Abstract :
[en] With the development of intensive culture systems to meet the increasing demand for global aquatic products, there has been an increasing incidence of diseases outbreak and substantial economic losses for farmers. Antibiotics are used as prophylaxis in this context causing serious drawbacks such as the surge of antibiotic resistance strains of common pathogens including humans. National bans for the administration of some antibiotics have been issued to minimize their overuse in aquaculture. As an alternative to antibiotics, probiotics have been proposed as a promising solution. Commensal gut microorganisms have been proposed as key suppliers of beneficial candidate probiotics. A substantial body of evidence supports that the gut microbiota plays critical roles in the regulation of metabolic, endocrine, and immunes functions. SCFAs, the main microbial metabolites produced by bacterial fermentation of indigestible carbohydrates in the intestines, are considered key candidate mediators in the microbiota-gut-brain communication directly or indirectly modulate processes associated with food intake, insulin secretion, and whole-body energy homeostasis. Currently, how to improve the ability of fish to use carbohydrates is another urgent scientific problem in aquaculture. With the development of aquaculture industry, high-carbohydrate diets were used to stimulate the protein-sparing effect and reduce feed costs. However, the utilization of carbohydrates in fish is limited compared to mammals, especially in carnivorous species. Fish fed with high levels of carbohydrates will lead to persistent postprandial hyperglycemia, and further induce low efficiency of feed utilization, abnormal fat deposition, and even high mortality. Here too, relevant probiotics could improve the utilization of carbohydrates in fish. Therefore, the aim of this work was to explore the potential host-associated probiotic, investigate the mechanism of indigenous probiotic regulate fish glucose homeostasis, and evaluate the beneficial effects of gut microbial metabolites on fish health.
In experiment 1, we explored host-associated probiotic for fish by performing proteomic analysis and 16S rRNA gene sequencing to investigate the mucus proteins and adhesive gut microbiota of different intestinal segments in adult zebrafish, from proximal, mid and distal gut segments. The results revealed that the three intestinal segments investigated exhibited significant differences in functions, and these differential proteins are more involved in metabolic processes. The composition of intestinal microbiota also showed differences in the three segments. At the genus level, Cetobacterium was the predominant taxa in midgut. Moreover, we also determined that the midgut mucus layer was the thickest among the segments, indicating that Cetobacterium might have better colonization capacity in zebrafish midgut. This finding has led us to further explore the role of this bacteria in the fish gut and its potential use as probiotic in aquaculture.
In experiment 2, we investigated the beneficial effects of commensal Cetobacterium on zebrafish glucose homeostasis. To explore the effect of feeding habits on the gut microbial composition and glucose homeostasis, 2-month-old zebrafish were fed with three formulated diets mimicking carnivorous diet (CD), omnivorous diet (OD) and herbivorous diet (HD) for 2 weeks. The results demonstrated that feeding habits could influence the composition of gut microbiota, and the Cetobacterium somerae was enriched in OD and HD groups. Zebrafish fed with omnivorous diet (OD) and herbivorous diet (HD) showed better glucose homeostasis compared with carnivorous diet (CD), suggesting a beneficial effect of enriched C. somerae on glucose utilization. To confirm the causative effects of C. somerae enrichment in glucose homeostasis in fish, conventionally raised (CONR) zebrafish were fed the basal diet with or without non-absorbable antibiotic mixture for 1 week. Then, exogenous C. somerae or Aeromonas veronii B565 (negative control) were applied to the rearing water of antibiotic-treated zebrafish. We observed significantly decreased postprandial blood glucose level and increased insulin concentration in C. somerae treated zebrafish. Furthermore, we mono-associated germ-free (GF) zebrafish with A. veronii B565 + Plesiomonas shigelloides, or A. veronii B565 + P. shigelloides + C. somerae. Consistently, gnotobiotic larval zebrafish with C. somerae colonization showed significantly lower glucose levels and increased insulin expression, and C. somerae colonization was associated with higher acetate level in whole-larvae zebrafish, which suggested the regulation of commensal C. somerae in zebrafish glucose homeostasis and a role for its metabolite acetate in this regulation. Dietary sodium acetate and intracerebroventricular (ICV) injection of NaAc or NaAc + atropine demonstrated that the effects of both acetate and C. somerae on glucose homeostasis was mediated through parasympathetic activation. This study highlights a specific C. somerae-brain axis in the regulation of glucose homeostasis in fish and suggests a role of acetate in mediating the axis function. Our results suggest potential strategies for improvement of fish carbohydrate utilization.
In experiment 3, we further evaluated the effects of acetate on fish growth performance. 1-month-old zebrafish were fed with 0.15% sodium acetate (NaAc) for 4 weeks. At the end of the feeding trial, zebrafish fed with 0.15% NaAc showed higher weight gain and daily feeding rate compared with the control group. Dietary supplementation of 0.15% NaAc significantly promoted the body fat mass, energy gain and energy conversion efficiency, as well as intestinal digestive enzyme (amylase) activity in fish. The expression of food intake-related genes (ghre, npy and npy7R) in the brain of fish fed on the diet contacting NaAc was higher than that of the control group. Furthermore, intracerebroventricular (ICV) injection of NaAc or NaAc + atropine showed that acetate played a direct role in controlling appetite through the parasympathetic nervous system. Finally, there was significant decrease in the relative abundance of Plesiomonas genus in the fish gut fed on the diet supplemented with NaAc. This study suggests that sodium acetate has a potential value as fish feed additive in aquaculture.
In conclusion, this thesis demonstrated that the commensal acetate-producing strain Cetobacterium has beneficial effects on fish glucose homeostasis, and provided evidence for benefits of a specific Cetobacterium-brain axis in the regulation of glucose homeostasis in fish, suggested a role of acetate in mediating the axis function. It also revealed the beneficial effects of sodium acetate on fish growth performance and health status, suggesting that sodium acetate might be a potential feed additive for aquaculture. This work not only may provide theoretical references for improvement of fish carbohydrates utilization targeting gut microbiota, but also promote the development of functional feed additives for fish health.
