Changes of bacterial communities and bile acid metabolism reveal the potential "intestine-hepatopancreas axis" in shrimp.

The interaction between the gut and the liver plays a significant role in individual health and diseases. Mounting evidence supports that bile acids are important metabolites in the bidirectional communication between the gut and the liver. Most of the current studies on the "gut-liver axis" have focused on higher vertebrates, however, few was reported on lower invertebrates such as shrimp with an open circulatory system. Here, microbiomic and metabolomic analyses were conducted to investigate the bacterial composition and bile acid metabolism in intestine, hemolymph and hepatopancreas of Penaeus vannamei fed diets supplemented with octanoic acid and oleic acid. After six days of feeding, the bacterial composition in intestine, hemolymph and hepatopancreas changed at different stages, with significant increases in the relative abundance of several genera such as Pseudomonas and Rheinheimera in intestine and hepatopancreas. Notably, there was a more similar bacterial composition in intestine and hepatopancreas at the genus level, which indicated the close communication between shrimp intestine and hepatopancreas. Meanwhile, higher content of some bile acids such as lithocholic acid (LCA) and α-muricholic acid (α-MCA) in intestine and lower content of some bile acids such as taurohyocholic acids (THCA) and isolithocholic acid (IsoLCA) in hepatopancreas were detected. Furthermore, Spearman correlation analysis revealed a significant correlation between bacterial composition and bile acid metabolism in intestine and hepatopancreas. The microbial source tracking analysis showed that there was a high proportion of intestine and hepatopancreas bacterial community as the source of each other. Collectively, these results showed a strong crosstalk between shrimp intestine and hepatopancreas, which suggests a unique potential "intestine-hepatopancreas axis" in lower invertebrate shrimp with an open circulatory system. Our finding contributed to the understanding of the interplay between shrimp intestine and hepatopancreas in the view of microecology and provided new ideas for shrimp farming and disease control.

Phage-prokaryote coexistence strategy mediates microbial community diversity in the intestine and sediment microhabitats of shrimp culture pond ecosystem.

Emerging evidence supports that the phage-prokaryote interaction drives ecological processes in various environments with different phage life strategies. However, the knowledge of phage-prokaryote interaction in the shrimp culture pond ecosystem (SCPE) is still limited. Here, the viral and prokaryotic community profiles at four culture stages in the intestine of Litopenaeus vannamei and cultural sediment microhabitats of SCPE were explored to elucidate the contribution of phage-prokaryote interaction in modulating microbial communities. The results demonstrated that the most abundant viral families in the shrimp intestine and sediment were Microviridae, Circoviridae, Inoviridae, Siphoviridae, Podoviridae, Myoviridae, Parvoviridae, Herelleviridae, Mimiviridae, and Genomoviridae, while phages dominated the viral community. The dominant prokaryotic genera were Vibrio, Formosa, Aurantisolimonas, and Shewanella in the shrimp intestine, and Formosa, Aurantisolimonas, Algoriphagus, and Flavobacterium in the sediment. The viral and prokaryotic composition of the shrimp intestine and sediment were significantly different at four culture stages, and the phage communities were closely related to the prokaryotic communities. Moreover, the phage-prokaryote interactions can directly or indirectly modulate the microbial community composition and function, including auxiliary metabolic genes and closed toxin genes. The interactional analysis revealed that phages and prokaryotes had diverse coexistence strategies in the shrimp intestine and sediment microhabitats of SCPE. Collectively, our findings characterized the composition of viral communities in the shrimp intestine and cultural sediment and revealed the distinct pattern of phage-prokaryote interaction in modulating microbial community diversity, which expanded our cognization of the phage-prokaryote coexistence strategy in aquatic ecosystems from the microecological perspective and provided theoretical support for microecological prevention and control of shrimp culture health management.

Sedimentary Nitrogen and Sulfur Reduction Functional-Couplings Interplay With the Microbial Community of Anthropogenic Shrimp Culture Pond Ecosystem.

Sediment nitrogen and sulfur cycles are essential biogeochemical processes that regulate the microbial communities of environmental ecosystems, which have closely linked to environment ecological health. However, their functional couplings in anthropogenic aquaculture sedimentary ecosystems remain poorly understood. Here, we explored the sediment functional genes in shrimp culture pond ecosystems (SCPEs) at different culture stages using the GeoChip gene array approach with 16S amplicon sequencing. Dissimilarity analysis showed that the compositions of both functional genes and bacterial communities differed at different phases of shrimp culture with the appearance of temporal distance decay (p < 0.05). During shrimp culture, the abundances of nitrite and sulfite reduction functional genes decreased (p < 0.05), while those of nitrate and sulfate reduction genes were enriched (p < 0.05) in sediments, implying the enrichment of nitrites and sulfites from microbial metabolism. Meanwhile, nitrogen and sulfur reduction genes were found to be linked with carbon degradation and phosphorous metabolism (p < 0.05). The influence pathways of nutrients were demonstrated by structural equation modeling through environmental factors and the bacterial community on the nitrogen and sulfur reduction functions, indicating that the bacterial community response to environmental factors was facilitated by nutrients, and led to the shifts of functional genes (p < 0.05). These results indicate that sediment nitrogen and sulfur reduction functions in SCPEs were coupled, which are interconnected with the SCPEs bacterial community. Our findings will be helpful for understanding biogeochemical cycles in anthropogenic aquaculture ecosystems and promoting sustainable management of sediment environments through the framework of an ecological perspective.

Environmental Water and Sediment Microbial Communities Shape Intestine Microbiota for Host Health: The Central Dogma in an Anthropogenic Aquaculture Ecosystem.

From increasing evidence has emerged a tight link among the environment, intestine microbiota, and host health status; moreover, the microbial interaction in different habitats is crucial for ecosystems. However, how the environmental microbial community assembly governs the intestinal microbiota and microbial communities of multiple habitats contribute to the metacommunity remain elusive. Here, we designed two delicate experiments from temporal and spatial scales in a shrimp culture pond ecosystem (SCPE). Of the SCPE metacommunity, the microbial diversity was mainly contributed to by the diversity of-ß IntraHabitats and ß InterHabitats , and water and sediment communities had a large contribution to the shrimp intestine community as shown by SourceTracker and Sloan neutral community model analyses. Also, phylogenetic bin-based null model results show that microbial assembly of three habitats in the SCPE appeared to be largely driven by stochastic processes. These results enrich our understanding of the environment-intestinal microbiota-host health closely linked relationship, making it possible to be the central dogma for an anthropogenic aquaculture ecosystem. Our findings enhance the mechanistic understanding of microbial assembly in the SCPE for further analyzing metacommunities, which has important implications for microbial ecology and animal health.

Distinct bacterial communities in the environmental water, sediment and intestine between two crayfish-plant coculture ecosystems.

Microorganisms are an important part of productivity, water quality, and biogeochemical cycles in an aquaculture ecosystems and play a key role in determining the growth and fitness of aquaculture animals. Coculture ecosystems are widely applied with great significance in agricultural production worldwide. The crayfish-rice coculture ecosystem (CRCE) and crayfish-waterweed coculture ecosystem (CWCE) are two high-profile artificial ecosystems for crayfish culture. However, the bacterial communities of the environmental water, sediment, and intestine in the CRCE and CWCE remain elusive. In this study, we investigated the diversity, composition, and function of bacterial communities in water, sediment, and intestine samples from the CRCE to CWCE. The physicochemical factors of water [such as ORP (oxidation-reduction potential), TC (total carbon), TOC (total oxygen carbon), and NO3--N] and sediment [such as TC, TOC, TN (total nitrogen), and TP (total phosphate)] were significantly different in the CRCE and CWCE. The abundances of Proteobacteria, Actinobacteria, Verrucomicrobia, Cyanobacteria, Chlorobi, Chloroflexi, and Firmicutes were significantly different in the water bacterial communities of the CRCE and CWCE. The abundance of Vibrio in the crayfish intestine was higher in the CRCE than in the CWCE. The most abundant phyla in the CRCE and CWCE sediment were Proteobacteria and Bacteroidetes. The abundances of genes involved in transporters and ABC transporters were different in water of CRCE and CWCE. The abundances of genes involved in oxidative phosphorylation were significantly higher in the crayfish intestine of the CRCE than in that of the CWCE. Furthermore, the functional genes associated with carbon metabolism were significantly more abundant in the sediment of the CRCE than in that of the CWCE. Spearman correlation analysis and redundancy analysis (RDA) showed that the bacterial communities of the water and sediment in the CRCE and CWCE were correlated with environmental factors (pH, total carbon (TC), total oxygen carbon (TOC), total nitrogen (TN), and total phosphorus (TP)). Our findings showed that the composition, diversity and function of the bacterial communities were distinct in the environmental water, sediment, and intestine of the CRCE and CWCE crayfish coculture ecosystems due to their different ecological patterns. These results can help guide healthy farming practices and deepen the understanding of bacterial communities in crayfish-plant coculture ecosystems from the perspective of bacterial ecology. KEY POINTS: • The composition of bacterial communities in the environmental water, sediment, and intestine of the CRCE and CWCE were distinct. ̉• The abundances of genes involved in transporters and ABC transporters were different in the water of the CRCE and CWCE. • The bacterial communities of the water and sediment in the CRCE and CWCE were correlated with some environmental factors.

Stochastic processes shape the bacterial community assembly in shrimp cultural pond sediments.

Sediment environments harbor a repertoire of microorganisms that contribute to animal health and the microecosystem in aquaculture ecosystems, but their community diversity and the potential factors that control it remain unclear. Here, we applied 16S rRNA gene amplicon sequencing to investigate bacterial diversity and assembly mechanisms in the sediments of shrimp cultural ponds at the mesoscale. Our results showed that sediment bacterial communities contained 10,333 operational taxonomic units (OTUs) but had only 34 core OTUs and that the relative abundances of these core OTUs were significantly correlated with the physicochemical properties of the sediments. Proteobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Acidobacteria, Firmicutes, Actinobacteria, Ignavibacteriae, Spirochaetae and Planctomycetes were the ten most abundant bacterial phyla. Notably, some opportunistic pathogens (e.g. Vibrio and Photobacterium) and potential functional microbes (e.g. Nitrospira, Nitrosomonas, Desulfobulbus and Desulfuromusa) were widely distributed in shrimp cultural pond sediments. More importantly, we found that there was a significant negative but weak distance-decay relationship among bacterial communities in shrimp culture pond sediments at the mesoscale, and that the spatial turnover of these bacterial communities appeared to be largely driven by stochastic processes. Additionally, environmental factors, such as pH and total nitrogen, also played important roles in influencing the sediment bacterial structure. Our findings enhance our understanding of microbial ecology in aquatic ecosystems and facilitate sediment microbiota management in aquaculture. KEY POINTS: • Core bacterial taxa in cultural pond sediments contributed to the shrimp health and element cycling. • There was a significant negative distance-decay relationship among bacterial communities in shrimp culture pond sediments at the mesoscale, and its spatial turnover appeared to be largely driven by stochastic processes. • Environmental factors (e.g. pH and total nitrogen) played important roles in influencing bacterial structure in shrimp cultural pond sediments.

Intestine Bacterial Community Composition of Shrimp Varies Under Low- and High-Salinity Culture Conditions.

Intestine microbiota is tightly associated with host health status. Increasing studies have focused on assessing how host intestine microbiota is affected by biotic factors but ignored abiotic factors. Here, we aimed to understand the effects of salinity on shrimp intestine microbiota, by comparing the differences of intestine bacterial signatures of shrimp under low-salinity (LS) and high-salinity (HS) culture conditions. Our results found that intestine core bacterial taxa of shrimp under LS and HS culture conditions were different and that under HS contained more opportunistic pathogen species. Notably, compared with LS culture conditions, opportunistic pathogens (e.g., Vibrio species) were enriched in shrimp intestine under HS. Network analysis revealed that shrimp under HS culture conditions exhibited less connected and lower competitive intestine bacterial interspecies interactions compared with LS. In addition, under HS culture conditions, several opportunistic pathogens were identified as keystone species of intestine bacterial network in shrimp. Furthermore, the ecological drift process played a more important role in the intestine bacterial assembly of shrimp under HS culture conditions than that under LS. These above traits regarding the intestine microbiota of shrimp under HS culture conditions might lead to host at a higher risk of disease. Collectively, this work aids our understanding of the effects of salinity on shrimp intestine microbiota and helps for shrimp culture.

Temporal variation of antibiotic resistance genes carried by culturable bacteria in the shrimp hepatopancreas and shrimp culture pond water.

The increasing prevalence of antibiotic resistance genes (ARGs) is a challenge to the health of humans, animals and the environments. Human activities and aquatic environments can increase ARGs. Few studies have focused on the temporal variation of aquatic bacteria with multiple ARGs in aquatic environments affected by human production activity. We studied culturable bacteria (CB) carrying ARGs, including sul1, sul2, floR, strA and gyrA in the shrimp hepatopancreas (HP) and in pond water during shrimp culture. The relative abundance of ARGs carried by CB in HP was higher than that in water (P < 0.05). However, CB carrying ARGs generally varied in random pattern. The correlation of sul2 abundance was significantly positive in HP, while that of strA abundance was significantly negative in water (P < 0.05) during shrimp culture. Among all of the CB, 33.59% carried multiple ARGs. Temporal distance-decay analysis indicated that CB carrying ARGs in water were more resistant to the effects of human activity. CB carrying ARGs varied temporally in HP and pond water during shrimp culture. These results demonstrate that multiple ARGs are carried by CB, and these varied with the phase of aquatic culture.

Identification of Multigene Biomarker for Shrimp White Feces Syndrome by Full-Length Transcriptome Sequencing.

The pacific white shrimp, Litopenaeus vannamei, with the largest shrimp industry production in the world, is currently threatened by a severe disease, white feces syndrome (WFS), which cause devastating losses globally, while its causal agents remain largely unknown. Herein, compared to the Control shrimp by metagenomic analysis, we firstly investigated that the altered functions of intestinal microbial community in WFS shrimp were the enrichment of bacterial chemotaxis and flagellar assembly pathways, hinting at a potential role of pathogenic bacteria for growth and development, which might be related to WFS occurrence. Single-molecule real-time (SMRT) sequencing was to further identify the gene structure and gene regulation for more clues in WFS aetiology. Totally 50,049 high quality transcripts were obtained, capturing 39,995 previously mapped and 10,054 newly detected transcripts, which were annotated to 30,554 genes. A total of 158 differentially expressed genes (DEGs) were characterized in WFS shrimp. These DEGs were strongly associated with various immune related genes that regulated the expression of multiple antimicrobial peptides (e.g., antilipopolysaccharide factors, penaeidins, and crustin), which were further experimentally validated using quantitative PCR on transcript level. Collectively, multigene biomarkers were identified to be closely associated with WFS, especially those functional alterations in microbial community and the upregulated immune related gene with antibacterial activities. Our finding not only inspired our cogitation on WFS aetiology from both microbial and host immune response perspectives with combined metagenomic and full-length transcriptome sequencing, but also provided valuable information for enhancing shrimp aquaculture.

Microecological Koch's postulates reveal that intestinal microbiota dysbiosis contributes to shrimp white feces syndrome.

BACKGROUND: Recently, increasing evidence supports that some complex diseases are not attributed to a given pathogen, but dysbiosis in the host intestinal microbiota (IM). The full intestinal ecosystem alterations, rather than a single pathogen, are associated with white feces syndrome (WFS), a globally severe non-infectious shrimp disease, while no experimental evidence to explore the causality. Herein, we conducted comprehensive metagenomic and metabolomic analysis, and intestinal microbiota transplantation (IMT) to investigate the causal relationship between IM dysbiosis and WFS. RESULTS: Compared to the Control shrimp, we found dramatically decreased microbial richness and diversity in WFS shrimp. Ten genera, such as Vibrio, Candidatus Bacilloplasma, Photobacterium, and Aeromonas, were overrepresented in WFS, whereas 11 genera, including Shewanella, Chitinibacter, and Rhodobacter were enriched in control. The divergent changes in these populations might contribute the observation that a decline of pathways conferring lipoic acid metabolism and mineral absorption in WFS. Meanwhile, some sorts of metabolites, especially lipids and organic acids, were found to be related to the IM alteration in WFS. Integrated with multiomics and IMT, we demonstrated that significant alterations in the community composition, functional potentials, and metabolites of IM were closely linked to shrimp WFS. The distinguished metabolites which were attributed to the IM dysbiosis were validated by feed-supplementary challenge. Both homogenous selection and heterogeneous selection process were less pronounced in WFS microbial community assembly. Notably, IMT shrimp from WFS donors eventually developed WFS clinical signs, while the dysbiotic IM can be recharacterized in recipient shrimp. CONCLUSIONS: Collectively, our findings offer solid evidence of the causality between IM dysbiosis and shrimp WFS, which exemplify the 'microecological Koch's postulates' (an intestinal microbiota dysbiosis, a disease) in disease etiology, and inspire our cogitation on etiology from an ecological perspective. Video abstract.

Occurrence of human pathogenic bacteria carrying antibiotic resistance genes revealed by metagenomic approach: A case study from an aquatic environment.

Antibiotic resistance genes (ARGs), human pathogenic bacteria (HPB), and HPB carrying ARGs are public issues that pose a high risk to aquatic environments and public health. Their diversity and abundance in water, intestine, and sediments of shrimp culture pond were investigated using metagenomic approach. A total of 19 classes of ARGs, 52 HPB species, and 7 species of HPB carrying ARGs were found. Additionally, 157, 104, and 86 subtypes of ARGs were detected in shrimp intestine, pond water, and sediment samples, respectively. In all the samples, multidrug resistance genes were the highest abundant class of ARGs. The dominant HPB was Enterococcus faecalis in shrimp intestine, Vibrio parahaemolyticus in sediments, and Mycobacterium yongonense in water, respectively. Moreover, E. faecalis (contig Intestine_364647) and Enterococcus faecium (contig Intestine_80272) carrying efrA, efrB and ANT(6)-Ia were found in shrimp intestine, Desulfosaricina cetonica (contig Sediment_825143) and Escherichia coli (contig Sediment_188430) carrying mexB and APH(3')-IIa were found in sediments, and Laribacter hongkongensis (contig Water_478168 and Water_369477), Shigella sonnei (contig Water_880246), and Acinetobacter baumannii (contig Water_525520) carrying sul1, sul2, ereA, qacH, OXA-21, and mphD were found in pond water. Mobile genetic elements (MGEs) analysis indicated that horizontal gene transfer (HGT) of integrons, insertion sequences, and plasmids existed in shrimp intestine, sediment, and water samples, and the abundance of integrons was higher than that of other two MGEs. The results suggested that HPB carrying ARGs potentially existed in aquatic environments, and that these contributed to the environment and public health risk evaluation.

Characterization of airborne antibiotic resistance genes from typical bioaerosol emission sources in the urban environment using metagenomic approach.

The wide spread of antibiotic resistance genes (ARGs) has attracted increasing concern. However, the occurrence and diversity of ARGs in airborne particles remains to be understood. In this study, total suspended particles (TSP) in the atmosphere were collected from typical sources of ARG pollution, including animal farms and wastewater treatment plant (WWTP), as well as the downtown area in Zhuhai, China. Metagenomic profiling demonstrated that ARGs were abundant and diverse in the TSP from animal farms and WWTP, but significant differences in ARG composition pattern between these samples were observed. ARGs associated with the resistance to aminoglycoside, macrolide-lincosamide-streptogramin (MLS) and tetracycline were dominant over other ARGs in the TSP of the animal farms, whereas multidrug and bacitracin resistance genes were more abundant than other ARGs in the TSP of the WWTP. In the animal farms, ARG profiles of the TSP were consistent with those of animal feces, indicating that animal feces could be one of the most contributing sources of airborne ARGs in animal farms. In contrast to representative sources of ARG pollution, ARG abundance and diversity in the TSP collected from the downtown area was relatively low, with multidrug resistance genes being predominant. This study suggests that metagenomic profiling of the ARGs in airborne TSP could enhance our comprehensive understanding of ARGs dissemination in the environment and their potential health threats.

Characterizing the antibiotic resistance genes in a river catchment: Influence of anthropogenic activities.

Previous studies on environmental antibiotics resistance genes (ARGs) have focused on the pollution sources such as wastewater treatment plants, aquaculture and livestock farms, etc. Few of them had addressed this issue in a regional scale such as river catchment. Hence, the occurrence and abundances of 23 ARGs were investigated in surface water samples collected from 38 sites which located from the river source to estuary of the Beijiang River. Among them, 11 ARGs were frequently detected in this region and 5 ARGs (sulI, sulII, tetB, tetC, and tetW) were selected for their distribution pattern analysis. The abundances of the selected ARGs were higher in the upstream (8.70×106copies/ng DNA) and downstream areas (3.17×106copies/ng DNA) than those in the midstream areas (1.23×106copies/ng DNA), which was positively correlated to the population density and number of pollution sources. Pollution sources of ARGs along the Beijiang River not only had a great impact on the abundances and diversity, but also on the distribution of specific ARGs in the water samples. Both sulI and sulII were likely originated from aquaculture farms and animal farms, tetW gene was possibly associated with the mining/metal melting industry and the electric waste disposal and tetC gene was commonly found in the area with multiple pollution sources. However, the abundance of tetB was not particularly related to anthropogenic impacts. These findings highlight the influence of pollution sources and density of population on the distribution and dissemination of ARGs at a regional scale.

Exploring the differences of antibiotic resistance genes profiles between river surface water and sediments using metagenomic approach.

To better understand the potential genic communication and dissemination of antibiotic resistance genes (ARGs) in different environmental matrices, the differences of ARG profiles between river surface water and sediments were explored. Metagenomic analysis was applied to investigate the comprehensive ARG profiles in water and sediment samples collected from the highly human-impacted catchment of the Beijiang River and its river source. A total of 135 ARG subtypes belonging to 18 ARG types were identified. Generally, ARGs in surface water were more diverse and abundant than those in sediments. ARG profiles in the surface water and sediment samples were distinct from each other, but some ARGs were shared by the surface water and sediments. Results revealed that multidrug and bacitracin resistance genes were the predominant ARGs types in both surface water (0.30, 0.17 copies/cell) and sediments (0.19, 0.15 copies/cell). 73 ARG subtypes were shared by the water and sediment samples and had taken over 90% of the total detected ARG abundance. Most of the shared ARGs are resistant to the clinically relevant antibiotics. Furthermore, significant correlations between the ARGs and 21 shared genera or mobile genetic elements (MGEs) (plasmids and integrons) were found in surface water and sediments, suggesting the important role of genera or MGEs in shaping ARGs profiles, propagation and distribution. These findings provide deeper insight into mitigating the propagation of ARGs and the associated risks to public health.

Acceleration of modern acidification in the South China Sea driven by anthropogenic CO2.

Modern acidification by the uptake of anthropogenic CO2 can profoundly affect the physiology of marine organisms and the structure of ocean ecosystems. Centennial-scale global and regional influences of anthropogenic CO2 remain largely unknown due to limited instrumental pH records. Here we present coral boron isotope-inferred pH records for two periods from the South China Sea: AD 1048-1079 and AD 1838-2001. There are no significant pH differences between the first period at the Medieval Warm Period and AD 1830-1870. However, we find anomalous and unprecedented acidification during the 20th century, pacing the observed increase in atmospheric CO2. Moreover, pH value also varies in phase with inter-decadal changes in Asian Winter Monsoon intensity. As the level of atmospheric CO2 keeps rising, the coupling global warming via weakening the winter monsoon intensity could exacerbate acidification of the South China Sea and threaten this expansive shallow water marine ecosystem.