Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.</p

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

Neutrophils and macrophage cell count in the gut area of zebrafish larvae treated with antibiotics or saponin or a combination of treatments

This data set contains data collected during zebrafish larvae experiments at the Wageningen University as part of Adrià López Nadal PhD Thesis. This study corresponds to the article: “Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae” published in Frontiers in Microbiology the 29th of October 2018

Corrigendum: Exposure to antibiotics affects Saponin immersion-induced immune stimulation and shift in microbial composition in Zebrafish larvae

<p>In the original article, there was an error. The name of the transgenic line used was incorrect. The correct name of the line is "mpeg1:mCherry/mpx:eGFPi114" Corrections have been made to the Materials and Methods subsection Animals: "Adult Tg(mpeg1:mCherry/mpx:eGFPi114) (Renshaw et al., 2006; Bernut et al., 2014) zebrafish (kindly provided by Prof. Meijer, Leiden University), expressing mCherry under the macrophage-specific mpeg1 promotor and GFP under the neutrophil-specific mpx promotor were housed in Zebtec family tanks (Tecniplast, Buguggiate, Italy) under continuous flow-through at 28°C (14/10-hour light/dark cycle) at Carus facilities (WUR, Wageningen, Netherlands). Zebrafish were fed with a mixture of Artemia 230.000 npg (Ocean Nutrition Europe, Essen, Belgium) and Tetramin Flakes (Tetra, Melle, Germany) twice per day. Embryos were obtained by natural spawning and raised with E3 water (0.10 mM NaCl in demineralized water, pH 7.6) in petri dishes at 28°C (12/12-hour light/dark cycle) (Westerfield, 2007). Dead or fungus-infected embryos were identified by microscopy and discarded in tricaine/E3 solution [8.4% (v/v) 24 mM Tricaine (Sigma-Aldrich, DL, United States) stock solution in E3]. Larval ages are expressed in days post-fertilization (dpf). From 5 dpf onward larvae were fed with live daily cultured Tetrahymena pyriformis." Materials and Methods, subsection Dose-Response Experiment Saponin Exposure: "Double Tg(mpeg1:mCherry /mpx:eGFPi114) zebrafish larvae were randomly distributed in 6 well plates (n = 20 fish/well) and exposed to different concentrations [0, 0.5, 0.7 and 1.0 mg/ml] of saponin [ultrapure Soy Saponin 95%, kindly provided by Trond Kortner NMBU Oslo Norway, origin: Organic Technologies, Coshocton, OH (Krogdahl et al., 2015)] dissolved in the E3 (10 ml solution/well) from 6-9 dpf. Mortality was registered and all media were refreshed daily. At 24 h (7 dpf) and 72 h (9 dpf) after the start of the immersion, zebrafish (n = 6-11/group) were anaesthetized embedded and imaged using fluorescent microscopy (as described below). Per time point several larvae were euthanized for further analysis with an overdose MS-222 (8.4 ml of 24 mM Tricaine (Sigma-Aldrich, DL, United States) in 100 ml E3). Pools of 5 larvae were used for RNA extraction (3 pools per group at 24 h, 7-9 pools per group at 72 h) and gene expression was measured on cDNA by Real Time PCR (as described below). Two independent experiments were performed and data were combined." Materials and Methods, subsection Fluorescent in vivo imaging: "Tg(mpeg1:mCherry/mpx:eGFPi₁₁₄) zebrafish larvae were anaesthetized with tricaine/E3 solution (4.2 ml of 24 mM Tricaine (Sigma-Aldrich, DL, United States) in 100 ml E3) and embedded in 1% low melting point agarose (Thermo Fisher Scientific, MA, United States). Larvae were imaged as whole mounts with a Leica M205 FA Fluorescence Stereo Microscope. After image acquisition, pictures were analyzed with ImageJ® software (United States National Institutes of Health, Bethesda, United States). The intestinal regions were manually selected per fish on the basis of the bright light picture and subsequently copied to the green and red channel pictures (Supplementary Figure S1). Within this intestinal region individual cells were counted for each fish. Furthermore, corrected total cell fluorescence (CTCF) was measured in ImageJ® on total fish larvae by using the following formula: Integrated density-(area of total fish x mean fluorescence of the background reading)." Material and Methods, subsection Experimental Design and Sampling Strategy Antibiotics and Saponin Exposure: A graphical representation of the experimental design and analysis performed per time-point is displayed in Figure 1. To assess the effect of antibiotics, 4 dpf Tg(mpeg1:mCherry/mpx:eGFPi114) fish were randomly distributed in five 6 well-plates (n = 20 fish/well) and 3 treatment conditions were established: (1) control (E3), (2) ciprofloxacin 5 μg/L (Sigma-Aldrich, DL, United States) or (3) oxytetracycline hydrochloride 5 μg/L (Sigma-Aldrich, DL, United States) (10 ml solution/well). The dose of antibiotics was based on several reviews and experimental papers summarizing environmental concentrations of antibiotics in water environments (Ding and He, 2010; Carvalho and Santos, 2016; Watts et al., 2017; Patrolecco et al., 2018; Zhou et al., 2018b) to be at a low dose (ng-μg/L range) and not acute dose (mg/L range). At 6 dpf, 4 pools of 5 larvae were sampled to assess changes in gene expression at baseline. Moreover, at 6 dpf DNA was isolated from 3 pools of 5 larvae to investigate microbiome composition at baseline. In vivo imaging was performed on n = 10 larvae/group to visualize innate immune cells. Subsequently, after sampling, at 6 dpf ultrapure soy saponin was applied to half of the remaining larvae at a concentration 0.5 mg/ml (to induce mild immune stimulation) so each treatment group was split into two, resulting in 6 treatment groups: (1) control, (2) ciprofloxacin (5 μg/L), (3) oxytetracycline hydrochloride (5 μg/L), (4) saponin (0.5 mg/ml), (5) ciprofloxacin + saponin (5 μg/L + 0.5 mg/ml), and (6) oxytetracycline hydrochloride + saponin (5 μg/L + 0.5 mg/ml). All treatment media were refreshed daily. At 9 dpf in vivo imaging was performed on n = 10 larvae/group to visualize innate immune cells. Gene expression was performed on 4 pools of 5 larvae to investigate immune gene expression and from 3 pools of 5 larvae DNA was isolated for microbiological analysis. Because of the error reported above, corrections have also been made to the Figure legends of Figure 2 and Figure 4. The correct legends appear below. Figure 2: Effect of saponin immersion on zebrafish larvae. (A) Percent survival of zebrafish exposed to control (E3), 0.5 mg/ml saponin, 0.7 mg/ml saponin and 1 mg/ml saponin from 6-9 dpf (n = 40 fish/treatment) (Log-rank Mantel-Cox Test for Chi-square, ∗∗∗p &lt; 0.0005). (B) Representative pictures of the saponin-treated Tg(mpeg1:mCherry/mpx:eGFPi114) fish displaying green neutrophils and red macrophages. (C) Quantification of neutrophils and macrophages in the intestinal area (n = 6-11 fish/group) (one way ANOVA Kruskal-Wallis test with Dunn's Multiple comparison Post-Test, mean ± SEM, ∗p &lt; 0.05 ∗∗p &lt; 0.01). Top: counted cells in intestinal area. Bottom: Corrected Total Cell Fluorescence (CTCF, measure for total fluorescent pixels in the whole fish). Two independent experiments were performed and data are combined. Figure 4: Effect of antibiotic exposure on saponin-immune-stimulation. (A) Percent survival of zebrafish exposed to control (E3), ciprofloxacin (4-9 dpf) (5 ug/L) or oxytetracycline (4-9 dpf) (5 ug/ml) + /- saponin (0.5 mg/ml) from 6-9 dpf (n = 100 fish / treatment) (Log-rank Mantel-Cox Test for Chi-square). (B) Representative pictures of the antibiotic/saponin-treated Tg(mpeg1:mCherry/mpx:eGFPi114) fish displaying green neutrophils and red macrophages. (C) Quantification of neutrophils and macrophages in the intestinal area (n = 10 fish/ group) (one way ANOVA Kruskal-Wallis test with Dunn's Multiple comparison Post-Test, mean ± SEM, ∗p &lt; 0.05). Two independent experiments were performed and one representative experiment is shown. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.</p

Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae

In the last decades, pollution of the environment by large scale use of antibiotics in agriculture and human medicine have led to increased antimicrobial resistance in both the environment and the host animal microbiome. Disturbances in the host microbiome can result in impaired immunity and reduced resilience of aquaculture species. Here, we investigated whether environmentally measured levels of the commonly used antibiotics ciprofloxacin and oxytetracycline influences the host microbiome and susceptibility toward saponin-induced immune stimulation in larval zebrafish. Firstly, neutrophil and macrophage reporter zebrafish larvae were exposed to different concentrations of soy saponin by immersion. A dose-dependent increase in neutrophil presence in the intestinal area was observed together with increased expression of immune genes il1b, tnfa, il22 and mmp9. To investigate the effect of antibiotics, larval zebrafish were immersed in ciprofloxacin or oxytetracycline in the presence or absence of a low dose of saponin. In vivo imaging revealed that antibiotic treatment did not reduce the number of neutrophils that were recruited to the intestinal area upon saponin exposure, although it did tend to lower pro-inflammatory cytokine levels. Microbial sequencing of whole larvae revealed that exposure to a low dose of saponin already shifted the microbial composition. The combination of oxytetracycline and saponin significantly increased α-diversity compared to the controls. In conclusion, the current study provides evidence that the combination of low levels of antibiotics with low levels of anti-nutritional factors (saponin) can induce inflammatory phenotypes and can modify the microbiota, which might lead to altered disease susceptibility

Corrigendum: Exposure to antibiotics affects Saponin immersion-induced immune stimulation and shift in microbial composition in Zebrafish larvae

In the original article, there was an error. The name of the transgenic line used was incorrect. The correct name of the line is "mpeg1:mCherry/mpx:eGFPi114" Corrections have been made to the Materials and Methods subsection Animals: "Adult Tg(mpeg1:mCherry/mpx:eGFPi114) (Renshaw et al., 2006; Bernut et al., 2014) zebrafish (kindly provided by Prof. Meijer, Leiden University), expressing mCherry under the macrophage-specific mpeg1 promotor and GFP under the neutrophil-specific mpx promotor were housed in Zebtec family tanks (Tecniplast, Buguggiate, Italy) under continuous flow-through at 28°C (14/10-hour light/dark cycle) at Carus facilities (WUR, Wageningen, Netherlands). Zebrafish were fed with a mixture of Artemia 230.000 npg (Ocean Nutrition Europe, Essen, Belgium) and Tetramin Flakes (Tetra, Melle, Germany) twice per day. Embryos were obtained by natural spawning and raised with E3 water (0.10 mM NaCl in demineralized water, pH 7.6) in petri dishes at 28°C (12/12-hour light/dark cycle) (Westerfield, 2007). Dead or fungus-infected embryos were identified by microscopy and discarded in tricaine/E3 solution [8.4% (v/v) 24 mM Tricaine (Sigma-Aldrich, DL, United States) stock solution in E3]. Larval ages are expressed in days post-fertilization (dpf). From 5 dpf onward larvae were fed with live daily cultured Tetrahymena pyriformis." Materials and Methods, subsection Dose-Response Experiment Saponin Exposure: "Double Tg(mpeg1:mCherry /mpx:eGFPi114) zebrafish larvae were randomly distributed in 6 well plates (n = 20 fish/well) and exposed to different concentrations [0, 0.5, 0.7 and 1.0 mg/ml] of saponin [ultrapure Soy Saponin 95%, kindly provided by Trond Kortner NMBU Oslo Norway, origin: Organic Technologies, Coshocton, OH (Krogdahl et al., 2015)] dissolved in the E3 (10 ml solution/well) from 6-9 dpf. Mortality was registered and all media were refreshed daily. At 24 h (7 dpf) and 72 h (9 dpf) after the start of the immersion, zebrafish (n = 6-11/group) were anaesthetized embedded and imaged using fluorescent microscopy (as described below). Per time point several larvae were euthanized for further analysis with an overdose MS-222 (8.4 ml of 24 mM Tricaine (Sigma-Aldrich, DL, United States) in 100 ml E3). Pools of 5 larvae were used for RNA extraction (3 pools per group at 24 h, 7-9 pools per group at 72 h) and gene expression was measured on cDNA by Real Time PCR (as described below). Two independent experiments were performed and data were combined." Materials and Methods, subsection Fluorescent in vivo imaging: "Tg(mpeg1:mCherry/mpx:eGFPi114) zebrafish larvae were anaesthetized with tricaine/E3 solution (4.2 ml of 24 mM Tricaine (Sigma-Aldrich, DL, United States) in 100 ml E3) and embedded in 1% low melting point agarose (Thermo Fisher Scientific, MA, United States). Larvae were imaged as whole mounts with a Leica M205 FA Fluorescence Stereo Microscope. After image acquisition, pictures were analyzed with ImageJ® software (United States National Institutes of Health, Bethesda, United States). The intestinal regions were manually selected per fish on the basis of the bright light picture and subsequently copied to the green and red channel pictures (Supplementary Figure S1). Within this intestinal region individual cells were counted for each fish. Furthermore, corrected total cell fluorescence (CTCF) was measured in ImageJ® on total fish larvae by using the following formula: Integrated density-(area of total fish x mean fluorescence of the background reading)." Material and Methods, subsection Experimental Design and Sampling Strategy Antibiotics and Saponin Exposure: A graphical representation of the experimental design and analysis performed per time-point is displayed in Figure 1. To assess the effect of antibiotics, 4 dpf Tg(mpeg1:mCherry/mpx:eGFPi114) fish were randomly distributed in five 6 well-plates (n = 20 fish/well) and 3 treatment conditions were established: (1) control (E3), (2) ciprofloxacin 5 μg/L (Sigma-Aldrich, DL, United States) or (3) oxytetracycline hydrochloride 5 μg/L (Sigma-Aldrich, DL, United States) (10 ml solution/well). The dose of antibiotics was based on several reviews and experimental papers summarizing environmental concentrations of antibiotics in water environments (Ding and He, 2010; Carvalho and Santos, 2016; Watts et al., 2017; Patrolecco et al., 2018; Zhou et al., 2018b) to be at a low dose (ng-μg/L range) and not acute dose (mg/L range). At 6 dpf, 4 pools of 5 larvae were sampled to assess changes in gene expression at baseline. Moreover, at 6 dpf DNA was isolated from 3 pools of 5 larvae to investigate microbiome composition at baseline. In vivo imaging was performed on n = 10 larvae/group to visualize innate immune cells. Subsequently, after sampling, at 6 dpf ultrapure soy saponin was applied to half of the remaining larvae at a concentration 0.5 mg/ml (to induce mild immune stimulation) so each treatment group was split into two, resulting in 6 treatment groups: (1) control, (2) ciprofloxacin (5 μg/L), (3) oxytetracycline hydrochloride (5 μg/L), (4) saponin (0.5 mg/ml), (5) ciprofloxacin + saponin (5 μg/L + 0.5 mg/ml), and (6) oxytetracycline hydrochloride + saponin (5 μg/L + 0.5 mg/ml). All treatment media were refreshed daily. At 9 dpf in vivo imaging was performed on n = 10 larvae/group to visualize innate immune cells. Gene expression was performed on 4 pools of 5 larvae to investigate immune gene expression and from 3 pools of 5 larvae DNA was isolated for microbiological analysis. Because of the error reported above, corrections have also been made to the Figure legends of Figure 2 and Figure 4. The correct legends appear below. Figure 2: Effect of saponin immersion on zebrafish larvae. (A) Percent survival of zebrafish exposed to control (E3), 0.5 mg/ml saponin, 0.7 mg/ml saponin and 1 mg/ml saponin from 6-9 dpf (n = 40 fish/treatment) (Log-rank Mantel-Cox Test for Chi-square, ∗∗∗p < 0.0005). (B) Representative pictures of the saponin-treated Tg(mpeg1:mCherry/mpx:eGFPi114) fish displaying green neutrophils and red macrophages. (C) Quantification of neutrophils and macrophages in the intestinal area (n = 6-11 fish/group) (one way ANOVA Kruskal-Wallis test with Dunn's Multiple comparison Post-Test, mean ± SEM, ∗p < 0.05 ∗∗p < 0.01). Top: counted cells in intestinal area. Bottom: Corrected Total Cell Fluorescence (CTCF, measure for total fluorescent pixels in the whole fish). Two independent experiments were performed and data are combined. Figure 4: Effect of antibiotic exposure on saponin-immune-stimulation. (A) Percent survival of zebrafish exposed to control (E3), ciprofloxacin (4-9 dpf) (5 ug/L) or oxytetracycline (4-9 dpf) (5 ug/ml) + /- saponin (0.5 mg/ml) from 6-9 dpf (n = 100 fish / treatment) (Log-rank Mantel-Cox Test for Chi-square). (B) Representative pictures of the antibiotic/saponin-treated Tg(mpeg1:mCherry/mpx:eGFPi114) fish displaying green neutrophils and red macrophages. (C) Quantification of neutrophils and macrophages in the intestinal area (n = 10 fish/ group) (one way ANOVA Kruskal-Wallis test with Dunn's Multiple comparison Post-Test, mean ± SEM, ∗p < 0.05). Two independent experiments were performed and one representative experiment is shown. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.</p

Environmental microbes determine macrophage response towards saponin-induced inflammation in zebrafish larvae

The microbial consortium within an organism is crucial for its development and immune status. Alteration of the host microbiome by antibiotics or antinutritional factors may contribute to increased disease susceptibility. Here, we investigated whether exposure to different microbes could influence zebrafish larval microbiota composition and modulate their immune response towards a saponin challenge. Adult zebrafish were exposed to the antibiotic oxytetracycline (OxyT) or control tank water and their intestinal content was harvested after 30 ​h (24 ​h exposure, 6 ​h wash-out). Subsequently, zebrafish embryos were exposed to either OxyT-treated content or non-treated content from 3 to 6 days post fertilization (dpf). At 6 dpf part of the group received a saponin challenge until 8 dpf. Zebrafish larvae exposed to OxyT-treated adult gut content (3–6 dpf) showed an altered microbiota composition compared to controls. Interestingly, larvae exposed to saponin-treated OxyT-content showed fewer macrophages (as visualized by fluorescent microscopy using mpx:GFP114;mpeg:mCherry transgenic fish) in the overall fish as well as around the gut area than saponin-treated control-exposed larvae. Fewer macrophages were associated with a decreased expression of interleukin 22 (il22) in larvae exposed to saponin-treated OxyT-content compared to controls. Overall, this study shows that exposure to different microbial environments early in life might affect disease susceptibility of larval zebrafish

Intestinal Microbiota and Immune Modulation in Zebrafish by Fucoidan From Okinawa Mozuku (Cladosiphon okamuranus)

Fucoidan represents fucose-rich sulfated polysaccharides derived from brown seaweeds, which exerts various biological activities applicable for functional foods and therapeutic agents. The objective of the present study was to investigate in vivo effects of fucoidan extracted from Okinawa mozuku (Cladosiphon okamuranus), common edible seaweed in Japan, on immune responses and microbiota composition in zebrafish. We treated larvae and adult zebrafish with Okinawa mozuku (OM) fucoidan by immersion (100 and 500 μg/mL, 3 days) and by feeding (3 weeks), respectively. The effect of OM fucoidan on immune responses in zebrafish larvae was evaluated by live imaging of neutrophils and macrophages as well as quantitative polymerase chain reaction of pro- and anti-inflammatory cytokine genes. Whole microbiota of zebrafish larvae and intestinal microbiota of adult zebrafish treated with OM fucoidan were analyzed by Illumina MiSeq pair-end sequencing of the V3–V4 region of 16S rRNA genes. Fucoidan treatment only slightly affected the composition of the larvae microbiota and the number of neutrophils and macrophages, while pro- and anti-inflammatory cytokine gene expression levels were upregulated in the larvae treated with 500 μg/mL OM fucoidan. In contrast, feeding of OM fucoidan clearly altered the intestinal microbiota composition of adult zebrafish, which was characterized by the emergence and predominance of multiple bacterial operational taxonomic units (OTUs) affiliated with Rhizobiaceae and Comamonadaceae at the expense of E. coli-related Enterobacteriaceae, the dominant OTUs throughout the studied samples. These changes were accompanied by decreased expression levels of pro-inflammatory cytokine il1b in the intestines of the adult zebrafish. Our current study provides the first insights into in vivo modulatory effects of fucoidan on microbiota and immune responses of unchallenged zebrafish, which underscores the potential of fucoidan to play a modulatory role in the diet–microbiota–host interplay.</p