A nomogram for predicting hemorrhagic shock in pediatric patients with multiple trauma.

The timely detection and management of hemorrhagic shock hold paramount importance in clinical practice. This study was designed to establish a nomogram that may facilitate early identification of hemorrhagic shock in pediatric patients with multiple-trauma. A retrospective study was conducted utilizing a cohort comprising 325 pediatric patients diagnosed with multiple-trauma, who received treatment at the Children's Hospital, Zhejiang University School of Medicine, Zhejiang, China. For external validation, an additional cohort of 144 patients from a children's hospital in Taizhou was included. The model's predictor selection was optimized through the application of the Least Absolute Shrinkage and Selection Operator (LASSO) regression. Subsequently, a prediction nomogram was constructed using multivariable logistic regression analysis. The performance and clinical utility of the developed model were comprehensively assessed utilizing various statistical metrics, including Harrell's Concordance Index (C-index), receiver operating characteristic (ROC) curve analysis, calibration curve analysis, and decision curve analysis (DCA). Multivariate logistic regression analysis identified systolic blood pressure (ΔSBP), platelet count, activated partial thromboplastin time (APTT), and injury severity score (ISS) as independent predictors for hemorrhagic shock. The nomogram constructed using these predictors demonstrated robust predictive capabilities, as evidenced by an impressive area under the curve (AUC) value of 0.963. The model's goodness-of-fit was assessed using the Hosmer-Lemeshow test (χ2 = 10.023, P = 0.209). Furthermore, decision curve analysis revealed significantly improved net benefits with the model. External validation further confirmed the reliability of the proposed predictive nomogram. This study successfully developed a nomogram for predicting the occurrence of hemorrhagic shock in pediatric patients with multiple trauma. This nomogram may serve as an accurate and effective tool for timely and efficient management of children with multiple trauma.

Effects of Scallop Visceral Mass and Mantle as Dietary Supplements on the Growth, Immune Response and Intestinal Microflora of Juvenile Sea Cucumber Apostichopus japonicus.

Scallop visceral mass and mantle are aquatic byproducts and waste, but they have high contents of protein. In this study, scallop visceral mass and mantle were used as supplements in the diet of juvenile sea cucumber (A. japonicus) and their effects on the growth, fatty acid and amino acid compositions, the non-specific immune responses and the intestinal microflora of A. japonicus were investigated through a 40 d feeding experiment. The results showed that dietary supplementation of scallop visceral mass significantly accelerated the specific growth rate (SGR) of juvenile A. japonicus by 3 times within 20 days, and also raised the contents of ω-3 fatty acids including EPA and DHA and the ω-3/ω-6 ratio of the sea cucumber tissue, which is favorable to the health and commercial value of the sea cucumber. Furthermore, it was found that the supplementation of scallop visceral mass and mantle stimulated the expression of immune-related genes and enhanced the immune defense in A. japonicus. Scallop visceral mass and mantle supplementation also increased the microbial diversity and the abundance of beneficial microbes including Bifidobacteriaceae, Streptomycetaceae, Clostridiaceae and Rhizobiales in the gut of A. japonicus. This study reveals the beneficial effects of dietary supplementation of scallop visceral mass and mantle on the growth of juvenile A. japonicus, which might be a promising way to reutilize this scallop waste and raise its economic value.

Effect of antibiotic and/or heavy metal on nitrogen cycle of sediment-water interface in aquaculture system: Implications from sea cucumber culture.

Antibiotics and heavy metals can have a negative impact on the nitrogen (N) cycle and microbial metabolism in coastal aquaculture environment. An indoor simulated culture experiment was conducted to explore how sulfadiazine and lead influence the N cycling in aquatic environment. Specifically, the experiment involved adding sulfadiazine (SDZ), lead (Pb), a combination of SDZ and Pb (SP), and a control group (CK). The fluxes and contents of ammonia nitrogen (NH4+-N), nitrate nitrogen (NO3--N) and nitrite nitrogen (NO2--N) in sediment-water interface and sediments, the abundance of N cycle function genes (amoA_AOB, hzsA, nar, nirK, nirS, norB and nosZ) and microbiota in sediments were analyzed. The results showed that the presence of SDZ and Pb inhibited the nitrification function gene and nitrifiers abundance in surface sediment, and thus leading to more accumulation of NH4+ and NO2- in overlying water. Pb exposure increased the abundances of denitrifying bacteria stimulated the first three steps of denitrification in the sediment, resulting in more removal of NO3- from the sediment, but possibly had the risk of releasing more greenhouse gas N2O. Conversely, the presence of SDZ ultimately inhibited denitrification and anammox bacterial activities in the sediment. This study revealed how heavy metal and antibiotic impair the microbial communities and N cycling function gene expression, leading to the deterioration of typical coastal aquaculture environments.

Probiotics Mediate Intestinal Microbiome and Microbiota-Derived Metabolites Regulating the Growth and Immunity of Rainbow Trout (Oncorhynchus mykiss).

Emerging evidence confirms using probiotics in promoting growth and immunity of farmed fish. However, the molecular mechanisms underlying the host-microbiome interactions mediated by probiotics are not fully understood. In this study, we used rainbow trout (Oncorhynchus mykiss) as a model to investigate the internal mechanisms of host-microbiome interactions influenced by two probiotic bacteria, Bacillus velezensis and Lactobacillus sakei. We carried out experiments, including intestinal histology, serum physiology, and transcriptome and combined intestinal microbiome and metabolite profiling. Our results showed that both probiotics had a positive effect on growth, immunity, serum enzyme activity, the gut microbiome, and resistance to Aeromonas salmonicida in rainbow trout. Moreover, the intestinal microbial structure was reshaped with increased relative abundance of potential beneficial bacteria, such as Ruminococcus, Lachnospiraceae ucg-004, Leptotrichia, Bacillus coagulans, Porphyromonadaceae, Anaerococcus, and Photobacterium in the B. velezensis group and Paenibacillaceae and Eubacterium hallii in the L. sakei group. Metabolomic profiling and transcriptome analysis revealed upregulated metabolites as biomarkers, i.e., sucrose and l-malic acid in the B. velezensis group, and N-acetyl-l-phenylalanine, N-acetylneuraminic acid, and hydroxyproline in the L. sakei group. Additionally, a multiomics combined analysis illustrated significant positive correlations between the relative abundance of microflora, metabolites, and gene expression associated with immunity and growth. This study highlights the significant role of probiotics as effectors of intestinal microbial activity and shows that different probiotics can have a species-specific effect on the physiological regulation of the host. These findings contribute to a better understanding of the complex host-microbiome interactions in rainbow trout and may have implications for the use of probiotics in aquaculture. IMPORTANCE Probiotics are kinds of beneficial live microbes that impart beneficial effects on the host. Recent studies have proven that when given supplementation with probiotics, farmed fish showed improved disease prevention and growth promotion. However, the underlying metabolic functions regarding their involvement in regulating growth phenotypes, nutrient utilization, and immune response are not yet well understood in the aquaculture field. Given the active interactions between the gut microbiota and fish immune and growth performance, we conducted the supplementation experiments with the probiotics Bacillus velezensis and Lactobacillus sakei. The results showed that probiotics mediated intestinal microbiome- and microbiota-derived metabolites regulating the growth and immunity of fish, and different probiotics participated in the species-specific physiological regulation of the host. This study contributed to a better understanding of the functional interactions associated with host health and gut microbiota species.

Adverse effects of dietary virgin (nano)microplastics on growth performance, immune response, and resistance to ammonia stress and pathogen challenge in juvenile sea cucumber Apostichopus japonicus (Selenka).

It has been well documented that micro- and nanoplastics are emerging pollutants in aquatic environments, and their potential toxic effects has attracted widespread concerns. Here, we evaluated the adverse effects of dietary polystyrene nanoplastics and microplastics (PS-N/MPs) on growth performance, oxidative stress induction, immune response, ammonia detoxification, and bacterial pathogen resistance of sea cucumber Apostichopus japonicus. After collection and acclimation, sea cucumbers were randomized into 3 groups (i.e., control, 100 nm PS-NPs and 20 µm PS-MPs at 100 mg kg-1 diet) for 60-day feeding experiment. Every group contained 360 sea cucumbers which were equally divided into 3 aquaria as biological triplicates. The results showed that the specific growth rate and final weight of the sea cucumbers fed with diets containing PS-N/MPs were significantly lower than those of control group. Dietary virgin PS-N/MPs significantly increased the reactive oxygen species production and malondialdehyde content in coelomic fluid, causing oxidative stress and damage to the growth and development of A. japonicus. During the experiment, 100 nm PS-NPs significantly induced the depletion in cellular and humoral immune parameters. The calculated IBR values based on multi-level biomarkers revealed the size-dependent toxic differences of PS-NPs > PS-MPs. The relative expression levels of GDH and GS mRNA showed first rise and then fall trends after exposure to ammonia, and 100 nm PS-NPs had a more profound impact on suppressing ammonia detoxification compared with 20 µm PS-MPs. Moreover, the expression of Hsp90, Hsp70, CL, TLR, and CASP2 genes were all down-regulated by ammonia exposure. Taken together of IBR results, ammonia stress test and pathogen challenge, we deduced that dietary 100 nm PS-NPs are more potentially hazardous than 20 µm PS-MPs. These findings provide valuable information for understanding the size-dependent toxic effects of PS-N/MPs and early risk warning on marine invertebrates.

Distribution, behavior and budget of Pb in suspended particles in the Changjiang Estuary and adjacent east China sea.

Content, isotopes and budget of Pb in suspended particulate matter (SPM) in the Changjiang Estuary and adjacent East China Sea (ECS) were determined to investigate the biogeochemical cycling of particulate Pb in coastal sea. The content of particulate Pb ranged from 11.3 to 669.4 µg/g in February (winter) and from 20.1 to 79.4 µg/g in August (summer). Except in surface water, particulate Pb content in August is higher than that in February. In lower water, particulate Pb and Th and SPM all decreased gradually from the estuary towards the sea, indicating their lithogenic origin from the Changjiang River. Particulate Pb displayed abnormally high concentration in February surface water, resulting from the atmospheric deposition of anthropogenic Pb in winter. 208Pb/206Pb and 207Pb/206Pb in surface water north to the estuary were higher than background values, suggesting anthropogenic disturbance of Pb. Particulate Pb content in the Changjiang River and the estuary had increased by 77-78% from the 1980s to 2016 due to pollution. Pb was also scavenged by organic matter, leading to higher Pb content in waters with high productivity. Budget of particulate Pb in the northern ECS was established. The Changjiang River contributed 97.0% and 58.1% of particulate Pb input in summer and winter half year, respectively. 88.8% of particulate Pb was deposited in the estuary and adjacent coastal sea in summer but 88.7% was exported with southward coastal currents in winter.

Biosynthesis and Isotopic Routing of Dietary Protein by Sea Cucumber Apostichopus japonicus (Selenka): Evidence from Compound-Specific Carbon Stable Isotope Analysis.

Compound-specific stable isotope analysis of individual amino acids (AAs) has been widely used in studies on food webs, resource tracing, and biochemical cycling. In the present study, juvenile sea cucumbers Apostichopus japonicus were fed the microalga Cylindrotheca fusiformis (CF) or Sargassum thunbergii (ST) during a 130-day experiment. The δ13C values of individual AAs in the experimental diet and body wall of sea cucumbers were determined to calculate the variability in carbon isotopic fractionation (Δ13C) and elucidate the isotopic routing of essential AAs and biosynthesis of nonessential AAs. The results showed that the sea cucumbers fed with diet CF had higher specific growth and ingestion rates but relatively lower feed conversion efficiency compared to those fed with diet ST. The experimental diets were generally less abundant in nonessential AAs (i.e., glycine, serine, aspartic acid, and arginine) but more abundant in essential AAs (i.e., isoleucine, leucine, lysine, phenylalanine, and histidine) than body walls. The fluctuations in the δ13C values of total AAs analyzed were 19.8 ± 4.6‰ for diets and 21.3 ± 2.7‰ for body walls. Serine and threonine were 13C-enriched AAs, while leucine and phenylalanine were 13C-depleted AAs. The diet ST treatment exhibited more positive Δ13C values of nonessential AAs (e.g., glycine, alanine, aspartic acid, and proline) compared to diet CF. There were significant negative relationships between Δ13C values and differences in nonessential AA percent abundance between the experimental diets and body walls of sea cucumber (for diet CF: y = -0.79 - 0.56x, r2 = 0.47; diet ST: y = 0.75 - 0.29x, r2 = 0.51), which implied the flexibility in the routing of various dietary macronutrients (protein, lipids, and carbohydrates) by sea cucumber. This study can greatly provide a new understanding of nutrient utilization and metabolism routing during juvenile sea cucumber culturing.

Galectins in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues.

Galectins, a family of evolutionary conserved ß-galactoside-binding proteins, have been characterized in a wide range of species. Many reports have indicated vital roles of galectins in innate immunity, especially in the mucosal tissues against infection. However, the systematic identification of galectin gene family is still lacking in teleost. Here, we characterized the galectin gene family and investigated their expression profiles post bacterial challenge in turbot (Scophthalmus maximus L.). In this study, a total of 13 galectin genes were characterized in turbot, phylogenetic analyses revealed their strong relationships to half smooth tongue sole and puffer fish, and syntenic analyses confirmed the orthology suggested by the phylogenetic analysis. In addition, the copy number of galectin genes is similar across a broad spectrum of species from fish to amphibians, birds, and mammals, ranging from 8 to 16 genes. Furthermore, the galectin genes were widely expressed in all the examined turbot tissues, and most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after Vibrio anguillarum infection in the intestine, gill and skin, suggesting that galectins were involved in the mucosal immune response to V. anguillarum infection in turbot. In addition, subcellular localization analysis showed lgals3a was distributed in the cytoplasm and nucleus. However, the knowledge of galectins are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Effects of aerial exposure on oxidative stress, antioxidant and non-specific immune responses of juvenile sea cucumber Apostichopus japonicus under low temperature.

Desiccation is a commonly stressful situation experienced by sea cucumber during transportation without/less water. The present study was conducted to investigate the effects of aerial exposure on the survival, oxidative damage, antioxidant capacity, immune-related response and gene expression of Apostichopus japonicus at different low temperatures. After acclimation, sea cucumbers were randomly divided into 3 groups, which were exposed to 5 °C, 10 °C and 15 °C in the closed laboratory condition, respectively. Each group has three parallel replicates. During the experiment, coelomic fluid and respiratory tree of A. japonicus were sampled at the time points of 0, 3, 6, 12, 24 and 48 h post-desiccation for further analysis. The results showed that the survival rates of sea cucumber significantly decreased as time prolonged, and those of 5 °C at 6-48 h of desiccation were significantly higher than 15 °C. Most oxidative damage parameters (e.g., O2- production, MDA, LPO and PC contents) significant increased after 6-12 h of desiccation. Antioxidant enzyme activities and T-AOC in coelomic fluid firstly increased and then decreased during aerial exposure, indicating that sea cucumber could adjust antioxidant defense to reduce the concentrations of ROS and MDA as a strategy for protecting organisms from oxidative damage in the early stage (0-6 h) of desiccation. The relative expression levels of Hsp90 and Hsp70 mRNA in respiratory tree of sea cucumber exhibited similar rise-fall trends with antioxidant parameters, while immune enzyme activities of ACP, AKP, LSZ and T-NOS, and gene expression of TLR, Rel and p105 all significantly decreased as time prolonged. Overall, low temperature postponed the process of ROS formation and the depression of antioxidant and non-specific immune responses of sea cucumber within a certain extent, which implied that it might play a positive role in improvement of desiccation tolerance. This study not only contribute to better understand the adaption mechanisms of A. japonicus to desiccation stress, but also provide valuable information for sea cucumber culture and transportation.

Characterization of a novel lncRNA (SETD3-OT) in turbot (Scophthalmus maximus L.).

LncRNAs have been demonstrated to play pivotal roles in virous biological processes, especially the gene expression regulation, including transcriptional regulation, posttranscriptional control and epigenetic processes. However, most of the current studies of lncRNAs are still limited in mammalian species, the investigations of functional roles of lncRNAs in teleost species are still lacking. In current study, we identified a novel lncRNA (SETD3-OT) in turbot, with 2,504 bp full-length obtained by 5' and 3' RACE, located in turbot chromosome 17, ranged from 20,933,835 to 20,936,302 bp. In addition, 8 neighboring genes of SETD3-OT were identified within 100 kbp in genome location. From the annotation of the neighboring adjacent genes, SETD3-OT might involve in regulation of cell apoptosis and cycle, the immune cell development, and the immune response against infection, and its expression pattern is similar to majority of the neighboring genes following Aeromonas salmonicida challenge. Intriguingly, SETD3-OT showed significant high expression levels in mucosal surfaces (intestine, gill and skin), and was dramatically down-regulated in these mucosal tissues following Vibrio anguillarum challenge, especially in gill and skin. In addition, SETD3-OT was distributed in nucleus, it might regulate the neighboring genes in cis or in trans. Taken together, our results provide insights for lncRNA in fish innate immunity, further studies should be conduct to explore the detailed molecular mechanism of the gene regulation between SETD3-OT and its neighboring genes.

Characterization of class B scavenger receptor type 1 (SRB1) in turbot (Scophthalmus maximus L.).

Class B scavenger receptor type 1 (SRB1) serves as a high-density lipoprotein (HDL) receptor essential for HDL metabolism, and plays vital roles in innate immunity. In this study, the turbot (Scophthalmus maximus) SRB1 was cloned and characterized. The gene structure consists of a coding region of 1,527 bp nucleotides dividing into 13 exons and 12 introns. Such genome structure is highly conserved among teleost fishes. The deduced SRB1 encodes 508 amino acids that mainly has a CD36 transmembrane domain. Tissue distribution of SRB1 showed the lowest expression in liver, while the highest expression was found in intestine. Significantly down-regulation pattern of SmSRB1 expression in intestine was shared after infection with Vibrio anguillarum and Streptococcus iniae. Brach and site models in CODEML program showed that SmSRB1 underwent a conservative evolutionary and three potential positive selected sites 470K, 496E, and 501Y were detected, which requires further investigation and confirmation using base-editing technologies. Subcellular localization demonstrated that turbot SRB1 was distributed in the membrane and cytoplasm. rSmSRB1 showed binding ability in vitro to bacteria, LPS, PGN, LTA and virus. Protein-protein interaction network agrees the function of SRB1 as lipoprotein receptor. Our results indicated SmSRB1 might act as co-receptors to TLRs and NLRs to modulate the immune response to pathogens. Further studies should pay attention to evaluate the specific co-receptor for SRB1 in recognition of different pathogens and selective mechanisms involved.

The efficacy of lactic acid bacteria usage in turbot Scophthalmus maximus on intestinal microbiota and expression of the immune related genes.

To understand the efficacy of lactic acid bacteria (LAB) as probiotics on the growth, immune response and intestinal microbiota of turbot Scophthalmus maximus, in this study, the Leuconostoc mesenteroides HY2 strain screened from wide caught fish was bath administrated for juvenile turbot with no bacteria administrated as control. The mRNA levels of toll-like receptors 3 (TLR3), interleukin 8 (IL-8) and interferon induced with helicase C domain 1 (IFIH1) in different organs (i.e. intestine, liver, spleen, kidney, brain and skin) were analyzed using RT-PCR technology. The intestinal microbiota was analyzed by 16S rRNA sequencing, in which principal co-ordinates analysis (PCoA) as well as cluster analysis was performed. The results showed that the specific growth rate of turbot in the LAB treatment was significantly higher than those of the control group (P < 0.05). The expression levels of TLR3, IL-8 and IFIH1 were significantly up-regulated in the organs of LAB treatment, except that IL-8 was slightly down-regulated in kidney. A total of 42 phyla in intestinal microbiota were identified. The composition of intestinal microbiota showed significant differences between LAB treatment and the control group. Shannon index in the LAB treatment was significantly increased while Simpson index significantly declined. The PCoA and cluster analysis exhibited significant differences in the composition and abundance between the two groups. Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria acted as biomarkers which may have effects to promote absorption and/or trigger the immune function. In conclusion, the administration of HY2 strain was capable of improving growth performance of turbot by enhancing immune response and optimizing structure and diversity of intestinal microbiota.

Regulation of growth, intestinal microbiota, non-specific immune response and disease resistance of sea cucumber Apostichopus japonicus (Selenka) in biofloc systems.

Bioflocs are not only a source of supplemental nutrition but also provide substantial probiotic bacteria and bioactive compounds, which play an important role in improving physiological health of aquatic organisms. A 60-day experiment was conducted to investigate the growth, intestinal microbiota, non-specific immune response and disease resistance of sea cucumber in biofloc systems with different carbon sources (glucose, sucrose and starch). Control (no biofloc) and three biofloc systems were set up, and each group has three replicates. The results showed that biofloc volume (BFV) and total suspended solids (TSS) increased in the sequences of glucose > sucrose > starch and green sea cucumber > white sea cucumber during the experiment. The highest specific growth rates (SGRs) were observed in biofloc system with glucose as carbon source, which also had relatively lower glucose, lactate and cortisol levels in coelomic fluid and higher glycogen content in muscle compared to other groups. There were significant increased Bacillus and Lactobacillus counts of sea cucumber intestine in biofloc systems, and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) also showed obvious ascending trends. Significant increases in total coelomocytes counts (TCC), phagocytosis, respiratory burst, complement C3 content and lysozyme (LSZ) and acid phosphatase (ACP) activities of sea cucumber were all found in biofloc system (glucose). The expression patterns of most immune-related genes (i.e. Hsp90, Hsp70, c-type lectin (CL), toll-like receptor (TLR)) were up-regulated, suggesting the promotion of pathogen recognition ability and immune signaling pathways activation by biofloc. Furthermore, green and white sea cucumber had significantly higher survival rates in biofloc systems during the 14-day challenge test. In conclusion, biofloc technology could improve growth and physiological health of A. japonicus, by optimizing intestinal microbiota, strengthening antioxidant ability, enhancing non-specific immune response and disease resistance against pathogens, meanwhile glucose was recommended as optimal carbon source in biofloc system of sea cucumber culturing.

Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka.

A feeding experiment was conducted to evaluate the effects of four strains of lactic acid bacteria (LAB) [i.e. Lactobacillus plantarum LL11 (LP), Weissella confuse LS13 (WC), Lactococcus lactis LH8 (LL) and Enterococcus faecalis LC3 (ES)] isolated from marine fish on growth, immune response and expression levels of immune-related gens in body wall of juvenile sea cucumber Apostichopus japonicus. As a result, sea cucumber had better growth performance fed supplementation of LP and ES than the control group (P < .05). Survival rate in each LAB supplementation group was significantly higher than that in control group after Vibrio splendidus challenge (P < .05). In regards to the enzyme activities, LP supplementation significantly imporved the enzyme activities of alkaline phosphatase (AKP) (P < .05), acid phosphatase (ACP) and superoxide dismutase (SOD), ES supplementation significantly imporved AKP activity (P < .05), and WC supplementation significantly imporved ACP activity (P < .05). However, lysozyme (LSZ) activity was not significantly changed in the four LAB supplementation treatments (P > .05). For the gene expression levels, different expression patterns were observed among four groups, heat shock proteins (HSP60, HSP70 and HSP90) and caspase-2 showed dramatic up-regulation at 30 d while NF-kappa-B transcription factor p65 was down-regulated at 15 d and up-regulated at 30 d, and nitric oxide synthase was down-regulated at both timepoints in almost all the four groups. In conclusion, the four LAB strains screened from marine fish supplemented in diets indicated positive effects on immune response for A. japonicus, especially, the L. plantarum LL11 and E. faecalis LC3 indicated better growth performance.

Dietary supplementation of biofloc influences growth performance, physiological stress, antioxidant status and immune response of juvenile sea cucumber Apostichopus japonicus (Selenka).

Bioflocs are rich in various probiotics and bioactive compounds, which play an important role in improving growth and health status of aquatic organisms. A 60-day experiment was conducted to investigate the effects of dietary supplementation of biofloc on growth performance, digestive enzyme activity, physiological stress, antioxidant status, expression of immune-related genes and disease resistance of sea cucumber Apostichopus japonicus. Juvenile sea cucumbers were fed five experimental diets containing graded levels of biofloc from 0% to 20% (referred as B0, B5, B10, B15 and B20, respectively). The results showed that the sea cucumbers at dietary supplementation levels of 10%-15% biofloc had significantly higher specific growth rate (SGR) compared to control group (diet B0). Digestive enzyme activity increased with the increasing of dietary biofloc level, while no significant difference was found between diets B15 and B20. Dietary supplementation of biofloc also had significant influences on physiological stress parameters except for lactate. There was no significant discrepancy in total coelomocytes counts (TCC) in coelomic fluid of sea cucumber between the treatments. Phagocytosis and respiratory burst of cellular immune at 15% and 20% biofloc levels were significantly higher than those of control group. Significant increases in superoxide dismutase (SOD), total nitric oxide synthase (T-NOS), lysozyme (LSZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of sea cucumber were found at highest dietary supplementation level of 20% biofloc. The expression patterns of immune-related genes (i.e., Hsp90, Hsp70, p105, Rel, NOS and LSZ) in tissues of sea cucumber were analyzed between the experimental diets, and a general trend of up-regulation was observed at higher biofloc levels. Furthermore, dietary 10%-20% biofloc significantly reduced cumulative mortality of sea cucumber after being challenged with Vibrio splendidus. In conclusion, dietary supplementation of biofloc could improve growth performance of A. japonicus, by increasing digestive enzyme activity, releasing physiological stress, enhancing immune response and disease resistance of sea cucumber. The suitable supplemental level of approximately 15% biofloc was recommended in the present study.

Toxic effects in juvenile sea cucumber Apostichopus japonicas (Selenka) exposure to benzo[a]pyrene.

To understand the physiological response of sea cucumber, Apostichopus japonicas, were exposed to different concentration of benzo[a]pyrene (B[a]P), and the growth, survival, antioxidant enzyme (SOD and T-AOC) activities were tested. Meanwhile, the quantitative real-time PCR technology was utilized to quantize the expression of immune related genes (i.e. innate immune genes, HSP genes and anti-oxidative genes). In our result, the SOD activity and T-AOC activity were induced at lower level of B[a]P (0.03 µg/L), however, a reduction of SOD activity and T-AOC activity were observed at relatively high B[a]P concentration (3 and 9 µg/L) for A. japonicas. Furthermore, the distinct expression patterns of selected immune-related genes were detected among different concentrations, and a general trend of down-regulation was observed at higher concentration. Especially, lysozyme almost showed the highest down-regulation at all concentrations, followed by NOS. Collectively, the growth, survival and expression signatures of immune related genes reflected an overall suppression of innate immunity in sea cucumber following exposure. Future functional studies should be carried out to characterize the detailed roles of immune genes and their related responses under B[a]P toxicity. Additionally, better understanding of the molecular indicators governing the healthy status under environmental toxicity would facilitate a healthy and sustainable culture program.

Transcriptomic profiling revealed the signatures of intestinal barrier alteration and pathogen entry in turbot (Scophthalmus maximus) following Vibrio anguillarum challenge.

The mucosal immune system serves as the frontline barriers of host defense against pathogen infection, especially for the fishes, which are living in the pathogen rich aquatic environment. The intestine constitutes the largest surface body area in constantly contact with the external pathogens, and plays a vital role in the immune defense against inflammation and pathogen infection. Previous studies have revealed that fish intestine might serves as the portal of entry for Vibrio anguillarum. To characterize the immune actors and their associated immune activities in turbot intestine barrier during bacterial infection, here we examined the gene expression profiles of turbot intestine at three time points following experimental infection with V. anguillarum utilizing RNA-seq technology. A total of 122 million reads were assembled into 183,101 contigs with an average length of 1151 bp and the N50 size of 2302 bp. Analysis of differential gene expression between control and infected samples at 1 h, 4 h, and 12 h revealed 2079 significantly expressed genes. Enrichment and pathway analysis of the differentially expressed genes showed the centrality of the pathogen attachment and recognition, antioxidant/apoptosis, mucus barrier modification and immune activation/inflammation in the pathogen entry and host immune responses. The present study reported the novel gene expression patterns in turbot mucosal immunity, which were overlooked in previous studies. Our results can help to understand the mechanisms of turbot host defense, and may also provide foundation to identify the biomarkers for future selection of disease-resistant broodstock and evaluation of disease prevention and treatment options.

Identification and expression analysis of TLR2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of the bacteria, play key roles in the mucosal surfaces for pathogen recognition and activation of immune signaling pathways. However, our understanding of the PRRs and their activities in mucosal surfaces in the critical early time points during pathogen infection is still limited. Towards to this end, here, we sought to identify the Toll-like receptor 2 (TLR2) in turbot as well as its expression profiles in mucosal barriers following bacterial infection in the early time points. The full-length TLR2 transcript consists of open reading frame (ORF) of 2451 bp encoding the putative peptide of 816 amino acids. The phylogenetic analysis revealed the turbot TLR2 showed the closest relationship to Paralichthys olivaceus. The TLR2 mRNA expression could be detected in all examined tissues, with the most abundant expression level in liver, and the lowest expression level in skin. In addition, TLR2 showed different expression patterns following Vibrio anguillarum and Streptococcus iniae infection, but was up-regulated following both challenge, especially post S. iniae challenge. Characterization of TLR2 will probably contribute to understanding of a number of infectious diseases and broaden the knowledge of interactions between host and pathogen, which will eventually help in the development of novel intervention strategies for farming turbot.

The mucosal expression signatures of g-type lysozyme in turbot (Scophthalmus maximus) following bacterial challenge.

The mucosal surfaces constitute the first line of host defense against infection, and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes, while in constantly contact with a wide range of pathogens. The lysozymes are considered as key components for innate immune response to pathogen infection with their strong antibacterial activities. But their activities in mucosal immune responses were always overlooked, especially for g-type lysozymes, whose expression patterns in mucosal tissues following bacterial challenge are still limited. Towards to this end, here, we characterized the g-type lysozymes, Lyg1 and Lyg2 in turbot, and determined their expression patterns in mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot g-type lysozyme genes showed the closest relationship to Cynoglossus semilaevis. The two lysozyme genes showed different expression patterns following challenge. Lyg2 was significantly up-regulated in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge, while Lyg1 showed a general trend of down-regulation. The significant mucosal expression signatures of g-type lysozyme genes indicated their key roles to prevent pathogen attachment and entry in the first line of host defense system. Further functional studies should be carried out to better characterize the availability of utilization of g-type lysozyme to increase the disease resistance in the mucosal surfaces and facilitate the disease resistant breeding selection.

Expression profiling analysis of immune-related genes in channel catfish (Ictalurus punctatus) skin mucus following Flavobacterium columnare challenge.

Fish are covered by a watery gel-mucus, mainly secreted by the goblet cells, serving as the physical and biochemical barrier between the external environment and the interior milieu, playing more important roles in fish that without scale. Despite the important roles of mucus in fish immunity, the knowledge of detailed molecular events happened during infection process is still limited. While most studies were focused on characterizing the protein and enzyme activities in the mucus following challenge, no studies have examined the gene expression profiles in fish mucus. In this regard, herein we carried out the first gene profiling analysis in catfish mucus using real-time PCR. Ten important immune-related genes were selected according to our previous studies. Their expression levels were examined in the early timepoints (namely, 1 h, 2 h, 4 h, 8 h, and 24 h) following Flavobacterium columnare challenge. Notably, expression levels of most of the selected genes were rapidly altered by the challenge. Seven genes were down-regulated, while only three genes were up-regulated. In addition, the gene expression patterns in mucus were very different from the mucosal surfaces (skin, gill and intestine) and the classical immune organs (liver, spleen and kidney). The unique expression patterns obtained here may be resulted from the great advantage of the large amount of attached bacteria in the mucus than the internal tissues, and resulted from the bacteria virulent actors to suppress the host immune response. Taken together, our results can expand our knowledge of fish mucosal immunity, and the un-lethal mucus sampling can provide early insight for developing the strategies for selection of disease resistant families and strains in catfish as well as other fish species.