activities. Villus height was significantly increased with dietary PHB supplementation, and villus width was increased at a 1% inclusion level. P0.25, P0.5, and P4.0 groups significantly increased phenoloxidase activity, and the P2.0 group significantly increased anti-protease activity compared to the Con group. The survival of shrimp challenged against V. parahaemolyticus was higher in P0.5, P1.0, and P2.0 groups than in the Con diet. Dietary PHB supplementation improved weight gain, digestive enzyme activity, intestinal morphology, non-specific immunity, and disease resistance against V. parahaemolyticus of shrimp. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp.

The combined analysis of transcriptomics and metabolomics reveals the mechanisms by which dietary quercetin regulates growth and immunity in Penaeus vannamei.

As a potent antioxidant, the flavonoid compound quercetin (QUE) has been widely used in the farming of aquatic animals. However, there are fewer reports of the beneficial effects, especially in improving immunity of Penaeus vannamei by QUE. The aim of this study was to investigate the effects of dietary QUE on growth, apoptosis, antioxidant and immunity of P. vannamei. It also explored the potential mechanisms of QUE in improving the growth and immunity of P. vannamei. P. vannamei were fed diets with QUE for 60 days. The results revealed that QUE (0.5 or 1.0 g/kg) ameliorated the growth, and the expressions of genes related to apoptosis, antioxidant, and immunity. The differentially expressed genes (DEGs) and differential metabolites (DMs) obtained through transcriptomics and metabolomics, respectively, enriched in pathways related to nutritional metabolism such as lipid metabolism, amino acid metabolism, and carbohydrate metabolism. After QUE addition, especially at 0.5 g/kg, DEGs were enriched into the functions of response to stimulus and antioxidant activity, and the pathways of HIF-1 signaling pathway, C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, and FoxO signaling pathway. In conclusion, dietary QUE can ameliorate growth, apoptosis, antioxidant and immunity of P. vannamei, the appropriate addition amount was 0.5 g/kg rather than 1.0 g/kg. Regulations of QUE on nutrient metabolism and immune-related pathways, and bioactive metabolites, were important factors for improving the aforementioned abilities in P. vannamei.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

BACKGROUND: Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. METHODS: Epithelial activation and/or barrier effects upon exposure to 2-50 µg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 µg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. RESULTS: Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. CONCLUSIONS: Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

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-h 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.

Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

[Novel tropomyosin consensus B and T epitopes involved in cross-reactivity between 10 different species. An in silico study]. / Nuevos epítopes B y T, consenso de tropomiosina involucradas en reactividad cruzada entre diez especies diferentes. Un estudio in silico.

OBJECTIVE: This study aimed to identify by in silico methods tropomyosin consensus B and T epitopes of shrimp species, house dust mites, insects, and nematodes associated with allergic diseases in tropical countries. METHODS: In silico analysis included tropomyosin from mites (Der p 10, Der f 10, Blo t 10), insects (Aed a 10, Per a 7, Bla g 7), shrimp (Lit v 1, Pen m 1, Pen a 1), and nematode (Asc l 3) all sequences were taken from the UniProt database. Linear IgE epitopes were predicted with AlgPred 2.0 and validated with BepiPred 3.0. MHC-II binding T cell epitopes were predicted using the IEDB server, which implements nine predictive methods (consensus method, combinatorial library, NN-align-2.3, NN- align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1, and NetMHCIIpan 3.2) these predictions focused on 10 HLA-DR and 2 HLA-DQ alleles associated with allergic diseases. Subsequently, consensus B and T epitopes present in all species were identified. RESULTS: We identified 12 sequences that behaved as IgE-epitopes and B-cell epitopes, three of them: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN215, 251KEVDRLEDELV261 were consensus in all species. Eleven peptides (T-epitopes) showed strong binding (percentile rank ≤ 2.0) to HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401, HLA-DQA1*03:01/DQB1*03:02, and HLA- DQA1*05:01/DQB1*02:01. Only two T-epitopes were consensus in all species: 167RKLAMVEADLERAEERAEt GEsKIVELEEELRV199, and 218EEeY KQQIKT LTaKLKEAEARAEFAERSV246. Subsequently, we identified 2 B and T epitope sequences and reached a consensus between species 167RKLAMVEA174 and 192ELEEELRV199. CONCLUSIONS: These data describe three sequences that may explain the IgE cross-reactivity between the analyzed species. In addition, the consensus B and T epitopes can be used for further in vitro investigations and may help to design multiple-epitope protein-based immunotherapy for tropomyosin-related allergic diseases.

OBJETIVO: Este estudio tuvo como objetivo identificar mediante métodos in silico epítopes B y T consenso de tropomiosina de especies de camarón, ácaros del polvo doméstico, insectos y nematodos asociados a enfermedades alérgicas en países tropicales. MÉTODOS: El análisis in silico incluyó tropomiosina de ácaros (Der p 10, Der f 10, Blo t 10), insectos (Aed a 10, Per a 7, Bla g 7), camarones (Lit v 1, Pen m 1, Pen a 1), y nematodo (Asc l 3). Todas las secuencias se tomaron de la base de datos UniProt. Los epítopes IgE lineales se predijeron con AlgPred 2.0 y se validaron con BepiPred 3.0. Los epítopes de células T de unión a MHC-II se predijeron utilizando el servidor IEDB, que implementa nueve métodos predictivos (método de consenso, biblioteca combinatoria, NN-align-2.3, NN-align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1 y NetMHCIIpan 3.2). Estas predicciones se centraron en diez alelos HLA-DR y 2 HLA-DQ asociados con enfermedades alérgicas. Posteriormente, se identificaron epítopes consenso B y T presentes en todas las especies. RESULTADOS: Se identificaron 12 secuencias que se comportaron como epítopes de IgE y, también, como epítopes de células B. Tres de ellas: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN213 y 251KEVDRLEDELV261, fueron consenso en todas las especies. Once péptidos mostraron una fuerte unión (rango percentil ≤ 2,0) a HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401 y a HLA HLA-DQA1*03:01/DQB1*03:02, o HLA-DQA1*05:01/DQB1*02:01. Solo se encontraron dos secuencias: 167RKLAMVEADLERAEERAEtGEsKIVELEEELRV199 con fuerte afinidad por HLA-DQA1*03:01/DQB1*03:02, y HLA-DQA1*05:01/DQB1*02:01. Se identificaron dos secuencias que son epítopos B y T, y son consenso entre especies: 167RKLAMVEA174 y 192ELEEELRV199. CONCLUSIONES: Estos datos describen tres secuencias que pueden explicar la reactividad cruzada de IgE entre las especies analizadas. Además, los epítopos B y T consenso se pueden usar para investigaciones in vitro adicionales, y pueden ayudar a diseñar inmunoterapia basada en proteínas de múltiepítopes para enfermedades alérgicas relacionadas con la tropomiosina.

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.