Gut microbiota of Pacific white shrimp (Litopenaeus vannamei) exhibits distinct responses to pathogenic and non-pathogenic Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a high-mortality-rate shrimp disease, is caused by specific Vibrio parahaemolyticus (Vp) strains with a plasmid encoding the PirABVp toxins. As a bacterial pathogen, the invasion of AHPND-causing Vp might impose pressure on commensal microbiota in the shrimp gut, while the relationship between the pathogenesis of AHPND and the dysbiosis of gut bacterial communities remains unclear. Here we explored the temporal changes of shrimp gut microbiota in response to AHPND-causing and non-AHPND-causing Vp strains, with the non-infected controls as a baseline of the shrimp gut microbiota. The diversity and composition of bacterial communities from 168 gut samples (covering three treatments at seven time points with eight individuals per set) were investigated using 16S rRNA gene metabarcoding with high-throughput sequencing. The results showed that (i) species diversity of gut bacterial communities declined in Vp-infected shrimp, independent of the strain pathogenicity; (ii) taxonomic compositions of gut bacterial communities were significantly different between shrimp infected by AHPND-causing and non-AHPND-causing Vp strains; (iii) short-term (within 6 hours) compositional shifts in the gut microbiota were found only in AHPND-causing Vp-infected shrimp; (iv) the gut microbiota of AHPND-causing Vp-infected shrimp was enriched with genera Photobacterium and Vibrio, with a decline in Candidatus Bacilliplasma; and (v) functional predictions suggested the loss of normal metabolism due to compositional shifts in the gut microbiota. Our work reveals distinct features of community dynamics in shrimp gut microbiota, associated with pathogenic versus non-pathogenic Vibrio infections, providing a new perspective of the pathogenesis of AHPND. IMPORTANCE Shrimp production is continually threatened by newly emerging diseases, such as AHPND, which is caused by specific Vp strains. Previous studies on the pathogenesis of AHPND have mainly focused on the histopathology and immune responses of the host. However, more attention needs to be paid to the gut microbiota, which acts as the first barrier to pathogen colonization. In this study, we revealed that shrimp gut microbiota responded differently to pathogenic and non-pathogenic Vp strains, with bacterial genera Photobacterium and Vibrio enriched in pathogenic Vp-infected shrimp, and Candidatus Bacilliplasma enriched in non-pathogenic Vp-infected shrimp. Moreover, functional predictions suggested that changes in taxonomic compositions would further affect normal metabolic functions, emphasizing the importance of sustaining an equilibrium in the gut microbiota. Several biomarkers associated with specific microbial taxa and functional pathways were identified in our data sets, which help predict the incidence of disease outcomes.

Resilience and probiotic interventions to prevent and recover from shrimp gut dysbiosis.

To counter the recurrent outbreaks of bacterial (acute hepatopancreatic necrosis disease; AHPND) and viral (white spot disease; WSD) shrimp diseases, which still remain a threat to the global industry, shrimp gut microbiota research has been gaining more attention in recent years, and the use of probiotics in aquaculture has had promising results in improving shrimp gut health and immunity. In this review based on our studies on AHPND and WSD, we summarize our current understanding of the shrimp gastrointestinal tract and the role of the microbiota in disease, as well as effects of probiotics. We focus particularly on the concept of microbiota resilience, and consider strategies that can be used to restore shrimp gut health by probiotic intervention at a crucial time during gut microbiota dysbiosis. Based on the available scientific evidence, we argue that the use of probiotics potentially has an important role in controlling disease in shrimp aquaculture.

Deterministic Assembly Processes Strengthen the Effects of ß-Diversity on Community Biomass of Marine Bacterioplankton.

The presence of more species in the community of a sampling site (α diversity) typically increases ecosystem functions via nonrandom processes like resource partitioning. When considering multiple communities, we hypothesize that higher compositional difference (ß diversity) increases overall functions of these communities. Further, we hypothesize that the ß diversity effect is more positive when ß diversity is increased by nonrandom assembly processes. To test these hypotheses, we collected bacterioplankton along a transect of 6 sampling sites in the southern East China Sea in 14 cruises. For any pairs of the 6 sites within a cruise, we calculated the Bray-Curtis index to represent ß diversity and summed bacterial biomass as a proxy to indicate the overall function of the two communities. We then calculated deviation of observed mean pairwise phylogenetic similarities among species in two communities from random to represent the influences of nonrandom processes. The bacterial ß diversity was found to positively affect the summed bacterial biomass; however, the effect varied among cruises. Cross-cruise comparison indicated that the ß diversity effect increased with the nonrandom processes selecting for phylogenetically dissimilar species. This study extends biodiversity-ecosystem functioning research to the scale of multiple sites and enriches the framework by considering community assembly processes. IMPORTANCE The implications of our analyses are twofold. First, we emphasize the importance of studying ß diversity. We expanded the current biodiversity-ecosystem functioning framework from single to multiple sampling sites and investigated the influences of species compositional differences among sites on the overall functioning of these sites. Since natural ecological communities never exist alone, our analyses allow us to more holistically perceive the role of biodiversity in natural ecosystems. Second, we took community assembly processes into account to attain a more mechanistic understanding of the impacts of biodiversity on ecosystem functioning.

Reconstructing large interaction networks from empirical time series data.

Reconstructing interactions from observational data is a critical need for investigating natural biological networks, wherein network dimensionality is usually high. However, these pose a challenge to existing methods that can quantify only small interaction networks. Here, we proposed a novel approach to reconstruct high-dimensional interaction Jacobian networks using empirical time series without specific model assumptions. This method, named "multiview distance regularised S-map," generalised the state space reconstruction to accommodate high dimensionality and overcome difficulties in quantifying massive interactions with limited data. When evaluating this method using time series generated from theoretical models involving hundreds of interacting species, estimated strengths of interaction Jacobians were in good agreement with theoretical expectations. Applying this method to a natural bacterial community helped identify important species from the interaction network and revealed mechanisms governing the dynamical stability of a bacterial community. The proposed method overcame the challenge of high dimensionality in large natural dynamical systems.

System Performance Corresponding to Bacterial Community Succession after a Disturbance in an Autotrophic Nitrogen Removal Bioreactor.

Performance of a bioreactor is affected by complex microbial consortia that regulate system functional processes. Studies so far, however, have mainly emphasized the selective pressures imposed by operational conditions (i.e., deterministic external physicochemical variables) on the microbial community as well as system performance, but have overlooked direct effects of the microbial community on system functioning. Here, using a bioreactor with ammonium as the sole substrate under controlled operational settings as a model system, we investigated succession of the bacterial community after a disturbance and its impact on nitrification and anammox (anaerobic ammonium oxidation) processes with fine-resolution time series data. System performance was quantified as the ratio of the fed ammonium converted to anammox-derived nitrogen gas (N2) versus nitrification-derived nitrate (npNO3 -). After the disturbance, the N2/npNO3 - ratio first decreased, then recovered, and finally stabilized until the end. Importantly, the dynamics of N2/npNO3 - could not be fully explained by physicochemical variables of the system. In comparison, the proportion of variation that could be explained substantially increased (tripled) when the changes in bacterial composition were taken into account. Specifically, distinct bacterial taxa tended to dominate at different successional stages, and their relative abundances could explain up to 46% of the variation in nitrogen removal efficiency. These findings add baseline knowledge of microbial succession and emphasize the importance of monitoring the dynamics of microbial consortia for understanding the variability of system performance.IMPORTANCE Dynamics of microbial communities are believed to be associated with system functional processes in bioreactors. However, few studies have provided quantitative evidence. The difficulty of evaluating direct microbe-system relationships arises from the fact that system performance is affected by convolved effects of microbiota and bioreactor operational parameters (i.e., deterministic external physicochemical forcing). Here, using fine-resolution time series data (daily sampling for 2 months) under controlled operational settings, we performed an in-depth analysis of system performance as a function of the microbial community in the context of bioreactor physicochemical conditions. We obtained statistically evaluated results supporting the idea that monitoring microbial community dynamics could improve the ability to predict system functioning, beyond what could be explained by operational physicochemical variables. Moreover, our results suggested that considering the succession of multiple bacterial taxa would account for more system variation than focusing on any particular taxon, highlighting the need to integrate microbial community ecology for understanding system functioning.

Body-size Scaling is Related to Gut Microbial Diversity, Metabolism and Dietary Niche of Arboreal Folivorous Flying Squirrels.

Thermal homeostasis of mammals is constrained by body-size scaling. Consequently, small mammals require considerable energy to maintain a high mass-specific metabolic rate (MSMR) and sustain target body temperature. In association with gut microbiota, mammalian hosts acquire absorbable molecules and fulfill their metabolic requirements. Our objective was to characterize gut microbes in wild mammals and relate those findings to host body-size scaling. Two large (Petaurista philippensis grandis and P. alborufus lena), one medium (Trogopterus xanthipes) and one small (Pteromys volans orii) species of flying squirrels (FS) were studied. Using 16S rRNA genes, 1,104 OTUs were detected from four FS, with 1.99% of OTUs shared among all FS. Although all FS gut microbiota were dominated by Firmicutes, they were constituted by different bacterial families. Moreover, Bacteroidetes accounted for up to 19% of gut microbiota in small FS, but was absent in large FS. Finally, based on metagenome predictions, carbohydrate and amino acid metabolism genes were enriched in small body-size FS. In conclusion, gut microbiota compositions and predictive metabolic functions were characteristic of body-size in FS, consistent with their adaptations to folivorous dietary niches.

Vertical Beta-Diversity of Bacterial Communities Depending on Water Stratification.

Many studies indicate that variation of marine bacterial beta diversity in the horizontal dimension is mainly attributable to environmental and spatial effects. However, whether and how these two effects drive bacterial beta diversity in the vertical dimension remains unclear, especially when considering seasonal variation in the strength of water stratification. Here, we used 78 paired bacterioplankton community samples from surface and deep chlorophyll maximum (DCM) layers along a transect in the Kuroshio region east of Taiwan across multiple seasons. Variance partitioning was used to evaluate the mechanisms driving the vertical beta diversity between surface-DCM bacterioplankton communities during weak stratification periods (i.e., spring and fall) versus strong stratification periods (i.e., summer). During strong periods of stratification, vertical beta diversity was shaped by both environmental and spatial effects; notably, the strength of stratification played an important role in enhancing environmental dissimilarity and creating a barrier to dispersal. In contrast, during periods of weak stratification, environmental effects dominate, with a non-significant spatial effect due to mixing. Variation of vertical beta diversity for bacterioplankton communities in the Kuroshio region east of Taiwan was structured by different mechanisms across seasons, and was further dependent on stratification strength of the water column.

Evolutionary constraints on species diversity in marine bacterioplankton communities.

Variation in microbial species diversity has typically been explained as the outcome of local ecological factors driving species coexistence, overlooking the roles of evolutionary constraints. Here, we argue that macro-evolutionary niche conservatism and unequal diversification rates among phylum-level lineages are strong determinants of diversity-environment relationships in bacterial systems. That is, apart from stochasticity, environmental effects operate most strongly on phylum composition, which in turn dictates the species diversity of bacterial communities. This concept is demonstrated using bacterioplankton in the surface seawaters of the East China Sea. Furthermore, we show that the species richness of a local bacterioplankton community can generally be estimated based on the relative abundances of phyla and their contributions of species numbers in the global seawater pool-highlighting the important influence of evolutionary constraints on local community diversity.

Contrasting the relative importance of species sorting and dispersal limitation in shaping marine bacterial versus protist communities.

A central challenge in microbial ecology is to understand the underlying mechanisms driving community assembly, particularly in the continuum of species sorting and dispersal limitation. However, little is known about the relative importance of species sorting and dispersal limitation in shaping marine microbial communities; especially, how they are related to organism types/traits and water depth. Here, we used variation partitioning and null model analysis to compare mechanisms driving bacterial and protist metacommunity dynamics at the basin scale in the East China Sea, based on MiSeq paired-end sequencing of 16S ribosomal DNA (rDNA) and 18S rDNA, respectively, in surface, deep chlorophyll maximum and bottom layers. Our analyses indicated that protist communities were governed more strongly by species sorting relative to dispersal limitation than were bacterial communities; this pattern was consistent across the three-depth layers, albeit to different degrees. Furthermore, we detected that bacteria exhibited wider habitat niche breadths than protists, whereas, passive dispersal abilities were not appreciably different between them. Our findings support the 'size-plasticity' hypothesis: smaller organisms (bacteria) are less environment filtered than larger organisms (protists), as smaller organisms are more likely to be plastic in metabolic abilities and have greater environmental tolerance.

Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling.

Mammalian herbivores rely on microbial activities in an expanded gut chamber to convert plant biomass into absorbable nutrients. Distinct from ruminants, small herbivores typically have a simple stomach but an enlarged cecum to harbor symbiotic microbes; however, knowledge of this specialized gut structure and characteristics of its microbial contents is limited. Here, we used leaf-eating flying squirrels as a model to explore functional characteristics of the cecal microbiota adapted to a high-fiber, toxin-rich diet. Specifically, environmental conditions across gut regions were evaluated by measuring mass, pH, feed particle size, and metabolomes. Then, parallel metagenomes and metatranscriptomes were used to detect microbial functions corresponding to the cecal environment. Based on metabolomic profiles, >600 phytochemical compounds were detected, although many were present only in the foregut and probably degraded or transformed by gut microbes in the hindgut. Based on metagenomic (DNA) and metatranscriptomic (RNA) profiles, taxonomic compositions of the cecal microbiota were dominated by bacteria of the Firmicutes taxa; they contained major gene functions related to degradation and fermentation of leaf-derived compounds. Based on functional compositions, genes related to multidrug exporters were rich in microbial genomes, whereas genes involved in nutrient importers were rich in microbial transcriptomes. In addition, genes encoding chemotaxis-associated components and glycoside hydrolases specific for plant beta-glycosidic linkages were abundant in both DNA and RNA. This exploratory study provides findings which may help to form molecular-based hypotheses regarding functional contributions of symbiotic gut microbiota in small herbivores with folivorous dietary habits.

Evaluating community-environment relationships along fine to broad taxonomic resolutions reveals evolutionary forces underlying community assembly.

We propose a method for detecting evolutionary forces underlying community assembly by quantifying the strength of community-environment relationships hierarchically along taxonomic ranks. This approach explores the potential role of phylogenetic conservatism on habitat preferences: wherein, phylogenetically related taxa are expected to exhibit similar environmental responses. Thus, when niches are conserved, broader taxonomic classification should not diminish the strength of community-environment relationships and may even yield stronger associations by summarizing occurrences and abundances of ecologically equivalent finely resolved taxa. In contrast, broader taxonomic classification should weaken community-environment relationships when niches are under great divergence (that is, by combining finer taxa with distinct environmental responses). Here, we quantified the strength of community-environment relationships using distance-based redundancy analysis, focusing on soil and seawater prokaryotic communities. We considered eight case studies (covering a variety of sampling scales and sequencing strategies) and found that the variation in community composition explained by environmental factors either increased or remained constant with broadening taxonomic resolution from species to order or even phylum level. These results support the niche conservatism hypothesis and indicate that broadening taxonomic resolution may strengthen niche-related signals by removing uncertainty in quantifying spatiotemporal distributions of finely resolved taxa, reinforcing the current notion of ecological coherence in deep prokaryotic branches.

Spatial heterogeneity of gut microbiota reveals multiple bacterial communities with distinct characteristics.

We analyzed bacterial communities of six distinct gut sites (the food bolus and mucus layer of the proximal small intestine, cecum and distal large intestine), using wild folivorous flying squirrels. We found significant spatial heterogeneity in composition, diversity, and species abundance distributions (SADs) of gut microbiota, corresponding to physicochemical conditions. High diversity was detected in the mucus layer of small intestine and the food bolus of cecum, followed by the food bolus of large intestine and the mucus layer of cecum, and relatively low diversity in the food bolus of small intestine and the mucus layer of large intestine, likely due to disturbance and resource partitioning. The SADs showed succession-like patterns in the food bolus communities from the proximal to distal gut. Notably, each mucus layer community had a unique pattern different from the food bolus community of the same compartment, with distinct relative abundances of dominant species. In combination with data from other mammalian fecal samples, we concluded that gut microbiota were apparently dynamic in community structure, from low species richness with unequal abundances to high species richness with equal abundances; these findings were interpreted as strong habitat effects on bacterial communities.

Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena).

BACKGROUND: Animals co-evolve with their gut microbiota; the latter can perform complex metabolic reactions that cannot be done independently by the host. Although the importance of gut microbiota has been well demonstrated, there is a paucity of research regarding its role in foliage-foraging mammals with a specialized digestive system. RESULTS: In this study, a 16S rRNA gene survey and metagenomic sequencing were used to characterize genetic diversity and functional capability of cecal microbiota of the folivorous flying squirrel (Petaurista alborufus lena). Phylogenetic compositions of the cecal microbiota derived from 3 flying squirrels were dominated by Firmicutes. Based on end-sequences of fosmid clones from 1 flying squirrel, we inferred that microbial metabolism greatly contributed to intestinal functions, including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins. Moreover, 33 polysaccharide-degrading enzymes and 2 large genomic fragments containing a series of carbohydrate-associated genes were identified. CONCLUSIONS: Cecal microbiota of the leaf-eating flying squirrel have great metabolic potential for converting diverse plant materials into absorbable nutrients. The present study should serve as the basis for future investigations, using metagenomic approaches to elucidate the intricate mechanisms and interactions between host and gut microbiota of the flying squirrel digestive system, as well as other mammals with similar adaptations.