Xian Li

Proteomic analysis in kidneys of Atlantic salmon infected with Aeromonas salmonicida by iTRAQ

Abstract Aeromonas salmonicida is a major etiologic agent which induces furunculosis and is globally harmful in salmonid and turbot cultures, especially in Atlantic salmon (Salmo salar) farming. In order to improve knowledge of its poorly understood pathogenesis, we utilized high‐throughput proteomics to display differentially expressed proteins in the kidney of Atlantic salmon challenged with high and low infection dose of A. salmonicida at 7 and 14 days. In quantitative proteomic assays, isobaric tags for relative and absolute quantitation (iTRAQ) combined with 2D LC‐MS/MS is emerging as a powerful methodology in the search for disease‐specific targets and biomarkers. In this study, 4009 distinct proteins (unused ≥ 1.3, which is a confidence ≥ 95%) were identified in three two‐dimensional LC/MS/MS analyses. Then we chose 140 proteins (fold change ratio ≥ 1.5 and P < 0.01) combined with protein–protein interaction analysis to ultimately obtain 39 proteins in network which could be considered as potential biomarkers for Atlantic salmon immune responses. Nine significant differentially expressed proteins were consistent with those at the proteomic level used to validate genes at the transcriptomic level by qPCR. Collectively, these data was first reported using an iTRAQ approach to provide additional elements for consideration in the pathophysiology of A. salmonicida and pave the way to resolve the influence of this disease in Atlantic salmon. Graphical abstract Figure. No Caption available. HighlightsIt’s first time by using iTRAQ to identify diferentially expressed proteins in the kidney of Atlantic salmon.A total of 4009 proteins were identified by the international standardized gene functional classification system of GO.2323 proteins were sub‐categorized into 24 COG classifications.2552 proteins were sub‐categorized into 264 KEGG classifications.39 significant differential expressed proteins were grouped into 13 major categories by protein‐protein network analysis.The results of our work provide a list of new protein targets that could be used to further explore disease mechanisms of A. salmonicida.

Nutrients removal and substrate enzyme activities in vertical subsurface flow constructed wetlands for mariculture wastewater treatment: Effects of ammonia nitrogen loading rates and salinity levels

This study aims to investigate the effects of ammonia nitrogen loading rates and salinity levels on nutrients removal rates and substrate enzyme activities of constructed wetland (CW) microcosms planted with Salicornia bigelovii treating mariculture wastewater. Activities of urease (UA), dehydrogenase (DA), protease (PrA) and phosphatase (PA) were considered. Using principal component analysis (PCA), nutrient removal index (NRI) and enzyme activity index (EAI) were developed to evaluate the effects. The results revealed that increasing ammonia nitrogen loading rates had positive effects on nitrogen removal rates (i.e. NH4-N and DIN) and enhanced substrate enzyme activities. Compared with low salinity (i.e. 15 and 22), high salinity levels (i.e. 29 and 36) enhanced nutrients removal rates, DA and UA, but weaken PA and PrA. In conclusion, CW microcosms with Salicornia bigelovii can be used for the removal of nutrients under a range of ammonia nitrogen loadings and high salinity levels.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L)

ABSTRACT Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co‐operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer‐2 (AI‐2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold‐water teleost, little information has been known about the function of AI‐2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI‐2 activity and luxS defective mutant strain fail to produce AI‐2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with &Dgr;luxS strain. Overall, we hypothesis that AI‐2 quorum sensing could regulate the expression of A‐layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida. HIGHLIGHTSAI‐2 activity exists in A. salmonicida and correlates to the expression of luxS.LuxS plays a dual role in cellular metabolism and virulent factors expression.LuxS deficiency affects bacterial survival ability during its infection to Atlantic salmon.

Comparison of time-restricted and ad libitum self-feeding on the growth, feeding behavior and daily digestive enzyme profiles of Atlantic salmon

Although it has been hypothesized that a predictable feeding regime in animals allows physiological variables to be adjusted to maximize nutrient utilization and, hence, better growth performance, the assumption has rarely been tested. This study compares the effects of time-restricted versus free access self-feeding on the growth, feeding behavior and daily digestive enzyme rhythms of Atlantic salmon (Salmo salar). In an experiment that lasted 6 weeks, fish (109.9 g) were divided into two groups: group 1 had free access to a self-feeder (FA); group 2 received three meals per day (2 h per meal) at dawn, midday and dusk via a time-restricted self-feeder (TR). At the end of the experiment, the fish were sampled every 3 h over a 24-h period. The results showed that the TR fish quickly synchronized their feeding behavior to the feeding window and their blood glucose showed a significant postprandial increase, while FA fish displayed no statistically significant rhythms (P>0.05). Pepsin activity of TR fish also showed a significant daily rhythm (P<0.05) with the acrophase at the second feed and a decrease over the next 12 h. Average daily trypsin, lipase and amylase levels of FA fish were significantly lower than those of TR fish (P<0.01); however, the growth performance of both groups was similar (P>0.05). In conclusion, the study failed to confirm a link between the entrainment of daily digestive enzyme profiles and growth performance, with the TR group showing comparatively poor blood glucose regulation.

Colour preferences of juvenile turbot (Scophthalmus maximus)

The background colour of aquaculture tanks is normally chosen based on practical experience and/or observations of fish behaviour and the growth rates achieved. However, some farmed species, including turbot, are sentient and can show a preference for a particular environment. In the current study, a self-referent colour preference device was developed and the self-referent colour preference of farmed fish investigated. In experiment 1, the background colour preference of juvenile turbot cultured under a grey background for >3months post-incubation was evaluated. Based on these results, in experiment 2, juvenile turbot were adapted to blue, pink, white, or black backgrounds for 50days and their preferences established. Meanwhile, the growth rates, feed intake, and metabolic rates (including oxygen consumption rate, and ammonia excretion rate) of the turbot were evaluated. The results showed that turbot farmed under a grey background, or after long-term white, blue, pink and black colour adaptation, always displayed a preference for a white background and a dislike for black, red, or brown backgrounds, although their body colour was greyish. Long-term adaptation influenced the frequency of juveniles selecting white, black, pink or blue backgrounds. They showed the highest growth rate, feed intake, and metabolic rates under blue and white backgrounds, and the lowest under a black background in accordance with their preferences shown in experiment 1. Although it is unclear how turbot determine their self-referent colour preferences over such a short period of time, these results indicate that dark colours are unsuitable for the aquaculture of turbot culture in terms of the welfare of the fish.

A preliminary stochastic model for managing microorganisms in a recirculating aquaculture system

Predicting the growth of key microorganisms is essential to improve the efficiency of wastewater treatment of recirculating aquaculture systems (RAS). We have developed a stochastic model to assess quantitatively the microbial populations in RAS. This stochastic model encompassed the growth model into the Monte Carlo simulation and was constructed with risk analysis software. A modified logistic model combined with the saturation growth-rate model was successfully developed to regress the growth curves of six microorganisms. Monte Carlo simulation was employed to model the effects of chemical oxygen demand (COD) on the maximum specific growth rate. Probabilistic distributions and predictions under the different COD ranges were generated for each simulated scenario. The coefficient of determination (R2) and bias factor (Bf) were used to assess the performance of an established model. Logistic model produced a good fit to the growth curve of Flavobacterium sp. (R2u2009=u20090.9511), Acinetobacter baumannii (R2u2009=u20090.9970), Sphingomonas paucimobilis (R2u2009=u20090.9086), Vibrio natriegens (R2u2009=u20090.9993), Lutimonas sp. (R2u2009=u20090.9872) and Bacillus pumilus (R2u2009=u20090.9816). Bacterial population structure was determined by the construction of 16S rRNA gene libraries. A regular variation trend was observed for the dominant groups during the entire process, with a decrease of Cytophaga–Flavobacterium–Bacteroidetes from 37.6 to 18.7xa0% and an increase in Gammaproteobacteria from 8.5 to 30.6xa0%. The predicted model agreed well with observed values except for Flavobacterium sp., and the results can be applied to predict key microorganisms in actual environments. The results of this study provide a method to monitor the dynamics of key microorganisms, which can also help to evaluate the impacts of microorganisms on the operations of RAS.

The effects of feeding Lactobacillus pentosus on growth, immunity, and disease resistance in Haliotis discus hannai Ino

ABSTRACT To study the effects of probiotic‐added food on the survival and growth of abalone (Haliotis discus hannai Ino), the expression levels of nonspecific immune genes and the anti‐Vibrio parahaemolyticus infection were examined. During an 8‐week culturing experiment in an indoor aquarium and a 2‐week V. parahaemolyticus artificial infection experiment, the control group was fed with untreated food once a day, while the experimental groups (L1, L2 and L3) were fed with Lactobacillus pentosus added food. The concentration of probiotics in the experimental food was 103cfu/g (L1), 105cfu/g (L2) and 107cfu/g (L3), respectively. The results showed that the survival rate, shell length‐specific growth rate, and the food conversion rate (FCR) of abalones in L1 and L2 were significantly higher than the control group. The food intake of abalones in L3 was significantly lower than that in L1, L2 and the control group, but there was no significant difference in FCR identified between L1, L2 and L3. In the L. pentosus‐added groups, the total number of blood lymphocytes, lysozyme activity, acid phosphatase, superoxide dismutase, and expression levels of Mn‐superoxide dismutase (Mn‐SOD) and thioredoxin peroxidase (TPx) were significantly higher than the control group, while the malondialdehyde (MDA) content was significantly lower than the control group. The phagocytic activity of blood lymphocytes, catalase activity and the expression levels of heat shock protein 70 (HSP70) of abalones in the control group were significantly lower than that in L1 and L2, but there was no significant difference when compared with L3. The levels of O2−, NO produced by respiratory burst of blood lymphocytes and the expression levels of catalase (CAT) in L1 and L2 were significantly higher than both L3 and the control group. Seven days after infection with V. parahaemolyticus, all abalones in the control group were dead. After 14 days the cumulative mortality rate of abalones in the L. pentosus‐added groups was significantly lower than that in the control group. Therefore, the 103cfu/g and 105cfu/g L. pentosus‐added food not only promoted food intake and growth of abalones, but also improved their non‐specific immunity and reduced V. parahaemolyticus infection, indicating that this strain is a good potential candidate for probiotic added food in the aquaculture industry. HIGHLIGHTSThree different concentrations of L. pentosus were added to the abalones basal feed.Dietary supplementation with L. pentosus for 8 weeks enhanced abalones growth.Dietary L. pentosus at 103cfu/g and 105cfu/g significantly improved immunity in abalones.L. pentosus enhanced disease resistance to Vibrio parahemolyticus in abalones.

Intestinal microbiota of healthy and unhealthy Atlantic salmon Salmo salar L. in a recirculating aquaculture system

The present study sampled the intestinal content of healthy and unhealthy Atlantic salmon (Salmo salar L.), the ambient water of unhealthy fish, and the biofilter material in the recirculating aquaculture system (RAS) to understand differences in the intestinal microbiota. The V4–V5 regions of the prokaryotic 16S rRNA genes in the samples were analyzed by MiSeq high-throughput sequencing. The fish were adults with no differences in body length or weight. Representative members of the intestinal microbiota were identified. The intestinal microbiota of the healthy fish included Proteobacteria (44.33%), Actinobacteria (17.89%), Bacteroidetes (15.25%), and Firmicutes (9.11%), among which the families Micrococcaceae and Oxalobacteraceae and genera Sphingomonas, Streptomyces, Pedobacter, Janthinobacterium, Burkholderia, and Balneimonas were most abundant. Proteobacteria (70.46%), Bacteroidetes (7.59%), and Firmicutes (7.55%) dominated the microbiota of unhealthy fish, and Chloroflexi (2.71%), and Aliivibrio and Vibrio as well as genera in the family Aeromonadaceae were most strongly represented. Overall, the intestinal hindgut microbiota differed between healthy and unhealthy fish. This study offers a useful tool for monitoring the health status of fish and for screening the utility of probiotics by studying the intestinal microbiota.

The response and osmotic pressure regulation mechanism of Haliotis discus hannai (Mollusca, Gastropoda) to sudden salinity changes

Salinity is one of the critical ecological factors which will impact the growth and development of marine shellfish. With the rapid expansion of aquaculture area for Haliotis discus hannai, the frequent summer rainstorms in South China, the influx of freshwater, or the strong volatility of seawater in coastal areas, inner bays have placed abalone into the dynamic environment where the salinity is changing drastically. This work examined the effects of sudden salinity changes on abalone’s survival, osmotic pressure regulation, energy metabolism, and related gene expression by simulating the salinity changes of water for breeding H. discus hannai caused by heavy storm. The salinity was gradually reduced from 30 to 20, then kept at 20 for 48xa0h, followed by gradual increase to 30, and was kept at 30 for 48xa0h. Samples were taken at 6, 12, 36, 60, 66, 72, 96, and 120xa0h after the start of the experiment, respectively. Results showed that the survival rate of abalone at 120xa0h was significantly lower than that at any other time except at 96xa0h (Pxa0<xa00.05). With the decrease and increase of salinity, the hemolymph osmotic pressure and the concentration of Na+, K+, Ca2+, Cl− in the hemolymph also followed the same trend, while the concentration of hemocyanin, total soluble protein, taurine, and free amino acids showed an inverse trend. The activity of Na+/K+-ATPase also increased then declined with salinity changes. Except at 0, 6, and 12xa0h, the activity of Na+/K+-ATPase in the salinity-changing group was significantly higher than that in the control group (Pxa0<xa00.05). In the salinity-changing group, the activity of pyruvate kinase, succinate dehydrogenase, and malate dehydrogenase reached a maximum at 72xa0h, but no significant difference was found at the end of the experiment compared to the control group (Pxa0>xa00.05). The expression levels of catalase, thioredoxin peroxidase, sigma-glutathione-s-transferase, and Mu-glutathione-s-transferase significantly rose with the salinity changes, and were significantly higher than that in the control group up to the end of the experiment (Pxa0<xa00.05). As sudden salinity changes may cause some abalone deaths, the enhanced activity of related enzymes and the increase of gene expression levels might be one of the effective methods for an organism to respond to salinity stress and regulate osmotic pressure.

Characterization of Microbial Communities in Pilot-Scale Constructed Wetlands with Salicornia for Treatment of Marine Aquaculture Effluents

Microorganisms play an essential role in the performance of constructed wetlands (CWs) for wastewater treatment. However, there has been limited discussion on the characteristics of microbial communities in CWs for treatment of effluents from marine recirculating aquaculture systems (RAS). This study is aimed at characterizing the microbial communities of pilot-scale CWs with Salicornia bigelovii for treatment of saline wastewater from a land-based Atlantic salmon RAS plant located in Northern China. Illumina high-throughput sequencing was employed to identify the profile of microbial communities of three CWs receiving wastewater under different total ammonia nitrogen (TAN) concentrations. Results of this study showed remarkable spatial variations in diversity and composition of microbial communities between roots and substrates in three CWs, with distinct response to different TAN concentrations. In particular, Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were predominant in roots, while Cyanobacteria, Proteobacteria, Firmicutes, Verrucomicrobia, and Bacteroidetes were prevalent in substrates. Moreover, redundancy analysis indicated that specific functional genera, such as Nitrosopumilus, Vibrio, Pseudoalteromonas, Nitrospina, and Planctomyces, played key roles in the removal of nitrogen/phosphorus pollutants and growth of wetland plants. From a microorganism perspective, the findings of this study could contribute to better understanding of contaminants removal mechanism and improved management of CWs for treatment of effluents from land-based marine aquaculture.