Zebrafish Gut Research Articles

Probiotic Escherichia coli Nissle 1917 alleviates the neurotoxicity caused by acrylamide in zebrafish.

Neurotoxicity is caused by damage to the brain tissue by neurotoxic agents present in the environment and artificial substances produced by human beings. Acrylamide (ACR) is one such chemical substance that causes neurotoxicity, affecting the brain cells. This neurotoxicity causes damage to the sensory and metabolic functions. The current research investigates the favourable effect of probiotic EcN (Escherichia coli Nissle 1917) on ACR-induced neurotoxicity in zebrafish. The protective role of EcN against ACR induced toxicity was assessed based on behaviour, biochemical, and gene expression analysis. Initially, the colonisation period of EcN in the zebrafish gut was determined and EcN was given orally to the zebrafish only once prior to the ACR treatment. Very interestingly, this dosage was able to ameliorate the adverse effects of ACR significantly in the brain cells. Quantification of oxidative stress and neuronal cell death clearly vindicate the efficiency of probiotic EcN in reversing the damages caused by ACR. EcN is being explored largely in recent days for its therapeutic applications. This study strongly supports the view that EcN can be developed as a supplement to the patients diagnosed with neuronal cell toxicity.

The probiotic SLAB51 as agent to counteract BPA toxicity on zebrafish gut microbiota -liver-brain axis

A plethora of studies have so far described the toxic effects of bisphenol A (BPA) on organism health, highlighting the urgent need to find new strategies not only to reduce the presence of this toxicant but also to counteract its adverse effects. In this context, probiotics emerged as a potential tool since they promote organism welfare. Using a multidisciplinary approach, this study explores the effects of SLAB51 dietary administration to counteract BPA toxicity using zebrafish as a model. Adult males and females were maintained under standard conditions (control group; C), exposed for 28 days via the water to an environmental relevant dose of BPA (10 μg/L; BPA), dietary treated with SLAB51 (109 CFU/g of body weight; P) and co-treated with BPA plus SLAB51 (BPA + P). In the gut, exposure to BPA resulted in altered architecture in both males and females, with females also experiencing an increase of pathogenic bacterial species. Co-administration of BPA + P led to the restoration of normal gut architecture, favored beneficial bacteria colonization, and decreased the abundance of pathogenic species. In the liver, male BPA exposure led to steatosis and glycogen depletion, which was partially mitigated by SLAB51 co-administration. In contrast, in females exposed to BPA, the lack of steatosis along with the greater glycogen depletion, suggested an increase in energy demand as supported by the metabolomic phenotype. The analysis of liver metabolites in BPA + P males revealed increased levels of anserine and reduced levels of glutamine, which could lie behind the counteraction of the brain histopathological damage caused by BPA. In BPA + P females, a reduction of retinoic acid was found in the liver, suggesting an increase in retinoids responsible for BPA detoxification. Overall, these results demonstrate that SLAB51 exerts its beneficial effects on the gut microbiota-brain-liver axis through distinct molecular pathways, effectively mitigating the pleiotropic toxicity of BPA.

Multi-omics analysis of zebrafish response to tick saliva reveals biological processes associated with alpha-Gal syndrome

The alpha-Gal syndrome (AGS) is a tick-borne allergy. A multi-omics approach was used to determine the effect of tick saliva and mammalian meat consumption on zebrafish gut transcriptome and proteome. Bioinformatics analysis using R software was focused on significant biological and metabolic pathway changes associated with AGS. Ortholog mapping identified highly concordant human ortholog genes for the detection of disease-enriched pathways. Tick saliva treatment increased zebrafish mortality, incidence of hemorrhagic type allergic reactions and changes in behavior and feeding patterns. Transcriptomics analysis showed downregulation of biological and metabolic pathways correlated with anti-alpha-Gal IgE and allergic reactions to tick saliva affecting blood circulation, cardiac and vascular smooth muscle contraction, behavior and sensory perception. Disease enrichment analysis revealed downregulated orthologous genes associated with human disorders affecting nervous, musculoskeletal, and cardiovascular systems. Proteomics analysis revealed suppression of pathways associated with immune system production of reactive oxygen species and cardiac muscle contraction. Underrepresented proteins were mainly linked to nervous and metabolic human disorders. Multi-omics data revealed inhibition of pathways associated with adrenergic signaling in cardiomyocytes, and heart and muscle contraction. Results identify tick saliva-related biological pathways supporting multisystemic organ involvement and linking α-Gal sensitization with other illnesses for the identification of potential disease biomarkers.

Single-cell resolution of the adult zebrafish intestine under conventional conditions and in response to an acute Vibrio cholerae infection

Vibrio cholerae is an aquatic bacterium that causes severe and potentially deadly diarrheal disease. Despite the impact on global health, our understanding of host mucosal responses to Vibrio remains limited, highlighting a knowledge gap critical for the development of effective prevention and treatment strategies. Using a natural infection model, we combine physiological and single-cell transcriptomic studies to characterize conventionally reared adult zebrafish guts and guts challenged with Vibrio. We demonstrate that Vibrio causes a mild mucosal immune response characterized by Tcell activation and enhanced antigen capture; Vibrio suppresses host interferon signaling; and ectopic activation of interferon alters the course of infection. We show that the adult zebrafish gut shares similarities with mammalian counterparts, including the presence of Best4+ cells, tuft cells, and a population of basal cycling cells. These findings provide important insights into host-pathogen interactions and emphasize the utility of zebrafish as a natural model of Vibrio infection.

Unveiling nanotubes-mediated communication: Enterococcus faecalis countering Salmonella ser. Typhi - In vitro and In vivo insights

Bacteria communicate with each other through contact-dependent and contact-independent mechanisms. While certain contact-dependent mechanisms, such as Type IV and Type VI, have received considerable attention, nanotubes-mediated communication among gut bacteria remains largely unknown. The purpose of this study is to demonstrate the presence of nanotube production in both gut commensal and gut pathogenic bacteria. And also aims to show how Enterococcus faecalis utilizes nanotubes to combat Salmonella ser. Typhi (S. Typhi), a pathogen in the gut. The research findings suggest that the formation of nanotubes is an inherent trait observed in both Gram-positive and Gram-negative bacteria. Interestingly, bacteria generate nanotubes in dynamic environments, biofilms, and even within the gut of zebrafish. These nanotubes develops over time in accordance with the duration of incubation. Furthermore, E. faecalis effectively combats S. Typhi through mechanisms that depend on physical contact rather than indirect methods. Notably, E. faecalis protects zebrafish larvae from S. Typhi infections by reducing reactive oxygen species and cell death, and concurrently boosting the production of antioxidant enzymes. It is hypothesized that E. faecalis might eliminate S. Typhi by transferring toxic metabolites into the pathogen via nanotubes. Gene expression analysis highlights that proinflammatory markers such as TNF-α, IL-1β, and IL-6 are elevated in Salmonella-infected larvae. However, co-treatment with E. faecalis counters this effect. Findings of this study underscores the significance of nanotubes as a vital machinery for bacterial communication and distribution of virulence factors. Exploring nanotubes-mediated communication at a molecular level could pave the way for innovative therapeutic interventions.

Disentangling the link between zebrafish diet, gut microbiome succession, and Mycobacterium chelonae infection

BackgroundDespite the long-established importance of zebrafish (Danio rerio) as a model organism and their increasing use in microbiome-targeted studies, relatively little is known about how husbandry practices involving diet impact the zebrafish gut microbiome. Given the microbiome’s important role in mediating host physiology and the potential for diet to drive variation in microbiome composition, we sought to clarify how three different dietary formulations that are commonly used in zebrafish facilities impact the gut microbiome. We compared the composition of gut microbiomes in approximately 60 AB line adult (129- and 214-day-old) zebrafish fed each diet throughout their lifespan.ResultsOur analysis finds that diet has a substantial impact on the composition of the gut microbiome in adult fish, and that diet also impacts the developmental variation in the gut microbiome. We further evaluated how 214-day-old fish microbiome compositions respond to exposure of a common laboratory pathogen, Mycobacterium chelonae, and whether these responses differ as a function of diet. Our analysis finds that diet determines the manner in which the zebrafish gut microbiome responds to M. chelonae exposure, especially for moderate and low abundance taxa. Moreover, histopathological analysis finds that male fish fed different diets are differentially infected by M. chelonae.ConclusionsOverall, our results indicate that diet drives the successional development of the gut microbiome as well as its sensitivity to exogenous exposure. Consequently, investigators should carefully consider the role of diet in their microbiome zebrafish investigations, especially when integrating results across studies that vary by diet.

Intestinal expression patterns of transcription factors and markers for interstitial cells in the larval zebrafish.

For the digestion of food, it is important for the gut to be differentiated regionally and to have proper motor control. However, the number of transcription factors that regulate its development is still limited. Meanwhile, the interstitial cells of the gastrointestinal (GI) tract are necessary for intestinal motility in addition to the enteric nervous system. There are anoctamine1 (Ano1)-positive and platelet-derived growth factor receptor α (Pdgfra)-positive interstitial cells in mammal, but Pdgfra-positive cells have not been reported in the zebrafish. To identify new transcription factors involved in GI tract development, we used RNA sequencing comparing between larval and adult gut. We isolated 40 transcription factors that were more highly expressed in the larval gut. We demonstrated expression patterns of the 13 genes, 7 of which were newly found to be expressed in the zebrafish larval gut. Six of the 13 genes encode nuclear receptors. The osr2 is expressed in the anterior part, while foxP4 in its distal part. Also, we reported the expression pattern of pdgfra for the first time in the larval zebrafish gut. Our data provide fundamental knowledge for studying vertebrate gut regionalization and motility by live imaging using zebrafish.

Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System.

The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.

Combined effects of lead and manganese on locomotor activity and microbiota in zebrafish

Exposure to lead (Pb) and manganese (Mn) during early life influences neurodevelopment and increases the risk of neurodegenerative disorders. However, the level of developmental neurotoxicity due to combined exposure to the two metals remains unclear. Although the microbiota plays an essential part in the development of the nervous system via the gut–brain axis, there is a paucity of information regarding the interactions between exposure to Pb and Mn, the destruction of the microbiome, and neurodevelopmental impacts. To fill in this knowledge gap, we investigated the developmental neurotoxicity and effects on the microbiota of Pb (0.05 mg·L−1) alone and in combination with Mn (0.3 mg·L−1) in zebrafish larvae. Our results revealed that combined exposure precipitated higher malformation rates and lower locomotor activity levels than exposure to either Pb or Mn alone. Additionally, when we separated the combined exposure group from the other groups by applying unsupervised principal coordinates analysis (PCoA) and linear discriminant analysis (LEfSe) of microflora sequencing results, we observed extensive alterations in microbial abundances under combined-exposure conditions. Functional prediction analysis showed that combined exposure contributed to altered amino acid and lipid metabolism, and also that combined exposure to Pb and Mn reflected the greatest number of differentially activated biological pathways compared to the other three groups. ATP-binding cassette G (ABCG) genes and genes related to serotonin signaling and metabolism were altered following combined Pb and Mn exposure and exhibited disparate trends vis-à-vis Pb or Mn exposure alone. According to the results, the combined exposure to Pb and Mn led to more severe effects on both zebrafish locomotor activity and gut microbial composition. We suggest that the microbiota contributes to the combined neurotoxicity by increasing ABCG5 and ABCG8 gene expression.

Comparison of the gill and gut microbiomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio) and their RAS environment

Recirculating aquaculture systems (RAS) are increasingly being used to grow fish, as intensive water reuse reduces water consumption and environmental impact. RAS use biofilters containing nitrogen-cycling microorganisms that remove ammonia from the aquaculture water. Knowledge of how RAS microbial communities relate to the fish-associated microbiome is limited, as is knowledge of fish-associated microbiota in general. Recently, nitrogen-cycling bacteria have been discovered in zebrafish and carp gills and shown to detoxify ammonia in a manner similar to the RAS biofilter. Here, we compared RAS water and biofilter microbiomes with fish-associated gut and gill microbial communities in laboratory RAS housing either zebrafish (Danio rerio) or common carp (Cyprinus carpio) using 16S rRNA gene amplicon sequencing. The phylogeny of ammonia-oxidizing bacteria in the gills and the RAS environment was investigated in more detail by phylogenetic analysis of the ammonia monooxygenase subunit A (amoA). The location from which the microbiome was sampled (RAS compartments and gills or gut) had a stronger effect on community composition than the fish species, but species-specific differences were also observed. We found that carp- and zebrafish-associated microbiomes were highly distinct from their respective RAS microbiomes, characterized by lower overall diversity and a small core microbiome consisting of taxa specifically adapted to the respective organ. The gill microbiome was also defined by a high proportion of unique taxa. Finally, we found that amoA sequences from the gills were distinct from those from the RAS biofilter and water. Our results showed that the gut and gill microbiomes of carp and zebrafish share a common and species-specific core microbiome that is distinct from the microbially-rich RAS environment.

Single and combined effects of polyethylene microplastics and acetochlor on accumulation and intestinal toxicity of zebrafish (Danio rerio)

The co-exposure of microplastics (MPs) and other contaminants has aroused extensive attention, but the combined impacts of MPs and pesticides remain poorly understood. Acetochlor (ACT), a widely used chloroacetamide herbicide, has raised concerns for its potential bio-adverse effects. This study evaluated the influences of polyethylene microplastics (PE-MPs) for acute toxicity, bioaccumulation, and intestinal toxicity in zebrafish to ACT. We found that PE-MPs significantly enhanced ACT acute toxicity. Also, PE-MPs increased the accumulation of ACT in zebrafish and aggravate the oxidative stress damage of ACT in intestines. Exposure to PE-MPs or/and ACT causes mild damage to the gut tissue of zebrafish and altered gut microbial composition. In terms of gene transcription, ACT exposure triggered a significant increase in inflammatory response-related gene expressions in the intestines, while some pro-inflammatory factors were found to be inhibited by PE-MPs. This study provides a new perspective on the fate of MPs in the environment and on the assessment of the combined effects of MPs and pesticides on organisms.

Chronic early life stress alters the neuroimmune profile and functioning of the developing zebrafish gut

Chronic early life stress (ELS) potently impacts the developing central nervous and immune systems and is associated with the onset of gastrointestinal disease in humans. Though the gut-brain axis is appreciated to be a major target of the stress response, the underlying mechanisms linking ELS to gut dysfunction later in life is incompletely understood. Zebrafish are a powerful model validated for stress research and have emerged as an important tool in delineating neuroimmune mechanisms in the developing gut. Here, we developed a novel model of ELS and utilized a comparative transcriptomics approach to assess how chronic ELS modulated expression of neuroimmune genes in the developing gut and brain. Zebrafish exposed to ELS throughout larval development exhibited anxiety-like behavior and altered expression of neuroimmune genes in a time- and tissue-dependent manner. Further, the altered gut neuroimmune profile, which included increased expression of genes associated with neuronal modulation, correlated with a reduction in enteric neuronal density and delayed gut transit. Together, these findings provide insights into the mechanisms linking ELS with gastrointestinal dysfunction and highlight the zebrafish model organism as a valuable tool in uncovering how “the body keeps the score.”

The Effects of Social Experience on Host Gut Microbiome in Male Zebrafish (Danio rerio).

AbstractAlthough the gut and the brain vastly differ in physiological function, they have been interlinked in a variety of different neurological and behavioral disorders. The bacteria that comprise the gut microbiome communicate and influence the function of various physiological processes within the body, including nervous system function. However, the effects of social experience in the context of dominance and social stress on gut microbiome remain poorly understood. Here, we examined whether social experience impacts the host zebrafish (Danio rerio) gut microbiome. We studied how social dominance during the first 2 weeks of social interactions changed the composition of zebrafish gut microbiome by comparing gut bacterial composition, diversity, and relative abundance between socially dominant, submissive, social isolates and control group-housed communal fish. Using amplicon sequencing of the 16S rRNA gene, we report that social dominance significantly affects host gut bacterial community composition but not bacterial diversity. At the genus level, Aeromonas and unclassified Enterobacteriaceae relative abundance decreased in dominant individuals while commensal bacteria (e.g., Exiguobacterium and Cetobacterium) increased in relative abundance. Conversely, the relative abundance of Psychrobacter and Acinetobacter was increased in subordinates, isolates, and communal fish compared to dominant fish. The shift in commensal and pathogenic bacteria highlights the impact of social experience and the accompanying stress on gut microbiome, with potentially similar effects in other social organisms.

The Outer Membrane Cytochrome OmcA Is Essential for Infection of Shewanella oneidensis by a Zebrafish-Associated Bacteriophage.

The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Combined exposure to microplastics and amitriptyline caused intestinal damage, oxidative stress and gut microbiota dysbiosis in zebrafish (Danio rerio)

The potential toxicity of microplastics (MPs) and hydrophilic pharmaceuticals to aquatic organisms has recently raised great public concern, yet their combined effects on aquatic organisms remain largely unknown. Herein, the combined effects of MPs and the commonly prescribed amitriptyline hydrochloride (AMI) on the intestinal tissue and gut microbiota of zebrafish (Danio rerio) were investigated. Adult zebrafish were exposed to microplastics (polystyrene, PS, 440 µg/L), AMI (2.5 µg/L), PS+AMI (440 µg/L PS + 2.5 µg/L AMI), and dechlorinated tap water (control) for 21 days, respectively. Our results showed that zebrafish rapidly ingested PS beads and accumulated them in the gut. Exposure to PS+AMI significantly enhanced the SOD and CAT activities compared to the control group, suggesting that combined exposure might increase ROS production in the zebrafish gut. Exposure to PS+AMI led to severe gut injuries, including cilia defects, partial absence and cracking of intestinal villi. Exposure to PS+AMI caused shifts in the gut bacterial communities, increasing the abundance of Proteobacteria and Actinobacteriota, and decreasing the abundance of Firmicutes, Bacteroidota and beneficial bacteria Cetobacterium, which caused dysbiosis in the gut microbiota, and subsequently may induce intestinal inflammation. Furthermore, exposure to PS+AMI disordered the predicted metabolic functions of gut microbiota, but functional changes in the PS+AMI group at KEGG level 1 and level 2 were not significantly different from those in the PS group. The results of this study extend our knowledge of the combined effects of MPs and AMI on the health of aquatic organisms, and will be helpful in assessing the combined effects of MPs and tricyclic antidepressants on aquatic organisms.

Gut-specific telomerase expression counteracts systemic aging in telomerase-deficient zebrafish

Telomere shortening is a hallmark of aging and is counteracted by telomerase. As in humans, the zebrafish gut is one of the organs with the fastest rate of telomere decline, triggering early tissue dysfunction during normal zebrafish aging and in prematurely aged telomerase mutants. However, whether telomere-dependent aging of an individual organ, the gut, causes systemic aging is unknown. Here we show that tissue-specific telomerase expression in the gut can prevent telomere shortening and rescues premature aging of tert−/−. Induction of telomerase rescues gut senescence and low cell proliferation, while restoring tissue integrity, inflammation and age-dependent microbiota dysbiosis. Averting gut aging causes systemic beneficial impacts, rescuing aging of distant organs such as reproductive and hematopoietic systems. Conclusively, we show that gut-specific telomerase expression extends the lifespan of tert−/− by 40%, while ameliorating natural aging. Our work demonstrates that gut-specific rescue of telomerase expression leading to telomere elongation is sufficient to systemically counteract aging in zebrafish.

Prolonged darkness attenuates imidacloprid toxicity through the brain-gut-microbiome axis in zebrafish, Danio rerio

The present study investigated the toxic effects of IMI on brain and gut of zebrafish (Danio rerio) by a combination of transcriptome and microbiome analysis. In addition, the involvement of light/dark period was also evaluated. An acute toxic test was conducted on adult zebrafish weighing 0.45 ± 0.02 g with 4 experimental groups (n = 15): 1) IMI group (Light: Dark = 12: 12 h), 2) prolonged light group (Light: Dark = 20: 4 h), 3) prolonged darkness group (Light: Dark = 4: 20 h) which received 20 mg/L of IMI, and 4) control group, which was not treated with IMI (Light: Dark = 12: 12 h). The results showed that prolonged darkness improved the survival rate of zebrafish upon IMI exposure for 96 h. In the sub-chronic test, zebrafish were divided into the same 4 groups and exposed to IMI at 1 mg/L for 14 d (n = 30). The results showed that IMI induced oxidative stress in both IMI and prolonged light groups by inhibition of antioxidant activities and accumulation of oxidative products. Transcriptome analysis revealed a compromise of antioxidation and tryptophan metabolism pathways under IMI exposure. Several genes encoding rate-limiting enzymes in serotonin and melatonin synthesis were all inhibited in both IMI and LL groups. Meanwhile, significant decrease (P < 0.5) of serotonin and melatonin levels was observed. However, there's remarkable improvement of biochemical and transcriptional status in prolonged darkness group. In addition, microbiome analysis showed great alteration of gut bacterial community structure and inhibition of tryptophan metabolism pathway. Similarly, the gut microbiota dysbiosis induced by IMI was alleviated in prolonged darkness. In summary, sub-chronic IMI exposure induced neurotoxicity and gut toxicity in zebrafish by oxidative stress and impaired the brain-gut-axis through tryptophan metabolism perturbation. Prolonged darkness could effectively attenuate the IMI toxicity probably through maintaining a normal tryptophan metabolism.

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

BackgroundPrebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature.ResultsWe observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area.ConclusionThe omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

Bacteriophage-based techniques for elucidating the function of zebrafish gut microbiota.

Bacteriophages (or phages) are unique viruses that can specifically infect bacteria. Since their discovery by Twort and d'Herelle, phages with bacterial specificity have played important roles in microbial regulation. The intestinal microbiota and host health are intimately linked with nutrient, metabolism, development, and immunity aspects. However, the mechanism of interactions between the composition of the microbiota and their functions in maintaining host health still needs to be further explored. To address the lack of methodology and functions of intestinal microbiota in the host, we first proposed that, with the regulations of special intestinal microbiota and applications of germ-free (GF) zebrafish model, phages would be used to infect and reduce/eliminate the defined gut bacteria in the conventionally raised (CR) zebrafish and compared with the GF zebrafish colonized with defined bacterial strains. Thus, this review highlighted the background and roles of phages and their functional characteristics, and we also summarized the phage-specific infection of target microorganisms, methods to improve the phage specificity, and their regulation within the zebrafish model and gut microbial functional study. Moreover, the primary protocol of phage therapy to control the intestinal microbiota in zebrafish models from larvae to adults was recommended including phage screening from natural sources, identification of host ranges, and experimental design in the animal. A well understanding of the interaction and mechanism between phages and gut bacteria in the host can potentially provide powerful strategies or techniques for preventing bacteria-related human diseases by precisely regulating in vitro and in vivo, which will provide novel insights for phages' application and combined research in the future. KEY POINTS: • Zebrafish models for clarifying the microbial and phages' functions were discussed • Phages infect host bacteria with exquisite specificity and efficacy • Phages can reduce/eliminate the defined gut bacteria to clarify their function.

Compositional Graphical Lasso Resolves the Impact of Parasitic Infection on Gut Microbial Interaction Networks in a Zebrafish Model

Understanding how microbes interact with each other is key to revealing the underlying role that microorganisms play in the host or environment and to identifying microorganisms as an agent that can potentially alter the host or environment. For example, understanding how the microbial interactions associate with parasitic infection can help resolve potential drug or diagnostic test for parasitic infection. To unravel the microbial interactions, existing tools often rely on graphical models to infer the conditional dependence of microbial abundances to represent their interactions. However, current methods do not simultaneously account for the discreteness, compositionality, and heterogeneity inherent to microbiome data. Thus, we build a new approach called “compositional graphical lasso” upon existing tools by incorporating the above characteristics into the graphical model explicitly. We illustrate the advantage of compositional graphical lasso over current methods under a variety of simulation scenarios and on a benchmark study, the Tara Oceans Project. Moreover, we present our results from the analysis of a dataset from the Zebrafish Parasite Infection Study, which aims to gain insight into how the gut microbiome and parasite burden covary during infection, thus, uncovering novel putative methods of disrupting parasite success. Our approach identifies changes in interaction degree between infected and uninfected individuals for three taxa, Photobacterium, Gemmobacter, and Paucibacter, which are inversely predicted by other methods. Further investigation of these method-specific taxa interaction changes reveals their biological plausibility. In particular, we speculate on the potential pathobiotic roles of Photobacterium and Gemmobacter in the zebrafish gut, and the potential probiotic role of Paucibacter. Collectively, our analyses demonstrate that compositional graphical lasso provides a powerful means of accurately resolving interactions between microbiota and can thus drive novel biological discovery. Supplementary materials for this article are available online.