Enhancement of immunity and disease resistance in Litopenaeus vannamei through injection of tyramine formulated with polyethylene glycol.

This study investigates the prolonged effect of immune disease resistance in Litopenaeus vannamei through the administration of tyramine (TA) formulated with polyethylene glycol (PEG). Facing the challenges of intensive farming, environmental stress, and global climate changes, innovative approaches to improve shrimp health are essential. The research focuses on the role of biogenic amines in stress response and immune regulation, demonstrating that TA, especially when combined with PEG, significantly prolongs immunity and resistance against Vibrio alginolyticus. The experimental design included administering TA, PEG, and TA-PEG, followed by evaluations of immunity, lactate and glucose levels, and immune-related gene expressions. Results showed notable prolonged effects in total hemocyte count, phenoloxidase activity, and phagocytic activity in the TA-PEG group, indicating enhanced immune activation period. Additionally, the expression of prophenoloxidase system-related genes was significantly upregulated in the TA-PEG group. Furthermore, the TA-PEG group exhibited a significantly higher survival rate in a susceptibility test against V. alginolyticus. The results of this study confirm that the combined use of PEG can effectively extend the immunostimulatory duration of TA.

Investigating dynamics, etiology, pathology, and therapeutic interventions of Caligus clemensi and Vibrio alginolyticus co-infection in farmed marine fish.

This study investigated a disease outbreak characterized by caligid copepod infestations and subsequent secondary bacterial infections in European seabass (Dicentrarchus labrax) and flathead grey mullet (Mugil cephalus) cultivated at a private facility in the Deeba Triangle region of Egypt. Moribund fish displayed brown spots on the skin, tongue, and gills, along with lethargy and excess mucus. The fish suffered severe infections, exhibiting external hemorrhages, ulcers, and ascites. The fish had pale, enlarged livers with hemorrhaging. Comprehensive parasitological, bacteriological, molecular, immunity and histopathological analyses were conducted to identify the etiological agents and pathological changes. Caligid copepod infestation was observed in wet mounts from the buccal and branchial cavities of all examined fish, and the caligids were identified as Caligus clemensi through COI gene sequencing and phylogenetic analysis. Vibrio alginolyticus was confirmed as a secondary bacterial infection through biochemical tests, recA gene sequencing, and phylogenetic analyses. Antibiotic susceptibility testing revealed resistance to ß-lactams, aminoglycosides, and trimethoprim-sulfamethoxazole in V. alginolyticus isolates. Upregulation of the inflammatory marker IL-1ß in gill and skin tissues indicated a robust cell-mediated immune response against the pathogens. Histopathological examination revealed severe tissue damage, hyperplasia, hemorrhage, and congestion in the gills, along with hepatocellular degeneration and steatosis in the liver, providing initial insights into this outbreak. A comprehensive therapeutic regimen was implemented, comprising prolonged hydrogen peroxide immersion baths, followed by the application of the nature-identical plant-based compound Lice-less and probiotic Sanolife Pro-W supplementation. This integrated approach effectively eliminated C. clemensi infestations, controlled secondary bacterial infections, and restored fish health, reducing morbidity and mortality rates to minimal levels.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus.

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer).

The study isolated two strains of intestinal autochthonous bacteria Lactiplantibacillus plantarum1 (MH155966.1) (L1) and Lactiplantibacillus plantarum2 (MH105076.1) (L2) from the Choobdeh Abadan region. The aim of this study was to investigate the effects of different strains of probiotic bacteria on the growth performance, digestive enzyme activity, histopathologic and histomorphometric characterization of the intestine, expression of immune and growth related genes, and evaluate Lates calcarifer resistance against Vibrio alginolyticus. To achieve this, for each treatment 60 L. calcarifer juveniles (75 ± 12 g) were randomly distributed in three fiberglass tanks (300 L) and fed for 45 days. The treatments were established as Diet 1 (control diet); L1 (diet with Lb. plantarum isolated 1); L2 (diet with Lb. plantarum isolated 2) with a bacterial concentration of 1 × 109 CFU/g. Nine fish from each treatment were sampled and examined, after euthanasia. The fish were placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium, with 3 treatments: The result showed differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed with L1 had higher (P < 0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, the digestive enzyme activities were higher (P < 0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor (EGF), and Transforming Growth Factor Beta (TGF-ß) genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 (76.2%) and L2 (80.95%) treatments than in the control (P < 0.05). These results indicate that host-derived probiotics (Lb. plantarum) have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of Lates calcarifer juveniles at 45 days of culture.

Climate challenges for fish larvae: Interactive multi-stressor effects impair acclimation potential of Atlantic herring larvae.

Fish early life stages are particularly vulnerable and heavily affected by changing environmental factors. The interactive effects of multiple climate change-related stressors on fish larvae remain, however, largely underexplored. As rising temperatures can increase the abundance and virulence of bacteria, we investigated the combination of a spring heat wave and bacterial exposure on the development of Atlantic herring larvae (Clupea harengus). Eggs and larvae of Western Baltic Spring-spawners were reared at a normal and high temperature ramp and exposed to Vibrio alginolyticus and V. anguillarum, respectively. Subsequently, mRNA and miRNA transcriptomes, microbiota composition, growth and survival were assessed. Both high temperature and V. alginolyticus exposure induced a major downregulation of gene expression likely impeding larval cell proliferation. In contrast, interactive effects of elevated temperature and V. alginolyticus resulted in minimal gene expression changes, indicating an impaired plastic response, which may cause cellular damage reducing survival in later larval stages. The heat wave alone or in combination with V. alginolyticus induced a notable shift in miRNA expression leading to the down- but also upregulation of predicted target genes. Moreover, both increased temperature and the Vibrio exposures significantly altered the larval microbiota composition, with warming reducing microbial richness and diversity. The outcomes of this study highlight the high sensitivity of herring early life stages towards multiple climate change-related stressors. Our results indicate that interactive effects of rapidly changing environmental factors may exceed the larval stress threshold impairing essential acclimation responses, which may contribute to the ongoing recruitment decline of Western Baltic Spring-Spawning herring.

Molecular characterization and gene expression of pattern recognition receptors in brown-marbled grouper (Epinephelus fuscoguttatus) fingerlings responding to vibriosis infection.

The pathogen recognition system involves receptors and genes that play a crucial role in activating innate immune response in brown-marbled grouper (Epinephelus fuscoguttatus) as a control agent against various infections including vibriosis. Here, we report the molecular cloning of partial open reading frames, sequences characterization, and expression profiles of Pattern Recognition Receptors (PRRs) in brown-marbled grouper. The PRRs, namely pglyrp5, tlr5, ctlD, and ctlE in brown-marbled grouper, possess conserved domains and showed shared evolutionary relationships with other fishes, humans, mammals, birds, reptilians, amphibians, and insects. In infection experiments, up to 50% mortality was found in brown-marbled grouper fingerlings infected with Vibrio alginolyticus compared to 27% mortality infected Vibrio parahaemolyticus and 100% survival of control groups. It is also demonstrated that all four PRRs had higher expression in samples infected with V. alginolyticus compared to V. parahaemolyticus. This PRRs gene expression analysis revealed that all four PRRs expressed rapidly at 4-h post-inoculation even though the Vibrio count was only detected earliest at 12-h post-inoculation in samples. The highest expression recorded was from V. alginolyticus inoculated fish spleen with up to 73-fold change for pglyrp5 gene, followed by 14 to 38-fold expression for the same treatment in spleen, head kidney, and blood samples for other PRRs, namely tlr5, ctlD, and ctlE genes. Meanwhile less than a 10% increase in expression of all four genes was detected in spleen, head kidney, and blood samples inoculated with V. parahaemolyticus. These findings indicated that pglyrp5, tlr5, ctlD, and ctlE play important roles in the early immune response to vibriosis infected, brown-marbled grouper fingerlings.

Quorum sensing positively regulates CPS-dependent Autographiviridae phage infection in Vibrio alginolyticus.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

Whole-genome DNA methylation profiling revealed epigenetic regulation of NF-κB signaling pathway involved in response to Vibrio alginolyticus infection in the Pacific oyster, Crassostrea gigas.

DNA methylation, an essential epigenetic alteration, is tightly linked to a variety of biological processes, such as immune response. To identify the epigenetic regulatory mechanism in Pacific oyster (Crassostrea gigas), whole-genome bisulfite sequencing (WGBS) was conducted on C. gigas at 0 h, 6 h, and 48 h after infection with Vibrio alginolyticus. At 6 h and 48 h, a total of 11,502 and 14,196 differentially methylated regions (DMRs) were identified (p<0.05, FDR<0.001) compared to 0 h, respectively. Gene ontology (GO) analysis showed that differentially methylated genes (DMGs) were significantly enriched in various biological pathways including immunity, cytoskeleton, epigenetic modification, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that transcription machinery (ko03021) is one of the most important pathways. Integrated transcriptome and methylome analyses allowed the identification of 167 and 379 DMG-related DEGs at 6 h and 48 h, respectively. These genes were significantly enriched in immune-related pathways, including nuclear factor kappa B (NF-κB) signaling pathway (ko04064) and tumor necrosis factor (TNF) signaling pathway (ko04668). Interestingly, it's observed that the NF-κB pathway could be activated jointly by TNF Receptor Associated Factor 2 (TRAF2) and Baculoviral IAP Repeat Containing 3 (BIRC3, the homolog of human BIRC2) which were regulated by DNA methylation in response to the challenge posed by V. alginolyticus infection. Through this study, we provided insightful information about the epigenetic regulation of immunity-related genes in the C. gigas, which will be valuable for the understanding of the innate immune system modulation and defense mechanism against bacterial infection in invertebrates.

Identification, functional characterization and immune response profiles of interleukin-10 in Nibea albiflora.

Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5'-UTR (untranslated region) of 350 bp, a 3'-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost.

The Ugd, a capsular polysaccharide synthesis protein, regulates the bacterial motility in Vibrio alginolyticus.

Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

Comparative proteome analysis revealed potential biomarkers and the underlying immune mechanisms in Vibrio-resistant hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

Vibrio alginolyticus is the causative agent of vibriosis, a common bacterial infection in grouper aquaculture that is associated with the development of haemorrhagic and non-haemorrhagic ulcerations on the fish. In the present study, comparative proteome analysis was performed on serum samples from Vibrio-resistant and Vibrio-susceptible grouper. Samples were analysed using high-throughput LC-MS/MS and identified 2770 unique peptides that corresponded to 344 proteins. Subsequent analysis identified 21 proteins that were significantly up-regulated in the resistant group compared to the control and the susceptible groups. Those proteins are associated with immunostimulatory effects, signalling and binding cascade, metabolism, and maintaining tissue integrity and physiological condition. Besides, potential protein biomarkers related to the immune system were identified, which could be associated with the disease-resistant phenotype. These data provide insights into the underlying immune mechanism of hybrid groupers upon Vibrio sp. infection.

Application of nZnO supported with nanoclay for improving shrimp immunity.

This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV-visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, ß-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.

Low-dose of formalin-inactivated Vibrio alginolyticus protects Crassostrea gigas from secondary infection and confers broad-spectrum Vibrio resistance on offspring.

An increasing number of evidences have shown that invertebrate taxa can be primed to produce immune memory to resist the secondary infection of pathogens, which was considered as a viable option to protect invertebrates from pathogens. In this work, we compared the protective effect of several different immune priming methods on the Vibrio alginolyticus secondary infection of the Crassostrea gigas. The results showed that C. gigas primed with live V. alginolyticus had higher ROS level, which led to hemocytes necrosis and higher mortality rate in the later stage. Low-dose of formalin-inactivated V. alginolyticus (including 5 × 104 CFU/mL and 5 × 105 CFU/mL) elicited appropriate immune response in C. gigas, protecting C. gigas from V. alginolyticus infection. Immersion with 5 × 104 CFU/mL formalin-inactivated V. alginolyticus was performed to prime C. gigas immunity in the trans-generational immune priming. Trans-generational immune priming significantly increased the resistance of larvae to various Vibrio species. Overall, these results suggested that low-dose of formalin-inactivated V. alginolyticus can protect C. gigas from secondary infection and confer broad-spectrum Vibrio resistance on offspring. This work provided valuable information toward a new direction for the protection of C. gigas from Vibrio infection.

Transcriptome analysis reveals tissue-specific responses of Mytilus unguiculatus to Vibrio alginolyticus infection.

Mytilus unguiculatus is an important economic bivalve species with wide consumption and aquaculture value. Disease is one of the primary limiting factors in mussel aquaculture, thus understanding the response of different tissues of M. unguiculatus to pathogens is crucial for disease prevention and control. In this study, we investigated the physiological and transcriptomic responses of the gills, adductor muscle, and mantle of M. unguiculatus infected with Vibrio alginolyticus. The results showed that V. alginolyticus infection caused inflammation and tissue structure changes in the gill, adductor muscle and mantle of M. unguiculatus. Meanwhile, the activities of superoxide dismutase and catalase in the three tissues increased, while the total antioxidant capacity decreased, suggesting that M. unguiculatus have an activated defense mechanism against infection-induced oxidative stress, despite a compromised total antioxidant capacity. Transcriptomic studies reveal that infected M. unguiculatus exhibits upregulation of endocytosis, lysosome activity, cellular apoptosis, and immune-related signaling pathways, indicating that M. unguiculatus responds to pathogen invasion by upregulating efferocytosis. Compared with the gill and adductor muscle, the mantle had a higher level of mytimycin, mytilin and myticin, and the three tissues also increased the expression of mytimycin to cope with the invasion of pathogens. In addition, the analysis of genes related to taste transduction pathways and muscle contraction and relaxation found that after infection with V. alginolyticus, M. unguiculatus may reduce appetite by inhibiting taste transduction in the gill, while improving muscle contraction of the adductor muscle and keeping the shell closed, to resist further invasion of pathogens and reduce the risk of pathogen transmission in the population.

Tyramine beta hydroxylase-mediated octopamine synthesis pathway in Litopenaeus vannamei under thermal, salinity, and Vibrio alginolyticus infection stress.

Stress responses impact the immune systems, growth, and reproduction of aquatic organisms. Neuroendocrine regulation involving biogenic amines, including octopamine (OA), plays a pivotal role in maintaining physiological balance during stress. This study focuses on the synthesis pathway of OA, particularly the role of tyramine beta hydroxylase (TBH), in Litopenaeus vannamei under stress. TBH catalyzes the conversion of tyramine to OA, a process critical for physiological responses. The present study demonstrated LvTBH at the protein level under different stress conditions during acute (0.5, 1, 2 h) and chronic stress (24, 72, 168 h) periods. LvTBH increased in thoracic ganglia within 2 h under hyperthermal stress, accompanied by elevated OA levels. Conversely, LvTBH decreased in the brain and circumesophageal connective tissues during acute and chronic hypothermal stress. Additionally, LvTBH increased in the brain and circumesophageal connective tissues under acute infection stress, coinciding with elevated OA levels. These findings collectively contribute to a more intricate understanding of the neuroendocrine dynamics within L. vannamei under stress, underscoring the role of TBH in orchestrating responses crucial for adaptation.

Physiological adaptation in flagellar architecture improves Vibrio alginolyticus chemotaxis in complex environments.

Bacteria navigate natural habitats with a wide range of mechanical properties, from the ocean to the digestive tract and soil, by rotating helical flagella like propellers. Species differ in the number, position, and shape of their flagella, but the adaptive value of these flagellar architectures is unclear. Many species traverse multiple types of environments, such as pathogens inside and outside a host. We investigate the hypothesis that flagellar architectures mediate environment-specific benefits in the marine pathogen Vibrio alginolyticus which exhibits physiological adaptation to the mechanical environment. In addition to its single polar flagellum, the bacterium produces lateral flagella in environments that differ mechanically from water. These are known to facilitate surface motility and attachment. We use high-throughput 3D bacterial tracking to quantify chemotactic performance of both flagellar architectures in three archetypes of mechanical environments relevant to the bacterium's native habitats: water, polymer solutions, and hydrogels. We reveal that lateral flagella impede chemotaxis in water by lowering the swimming speed but improve chemotaxis in both types of complex environments. Statistical trajectory analysis reveals two distinct underlying behavioral mechanisms: In viscous solutions of the polymer PVP K90, lateral flagella increase the swimming speed. In agar hydrogels, lateral flagella improve overall chemotactic performance, despite lowering the swimming speed, by preventing trapping in pores. Our findings show that lateral flagella are multipurpose tools with a wide range of applications beyond surfaces. They implicate flagellar architecture as a mediator of environment-specific benefits and point to a rich space of bacterial navigation behaviors in complex environments.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) negatively regulates the immunity and resistance to Vibrio alginolyticus of white shrimp, Penaeus vannamei.

Shrimp immunology is vital in establishing prophylactic and therapeutic strategies for controlling pathological problems that threaten shrimp production. Apart from dietary treatments, the adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulatory enzyme that restores cellular energy balance during metabolic and physiological stress, is known to have therapeutic potential to improve shrimp's defense mechanism. Despite this, studies targeting the AMPK pathway in shrimp exposed to stressful conditions are vastly limited. In this study, AMPK was knocked down to assess the immunological changes and white shrimp, Penaeus vannamei resistance to Vibrio alginolyticus infection. Shrimps were injected individually and simultaneously with dsRNA targeting specific genes such as AMPK, Rheb, and TOR, after which the hepatopancreas was analyzed for the different gene expressions. The gene expressions of AMPK, Rheb, and TOR were effectively suppressed after being treated with dsRNAs. The Western blot analysis further confirmed a reduction in the protein concentration of AMPK and Rheb in the hepatopancreas. The suppression of AMPK gene led to a robust increase in the shrimp's resistance to V. alginolyticus, whereas the activation of AMPK by metformin decreased the shrimp's disease resistance. Among the mTOR downstream targets, the HIF-1α expression in shrimp treated with dsAMPK significantly increased at 48 h but returned to normal levels when shrimp were treated with dsAMPK and either dsRheb or dsTOR. Immune responses such as respiratory burst, lysozyme activity, and phagocytic activity increased, while superoxide dismutase activity decreased following the knockdown of the AMPK gene compared to the control group. However, co-injection with dsAMPK and dsTOR or dsRheb restored immune responses to normal levels. Collectively, these results demonstrate that the inactivation of AMPK may ameliorate shrimp's innate immune response to recognize and defend against pathogens via the AMPK/mTOR1 pathway.

Identification and characterization of mitogen-activated protein kinase kinase 4 (MKK4) from the mud crab Scylla paramamosain in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV).

Mitogen-activated protein kinase kinase 4 (MKK4), serves as a critical component of the mitogen-activated protein kinase signaling pathway, facilitating the direct phosphorylation and activation of the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stresses. In the current research, we identified two MKK4 subtypes, namely SpMKK4-1 and SpMKK4-2, from Scylla paramamosain, followed by the analysis of their molecular characteristics and tissue distributions. The expression of SpMKK4s was induced upon WSSV and Vibrio alginolyticus challenges, and the bacteria clearance capacity and antimicrobial peptide (AMP) genes' expression upon bacterial infection were significantly decreased after knocking down SpMKK4s. Additionally, the overexpression of both SpMKK4s remarkably activated NF-κB reporter plasmid in HEK293T cells, suggesting the activation of the NF-κB signaling pathway. These results indicated the participation of SpMKK4s in the innate immunity of crabs, which shed light on a better understanding of the mechanisms through which MKK4s regulate innate immunity.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.