Nitrite nitrogen stress disrupts the intestine bacterial community by altering host-community interactions in shrimp.

Environmental stress can disrupt the intricate interactions between the host and intestine microbiota, thereby impacting the host health. In this study, we aimed to elucidate the dynamic changes in the bacterial community within shrimp intestines under nitrite nitrogen (nitrite-N) stress and investigate potential host-related factors influencing these changes. Our results revealed a significant reduction in community diversity within the intestine exposed to nitrite-N compared to control conditions. Furthermore, distinct differences in community structures were observed between these two groups at 72 h and 120 h post-stress induction. Nitrite-N stress also altered the abundances of some bacterial species in the intestine dramatically. It is noteworthy that, in comparison to the 72 h, intestine bacterial community structure of stressed shrimp exhibited a significantly higher degree of dispersion after 120 h of nitrite-N stress when compared to control shrimp, and the relative abundance of numerous bacterial species experienced a substantial decrease or even reached 0 %. Moreover, it led to a reduction in bacterial community interactions and decreased competitiveness within the intestine microbiota. Notably, the influence of bacterial community assemblies in the shrimp intestine shifted from a stochastic process to a deterministic one after 24 h and 72 h of nitrite-N stress, returning to a stochastic process at 120 h. We further observed a close association between this phenomenon and host's response to nitrite-N stress. Expression levels of differentially expressed genes in the intestinal tissue significantly impact the intestine bacterial diversity and abundance of species. In particular, the significant decline in bacterial diversity and abundances of quite a few species in intestine was attributed to the up-regulation of peritrophin-48-like. Overall, nitrite-N stress indeed disrupted the intestine microbiota and changed the host-microbiota interactions of shrimp. This study offered novel insights into environment-host-microbiota interactions and also provided practical guidance for promoting healthy shrimp cultivation practices.

Intestine bacterial community affects the growth of the Pacific white shrimp (Litopenaeus vannamei).

Increasing evidence suggests that intestine microorganisms are closely related to shrimp growth, but there is no existing experiment to prove this hypothesis. Here, we compared the intestine bacterial community of fast- and slow-growing shrimp at the same developmental stage with a marked difference in body size. Our results showed that the intestine bacterial communities of slow-growing shrimp exhibited less diversity but were more heterogeneous than those of fast-growing shrimp. Uncultured_bacterium_g_Candidatus Bacilloplasma, Tamlana agarivorans, Donghicola tyrosinivorans, and uncultured_bacterium_f_Flavobacteriaceae were overrepresented in the intestines of fast-growing shrimp, while Shimia marina, Vibrio sp., and Vibrio campbellii showed the opposite trends. We further found that the bacterial community composition was significantly correlated with shrimp length, and some bacterial species abundances were found to be significantly correlated with shrimp weight and length, including T. agarivorans and V. campbellii, which were chosen as indicators for a reverse gavage experiment. Finally, T. agarivorans was found to significantly promote shrimp growth after the experiment. Collectively, these results suggest that intestine bacterial community could be important factors in determining the growth of shrimp, indicating that specific bacteria could be tested in further studies against shrimp growth retardation. KEY POINTS: • A close relationship between intestine bacterial community and shrimp growth was proven by controllable experiments. • The bacterial signatures of the intestine were markedly different between slow- and fast-growing shrimp, and the relative abundances of some intestine bacterial species were correlated significantly with shrimp body size. • Reverse gavage by Tamlana agarivorans significantly promoted shrimp growth.

Integration of microbiome and Koch's postulates to reveal multiple bacterial pathogens of whitish muscle syndrome in mud crab, Scylla paramamosain.

BACKGROUND: For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch's postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch's postulates to determinate causative agents of multiple pathogens-one disease. RESULTS: Microbiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch's postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently. CONCLUSIONS: This study fully demonstrated that our research strategy that integrates the microbiome and Koch's postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease. Video Abstract.

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.

Whole-Genome Sequencing and Analysis of the White-Rot Fungus Ceriporia lacerata Reveals Its Phylogenetic Status and the Genetic Basis of Lignocellulose Degradation and Terpenoid Synthesis.

Ceriporia lacerata is an endophytic white-rot fungus that has lignocellulolytic and terpenoid-biosynthetic abilities. However, little is known about the genomic architecture of this fungus, even at the genus level. In this study, we present the first de novo genome assembly of C. lacerata (CGMCC No. 10485), based on PacBio long-read and Illumina short-read sequencing. The size of the C. lacerata genome is approximately 36 Mb (N50, 3.4 Mb). It encodes a total of 13,243 genes, with further functional analysis revealing that these genes are primarily involved in primary metabolism and host interactions in this strain's saprophytic lifestyle. Phylogenetic analysis based on ITS demonstrated a primary evolutionary position for C. lacerata, while the phylogenetic analysis based on orthogroup inference and average nucleotide identity revealed high-resolution phylogenetic details in which Ceriporia, Phlebia, Phlebiopsis, and Phanerochaete belong to the same evolutionary clade within the order Polyporales. Annotation of carbohydrate-active enzymes across the genome yielded a total of 806 genes encoding enzymes that decompose lignocellulose, particularly ligninolytic enzymes, lytic polysaccharides monooxygenases, and enzymes involved in the biodegradation of aromatic components. These findings illustrate the strain's adaptation to woody habitats, which requires the degradation of lignin and various polycyclic aromatic hydrocarbons. The terpenoid-production potential of C. lacerata was evaluated by comparing the genes of terpenoid biosynthetic pathways across nine Polyporales species. The shared genes highlight the major part of terpenoid synthesis pathways, especially the mevalonic acid pathway, as well as the main pathways of sesquiterpenoid, monoterpenoid, diterpenoid, and triterpenoid synthesis, while the strain-specific genes illustrate the distinct genetic factors determining the synthesis of structurally diverse terpenoids. This is the first genomic analysis of a species from this genus that we are aware of, and it will help advance functional genome research and resource development of this important fungus for applications in renewable energy, pharmaceuticals, and agriculture.

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.

Sediment microbiota in polyculture of shrimp and fish pattern is distinctive from those in monoculture intensive shrimp or fish ponds.

Sediment microbial community plays a crucial role in aquaculture ecosystem. In aquaculture practice, rather than monoculture intensive shrimp (IS) or intensive fish (IF) patterns, polyculture of shrimp and fish (PolySF) pattern leads to a more reliable production. However, knowledge is still limited about the characteristics of sediment microbiota and its potential functions in the PolySF ponds compared to monoculture patterns (IS and IF). Herein, we collected sediment samples from these three patterns in seven cities to evaluate microbial variations among patterns. The highest oxidation reduction potential (ORP), total phosphate (TP) and total organic carbon (TOC) were detected in the PolySF pattern, representing a relatively less anoxic environment, while the highest iron (Fe) was detected in IS pattern. Proteobacteria was the most abundant phylum among three patterns, followed by Bacteroidetes and Chloroflexi. The microbial alpha diversity in the PolySF was higher than those in the IF, but lower than those in the IS. Microbial communities of these three patterns were significantly distinct from each other, and 23 distinguished taxa for each pattern were further characterized. In additional, the relative abundances of genes involved in nitrogen metabolism, fatty acid biosynthesis and carbon fixation pathways were markedly shifted. Moreover, ORP, TOC and Fe were the shaping factors for sediment microbiota, which significantly varied among three patterns. Collectively, these findings demonstrated that sediment microbial communities in the PolySF were distinctive from those in the IS and IF, which enlarged our understanding for the underlying mechanism of advances in the PolySF pattern from ecological perspective.

Host development overwhelms environmental dispersal in governing the ecological succession of zebrafish gut microbiota.

Clarifying mechanisms underlying the ecological succession of gut microbiota is a central theme of gut ecology. Under experimental manipulations of zebrafish hatching and rearing environments, we test our core hypothesis that the host development will overwhelm environmental dispersal in governing fish gut microbial community succession due to host genetics, immunology, and gut nutrient niches. We find that zebrafish developmental stage substantially explains the gut microbial community succession, whereas the environmental effects do not significantly affect the gut microbiota succession from larvae to adult fish. The gut microbiotas of zebrafish are clearly separated according to fish developmental stages, and the degree of homogeneous selection governing gut microbiota succession is increasing with host development. This study advances our mechanistic understanding of the gut microbiota assembly and succession by integrating the host and environmental effects, which also provides new insights into the gut ecology of other aquatic animals.

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.

Antibiotic supplement in feed can perturb the intestinal microbial composition and function in Pacific white shrimp.

The intestinal microbiota plays crucial roles in host health. The Pacific white shrimp is one of the most profitable aquaculture species in the world. Antibiotic supplement in feed is an optional practice to treat shrimp bacterial diseases. However, little is known about antibiotic effects on intestinal microbiota in pacific white shrimp. Here, shrimps were given feed supplemented with ciprofloxacin (Cip) (40 and 80 mg kg-1) and sulfonamide (Sul) (200 and 400 mg kg-1) to investigate the microbial community by targeting the V4 region of 16S rRNA genes. Within 4 days after feeding with normal feed and with antibiotics, antibiotic concentrations of Cip and Sul groups in the intestine dropped sharply. Significantly, increased abundance of antibiotic resistance genes (ARGs) of ciprofloxacin (qnrB, qnrD, and qnrS) and sulfonamide (sul1, sul2, and sul3) was observed in Cip and Sul groups (P < 0.05). A total of 3191 operational taxonomic units (OTUs) were obtained and 41 phyla were identified from 63 samples in shrimp intestine. The numbers of OTUs and Shannon index decreased rapidly at day 1 (the first day after feeding with antibiotics) and increased at day 3 (the third day after feeding with antibiotics). The relative abundance of dominant phyla and genera in Cip and Sul groups were significantly different from that in the control group (Ctrl). Furthermore, functional potentials that were related to amino acid metabolism, carbohydrate metabolism, and cellular processes and signaling varied significantly in Cip and Sul groups. These results point to an antibiotic-induced shift in shrimp intestinal microbiota, which highlights the importance of considering the microbiota in shrimp health management.

Intestinal bacterial signatures of white feces syndrome in shrimp.

Increasing evidence suggests that the intestinal microbiota is closely correlated with the host's health status. Thus, a serious disturbance that disrupts the stability of the intestinal microecosystem could cause host disease. Shrimps are one of the most important products among fishery trading commodities. However, digestive system diseases, such as white feces syndrome (WFS), frequently occur in shrimp culture and have led to enormous economic losses across the world. The WFS occurrences are unclear. Here, we compared intestinal bacterial communities of WFS shrimp and healthy shrimp. Intestinal bacterial communities of WFS shrimp exhibited less diversity but were more heterogeneous than those of healthy shrimp. The intestinal bacterial communities were significantly different between WFS shrimp and healthy shrimp; compared with healthy shrimp, in WFS shrimp, Candidatus Bacilloplasma and Phascolarctobacterium were overrepresented, whereas Paracoccus and Lactococcus were underrepresented. PICRUSt functional predictions indicated that the relative abundances of genes involved in energy metabolism and genetic information processing were significantly greater in WFS shrimp. Collectively, we found that the composition and predicted functions of the intestinal bacterial community were markedly shifted by WFS. Significant increases in Candidatus Bacilloplasma and Phascolarctobacterium and decreases in Paracoccus and Lactococcus may contribute to WFS in shrimp.

Environmental Factors Shape Water Microbial Community Structure and Function in Shrimp Cultural Enclosure Ecosystems.

The effects of environmental factors on water microbial communities have been extensively studied, but little is known about the effects in shrimp cultural enclosure ecosystems. We analyzed 16S rRNA gene amplicons to determine the principal environmental factors that shape the structure and function of microbial communities in shrimp cultural enclosure ecosystems from Guangdong and Hainan provinces, in China. High quality sequences were clustered into operational taxonomic units (OTUs) at the 97% similarity level, generating 659-1,835 OTUs per sample. The 10 most abundant phyla were Proteobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, Actinobacteria, Verrucomicrobia, Firmicutes, Chlorobi, Chloroflexi, and Chlamydiae. The results of canonical correspondence analyses (CCA) indicated that salinity, total phosphate (TP), total nitrogen (TN), temperature, and pH were the most important factors shaping microbial community structure. Differences in microbial community structure between high and low salinity samples were explained by changes in the relative abundances of some OTUs (e.g., OTU5, OTU19, OTU21, OTU39, and OTU71). Moreover, the contribution of spatial distribution to the microbial community assembly was investigated via aggregated boosted tree (ABT) analyses, and the results indicated spatial isolation was not a major factor affecting the phylogenetic diversity and phylotypes of water microbial communities. Furthermore, we predicted water microbial community functional profiling using the PICRUSt program and principal component analyses (PCA) suggested that salinity was a major contributor to the structure and function of the microbial communities. Collectively, these results showed that environmental factors influenced the structure and function of water microbial communities, while salinity was the principal environmental factor instead of temperature, TP, TN, and pH in shrimp cultural enclosure ecosystems.

Composition, diversity and function of intestinal microbiota in pacific white shrimp (Litopenaeus vannamei) at different culture stages.

Intestinal microbiota is an integral component of the host and plays important roles in host health. The pacific white shrimp is one of the most profitable aquaculture species commercialized in the world market with the largest production in shrimp consumption. Many studies revealed that the intestinal microbiota shifted significantly during host development in other aquaculture animals. In the present study, 22 shrimp samples were collected every 15 days from larval stage (15 day post-hatching, dph) to adult stage (75 dph) to investigate the intestinal microbiota at different culture stages by targeting the V4 region of 16S rRNA gene, and the microbial function prediction was conducted by PICRUSt. The operational taxonomic unit (OTU) was assigned at 97% sequence identity. A total of 2,496 OTUs were obtained, ranging from 585 to 1,239 in each sample. Forty-three phyla were identified due to the classifiable sequence. The most abundant phyla were Proteobacteria, Cyanobacteria, Tenericutes, Fusobacteria, Firmicutes, Verrucomicrobia, Bacteroidetes, Planctomycetes, Actinobacteria and Chloroflexi. OTUs belonged to 289 genera and the most abundant genera were Candidatus_Xiphinematobacter, Propionigenium, Synechococcus, Shewanella and Cetobacterium. Fifty-nine OTUs were detected in all samples, which were considered as the major microbes in intestine of shrimp. The intestinal microbiota was enriched with functional potentials that were related to transporters, ABC transporters, DNA repair and recombination proteins, two component system, secretion system, bacterial motility proteins, purine metabolism and ribosome. All the results showed that the intestinal microbial composition, diversity and functions varied significantly at different culture stages, which indicated that shrimp intestinal microbiota depended on culture stages. These findings provided new evidence on intestinal microorganism microecology and greatly enhanced our understanding of stage-specific community in the shrimp intestinal ecosystem.