An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Characterizing sediment functional traits and ecological consequences respond to increasing antibiotic pollution.

Current studies have shown that the taxonomic structures of ecologically important microbial communities are altered by antibiotic exposure, but the resulting effects on functional potentials and subsequent biogeochemical processes are poorly understood. However, this knowledge is indispensable for developing an accurate projection of nutrient dynamics in the future. Using metagenomic analyses, here we explored the responses of taxonomical and functional structures of a sediment microbial community, and their links with key biogeochemical processes to increasing antibiotic pollution from the pristine inlet to the outfall sites along an aquaculture discharge channel. We identified sharply contrasting sedimentary microbial communities and functional traits along increasing antibiotic pollution. Functional structures exhibited steeper distance-decay relationships than taxonomical structures along both the antibiotic distance and physicochemical distance, revealing higher functional sensitivity. Sediment enzyme activities were significantly and positively coupled with the relative abundances of their coding genes, thus the abundances of genes were indicative of functional potentials. The nitrogen cycling pathways were commonly inhibited by antibiotics, but not for the first step of nitrification, which could synergistically mitigate nitrous oxide emission. However, antibiotic pollution stimulated methanogens and inhibited methanotrophs, thereby promoting methane efflux. Furthermore, microbes could adapt to antibiotic pollution through enriched potential of sulfate uptake. Antibiotics indirectly affected taxonomic structures through alterations in network topological features, which in turn affected sediment functional structures and biogeochemical processes. Notably, only 13 antibiotics concentration-discriminatory genes contributed an overall 95.9% accuracy in diagnosing in situ antibiotic concentrations, in which just two indicators were antibiotic resistance genes. Our study comprehensively integrates sediment compositional and functional traits, biotic interactions, and enzymatic activities, thus generating a better understanding about ecological consequences of increasing antibiotics pollution. KEY POINTS: • Contrasting functional traits respond to increasing antibiotic pollution. • Antibiotics pollution stimulates CH4 efflux, while mitigating N2O emission and may drive an adaptive response of enriched sulfate uptake. • Indicator genes contribute 95.9% accuracy in diagnosing antibiotic concentrations.

The assembly of gut microbiota implicates shrimp acute hepatopancreas necrosis disease progression.

Ample evidence shows dysbiosis in the gut microbiota when comparing healthy shrimp with those affected by severe acute hepatopancreatic necrosis disease (AHPND). However, the static comparison used in available studies leads to the uncertainties regarding how and to what extent the gut microbiota responds to the progressive severity of AHPND. In addition, shrimp AHPND is featured by rapid and massive mortality, thus the initiation of AHPND must be diagnosed for preemptive therapy. For these reasons, we explored the ecological assembly of gut microbiota over shrimp AHPND progression. Increasing AHPND severity was associated with linear increase in the copies of pirAB genes, relative abundance of gut Vibrio and potentially pathogenic, and reduction in the gut bacterial diversity, stability, and relative abundance of Bdellovibrio. Negative and significant association between gut Vibrio and Bdellovibrio were noted, indicating that compromised predation exerts a role in AHPND progression. Notably, the extents of departure to the healthy shrimp gut microbiota were positively coupled with the increasing severity of AHPND. After controlling the temporal variation in the gut microbiota as healthy shrimp age, we constructed a diagnosis model that accurately diagnosed the initial, progressed or moribund stages of AHPND, with an overall accuracy of 86.5%. Shrimp AHPND induced more stochastic gut microbiotas as a consequence of the attenuated ability of diseased shrimp to select their commensals, resulting in convergent bacterial communities between gut and rearing water over AHPND progression. Collectively, our findings provide important step toward the ecological assembly of gut microbiota implicating in AHPND etiology and in diagnosing AHPND stages. KEY POINTS: • The departure of shrimp gut microbiota positively linked with AHPND severity. • The diagnosis model accurately diagnosed the stages of AHPND. • Shrimp AHPND induced more stochastic gut microbiota.

Significant tipping points of sediment microeukaryotes forewarn increasing antibiotic pollution.

Antibiotic pollution imposes urgent threats to public health and microbial-mediated ecological processes. Existing studies have primarily focused on bacterial responses to antibiotic pollution, but they ignored the microeukaryotic counterpart, though microeukaryotes are functionally important (e.g., predators and saprophytes) in microbial ecology. Herein, we explored how the assembly of sediment microeukaryotes was affected by increasing antibiotic pollution at the inlet (control) and across the outlet sites along a shrimp wastewater discharge channel. The structures of sediment microeukaryotic community were substantially altered by the increasing nutrient and antibiotic pollutions, which were primarily controlled by the direct effects of phosphate and ammonium (-0.645 and 0.507, respectively). In addition, tetracyclines exerted a large effect (0.209), including direct effect (0.326) and indirect effect (-0.117), on the microeukaryotic assembly. On the contrary, the fungal subcommunity was relatively resistant to antibiotic pollution. Segmented analysis depicted nonlinear responses of microeukaryotic genera to the antibiotic pollution gradient, as supported by the significant tipping points. We screened 30 antibiotic concentration-discriminatory taxa of microeukaryotes, which can quantitatively and accurately predict (98.7% accuracy) the in-situ antibiotic concentration. Sediment microeukaryotic (except fungal) community is sensitive to antibiotic pollution, and the identified bioindicators could be used for antibiotic pollution diagnosis.

A meta-analysis study of the robustness and universality of gut microbiota-shrimp diseases relationship.

Intensive case study has established dysbiosis in the gut microbiota-shrimp disease relationship; however, variability in experimental design and the diversity of diseases arise the question of whether some gut indicators are robust and universal in response to shrimp health status, irrespective of causal agents. Through an unbiased subject-level meta-analysis framework, we re-analysed 10 studies, including 261 samples, four lifestages and six different diseases (the causal agents are virus, bacterial, eukaryotic pathogens, or unknown). Results showed that shrimp diseases reproducibly altered the structure of gut bacterial community, but not diversity. After ruling out the lifestage- and disease specific- discriminatory taxa (different diseases dependent indicators), we identify 18 common disease-discriminatory taxa (indicative of health status, irrespective of causal agents) that accurately diagnosed (90.0% accuracy) shrimp health status, regardless of different diseases. These optimizations substantially improved the performance (62.6% vs. 90.0%) diagnosing model. The robustness and universality of model were validated for effectiveness via leave-one-dataset-out validation and independent cohorts. Interspecies interaction and stability of the gut microbiotas were consistently compromised in diseased shrimp compared with corresponding healthy cohorts, while stochasticity and beta-dispersion exhibited the opposite trend. Collectively, our findings exemplify the utility of microbiome meta-analyses in identifying robust and reproducible features for quantitatively diagnosing disease incidence, and the downstream consequences for shrimp pathogenesis from an ecological prospective.

High nutrient induces virulence in the AHPND-causing Vibrio parahaemolyticus, interpretation from the ecological assembly of shrimp gut microbiota.

Shrimp diseases frequently occur during the later farming stages, when the rearing water is eutrophic. This observation provides clue that the virulence of pathogens could be induced by elevated nutrient, whereas the underlying ecological mechanism remains limited. To address this pressing knowledge, we explored how gut microbiota responded to the infection of oligotrophic (OVp) or eutrophic (EVp) pre-cultured Vibrio parahaemolyticus, a causing pathogen of shrimp acute hepatopancreatic necrosis disease (AHPND). Resulted revealed that OVp and EVp infections caused dysbiosis in the gut microbiota and compromised shrimp immunity, while the later infection led to earlier and higher mortality. Significant associations were detected between the gut microbiota and each of the measured immune activities. Neutral community model showed that the assembly of gut microbiota was more strongly governed by deterministic processes in EVp infection, followed by EVp infected and control shrimp. Additionally, there were significantly lower temporal turnover rate and average variation degree in the gut microbiota in EVp infected shrimp compared with control individuals. Notably, we identified 22 infection-discriminatory taxa after ruling out the ontogenic effect. Using profiles of the 22 indicators as independent variables, the diagnosis model accurately distinguished (an overall 85.9% accuracy) the infected status (control, OVp or EVp infected shrimp), with 81.3% accuracy at the initial infection stage. The convergent and deterministic gut microbiota in EVp infected shrimp could partially explain why it is challenge to cure APHND from an ecological viewpoint. In addition, we provided a sensitive approach for diagnosing the onset of infection, when disease symptom is unobservable.

Wocania indica gen. nov., sp. nov., isolated from deep sea hydrothermal sulfide in the northwest Indian Ocean, and proposal to reclassify Flaviramulus ichthyoenteri as Wocania ichthyoenteri comb. nov.

A novel Gram-stain-negative, non-motile, strictly aerobic, rod-shaped bacterium was isolated from deep-sea hydrothermal sulfide in the northwest Indian Ocean Ridge and designated as strain IOP_32T. Strain IOP_32T could grow at 4-40 °C (optimum, 37 °C), pH 5-9 (optimum, pH 7-8) and salinity of 0-12 % NaCl (w/v; optimum, 2-3 %). The 16S rRNA gene sequence of strain IOP_32T is most similar to Bizionia fulviae EM7T, Bizionia berychis RA3-3-1T, Bizionia paragorgiae KMM 6029T and Oceanihabitans sediminis S9_10T with 95.5-95.3 % similarity. The phylogenomic analysis indicated that strain IOP_32T forms a distinct lineage with Flaviramulus ichthyoenteri Th78T within the family Flavobacteriaceae. The average nucleotide identity, average amino acid identity and percentage of conserved protein values between strain IOP_32T and the type strains of close genera were 72.3-78.5 %, 67.4-76.9 % and 56.3-61.6 %, respectively. The major cellular fatty acid was anteiso-C15 : 0. The respiratory quinone was MK-6. The polar lipids were mainly composed of phosphatidylethanolamine, an unidentified aminophospholipid and five unidentified polar lipids. Strain IOP_32T is significantly different from related genera, which is reflected by the wide adaptability to temperature and salinity levels, the composition of phospholipids and fatty acids, and the results of phylogenetic analyses. The phenotypic properties and phylogenetic data suggest that the lineage represents a novel genus and species within the family Flavobacteriaceae, for which the name Wocania indica gen. sp. nov. is proposed, with the type strain IOP_32T (=MCCC 1A14017 T=KCTC 62660 T). We also propose the reclassification of Flaviramulus ichthyoenteri as Wocania ichthyoenteri comb. nov. (Th78T=DSM 26285T=JCM 18634T=KCTC 32142T).

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.

Immune and gut bacterial successions of large yellow croaker (Larimichthys crocea) during Pseudomonas plecoglossicida infection.

Large yellow croaker (Larimichthys crocea, LYC) aquaculture is being threatened by intensive infectious diseases. Relevant studies have focused on LYC immune responses to infection. By contrast, little is known how and to what extent the gut microbiota responds to infection. Here, we explored the interactions between LYC immune responses and gut bacterial communities during Pseudomonas plecoglossicida infection. P. plecoglossicida successfully colonized into LYC gut microbiota, resulting in an increasing mortality rate. Relative gene expressions of pro-inflammatory cytokines (TNF-α1, TNF-α2 and IL-1ß) and anti-inflammatory cytokine (IL-10) were consistently and significantly induced by P. plecoglossicida infection, whereas non-specific immune enzymes activities were only enhanced at the early infection stages. P. plecoglossicida infection caused an irreversible disruption in the gut microbiota, of which infection and hours post infection constrained 16.2% and 5.6% variations, respectively. In addition, top 18 discriminatory taxa that were responsible for the difference between treatments were identified, whose abundances were significantly associated with the immune activities of LYC. Using a structural equation modeling (SEM), we found that gut bacterial communities were primarily governed by the conjointly direct (-0.33) and indirect (0) effects of infection, which subsequently affect host immune responses. Our results suggest that an irreversible dysbiosis in gut microbiota could be the causality of increasing mortality. To our knowledge, this is the first study to provide an integrated overview among pathogen infection, immune response and gut microbiota of LYC.

Effect of Rhizophora apiculata plantation for improving water quality, growth, and health of mud crab.

A deteriorated water quality is closely associated with disease outbreaks in aquaculture, where microorganisms play indispensable roles in improving water quality and aquatic animals' health. Mangrove is known to be a natural water quality filter and microbiological buffer of pathogen and prebiotics. However, it is unclear how and to what extent Rhizophora apiculata plantation is of benefits to the gut microbiota and growth over mud crab (Scylla paramamosain) aging. To address these concerns, we explored the bacterial communities in mud crab gut and rearing water at 45, 114, and 132 days after incubation, roughly corresponding to juvenile, pre-adult, and adult stages of mud crab. Results showed that 1-year R. apiculata plantation slightly increased the body weight of mud crab and improved water quality to a certain extent. Both bacterioplankton and gut bacterial communities were highly temporal dynamic, while the two communities were significantly distinct (ANOSIM r = 0.90, P = 0.0001). Relative abundances of dominant taxa in water and gut significantly varied between the plantation and the control conditions over mud crab aging. R. apiculata plantation promoted the stability of gut microbiota, as evidenced by more diverse core species. Furthermore, R. apiculata plantation led to the dominance of Verrucomicrobiae species in water and probiotic Bacteroidetes and Lactobacillales taxa in gut. A structural equation model revealed that water variables directly constrained gut microbiota, which in turn affected the body weight of mud crab (r = 0.52, P < 0.001). In addition, functional pathways facilitating immunity and lipid metabolism significantly increased in mud crab gut under the plantation, while those involved in infectious diseases exhibited the opposing trend. These findings greatly expand our understanding of the R. apiculata plantation effects on water quality, gut microbiota, and growth feature of mud crab. Overall, R. apiculata plantation is beneficial for mud crab growth and health. KEY POINTS: • A short-term R. apiculata plantation could potentially improve water quality. • Bacterioplankton is more sensitive than mud crab gut microbiota in response to R. apiculata plantation. • R. apiculata plantation enhances mud crab resistance against pathogen invasion. • R. apiculata plantation alters mud crab gut microbiota, which in turn promotes their body weight.

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.

Concept of microbial gatekeepers: Positive guys?

Microorganisms contribute diverse and fundamental roles in biogeochemical processes. In a given microbial community, individuals interact with one another to form complex regulatory networks in which gatekeepers contribute disproportional roles in sustaining stability, dynamics, and function. Owing to the ecological and functional importance of microbial gatekeeper, this review provides an overview on its history, identification, roles, application in biological sciences, and clinical diagnostics. The roles of microbial gatekeepers can be beneficial or detrimental, which depends on our purpose. As the field is rather new, some limitations are raised, and further efforts devoted to solving these concerns are proposed. Collectively, gatekeepers provide promising targets for sustaining and re-establishing a desired microbial community. However, substantial obstacles, such as factors governing gatekeepers, must be overcome to manipulate gatekeepers as positive guys.

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.

Progress in the gut microbiota in exploring shrimp disease pathogenesis and incidence.

It is now recognized that gut microbiota contributes indispensable roles in safeguarding host health. Shrimp is being threatened by newly emerging diseases globally; thus, understanding the driving factors that govern its gut microbiota would facilitate an initial step to reestablish and maintain a "healthy" gut microbiota. This review summarizes the factors that assemble the shrimp gut microbiota, which focuses on the current progresses of knowledge linking the gut microbiota and shrimp health status. In particular, I propose the exploration of shrimp disease pathogenesis and incidence based on the interplay between dysbiosis in the gut microbiota and disease severity. An updated research on shrimp disease toward an ecological perspective is discussed, including host-bacterial colonization, identification of polymicrobial pathogens and diagnosing disease incidence. Further, a simple conceptual model is offered to summarize the interplay among the gut microbiota, external factors, and shrimp disease. Finally, based on the review, current limitations are raised and future studies directed at solving these concerns are proposed. This review is timely given the increased interest in the role of gut microbiota in disease pathogenesis and the advent of novel diagnosis strategies.

Integrating molecular and ecological approaches to identify potential polymicrobial pathogens over a shrimp disease progression.

It is now recognized that some gut diseases attribute to polymicrobial pathogens infections. Thus, traditional isolation of single pathogen from disease subjects could bias the identification of causal agents. To fill this gap, using Illumina sequencing of the bacterial 16S rRNA gene, we explored the dynamics of gut bacterial communities over a shrimp disease progression. The results showed significant differences in the gut bacterial communities between healthy and diseased shrimp. Potential pathogens were inferred by a local pathogens database, of which two OTUs (affiliated with Vibrio tubiashii and Vibrio harveyi) exhibited significantly higher abundances in diseased shrimp as compared to healthy subjects. The two OTUs cumulatively contributed 64.5% dissimilarity in the gut microbiotas between shrimp health status. Notably, the random Forest model depicted that profiles of the two OTUs contributed 78.5% predicted accuracy of shrimp health status. Removal of the two OTUs from co-occurrence networks led to network fragmentation, suggesting their gatekeeper features. For these evidences, the two OTUs were inferred as candidate pathogens. Three virulence genes (bca, tlpA, and fdeC) that were coded by the two candidate pathogens were inferred by a virulence factor database, which were enriched significantly (P < 0.05 in the three cases, as validated by qPCR) in diseased shrimp as compared to healthy ones. The two candidate pathogens were repressed by Flavobacteriaceae, Garvieae, and Photobacrerium species in healthy shrimp, while these interactions shifted into synergy in disease cohorts. Collectively, our findings offer a frame to identify potential polymicrobial pathogen infections from an ecological perspective.

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.