Identifying Potential Polymicrobial Pathogens: Moving Beyond Differential Abundance to Driver Taxa.

It is now recognized that some diseases of aquatic animals are attributed to polymicrobial pathogens infection. Thus, the traditional view of "one pathogen, one disease" might mislead the identification of multiple pathogens, which in turn impedes the design of probiotics. To address this gap, we explored polymicrobial pathogens based on the origin and timing of increased abundance over shrimp white feces syndrome (WFS) progression. OTU70848 Vibrio fluvialis, OTU35090 V. coralliilyticus, and OTU28721 V. tubiashii were identified as the primary colonizers, whose abundances increased only in individuals that eventually showed disease signs but were stable in healthy subjects over the same timeframe. Notably, the random Forest model revealed that the profiles of the three primary colonizers contributed an overall 91.4% of diagnosing accuracy of shrimp health status. Additionally, NetShift analysis quantified that the three primary colonizers were important "drivers" in the gut microbiotas from healthy to WFS shrimp. For these reasons, the primary colonizers were potential pathogens that contributed to the exacerbation of WFS. By this logic, we further identified a few "drivers" commensals in healthy individuals, such as OUT50531 Demequina sediminicola and OTU_74495 Ruegeria lacuscaerulensis, which directly antagonized the three primary colonizers. The predicted functional pathways involved in energy metabolism, genetic information processing, terpenoids and polyketides metabolism, lipid and amino acid metabolism significantly decreased in diseased shrimp compared with those in healthy cohorts, in concordant with the knowledge that the attenuations of these functional pathways increase shrimp sensitivity to pathogen infection. Collectively, we provide an ecological framework for inferring polymicrobial pathogens and designing antagonized probiotics by quantifying their changed "driver" feature that intimately links shrimp WFS progression. This approach might generalize to the exploring disease etiology for other aquatic animals.

Comparable Ecological Processes Govern the Temporal Succession of Gut Bacteria and Microeukaryotes as Shrimp Aged.

Understanding the rules that govern the successions of gut microbiota is prerequisite for testing general ecological theories and sustaining a desirable microbiota. However, the ignorance of microeukaryotes raises the question of whether gut microeukaryotes are assembled according to the same rules as bacteria. We tracked the shrimp gut bacterial and microeukaryotic communities by a longitudinal dense sampling. The successions of both domains were significantly correlated with host age, with relatively stable microeukaryotic communities in adult shrimp. Gut microeukaryotes exhibited significantly higher turnover rate, but fewer transient species, lower proportion of temporal generalists, and narrower habitat niche breadth than bacteria. The γ-diversity partitioning analysis revealed that the successions of gut microbiotas were primarily ascribed to the high dissimilarity as shrimp aged ([Formula: see text]IntraTimes), whereas the relative importance of [Formula: see text]IntraTimes was significantly higher for microeukaryotes than that for bacteria. Compared with contrasting ecological processes in governing free-living bacteria and microeukaryotes, the ecological patterns were comparable between host-associated gut counterparts. However, the gut microeukaryotes were governed more strongly by deterministic selection relative to nestedness compared with the gut bacteria, which supports the "size-plasticity" hypothesis. Our results highlight the importance of independently interpreting free-living and host-associated meta-communities for a comprehensive understanding of the processes that govern microbial successions.

Spatiotemporal successions of shrimp gut microbial colonization: high consistency despite distinct species pool.

Aquatic animals encounter suites of novel planktonic microbes during their development. Although hosts have been shown to exert strong selection on their gut microbiota from surrounding environment, to what extent and the generality that the gut microbiota and the underlying ecological processes are affected by biotic and abiotic variations are largely unclear. Here, these concerns were explored by coupling spatiotemporal data on gut and rearing water bacterial communities with environmental variables over shrimp life stages at spatially distant locations. Shrimp gut microbiotas significantly changed mirroring their development, as evidenced by gut bacterial signatures of shrimp life stage contributing 95.5% stratification accuracy. Shrimp sourced little (2.6%-15.8%) of their gut microbiota from their rearing water. This microbial resistance was reflected by weak compositional differences between shrimp farming spatially distinct locations where species pools were distinct. Consistently, the assembly of shrimp gut microbiota was not adequately explained by the rearing water variables and bacterial community, but rather by host-age-associated biotic features. The successions of shrimp gut microbiota were droved by replacement (ßsim), rather than by nestedness (ßnes), while those of bacterioplankton communities were equally governed by replacement and nestedness. Our study highlights how shrimp gut bacterial community assembly is coupled to their development, rearing species pool, and that the successional pattern of host-associated communities is differed from that of free-living bacteria.

Quantitative PCR Analysis of Gut Disease-Discriminatory Phyla for Determining Shrimp Disease Incidence.

There is evidence that gut microbial signatures are indicative of host health status. However, few efforts have been devoted to establishing an applicable technique for determining disease incidence by using gut microbial signatures. Herein, we established a quantitative PCR (qPCR)-based approach to detect the relative abundances of gut disease-discriminatory phyla, which in turn afforded independent variables for quantitatively determining the incidence of shrimp disease. Given the temporal dynamics of gut bacterial communities as healthy shrimp aged, we identified disease-discriminatory phyla after ruling out age-discriminatory phyla. The top 10 disease-discriminatory phyla contributed to an overall 93.2% accuracy in diagnosis (n = 103 samples from shrimp that were determined with high confidence to be healthy or that exhibited apparent disease symptoms and subsequent death), with 70% diagnosis accuracy at the disease onset stage, when symptoms or signs of disease were not apparent. 16S rRNA gene-targeted group-specific primers of five disease-discriminatory phyla were then designed according to their compositions within shrimp gut microbiota, and other primers were borrowed from previous studies. The relative abundances of the 10 disease-discriminatory phyla assayed by qPCR exhibited a high consistency (r = 0.946, P < 0.001) with those detected by Illumina sequencing. Notably, using the profiles of disease-discriminatory phyla assayed by qPCR and the corresponding weight coefficients as independent variables, we were able to accurately estimate the incidences of future disease outcome. This work establishes an applicable technique to quantitatively determine the incidence and onset of shrimp disease, which is a valuable attempt to translate scientific research into a practical application.IMPORTANCE Current studies have identified gut microbial signatures of host health using high-throughput sequencing (HTS) techniques. However, HTS is still expensive and time-consuming and requires a high technical ability, thereby impeding its application in routine monitoring in aquaculture. Hence, it is necessary to seek an alternative strategy to overcome these shortcomings. Herein, we establish a qPCR-based approach to detect the relative abundances of gut disease-discriminatory phyla, which in turn afford independent variables to quantitatively determine the incidence and onset of shrimp disease. Notably, there is a high consistency between the accuracies of disease diagnosis achieved by qPCR and HTS. This applicable technique makes important progress toward defining a diseased state in shrimp and toward solving an important animal health management-driven economic problem.

Response of host-bacterial colonization in shrimp to developmental stage, environment and disease.

The host-associated microbiota is increasingly recognized to facilitate host fitness, but the understanding of the underlying ecological processes that govern the host-bacterial colonization over development and, particularly, under disease remains scarce. Here, we tracked the gut microbiota of shrimp over developmental stages and in response to disease. The stage-specific gut microbiotas contributed parallel changes to the predicted functions, while shrimp disease decoupled this intimate association. After ruling out the age-discriminatory taxa, we identified key features indicative of shrimp health status. Structural equation modelling revealed that variations in rearing water led to significant changes in bacterioplankton communities, which subsequently affected the shrimp gut microbiota. However, shrimp gut microbiotas are not directly mirrored by the changes in rearing bacterioplankton communities. A neutral model analysis showed that the stochastic processes that govern gut microbiota tended to become more important as healthy shrimp aged, with 37.5% stochasticity in larvae linearly increasing to 60.4% in adults. However, this defined trend was skewed when disease occurred. This departure was attributed to the uncontrolled growth of two candidate pathogens (over-represented taxa). The co-occurrence patterns provided novel clues on how the gut commensals interact with candidate pathogens in sustaining shrimp health. Collectively, these findings offer updated insight into the ecological processes that govern the host-bacterial colonization in shrimp and provide a pathological understanding of polymicrobial infections.

Starvation stress affects the interplay among shrimp gut microbiota, digestion and immune activities.

Aquatic animals are frequently suffered from starvation due to restricted food availability or deprivation. It is currently known that gut microbiota assists host in nutrient acquisition. Thus, exploring the gut microbiota responses would improve our understanding on physiological adaptation to starvation. To achieve this, we investigated how the gut microbiota and shrimp digestion and immune activities were affected under starvation stress. The results showed that the measured digestion activities in starved shrimp were significantly lower than in normal cohorts; while the measured immune activities exhibited an opposite trend. A structural equation modeling (SEM) revealed that changes in the gut bacterial community were directly related to digestive and immune enzyme activities, which in turn markedly affected shrimp growth traits. Notably, several gut bacterial indicators that characterized the shrimp nutrient status were identified, with more abundant opportunistic pathogens in starved shrimp, although there were no statistical differences in the overall diversity and the structures of gut bacterial communities between starved and normal shrimp. Starved shrimp exhibited less connected and cooperative interspecies interaction as compared with normal cohorts. Additionally, the functional pathways involved in carbohydrate and protein digestion, glycan biosynthesis, lipid and enzyme metabolism remarkably decreased in starved shrimp. These attenuations could increase the susceptibility of starved shrimp to pathogens infection. In summary, this study provides novel insights into the interplay among shrimp digestion, immune activities and gut microbiota in response to starvation stress.

Quantitative prediction of shrimp disease incidence via the profiles of gut eukaryotic microbiota.

One common notion is emerging that gut eukaryotes are commensal or beneficial, rather than detrimental. To date, however, surprisingly few studies have been taken to discern the factors that govern the assembly of gut eukaryotes, despite growing interest in the dysbiosis of gut microbiota-disease relationship. Herein, we firstly explored how the gut eukaryotic microbiotas were assembled over shrimp postlarval to adult stages and a disease progression. The gut eukaryotic communities changed markedly as healthy shrimp aged, and converged toward an adult-microbiota configuration. However, the adult-like stability was distorted by disease exacerbation. A null model untangled that the deterministic processes that governed the gut eukaryotic assembly tended to be more important over healthy shrimp development, whereas this trend was inverted as the disease progressed. After ruling out the baseline of gut eukaryotes over shrimp ages, we identified disease-discriminatory taxa (species level afforded the highest accuracy of prediction) that characteristic of shrimp health status. The profiles of these taxa contributed an overall 92.4% accuracy in predicting shrimp health status. Notably, this model can accurately diagnose the onset of shrimp disease. Interspecies interaction analysis depicted how the disease-discriminatory taxa interacted with one another in sustaining shrimp health. Taken together, our findings offer novel insights into the underlying ecological processes that govern the assembly of gut eukaryotes over shrimp postlarval to adult stages and a disease progression. Intriguingly, the established model can quantitatively and accurately predict the incidences of shrimp disease.

Integrating gut microbiota immaturity and disease-discriminatory taxa to diagnose the initiation and severity of shrimp disease.

Increasing evidence has emerged a tight link among the gut microbiota, host age and health status. This osculating interplay impedes the definition of gut microbiome features associated with host health from that in developmental stages. Consequently, gut microbiota-based prediction of health status is promising yet not well established. Here we firstly tracked shrimp gut microbiota (N = 118) over an entire cycle of culture; shrimp either stayed healthy or progressively transitioned into severe disease. The results showed that the gut microbiota were significantly distinct over shrimp developmental stages and disease progression. Null model and phylogenetic-based mean nearest taxon distance (MNTD) analyses indicated that deterministic processes that governed gut community became less important as the shrimp aged and disease progressed. The predicted gut microbiota age (using the profiles of age-discriminatory bacterial species as independent variables) fitted well (r = 0.996; P < 0.001) with the age of healthy subjects, while this defined trend was disrupted by disease. Microbiota-for-age Z-scores (MAZ, here defined as immaturity) were relative stable among healthy shrimp, but sharply decreased when disease emerged. By distinguishing between age- and disease- discriminatory taxa, we developed a model, bacterial indicators of shrimp health status, to diagnose disease from healthy subjects with 91.5% accuracy. Notably, the relative abundances of the bacterial indicators were indicative for shrimp disease severity. These findings, in aggregate, add our understanding on the gut community assembly patterns over shrimp developmental stages and disease progression. In addition, shrimp disease initiation and severity can be accurately diagnosed using gut microbiota immaturity and bacterial indicators.

The Underlying Ecological Processes of Gut Microbiota Among Cohabitating Retarded, Overgrown and Normal Shrimp.

Increasing evidence of tight links among the gut microbiota, obesity, and host health has emerged, but knowledge of the ecological processes that shape the variation in microbial assemblages across growth rates remains elusive. Moreover, inadequately control for differences in factors that profoundly affect the gut microbial community, hampers evaluation of the gut microbiota roles in regulating growth rates. To address this gap, we evaluated the composition and ecological processes of the gut bacterial community in cohabitating retarded, overgrown, and normal shrimps from identically managed ponds. Gut bacterial community structures were distinct (P = 0.0006) among the shrimp categories. Using a structural equation modeling (SEM), we found that changes in the gut bacterial community were positively related to digestive activities, which subsequently affected shrimp growth rate. This association was further supported by intensified interspecies interaction and enriched lineages with high nutrient intake efficiencies in overgrown shrimps. However, the less phylogenetic clustering of gut microbiota in overgrown and retarded subjects may offer empty niches for pathogens invasion, as evidenced by higher abundances of predicted functional pathways involved in disease infection. Given no differences in biotic and abiotic factors among the cohabitating shrimps, we speculated that the distinct gut community assembly could be attributed to random colonization in larval shrimp (e.g., priority effects) and that an altered microbiota could be a causative factor in overgrowth or retardation in shrimp. To our knowledge, this is the first study to provide an integrated overview of the direct roles of gut microbiota in shaping shrimp growth rate and the underlying ecological mechanisms.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus.

Glutathione S-transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST-θ) by RACE approaches. The full-length cDNA of AjGST-θ was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST-θ processed the characteristic N-terminal GSH-binding site (G-site) and the C-terminal hydrophobic substrate binding site (H-site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST-θ belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST-θ was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up-regulate the mRNA expression of AjGST-θ when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST-θ showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST-θ protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST-θ could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST-θ might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus.

Contrasting Ecological Processes and Functional Compositions Between Intestinal Bacterial Community in Healthy and Diseased Shrimp.

Intestinal bacterial communities play a pivotal role in promoting host health; therefore, the disruption of intestinal bacterial homeostasis could result in disease. However, the effect of the occurrences of disease on intestinal bacterial community assembly remains unclear. To address this gap, we compared the multifaceted ecological differences in maintaining intestinal bacterial community assembly between healthy and diseased shrimps. The neutral model analysis shows that the relative importance of neutral processes decreases when disease occurs. This pattern is further corroborated by the ecosphere null model, revealing that the bacterial community assembly of diseased samples is dominated by stochastic processes. In addition, the occurrence of shrimp disease reduces the complexity and cooperative activities of species-to-species interactions. The keystone taxa affiliated with Alphaproteobacteria and Actinobacteria in healthy shrimp gut shift to Gammaproteobacteria species in diseased shrimp. Changes in intestinal bacterial communities significantly alter biological functions in shrimp. Within a given metabolic pathway, the pattern of enrichment or decrease between healthy and deceased shrimp is correlated with its functional effects. We propose that stressed shrimp are more prone to invasion by alien strains (evidenced by more stochastic assembly and higher migration rate in diseased shrimp), which, in turn, disrupts the cooperative activity among resident species. These findings greatly aid our understanding of the underlying mechanisms that govern shrimp intestinal community assembly between health statuses.

RNA-seq analysis revealed ROS-mediated related genes involved in cadmium detoxification in the razor clam Sinonovacula constricta.

The razor clam Sinonovacula constricta is a eukaryotic benthic intertidal bivalve species that is tolerant to different heavy metals, such as cadmium ion (Cd(2+)). However, the mechanism by which S. constricta responds to Cd(2+)-induced stress remains unclear. In this study, eight transcriptome libraries were constructed and sequenced using razor clams exposed to Cd(2+) for 12 and 48 h. A total of 18,330 unigenes with an average length of 500 bp were annotated. Among these 18,330 unigenes, 582 and 649 displayed differential expression profiles at 12 and 48 h in the gill, respectively. The corresponding differential unigenes in the hepatopancreas were 1056 and 382. Gene Ontology annotation revealed that these unigenes were highly pronounced in metabolic process, cellular process, binding, and catalytic activity. Notably, ROS production-related genes, such as heat shock proteins 32, metallothionein, and glutathione, were synchronously enriched in all experimental samples with induced expression profiles, which was also validated by qPCR. Our results highlighted the relation between immune regulation and Cd(2+)-induced stress in razor clam and provided new insights into the molecular mechanisms of heavy toxicology.

Molecular cloning and characterization of four caspases members in Apostichopus japonicus.

The caspase family representing aspartate-specific cysteine proteases have been demonstrated to possess key roles in apoptosis and immune response. We previously demonstrated that LPS challenged Apostichopus japonicus coelomocyte could significantly induced apoptosis in vitro. However, apoptosis related molecules were scarcely investigated in this economic species. In the present work, we cloned and characterized four members caspase family from A. japonicus (designated as Ajcaspase-2, Ajcaspase-3, Ajcaspase-6, and Ajcaspase-8, respectively) by RACE. Multiple sequence alignment and structural analysis revealed that all Ajcaspases contained the conservative CASC domain at C terminal, in which some unique features for each Ajcaspase made them different from each other. These specific domains together with phylogenetic analysis supported that all these four identified proteins belonged to novel members of apoptotic signaling pathway in sea cucumber. Tissue distribution analysis revealed that four Ajcaspase genes were constitutively expressed in all examined tissues. The expression of Ajcaspase-2 was tightly correlated with that of Ajcaspase-8 in each detected tissues. Ajcaspase-3 and Ajcaspase-6 transcripts were both highly expressed in immune tissue of coelomocytes. Furthermore, the Vibrio splendidus challenged sea cucumber coelomocytes could significantly up-regulate the mRNA expressions of four genes. The expression levels of Ajcaspase-2 and Ajcaspase-8 were relative earlier than those of Ajcaspase-6 and Ajcaspase-3, respectively, which could be inferred that Ajcapase-2 might directly modulate Ajcaspase-6, and Ajcaspase-8 initiate the expression of Ajcaspase-3. The induce expressions differed among each Ajcaspase depending upon their roles such as initiator or effector caspase. All our results demonstrated that four Ajcaspases present diversified functions in apoptotic cascade signaling pathway of sea cucumber under immune response.

Effects of different fertilizers on the abundance and community structure of ammonia oxidizers in a yellow clay soil.

Yellow clay paddy soil (Oxisols) is a typical soil with low productivity in southern China. Nitrification inhibitors and slow release fertilizers have been used to improve nitrogen fertilizer utilization and reduce environmental impaction of the paddy soil. However, their effects on ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in paddy soil have rarely been investigated. In the present work, we compared the influences of several slow release fertilizers and nitrification inhibitors on the community structure and activities of the ammonia oxidizers in yellow clay soil. The abundances and community compositions of AOA and AOB were determined with qPCR, terminal restriction fragment length polymorphism (T-RFLP), and clone library approaches. Our results indicated that the potential nitrification rate (PNR) of the soil was significantly related to the abundances of both AOA and AOB. Nitrogen fertilizer application stimulated the growth of AOA and AOB, and the combinations of nitrapyrin with urea (NPU) and urea-formaldehyde (UF) inhibited the growth of AOA and AOB, respectively. Compared with other treatments, the applications of NPU and UF also led to significant shifts in the community compositions of AOA and AOB, respectively. NPU showed an inhibitory effect on AOA T-RF 166 bp that belonged to Nitrosotalea. UF had a negative effect on AOB T-RF 62 bp that was assigned to Nitrosospira. These results suggested that NPU inhibited PNR and increased nitrogen use efficiency (NUE) by inhibiting the growth of AOA and altering AOA community. UF showed no effect on NUE but decreased AOB abundance and shifted AOB community.

Syntrophic oxidation of propionate in rice field soil at 15 and 30°C under methanogenic conditions.

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H(2)-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-(13)C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating (13)C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating (13)C into their nucleic acids after [(13)C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with (13)C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales, Methanocellales, and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae, Acidobacteria, Actinobacteria, and Thermomicrobia probably also assimilated propionate-derived (13)C. The mechanisms for the involvement of these organisms remain unclear.

Syntrophomonadaceae-affiliated species as active butyrate-utilizing syntrophs in paddy field soil.

DNA-based stable-isotope probing was applied to identify the active microorganisms involved in syntrophic butyrate oxidation in paddy field soil. After 14 and 21 days of incubation with [U-(13)C]butyrate, the bacterial Syntrophomonadaceae and the archaeal Methanosarcinaceae and Methanocellales incorporated substantial amounts of (13)C label into their nucleic acids. Unexpectedly, members of the Planctomycetes and Chloroflexi were also labeled with (13)C by yet-unclear mechanisms.

Quantifying the Importance of Abiotic and Biotic Factors Governing the Succession of Gut Microbiota Over Shrimp Ontogeny.

Intensive studies have evaluated abiotic factors in shaping host gut microbiota. In contrast, little is known on how and to what extent abiotic (geochemical variables) and biotic (i.e., surrounding microbes, younger shrimp, and age) factors assemble the gut microbiota over shrimp ontogeny. Considering the functional importance of gut microbiota in improving host fitness, this knowledge is fundamental to sustain a desirable gut microbiota for a healthy aquaculture. Here, we characterized the successional rules of both the shrimp gut and rearing water bacterial communities over the entire shrimp farming. Both the gut and rearing water bacterial communities exhibited the time decay of similarity relationship, with significantly lower temporal turnover rate for the gut microbiota, which were primarily governed by shrimp age (days postlarval inoculation) and water pH. Gut commensals were primary sourced (averaged 60.3%) from their younger host, rather than surrounding bacterioplankton (19.1%). A structural equation model revealed that water salinity, pH, total phosphorus, and dissolve oxygen directly governed bacterioplankton communities but not for the gut microbiota. In addition, shrimp gut microbiota did not simply mirror the rearing bacterioplankton communities. The gut microbiota tended to be governed by variable selection over shrimp ontogeny, while the rearing bacterioplankton community was shaped by homogeneous selection. However, the determinism of rare and stochasticity of abundant subcommunities were consistent between shrimp gut and rearing water. These findings highlight the importance of independently interpreting host-associated and free-living communities, as well as their rare and abundant subcommunities for a comprehensive understanding of the ecological processes that govern microbial successions.

Shrimp AHPND Causing Vibrio anguillarum Infection: Quantitative Diagnosis and Identifying Antagonistic Bacteria.

Acute hepatopancreatic necrosis disease (AHPND) is one of the most common and serious diseases in shrimp aquaculture. Relevant works have focused on the gut microbiota-disease relationship when serious AHPND occurs. In contrast, little is known about how the gut microbiota responds to pathogen infection over AHPND progression, whereas this knowledge is fundamental to uncover the etiology of AHPND. Here, we explored the temporal succession of shrimp gut microbiota during Vibrio anguillarum (a causal pathogen of AHPND) challenge. The successful infection of V. anguillarum was confirmed by linearly increased abundance of the pathogen in the shrimp gut over AHPND progression. V. anguillarum infection caused an irreversible disruption in the shrimp gut microbiota, of which infection and hours post infection (hpi) respectively constrained 6.2% and 10.2% of variation in the data. Furthermore, the predicted functional pathways involved in immunity and metabolism significantly decreased, while those facilitating infectious diseases significantly enriched in the infected shrimp. Intriguingly, after ruling out the effect of background changes in gut microbiota, we identified 20 infection-discriminatory taxa that could be served as independent variables for accurately (89.4%) diagnosing V. anguillarum infection, even at the early infection stage, i.e., 24 hpi. Using a consensus network, we identified several Vibrio and Pseudoalteromonas taxa that directly antagonized V. anguillarum, following the Darwin's niche theory. This is one of the few attempts to identify gut bioindicators for diagnosing pathogen infection. In addition, the antagonistic commensals of V. anguillarum might be the candidate probiotics for preventing AHPND.

[Bacterial Community Composition in Channel Sediment in Response to Mariculture Wastewater Drainage].

The impact of mariculture on the ecological environment of sediments in surrounding waters is intensely debated. Microbial communities are considered to be keystone indicators of lineage responses to changes in environmental quality. To examine the mariculture effects, we collected channel sediment from four sites in the culture area including oneintake canal, one outfall, and two outlet canals. On the basis of bacterial 16S rRNA gene and phospholipid fatty acid (PLFA) technology, we used Illumina MiSeq sequencing applied to the analysis results to explore the effects of mariculture wastewater drainage on the bacterial community structures in the sediment. The results revealed that Proteobacteria, including γ-Proteobacteria, δ-Proteobacteria, and α-Proteobacteria, were the dominant lineages of bacteria at the four sites. The microbial biomass of the sediment increased significantly owing to the effects of mariculture wastewater drainage. The relative abundance of Actinobacteria and ammoniated microorganisms decreased, whereas the relative abundance of Bacteroidetes increased. The results of non-metric multidimensional scaling (NMDS) analysis indicated that most of the sediment bacterial communities clustered by influent and effluent. The diversity indicated that the mariculture wastewater drainage decreased the bacterial diversity, and redundancy analysis (RDA) indicated that the most obvious correlation occurred between ammonia nitrogen and bacterial community structures. In summary, the mariculture wastewater drainage resulted in significant changes in the physical and chemical properties and structures of bacterial communities in the aquaculture channel sediment. This indicates that the long-term direct discharge of aquaculture wastewater would excessively contaminate a channel environment and might further pose a threat to the offshore ecological environment.

Bacterioplankton community responses and the potential ecological thresholds along disturbance gradients.

Increasing intensity and frequency of coastal pollutions are the trajectory to be expected due to anthropogenic pressures. However, it is still unclear how and to what extent bacterioplankton communities respond to the two factors, despite the functional importance of bacterioplankton in biogeochemical cycles. In this study, significant organic pollution index (OPI) and offshore distance gradients, as respective proxies of disturbance intensity and disturbance frequency, were detected in a regional scale across the East China Sea. A multiple regression on matrices (MRM) revealed that the biogeography of bacterioplankton community depended on spatial scale, which was governed by local characters. Bacterioplankton community compositions (BCCs) were primarily governed by the conjointly direct (-0.28) and indirect (-0.48) effects of OPI, while offshore distance contributed a large indirectly effect (0.52). A SEGMENTED analysis depicted non-linear responses of BCCs to increasing disturbance intensity and disturbance frequency, as evidenced by significant tipping points. This was also true for the dominant bacterial phyla. Notably, we screened 30 OPI-discriminatory taxa that could quantitatively diagnose coastal OPI levels, with an overall 79.3% accuracy. Collectively, the buffer capacity of bacterioplankton communities to increasing disturbance intensity and disturbance frequency is limited, of which the significant tipping points afford a warning line for coastal management. In addition, coastal pollution level can be accurately diagnosed by a few OPI-discriminatory taxa.