Nitrogen Removal Performance and Microbial Community Structure of IMTA Ponds (Apostistius japonicus-Penaeus japonicus-Ulva).

Denitrification and anaerobic ammonium oxidation (anammox) are key processes for nitrogen removal in aquaculture, reducing the accumulated nitrogen nutrients to nitrogen gas or nitrous oxide gas. Complete removal of nitrogen from aquaculture systems is an important measure to solve environmental pollution. In order to evaluate the nitrogen removal potential of marine aquaculture ponds, this study investigated the denitrification and anammox rates, the flux of nitrous oxide (N2O) at the water-air interface, the sediment microbial community structure, and the gene expression associated with the nitrogen removal process in integrated multi-trophic aquaculture (IMTA) ponds (Apostistius japonicus-Penaeus japonicus-Ulva) with different culture periods. The results showed that the denitrification and anammox rates in sediments increased with the increase of cultivation periods and depth, and there was no significant difference in nitrous oxide gas flux at the water-air interface between different cultivation periods (p > 0.05). At the genus and phylum levels, the abundance of microorganisms related to nitrogen removal reactions in sediments changed significantly with the increase of cultivation period and depth, and was most significantly affected by the concentration of particulate organic nitrogen (PON) in sediments. The expression of denitrification gene (narG, nirS, nosZ) in surface sediments was significantly higher than that in deep sediments (p < 0.05), and was negatively correlated with denitrification rate. All samples had a certain anammox capacity, but no known anammox bacteria were found in the microbial diversity detection, and the expression of gene (hzsB) related to the anammox process was extremely low, which may indicate the existence of an unknown anammox bacterium. The data of this study showed that the IMTA culture pond had a certain potential for nitrogen removal, and whether it could make a contribution to reducing the pollution of culture wastewater still needed additional practice and evaluation, and also provided a theoretical basis for the nitrogen removal research of coastal mariculture ponds.

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.

Functional and mechanistic studies of a phytogenic formulation, Shrimp Best, in growth performance and vibriosis in whiteleg shrimp.

Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp. Functional studies showed that SB dose-dependently increased shrimp body weight and decreased feed conversion ratio. We found that SB protected against Vibrio parahaemolyticus as evidenced by survival rate, bacterial load, and hepatopancreatic pathology in shrimp. Finally, we explored the likely mechanism by which SB affects growth performance and vibriosis in shrimp. The 16S rRNA sequencing data showed that SB increased 6 probiotic genera and decreased 6 genera of pathogenic bacteria in shrimp. Among these, SB increased the proportion of Lactobacillus johnsonii and decreased that of V. parahaemolyticus in shrimp guts. To dissect the relationship among SB, Lactobacillus and Vibrio, we investigated the in vitro regulation of Lactobacillus and Vibrio by SB. SB at ≥ 0.25 µg/mL promoted L. johnsonii growth. Additionally, L. johnsonii and its supernatant could inhibit V. parahaemolyticus. Furthermore, SB could up-regulate five anti-Vibrio metabolites of L. johnsonii, which caused bacterial membrane destruction. In parallel, we identified 3 fatty acids as active compounds from SB. Overall, this work demonstrated that SB improved growth performance and vibriosis protection in shrimp via the regulation of gut microbiota.

Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp.

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

Interactions between the gut bacterial community of Exopalaemon carinicauda and infection by Enterocytozoon hepatopenaei.

To explore the relationship between the intestinal flora of Exopalaemon Carinicauda and infection by Enterocytozoo Hepatopenaei (EHP), we analyzed the species and richness of gut microbiota in infected individuals in different EHP load groups [i.e., control (C), high load (H), and low load (L)] using gene sequencing after infection. The results showed that the abundance of intestinal flora in the high-load EHP group was significantly lower than that in the healthy group. Based on the UPGMA cluster tree and PCoA analysis, with comparisons to healthy shrimp, the gut microbiota of the EHP high load and low load groups were clustered into one branch, which indicated that EHP infection changed the composition of the gut microbiota of infected shrimps. The heat map analysis of species abundance clustering revealed that the dominant bacteria in the low EHP load group and the control group were beneficial genera such as Lactococcus, Ligilactobacillius, and Bifidobacterium, but the dominant bacteria in the high EHP load group were harmful genera such as Pseudomonas, Photobacterium, and Candidatus hepatincola. The functions of the intestinal flora predicted that most genes related to metabolism were more abundant in healthy shrimp, most genes related to metabolism and the organisms' system were more abundant in the low EHP load group, and most genes related to diseases and environmental information processing were more abundant in the high EHP load group. After separation and purification, the dominant bacteria (Bifidobacterium animalis in healthy shrimp and Lactococcus garvieae in the low EHP load group) and the non-dominant bacteria (Macrococus caseolyticus in the low EHP load group) were obtained. Each of these isolated strains were used together with EHP to infect E. carinicauda, and the results showed that Bifidobacterium animali and Lactococcus garvieae significantly reduced the EHP load in EHP-infected individuals. At the same time, the morphology and structure of the hepatopancreas and intestinal tissue of EHP-infected E. carinicauda were improved. No improvement was seen in tissue that was infected with Macrococus caseolyticus.

Whole-genome sequencing of Pseudoalteromonas piscicida 2515 revealed its antibacterial potency against Vibrio anguillarum: a preliminary invitro study.

Pseudoalteromonas piscicida 2515, isolated from Litopenaeus vannamei culture water, is a potential marine probiotic with broad anti-Vibrio properties. However, genomic information on P. piscicida 2515 is scarce. In this study, the general genomic characteristics and probiotic properties of the P. piscicida 2515 strain were analysed. In addition, we determined the antibacterial mechanism of this bacterial strain by scanning electron microscopy (SEM). The results indicated that the whole-genome sequence of P. piscicida 2515 contained one chromosome and one plasmid, including a total length of 5,541,406 bp with a G + C content of 43.24%, and 4679 protein-coding genes were predicted. Various adhesion-related genes, amino acid and vitamin metabolism and biosynthesis genes, and stress-responsive genes were found with genome mining tools. The presence of genes encoding chitin, bromocyclic peptides, lantibiotics, and sactipeptides showed the strong antibacterial activity of the P. piscicida 2515 strain. Moreover, in coculture with Vibrio anguillarum, P. piscicida 2515 displayed vesicle/pilus-like structures located on its surface that possibly participated in its bactericidal activity, representing an antibacterial mechanism. Additionally, 16 haemolytic genes and 3 antibiotic resistance genes, including tetracycline, fluoroquinolone, and carbapenem were annotated, but virulence genes encoding enterotoxin FM (entFM), cereulide (ces), and cytotoxin K were not detected. Further tests should be conducted to confirm the safety characteristics of P. piscicida 2515, including long-term toxicology tests, ecotoxicological assessment, and antibiotic resistance transfer risk assessment. Our results here revealed a new understanding of the probiotic properties and antibacterial mechanism of P. piscicida 2515, in addition to theoretical information for its application in aquaculture.

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.

Effects of 'bask in sunlight and dewed at night' on the formation of fermented flavor in shrimp paste after maturation.

The purpose of the study was to illustrate the roles of three primary indexes, namely sunlight, ventilation and stirring, in the 'bask in sunlight and dewed at night' technique on the quality of shrimp paste, through a laboratory-scale design. The results showed that changes in the post-ripening fermentation conditions, especially sunlight, was instrumental in the physicochemical properties of the shrimp paste. E-nose and SPME-GC-MS were employed to assess the volatile flavor of post-ripening fermentation. A total of 29 key volatile aroma components played a crucial role in the development of post-ripening flavor in shrimp paste with or without sunlight. Lipidomic analysis revealed that sunlight promoted the oxidative degradation of FA, resulting in the production of a diverse range of flavor compounds that imparted the unique aroma of shrimp paste. The findings of this study will establish a theoretical basic for better control of the post-ripening fermentation of traditional shrimp paste.

Effect of microplastics on oxytetracycline trophic transfer: Immune, gut microbiota and antibiotic resistance gene responses.

Microplastics and antibiotics are prevalent and emerging pollutants in aquatic ecosystems, but their interactions in aquatic food chains remain largely unexplored. This study investigated the impact of polypropylene microplastics (PP-MPs) on oxytetracycline (OTC) trophic transfer from the shrimp (Neocaridina denticulate) to crucian carp (Carassius auratus) by metagenomic sequencing. The carrier effects of PP-MPs promoted OTC bioaccumulation and trophic transfer, which exacerbated enterocyte vacuolation and hepatocyte eosinophilic necrosis. PP-MPs enhanced the inhibitory effect of OTC on intestinal lysozyme activities and complement C3 levels in shrimp and fish, and hepatic immunoglobulin M levels in fish (p < 0.05). Co-exposure of MPs and OTC markedly increased the abundance of Actinobacteria in shrimp and Firmicutes in fish, which caused disturbances in carbohydrate, amino acid, and energy metabolism. Moreover, OTC exacerbated the enrichment of antibiotic resistance genes (ARGs) in aquatic animals, and PP-MPs significantly increased the diversity and abundance of ARGs and facilitated the trophic transfer of teta and tetm. Our findings disclosed the impacts of PP-MPs on the mechanism of antibiotic toxicity in aquatic food chains and emphasized the importance of gut microbiota for ARGs trophic transfer, which contributed to a deeper understanding of potential risks posed by complex pollutants on aquatic ecosystems.

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.

Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

The Use of the Internal Transcribed Spacer Region for Phylogenetic Analysis of the Microsporidian Parasite Enterocytozoon hepatopenaei Infecting Whiteleg Shrimp (Penaeus vannamei) and for the Development of a Nested PCR as Its Diagnostic Tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

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.

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.

Nitrite nitrogen stress disrupts the intestine bacterial community by altering host-community interactions in shrimp.

Environmental stress can disrupt the intricate interactions between the host and intestine microbiota, thereby impacting the host health. In this study, we aimed to elucidate the dynamic changes in the bacterial community within shrimp intestines under nitrite nitrogen (nitrite-N) stress and investigate potential host-related factors influencing these changes. Our results revealed a significant reduction in community diversity within the intestine exposed to nitrite-N compared to control conditions. Furthermore, distinct differences in community structures were observed between these two groups at 72 h and 120 h post-stress induction. Nitrite-N stress also altered the abundances of some bacterial species in the intestine dramatically. It is noteworthy that, in comparison to the 72 h, intestine bacterial community structure of stressed shrimp exhibited a significantly higher degree of dispersion after 120 h of nitrite-N stress when compared to control shrimp, and the relative abundance of numerous bacterial species experienced a substantial decrease or even reached 0 %. Moreover, it led to a reduction in bacterial community interactions and decreased competitiveness within the intestine microbiota. Notably, the influence of bacterial community assemblies in the shrimp intestine shifted from a stochastic process to a deterministic one after 24 h and 72 h of nitrite-N stress, returning to a stochastic process at 120 h. We further observed a close association between this phenomenon and host's response to nitrite-N stress. Expression levels of differentially expressed genes in the intestinal tissue significantly impact the intestine bacterial diversity and abundance of species. In particular, the significant decline in bacterial diversity and abundances of quite a few species in intestine was attributed to the up-regulation of peritrophin-48-like. Overall, nitrite-N stress indeed disrupted the intestine microbiota and changed the host-microbiota interactions of shrimp. This study offered novel insights into environment-host-microbiota interactions and also provided practical guidance for promoting healthy shrimp cultivation practices.

In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei.

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

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

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

Proteome analysis of outer membrane vesicles from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease.

A specific strain of Vibrio parahaemolyticus (VpAHPND) causes acute hepatopancreatic necrosis disease (AHPND), leading to significant losses in shrimp aquaculture. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, and their significant roles in host-pathogen interactions and pathogenicity have been recognized. In the present study, OMVs were isolated from VpAHPND by differential-ultracentrifugation and used for proteomics analysis. In the Nano-HPLC-MS/MS analysis, totally 645 proteins were determined, including virulence factors, immunogenic proteins, outer membrane protein, bacterial secretory proteins, ribosomal proteins, protease, and iron regulation proteins. Furthermore, GO and KEGG annotations indicated that proteins identified in VpAHPND-OMVs are involved in metabolism, regulation of multiple biological processes, genetic information processes, immunity and more. Meanwhile, toxin proteins PirAvp and PirBvp, associated with VpAHPND pathogenicity, were also identified in the proteome of VpAHPND-OMVs. Our objective is to identify the protein composition of OMVs released by VpAHPND, analyzing the potential for cytotoxicity and immunomodulatory activity of these granule hosts. This study is crucial for understanding the roles played by bacterial-derived vesicles in the disease process, given that these vesicles carry relevant activities inherent to the bacteria that produce them.

Vibrio Species and Cyanobacteria: Understanding Their Association in Local Shrimp Farm Using Canonical Correspondence Analysis (CCA).

In aquatic environments, Vibrio and cyanobacteria establish varying relationships influenced by environmental factors. To investigate their association, this study spanned 5 months at a local shrimp farm, covering the shrimp larvae stocking cycle until harvesting. A total of 32 samples were collected from pond A (n = 6), pond B (n = 6), effluent (n = 10), and influent (n = 10). Vibrio species and cyanobacteria density were observed, and canonical correspondence analysis (CCA) assessed their correlation. CCA revealed a minor correlation (p = 0.847, 0.255, 0.288, and 0.304) between Vibrio and cyanobacteria in pond A, pond B, effluent, and influent water, respectively. Notably, Vibrio showed a stronger correlation with pH (6.14-7.64), while cyanobacteria correlated with pH, salinity (17.4-24 ppt), and temperature (30.8-31.5 °C), with salinity as the most influential factor. This suggests that factors beyond cyanobacteria influence Vibrio survival. Future research could explore species-specific relationships, regional dynamics, and multidimensional landscapes to better understand Vibrio-cyanobacteria connections. Managing water parameters may prove more efficient in controlling vibriosis in shrimp farms than targeting cyanobacterial populations.

Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.