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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/m 2 and high density (HD) with 200 organisms/m 2 . 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.

The Mab21/cGAS protein family has diversified across metazoans to regulate development and innate immunity. In vertebrates, cGAS detects cytosolic DNA and synthesizes 2′3′-cGAMP to activate STING–TBK1–IRF signaling, while invertebrate cGAS-like receptors (cGLRs) recognize RNA or DNA and generate non-canonical cyclic dinucleotides. However, whether shrimp Mab21 proteins function as canonical nucleic acid sensors remains unresolved. Here, we identified three Mab21 proteins from Litopenaeus vannamei —LvMab21–1, LvMab21–2, and LvMab21–3. Although they are phylogenetically related to cGAS-like proteins, none bound dsDNA or dsRNA or synthesized cGAMP in response to ISD or poly(I:C). Instead, all three interacted directly with the TBK1 homolog LvIKKε, promoted its phosphorylation at serine 175, and thereby activated the downstream IRF–Vago4 signaling axis. This mechanism defines a non-canonical nucleic acid sensing paradigm, whereby Mab21 proteins act as protein-based enhancers of kinase activation rather than as nucleic acid–dependent CDN synthases. We further show that these proteins display tissue-specific antiviral functions: all three act in hemocytes, LvMab21–1 predominates in hepatopancreas, LvMab21–2 and LvMab21–3 are most critical in gills, and LvMab21–1 and LvMab21–3 cooperate in intestine. Silencing any Mab21 paralog reduced survival and increased white spot syndrome virus (WSSV) burden, underscoring their physiological relevance. Together, our findings expand the known repertoire of innate immune strategies within the Mab21 family, highlight a previously unrecognized non-canonical mechanism of interferon-like activation, and reveal tissue-specific specialization that tailors antiviral responses across shrimp organs. These insights provide both evolutionary context and candidate targets for breeding disease-resistant shrimp.

Intracellular recognition of viral nucleic acids by NLRs and subsequent activation of antiviral immunity are crucial for host defense against virus infection in vertebrates. However, understanding on these processes is very limited in invertebrates, especially for crustaceans. In the present study, an NLR gene belonging to the NLRC subfamily ( LvNLRC ) was identified in the Pacific whiteleg shrimp Litopenaeus vannamei and its functions in intracellular recognition to DNA virus and antiviral immunity during WSSV infection were elucidated. LvNLRC possesses an ability to detect DNA viral mimics such as poly(dA:dT) with a dose-dependent manner through its leucine-rich repeat (LRR) domain. The LRRs domain also recognizes the partial DNA encoding VP24 of WSSV. LvNLRC could modulate the interferon system-like antiviral response in shrimp through direct interaction between its NACHT domain and LvSTING. Different from the regulatory mechanism in vertebrates, the presence of poly(dA:dT) does not affect the release of STING from the NACHT domain of LvNLRC in shrimp. Interestingly, shrimp Cyclophilin A (LvCypA) can directly interact with the NACHT domain of LvNLRC in a dose-dependent manner in the presence of poly(dA:dT). Furthermore, LvCypA also plays an important role in the regulation of the interferon system-like antiviral immunity in shrimp. The present results provide the first evidence to show that Cyclophilin A can modulate NLR-mediated cytokine-like antiviral immunity in animals. These findings shed light on the roles of NLRs in regulating host innate immunity, contributing valuable insights into this area of study.

Resistant starch (RS) is a constituent of dietary fibre that has beneficial effects on the intestine physiological function of animals. However, the roles of RS on shrimp intestine health is unknown. In this study, we investigated the the effects of dietary RS on the microbial composition, and digestive and immune-related indices in the intestine of Litopenaeus vannamei . The shrimp were fed with diets containing different levels of RS: 0 g/kg (Control), 10 g/kg (RS1), 30 g/kg (RS2) and 50 g/kg (RS3) for 56 days. The results showed that dietary RS improved the morphology of the intestine mucosa. RS also increased the activity of digestive enzymes (AMS, LPS, Tryp, and Pep) and immune enzymes (PO, T-AOC, T-NOS, and NO), and the expression levels of immune-related genes ( proPO , ALF , Lys , HSP70 , Trx , Muc-1 , Muc-2 , Muc-5AC , Muc-5B , and Muc-19 ). A microbiome analysis indicated that dietary RS increased the short-chain fatty acids (SCFAs) contents and altered the composition of the intestine microbial. Specifically, RS increased the abundances of Proteobacteria and decreased the abundance of Bacteroidetes. At the genus level, the beneficial bacteria ( Lutimonas , Ruegeria , Shimia , Mesoflavibacter , and Mameliella ) were enriched, which might be involved in degrading toxins and producing beneficial metabolites; while potential pathogens ( Formosa and Pseudoalteromonas ) were decreased in response to dietary RS. Our results revealed that dietary RS could improve the intestine health of L . vannamei , probably via modulating the intestine microbial composition and SCFAs contents, and enhancing the digestion and immunity of the shrimp.

Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp ( Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum . FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish , the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C ( Src ), and two small GTPases, Rab5 and Rab7 , which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates.

Nutritional deficiencies during the early developmental stages of Penaeus vannamei often lead to poor growth, weak immunity and high mortality. To address these issues, this study investigated the effects of live feeds enriched with Schizochytrium sp. (SCH), taurine and inosine monophosphate (IMP) on the growth, lipid metabolism and immune response of shrimp post-larvae. Two feeding trials were conducted using rotifers (Trial 1) and Artemia (Trial 2) as live feeds. In trial 1, post-larvae (PL1–2) were fed unenriched rotifers (RN) or rotifers enriched with 0.5% SCH (RS), RS + 0.1% taurine (RS + T), RS + 0.1% IMP (RS + I) and 0.5% Chlorella powder (RCL) for 9 days. In trial 2, post-larvae (PL12–14) were fed unenriched Artemia (AN) or Artemia enriched with 0.5% SCH (AS), AS + 0.1% taurine (AS+T), AS + 0.1% IMP (AS+I) and 0.5% Chlorella powder (ACL) for 12 days. In trial 1, growth was significantly higher in RS, RS + T and RS + I groups compared to RN and RCL groups. Survival was significantly higher in RS, RS + T and RS + I groups than in RN group. In trial 2, growth performance was significantly higher in all SCH-enriched groups compared to AN and ACL groups whereas, survival did not differ significantly among the experimental groups. In both trials, whole-body docosahexaenoic acid levels increased markedly in all SCH-supplemented treatments, and the expression of lipid metabolism-related genes ( FAS , FABP , FATP and CPT1 ) and immune-related genes ( lysozyme , crustin , LGBP , Pen-3a and proPO ) was significantly upregulated. These results demonstrate that enrichment of live feeds with SCH effectively enhances growth, lipid utilization and immune function in P. vannamei post-larvae. Furthermore, combined supplementation with taurine and IMP produced synergistic immunostimulatory effects. Overall, SCH-based enrichment can serve as a promising functional nutritional fortifier for shrimp hatchery diets.

The rapid growth of the aquaculture industry has increased the demand for feed ingredients, resulting in a shortage of fishmeal and fish oil, the latter of which serves as a source of essential fatty acids in aquaculture feed. As dietary supplementation of long-chain polyunsaturated fatty acids (LC-PUFA) improved growth and strengthened immunity in marine larvae, alternative sources for LC-PUFA are required to maintain sustainable aquaculture practice. This study explored the use of Aurantiochytrium limacinum BCC52274 (AL) and oil extracted from Mortierella sp. (ARASCO) as the source of LC-PUFA for the Pacific white shrimp Litopenaeus vannamei post-larvae (PLs) by using Artemia as a carrier. The Artemia were first enriched with combinations of AL and ARASCO with varying ratios of DHA:ARA at 100:0, 75:25, 50:50, 25:75, and 0:100, which were designated as Groups A, B, C, D, and E, respectively. The unenriched Artemia in Group R served as a control. The L. vannamei PL1 were fed with these Artemia for 18 days, revealing that the PL18 in Group A contained the highest biomass and average body weight among all feed groups. Meanwhile, other ratios of DHA:ARA supplementation were more beneficial to shrimp immunity, as the PL18 from Group D expressed the highest levels of prophenoloxidase II transcripts among all feed groups. The increasing proportion of ARA in the enriched Artemia also increased the levels of anti-inflammatory eicosanoids, including 15-deoxy-Δ 12,14 -prostaglandin J 2 , 11-hydroxyeicosatetraenoic acid (11-HETE), and 12-HETE. When these PLs were infected with white spot syndrome virus (WSSV), the PLs from Groups A, C, D, and E contained lower WSSV copy numbers compared with Group R, suggesting that the supplementation of LC-PUFAs from AL and ARASCO strengthened the immunity of these PLs against viral infection. However, the challenge with Vibrio harveyi resulted in no significant difference in the mean survival rates of PLs in all feed groups. Our results indicate that AL and ARASCO are more sustainable alternative sources of essential fatty acids that can be used strategically to enhance the growth and immunity of L. vannamei PLs.

Vitamin E (tocopherols, VE) is a lipid-soluble antioxidant involved in neutralizing reactive oxygen species and maintaining immune function in animals. This study aimed to determine the optimum dietary VE requirement of juvenile Pacific white shrimp ( Penaeus vannamei ) for growth, feed utilization, immune responses, antioxidative capacity, diet digestibility, intestinal histomorphology and disease resistance against Vibrio parahaemolyticus . Eight experimental diets were formulated to contain graded levels of VE (0, 20, 40, 60, 80, 100, 120 and 240 mg/kg; designated as VE0, VE20, VE40, VE60, VE80, VE100, VE120 and VE240). Four replicate groups, each containing 30 shrimp (0.20 ± 0.04 g), were fed one of the diets six times daily for 56 days. Shrimp fed VE80 diet exhibited significantly increased growth performance compared to shrimp fed VE0, VE20 and VE240 diets. Non-specific immune responses were significantly enhanced in shrimp fed VE60-VE80 diets. Hepatopancreatic lipid peroxidation in VE80 group was significantly lower compared to the VE0 group. The expression of Crustin , C-MnSOD and GPx genes in the hepatopancreas was significantly upregulated in VE80 group. Graded dietary VE levels significantly linearly increased hemolymph and hepatopancreas VE concentrations. Intestinal villi height and width were significantly improved with dietary VE supplementation. The digestibility of protein, lipid and dry matter was significantly higher in shrimp fed VE80 diet compared to those fed VE0 diet. The resistance against V. parahaemolyticus was significantly higher in shrimp fed VE80, VE100 and VE120 diets compared to those fed VE0 and VE20 diets. The optimal dietary VE level for Pacific white shrimp was estimated to be 72.17 mg/kg for weight gain and 72.21 mg/kg specific growth rate, based on broken-line analysis. In conclusion, optimal dietary VE supplementation enhances shrimp growth, immunity, antioxidative defense and disease resistance against V. parahaemolyticus , thereby reducing the risk of early mortality syndrome caused by acute hepatopancreatic necrosis disease.

Penaeus vannamei hemocyanin (PvHMC) exhibits multifunctional roles in immunity, often mediated by various post-translational modifications. While linear ubiquitination catalyzed by LUBAC in mammals regulates immune signaling, its role in crustacean immunity remains unclear. Here, we investigated the regulatory mechanism of PvHMC linear ubiquitination mediated by an E3 ligase PvLUBEL (a HOIP homolog), with emphasis on its role in shrimp immunity defending against diverse pathogens. We detected linear ubiquitination of PvHMC in multiple tissues, including hemocytes and the hepatopancreas. During Vibrio parahaemolyticus infection, the expression of PvLUBEL and the level of PvHMC linear ubiquitination were suppressed, whereas infection by white spot syndrome virus (WSSV) led to their upregulation. Structural analyses revealed that PvLUBEL, which shares a conserved RING-IBR-RING (RBR) domain with mammalian HOIP, serves as the catalytic subunit. Notably, inhibition of PvLUBEL via HOIPIN-1 (a covalent inhibitor) or RNA interference (RNAi) significantly reduced PvHMC linear ubiquitination, thereby increasing pathogen proliferation and decreasing host survival. These findings unveil a novel post-translational regulatory mechanism in which PvLUBEL-mediated linear ubiquitination of PvHMC underpins the shrimp immune response against aquaculture pathogens.

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.