Suxu He

Comparison of adhesive gut bacteria composition, immunity, and disease resistance in juvenile hybrid tilapia fed two different Lactobacillus strains

This study compares the effects of two Lactobacillus strains, highly adhesive Lactobacillus brevis JCM 1170 (HALB) and less-adhesive Lactobacillus acidophilus JCM 1132 (LALB), on the survival and growth, adhesive gut bacterial communities, immunity, and protection against pathogenic bacterial infection in juvenile hybrid tilapia. During a 5-week feeding trial the fish were fed a diet containing 0 to 10(9) cells/g feed of the two Lactobacillus strains. Samples of intestine, kidney, and spleen were taken at the start and at 10, 20, and 35 days for analysis of stress tolerance and cytokine gene mRNA levels and to assess the diversity of adhesive gut bacterial communities. A 14-day immersion challenge with Aeromonas hydrophila NJ-1 was also performed following the feeding trial. The results showed no significant differences in survival rate, weight gain, or feed conversion in the different dietary treatments. The adhesive gut bacterial communities were strikingly altered in the fish fed either the HALB or the LALB, but the response was more rapid and substantial with the adhesive strain. The two strains induced similar changes in the patterns (upregulation or downregulation) of intestinal, splenic or kidney cytokine expression, but they differed in the degree of response for these genes. Changes in intestinal HSP70 expression levels coincided with changes in the similarity coefficient of the adhesive gut bacterial communities between the probiotic treatments. The highest dose of the HALB appeared to protect against the toxic effects of immersion in A. hydrophila (P < 0.05). In conclusion, the degree to which Lactobacillus strains adhere to the gut may be a favorable criterion in selecting probiotic strain for aquaculture.

Comparison of fecundity and offspring immunity in zebrafish fed Lactobacillus rhamnosus CICC 6141 and Lactobacillus casei BL23.

To increase the knowledge of probiotic effects on zebrafish (Danio rerio), we compare the effects of two probiotic strains, Lactobacillus rhamnosus CICC 6141 (a highly adhesive strain) and Lactobacillus casei BL23 (a weakly adhesive strain), on zebrafish reproduction and their offsprings innate level of immunity to water-borne pathogens. During probiotics treatments from 7 to 28 days, both the Lactobacillus strains, and especially L. casei BL23, significantly increased fecundity in zebrafish: higher rates of egg ovulation, fertilization, and hatching were observed. Increased densities of both small and large vitellogenic follicles, seen in specimens fed either Lactobacillus strain, demonstrated accelerated oocyte maturation. Feeding either strain of Lactobacillus upregulated gene expression of leptin, kiss2, gnrh3, fsh, lh, lhcgr, and paqr8, which were regarded to enhance fecundity and encourage oocyte maturation. Concomitantly, the gene expression of bmp15 and tgfb1 was inhibited, which code for local factors that prevent oocyte maturation. The beneficial effects of the Lactobacillus strains on fecundity diminished after feeding of the probiotics was discontinued, even for the highly adhesive gut Lactobacillus strain. Administering L. rhamnosus CICC 6141 for 28 days was found to affect the innate immunity of offspring derived from their parents, as evinced by a lower level of alkaline phosphatase activity in early larval stages. This study highlights the effects of probiotics both upon the reproductive process and upon the offsprings immunity during early developmental stages.

Lactobacillus planarum subsp. plantarum JCM 1149 vs. Aeromonas hydrophila NJ-1 in the anterior intestine and posterior intestine of hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂: An ex vivo study

To investigate the ex vivo interactions of probiotic-pathogen-host in warm-water fish, hybrid tilapia (Oreochromis niloticus♀ × Oreochromis aureus♂) were sacrificed to isolate anterior and posterior intestine for incubation with phosphate-buffered saline (PBS; pH 7.2) as the control, Lactobacillus plantarum JCM 1149 at 1.0 × 10(9) CFU/ml, Aeromonas hydrophila NJ-1 at 1.0 × 10(8) CFU/ml, or the both combination. Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint and consequent sequence analysis confirmed anterior intestine sac was more prone to the colonization of L. plantarum JCM 1149 and A. hydrophila NJ-1 than the posterior part. L. plantarum JCM 1149 and A. hydrophila NJ-1 inhibited the population each other in anterior or posterior sac, indicating their competition for the colonization. The induced expression of HSP70, IL-1β and TNF-α in the anterior sac by the addition of L. plantarum JCM 1149 or A. hydrophila NJ-1 demonstrated the activity and a local immune response of ex vivo anterior sac. Compared with posterior intestine, higher population colonization and more sensitive immune response of anterior sac indicated differential patterns for the probiotic-pathogen-host interactions. Scanning electronic microscopy (SEM) observation showed that pathogen A. hydrophila NJ-1 damaged the anterior intestine, which was alleviated by the pretreatment of L. plantarum JCM 1149, showing its probiotic effect.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model

BACKGROUNDnEssential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host.nnnOBJECTIVEnThe purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model.nnnMETHODSnJuvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d.nnnRESULTSnNE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1β (Il1β; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1β (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05).nnnCONCLUSIONnNE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

Effects of dietary yeast nucleotides on growth, non-specific immunity, intestine growth and intestinal microbiota of juvenile hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂

This study investigated the effect of dietary supplementation of yeast nucleotides on the growth, non-specific immunity, intestine growth and intestinal microbiota of juvenile hybrid tilapia. Tilapia (initial average weight of 8.02 g) was fed test diets supplemented with a yeast-originated nucleotide mixture (0, 0.15, 0.30, 0.60, and 1.20 g/100 g diet) for 8 weeks. Fish fed the diet with 0.60% nucleotide had significantly higher weight gain than the control group (P < 0.05). Feed efficiency was improved in the fish fed 0.60 and 1.20% nucleotide compared with that in the control group. The optimal doses of nucleotides supplementation for growth and feed efficiency of fish were determined as 0.63 and 0.81%, respectively. Intestinal growth was improved in the 0.30 and 0.60% groups, as indicated by significant increase in intestine length. The fish fed 0.60 and 1.20% nucleotide showed higher super oxide dismutase (SOD) activity and lower malondialdehyde (MDA) level in the liver than the control fish, indicating enhancement of the anti-oxidant status. Serum lysozyme activity was significantly increased in the 0.15 and 0.3% nucleotide supplementation groups, suggesting an enhancement effect on the non-specific immune response. Lastly, dietary nucleotides supplementation exerted moderate influence on the intestinal microbiota of hybrid tilapia. A reduction in the cumulative abundance of putative butyrate-producing species was observed in the intestinal microbiota of fish fed diets with 0.60% nucleotide compared with the control, implying an interaction between dietary nucleotides and butyrate production. Briefly, dietary supplementation with 0.60% nucleotide improve the growth performance, immune activity and intestine growth in tilapia.

Effects of dietary live and heat-inactive baker's yeast on growth, gut health, and disease resistance of Nile tilapia under high rearing density.

In this study, the effects of bakers yeast as probiotics was evaluated in Nile tilapia reared at high density. Juvenile tilapia were distributed to tanks at high density (436xa0fish/m(3)) and fed with basal diet (CK) or diets supplemented with live (LY) or heat-inactivated yeast (HIY). Another group of fish reared at low density (218xa0fish/m(3)) and fed with basal diet was also included (LowCK). After 8 weeks of feeding, growth, feed utilization, gut microvilli morphology, digestive enzymes, and expressions of hsp70 and inflammation-related cytokines in the intestine were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Fish were challenged with Aeromonas hydrophila to evaluate disease resistance. High rearing density significantly decreased the growth, feed utilization, microvilli length, and disease resistance of fish (CK versus LowCK). Moreover, the intestinal hsp70 expression was increased in fish reared at high density, supporting a stress condition. Compared to CK group, supplementation of live yeast significantly increased gut microvilli length and trypsin activity, decreased intestinal hsp70 expression, and enhanced resistance of fish against A.xa0hydrophila (reflected by reduced intestinal alkaline phosphatase activity 24xa0h post infection). The gut microbiota was not markedly influenced by either rearing density or yeast supplementation. Heat-inactivated yeast (HIY) didnt display the beneficial effects observed in LY except an increase in gut trypsin activity, suggesting the importance of yeast viability and thus secretory metabolites of yeast. In conclusion, live bakers yeast may alleviate the negative effects induced by crowding stress, and has the potential to be used as probiotics for tilapia reared at high density.

The Growth-Promoting Effect of Dietary Nucleotides in Fish Is Associated with an Intestinal Microbiota-Mediated Reduction in Energy Expenditure

Background: Nucleotides have been used as functional nutrients to improve the growth and health of animals, including fish. The mechanism involved in the growth-promotion effect of nucleotides is still unclear.Objective: We investigated the bioenergetic mechanism underlying the growth-promotion effect of nucleotides in zebrafish and the associated roles played by the intestinal microbiota.Methods: Larval zebrafish were fed a control or a 0.1% mixed nucleotides-supplemented diet for 2 wk. Standard metabolic rate, the minimal rate of energy expenditure by animals at rest, was evaluated by oxygen consumption with the use of a respirometer. The expressions of fasting-induced adipose factor (Fiaf), inflammatory cytokines, and genes involved in fatty acid (FA) oxidation were tested by quantitative reverse transcriptase-polymerase chain reaction. The intestinal microbiota from the nucleotide-fed fish (NT fish) or control fish was transferred to 3-d postfertilization germ-free zebrafish in which oxygen consumption and expression of cytokines and fiaf were evaluated.Results: Compared with controls, nucleotide supplementation at 0.1% increased the weight and energy gains of zebrafish by 10% and 25%, respectively (P < 0.01). Standard metabolic rate was 28% lower in NT fish than in controls (P < 0.001). Nucleotide supplementation downregulated the inflammatory tone in the head kidney of the fish. Moreover, NT fish had a 51% lower intestinal expression of fiaf than did controls (P < 0.05), which was consistent with decreased expression of key genes involved in FA oxidation [carnitine:palmitoyl transferase 1a (cpt1a) and medium-chain acyl coenzyme A dehydrogenase (mcad)] in liver and muscle. Germ-free zebrafish colonized with microbiota from NT fish had a 25% lower standard metabolic rate than did those colonized by control microbiota (P < 0.01), whereas direct nucleotide feeding of germ-free zebrafish did not affect standard metabolic rate relative to germ-free controls that were not fed nucleotides. Furthermore, germ-free zebrafish colonized with nucleotide microbiota exhibited downregulated inflammatory tone and 33% lower fiaf expression compared with their control microbiota-colonized counterparts.Conclusions: The growth-promoting effect of dietary nucleotides in zebrafish involves 2 intestinal microbiota-mediated mechanisms that result in reduced standard metabolic rate: 1) lower inflammatory tone and 2) reduced FA oxidation associated with increased microbial suppression of intestinal fiaf.

Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis

Low dose antibiotics have been used as growth promoters in livestock and fish. The use of antibiotics has been associated with reduced pathogen infections in livestock. In contrast, antibiotic growth promoter has been suspected of leading to disease outbreaks in aquaculture. However, this phenomenon is circumstantial and has not been confirmed in experimental conditions. In this study, we showed that antibiotic olaquindox increased the susceptibility of zebrafish to A. hydrophila infection. Olaquindox led to profound alterations in the intestinal microbiota of zebrafish, with a drastic bloom of Enterobacter and diminishing of Cetobacterium. Moreover, the innate immune responses of zebrafish were compromised by olaquindox (P<0.05). Transfer of microbiota to GF zebrafish indicated that while the immuo-suppression effect of olaquindox is a combined effect mediated by both OLA-altered microbiota and direct action of the antibiotic (P<0.05), the increased pathogen susceptibility was driven by the OLA-altered microbiota and was not dependent on direct antibiotic effect. Taken together, these data indicate that low level of OLA induced gut microbiota dysbiosis in zebrafish, which led to increased pathogen susceptibility.

Impact of Lactobacillus casei BL23 on the Host Transcriptome, Growth and Disease Resistance in Larval Zebrafish

In this study, zebrafish were treated with Lactobacillus strains as probiotics from hatching to puberty, and the effect of treatment with L. casei BL23 on the development and immunity response of the host was investigated. Genes that were differentially expressed (DEGs) in the overall body and intestine were detected at 14 days post fertilization (dpf) and 35 dpf, respectively, using whole transcriptome sequencing (mRNAseq). We showed that zebrafish raised by continuous immersion with L. casei BL23 showed a higher final body weight at 14 dpf (P < 0.05), and 35 dpf (P < 0.01). DEGs between L. casei BL23 treatment and control group at 14 dpf were involved in myogenesis, cell adhesion, transcription regulation and DNA-binding and activator. At 35 dpf, 369 genes were DEGs in the intestine after treatment with L. casei BL23, which were involved in such categories as signaling, secretion, motor proteins, oxidoreductase and iron, tight junctions, lipid metabolism, growth regulation, proteases, and humoral and cellular effectors. KEGG analysis showed DEGs to be involved in such pathways as those associated with tight junctions and the PPAR signal pathway. RT-qPCR analysis showed that expression of insulin-like growth factors-I (igf1), peroxisome proliferator activated receptors-α (ppar-α) and -β (ppar-β), Vitamin D receptor-α (vdr-α), and retinoic acid receptor-γ (rar-γ) was up-regulated in fish treated with L. casei BL23 at 35 dpf. After 35 days of treatment, the mortality rate in L. casei BL23 treated group was lower than the control after challenge with A. hydrophila (P < 0.05), and the pro-inflammatory cytokine il-1β, anti-inflammatory cytokine il-10 and complement component 3a (c3a) showed more expression in L. casei BL23 group at 8h after challenge, 24 h after challenge, or both.. Together, these data suggest that specific Lactobacillus probiotic strains can accelerate the development profile and enhance immunity in zebrafish, which supports the rationale of early administration of probiotics in aquaculture.

The Hepatotoxicity of Palmitic Acid in Zebrafish Involves the Intestinal Microbiota

BackgroundnPalmitic acid (PA) is the main saturated fatty acid naturally occurring in animal fats and vegetable oils. In recent decades, palm oil, an alternative lipid source containing high amounts of PA, has been widely used to replace fish oil in aquafeed.nnnObjectivenWe investigated the hepatotoxicity of PA in zebrafish and the underlying mechanism.nnnMethodsnOne-month-old zebrafish fed a high-fat diet (HFD) containing 16% soybean oil and 3 PA-incorporated HFDs [4%, 8%, and 12% PA (12PA)] for 2 wk (experiment 1) and 4 wk (experiment 2) were used to evaluate PA-induced liver damage and endoplasmic reticulum (ER) stress. Germ-free (GF) zebrafish fed low-fat, high-fat, or 12PA diets for 5 d were used to study the direct effects of PA on liver damage (experiment 3). GF zebrafish colonized with HFD or 12PA microbiota for 48 h were used to elucidate the indirect effects of PA-altered microbiota on liver damage (experiment 4). Last, GF zebrafish colonized with HFD or 12PA microbiota were used to evaluate the effects of different microbiotas on PA absorption (experiment 5).nnnResultsnIn experiment 1, the proportion of PA in the liver linearly increased as its percentage in dietary lipid increased (r2xa0=xa00.83, Pxa0<xa00.05). In experiment 2, the expression of glucose-regulated protein 78 (Grp78) and C/EBP-homologous protein (Chop) was higher in the 12PA group than in the HFD group (2.2- and 2.7-fold, respectively; Pxa0<xa00.05). The activity of caspase-12 was increased by 61.1% in the 12PA group compared with the HFD group (Pxa0<xa00.05). In experiment 3, caspase-12 activity was higher in the 12PA group than in the HFD group (Pxa0<xa00.05). In experiment 4, GF zebrafish colonized with PA-altered microbiota had higher caspase-12 activity (Pxa0<xa00.05) than those colonized by HFD microbiota. In experiment 5, PA-altered microbiota promoted PA absorption (Pxa0<xa00.05) and aggravated ER stress and liver damage in the context of high-PA feeding.nnnConclusionsnThe PA-altered microbiota indirectly induced ER stress and liver damage in zebrafish. Moreover, the PA microbiota promoted the absorption of PA, leading to enhanced PA overflow into the liver and aggravated hepatotoxicity of PA in zebrafish.