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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.

Penaeus monodon is a major marine aquaculture species; however, production intensification has increased water-quality deterioration and disease pressure. Copper-loaded montmorillonite (Cu-MMT) is a functional clay additive with adsorption and antimicrobial properties, yet the optimal application mode remains unclear. We compared a control (KZ), water application (PZ), and dietary inclusion (BZ) of Cu-MMT in P. monodon. BZ was associated with higher survival and a numerically higher specific growth rate, whereas final body weight did not differ among treatments. Antioxidant status improved in BZ, with higher catalase (CAT) and total superoxide dismutase (T-SOD) activities (both p < 0.05). Hepatopancreas RNA-seq identified 949 differentially expressed genes (DEGs) for KZ vs. PZ and 814 DEGs for KZ vs. BZ. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that PZ was enriched for redox processes, transporter activity, and amino-acid biosynthesis—indicative of a stress-defense state—whereas BZ was enriched for proteolysis, endoplasmic-reticulum protein processing, and proteasome pathways, consistent with an anabolic, protein-quality-control–oriented mode. Intestinal 16S rRNA profiling indicated higher diversity and reduced putative pathogens in BZ. Overall, dietary Cu-MMT is the preferred application, shifting shrimp from an energy-consuming stress response to efficient anabolism and thereby improving performance and survival.

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.

Beneficial microorganisms maintain the ecosystems, plants, animals and humans working in healthy conditions. In nature, around 95% of all microorganisms produce beneficial effects by increasing nutrients digestion and assimilation, preventing pathogens development and by improving environmental parameters. However, increase in human population and indiscriminate uses of antibiotics have been exerting a great pressure on agriculture, livestock, aquaculture, and also to the environment. This pressure has induced the decomposition of environmental parameters and the development of pathogenic strains resistant to most antibiotics. Therefore, all antibiotics have been restricted by corresponding authorities; hence, new and healthy alternatives to prevent or eliminate these pathogens need to be identified. Thus, probiotic bacteria utilization in aquaculture systems has emerged as a solution to prevent pathogens development, to enhance nutrients assimilation and to improve environmental parameters. In this sense, B. subtilis is an ideal multifunctional probiotic bacterium, with the capacity to solve these problems and also to increase aquaculture profitability.

Stocking density plays an important role in P. monodon aquaculture. However, its underlying molecular mechanisms remain unknown. In this work, after a 40-day culture period, we studied the effects of different stocking densities (G1: 50, G2: 100, G3: 150 ind/m3) on P. monodon, assessing their growth performances, physiological indexes, and transcriptome profiles. It was found that increased stocking density greatly decreased the growth performance and survival rate as well as the level of immune enzymes (alkaline phosphatase, acid phosphatase) and antioxidant component (total antioxidant capacity, superoxide dismutase, reduced glutathione) activities. Transcriptomic analysis showed that there are 2284 differentially expressed genes in all groups. Enrichment analysis of WGCNA results indicated that the green module associated with G2 was enriched for amino acid metabolism, lipid metabolism and immunity-related pathway. Furthermore, the G3 associated turquoise module was dominated by stress response, detoxification and energy metabolism pathways. Together, high stocking density causes the occurrence of oxidative stress, disturbance to immune system and alteration of metabolism profiles in P. monodon, whereas medium density (G2) is favorable to maintain physiological homeostasis. The results provide theoretical support to optimize aquaculture practice and contribute valuable information for subsequent studies.

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 function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins to inhibit viral infection. Taken together, we therefore identify that the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs.

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.

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.

Aquaculture contributes more than one-third of the animal protein from marine sources worldwide. A significant proportion of aquaculture products are derived from marine protostomes that are commonly referred to as ‘marine invertebrates’. Among them, penaeid shrimp (Ecdysozosoa, Arthropoda) and bivalve molluscs (Lophotrochozoa, Mollusca) are economically important. Mass rearing of arthropods and molluscs causes problems with pathogens in aquatic ecosystems that are exploited by humans. Remarkably, species of corals (Cnidaria) living in non-exploited ecosystems also suffer from devastating infectious diseases that display intriguing similarities with those affecting farmed animals. Infectious diseases affecting wild and farmed animals that are present in marine environments are predicted to increase in the future. This paper summarizes the role of the main pathogens and their interaction with host immunity, with a specific focus on antimicrobial peptides (AMPs) and pathogen resistance against AMPs. We provide a detailed review of penaeid shrimp AMPs and their role at the interface between the host and its resident/pathogenic microbiota. We also briefly describe the relevance of marine invertebrate AMPs in an applied context. This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.