Impact of a novel protein meal on the gastrointestinal microbiota and the host transcriptome of larval zebrafish Danio rerio.

Larval zebrafish was subjected to a methodological exploration of the gastrointestinal microbiota and transcriptome. Assessed was the impact of two dietary inclusion levels of a novel protein meal (NPM) of animal origin (ragworm Nereis virens) on the gastrointestinal tract (GIT). Microbial development was assessed over the first 21 days post egg fertilization (dpf) through 16S rRNA gene-based microbial composition profiling by pyrosequencing. Differentially expressed genes in the GIT were demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq). Larval zebrafish showed rapid temporal changes in microbial colonization but domination occurred by one to three bacterial species generally belonging to Proteobacteria and Firmicutes. The high iron content of NPM may have led to an increased relative abundance of bacteria that were related to potential pathogens and bacteria with an increased iron metabolism. Functional classification of the 328 differentially expressed genes indicated that the GIT of larvae fed at higher NPM level was more active in transmembrane ion transport and protein synthesis. mRNAseq analysis did not reveal a major activation of genes involved in the immune response or indicating differences in iron uptake and homeostasis in zebrafish fed at the high inclusion level of NPM.

Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut microbiota of juvenile Siberian sturgeon (Acipenser baerii).

We investigated the effects of administration of putative endogenous probiotics Lactococcus lactis spp. lactis or Bacillus circulans, alone and in combination with arabinoxylan-oligosaccharides (AXOS), a new class of candidate prebiotics, in juvenile Siberian sturgeon (Acipenser baerii). Eight experimental diets were tested: basal diet (Diet 1), basal diet supplemented with 2% AXOS (Diet 2), or L. lactis ST G81 (Diet 3), L. lactis ST G45 (Diet 4), B. circulans ST M53 (Diet 5), L. lactis ST G81 + 2% AXOS (Diet 6), L. lactis ST G45 + 2% AXOS (Diet 7), B. circulans ST M53 + 2% AXOS (Diet 8). After four weeks, growth performance and feed conversion ratio significantly improved in fish fed diet 7. Innate immune responses of fish were boosted with both AXOS and probiotic diets, however synergistic effects of AXOS and probiotic diets were only observed for phagocytic and alternative complement activity. Phagocytic and respiratory burst activity of fish macrophage increased in fish fed diet 2 and 7, while humoral immune responses only increased in fish fed diet 7. Pyrosequencing analysis (16S rDNA) of the hindgut microbiota demonstrated that AXOS improved the colonization or/and growth capacity of L. lactis, as a higher relative abundance of L. lactis was observed in fish receiving diet 7. However, no observable colonization of B. circulans was found in the hindgut of fish fed diet 5 or 8, containing this bacterium. The dietary L. lactis ST G45 + 2% AXOS caused significant alterations in the intestinal microbiota by significantly decreasing in bacterial diversity, demonstrated by the fall in richness and Shannon diversity, and improved growth performance and boosted immune responses of Siberian sturgeon.

Prebiotic effects of arabinoxylan oligosaccharides on juvenile Siberian sturgeon (Acipenser baerii) with emphasis on the modulation of the gut microbiota using 454 pyrosequencing.

The potential of a novel class of prebiotics, arabinoxylan oligosaccharides (AXOS), was investigated on growth performance and gut microbiota of juvenile Acipenser baerii. Two independent feeding trials of 10 or 12 weeks were performed with basal diets supplemented with 2% or 4% AXOS-32-0.30 (trial 1) and 2% AXOS-32-0.30 or AXOS-3-0.25 (trial 2), respectively. Growth performance was improved by feeding 2% AXOS-32-0.30 in both trials, although not significantly. Microbial community profiles were determined using 454-pyrosequencing with barcoded primers targeting the V3 region of the 16S rRNA gene. AXOS significantly affected the relative abundance of bacteria at the phylum, family, genus and species level. The consumption of 2% AXOS-32-0.30 increased the relative abundance of Eubacteriaceae, Clostridiaceae, Streptococcaceae and Lactobacillaceae, while the abundance of Bacillaceae was greater in response to 4% AXOS-32-0.30 and 2% AXOS-3-0.25. The abundance of Lactobacillus spp. and Lactococcus lactis was greater after 2% AXOS-32-0.30 intake. Redundancy analysis showed a distinct and significant clustering of the gut microbiota of individuals consuming an AXOS diet. In both trials, concentration of acetate, butyrate and total short-chain fatty acids (SCFAs) increased in fish fed 2% AXOS-32-0.30. Our data demonstrate a shift in the hindgut microbiome of fish consuming different preparation of AXOS, with potential application as prebiotics.

Effects of arabinoxylan-oligosaccharides (AXOS) on juvenile Siberian sturgeon (Acipenser baerii) performance, immune responses and gastrointestinal microbial community.

Arabinoxylan-oligosaccharides (AXOS) are a newly discovered class of candidate prebiotics that exert different properties depending on their structure. In this study the effects of two different structures of AXOS, namely AXOS-32-0.30 (average degree of polymerization: 32, average degree of substitution: 0.30) and AXOS-3-0.25, were investigated on growth performance, immune responses, gut microbial fermentation and gut bacterial composition of juvenile Siberian sturgeon (Acipenser baerii). After a two weeks acclimation, fish (25.9 ± 0.9 g) were distributed over 24 aquariums (8 replicates per treatment) and fed a control diet or a diet containing 2% AXOS-32-0.30 or AXOS-3-0.25 for 12 weeks. Growth performance and feed utilization tend to improve in sturgeon fed on diets supplemented with AXOS-32-0.30, however not significant. Survival was high in all groups. Both AXOS preparations significantly enhanced the phagocytic activity of fish macrophages compared to the control group, while the alternative haemolytic complement activity and total serum peroxidase content improved only in the group fed AXOS-32-0.30 (P < 0.05). The lysozyme activity was not affected by AXOS addition. Simultaneously, the amount of short-chain fatty acids (SCFAs) was highest in the hind gut of sturgeon fed AXOS-32-0.30. The concentrations of acetate, butyrate and total SCFAs in fish fed AXOS-32-0.30 was significantly higher than in the groups fed the control diet or AXOS-3-0.25. Study of the bacterial community in the sturgeon hindgut using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that both preparations of AXOS induced changes in the bacterial composition. According to redundancy analysis (RDA), hindgut microbiota of each treatment group clustered apart from one another (P = 0.001). DNA sequencing of the dominant DGGE bands recovered from the different treatments showed that AXOS mainly stimulated the growth of lactic acid bacteria and Clostridium sp., with more pronounced effects of AXOS-32-0.30. It is concluded that AXOS improves sturgeon health through prebiotic action, but the induced effects depend on the specific structure of AXOS. A higher degree of polymerization of AXOS had a stronger beneficial impact in this sturgeon species.

The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors.

The importance of thyroid hormones (TH) for embryonic development has long been shown in many vertebrates, but is not yet established in pre-hatch teleost models despite the presence of TH, TH receptors and iodothyronine deiodinases. Lack of data about the dynamics of TH metabolism in embryonic stages of fish does not allow to speculate about the involvement and/or role of TH in fish embryonic development. We therefore set up an experiment to examine the effect of 3,5,3'-triiodothyronine (T(3)) on zebrafish (Danio rerio) embryonic development and on the expression of some thyroid hormone-regulated genes as measured by real-time PCR. Maternally derived thyroxine (T(4)) and T(3) were detected throughout embryonic development and total levels remained stable. Thyroid hormone receptor (TR) alpha and beta mRNA were found to be present in early embryos. After an initial fall, TRalpha mRNA levels in the control group increased gradually from 12h post fertilization (HPF) onwards. TRbeta mRNA levels of control embryos were relatively stable during embryonic development, but increased around the hatching period. We also quantified type I (D1) and type II (D2) deiodinase mRNA expression in zebrafish embryos. D1 mRNA levels in the control group gradually increased during development while D2 levels were kept at a low and stable level until hatching. At 75 HPF, a fivefold increase of D2 expression was observed compared to embryonic levels. Exogenous T(3) added to the water (5nM) was taken up by the embryos, causing increased pigmentation and accelerated hatching. T(3) treatment significantly up regulated TRalpha mRNA levels at 48 HPF, while D2 mRNA was significantly down regulated at 75 HPF. Neither TRbeta nor D1 mRNA levels seemed responsive to the treatment. Taken together, these data show that during embryonic development zebrafish already have the necessary regulatory machinery for TH activation and signaling, and that T(3) treatment at that stage indeed influence embryonic development.

Long-term exposure to environmentally relevant doses of methyl-tert-butyl ether causes significant reproductive dysfunction in the zebrafish (Danio rerio).

Methyl-tert-butyl ether (MTBE), an anthropogenic chemical used as a gasoline additive, is being detected at an increasing frequency in the environment. The acute lethal concentration that kills 50% of the fish test population and the chronic effects of exposure to MTBE were investigated in the zebrafish (Danio rerio). Chronic exposure over three weeks to effective MTBE conceritrations as low as 0.11 mg/L induced a significant increase in the vitellogenin concentration of male fish. The impact of a chronic, eight-week exposure at effective concentrations ranging from 0.44 to 220 mg/L had no significant effect on fecundity, fertilization, or hatch rate but highly significant impacts on sperm motility. Spermatozoa of all MTBE-exposure groups showed a significantly lower straight-line velocity and lower average path velocity compared to those of the nonexposed group. These results suggest that chronic exposure to MTBE negatively affects fish sperm motility at concentrations that are environmentally relevant and several orders of magnitude lower than concentrations inducing acute effects.

Effect of short-term exposure to methyl-tert-butyl ether and tert-butyl alcohol on the hatch rate and development of the African catfish, Clarias gariepinus.

Methyl tert-butyl ether (MTBE), a synthetic chemical used as a fuel additive, has been detected more frequently in the environment than previously. In this study, we examine the effects of MTBE (up to 100 mg/L) and its primary metabolite tertbutyl alcohol (TBA) (up to 1,400 mg/L) on the hatch rate and larval development of the African catfish Clarias gariepinus. Exposure to higher MTBE concentrations resulted in deformed eyes, mouthparts, and spinal cord and in increased larval mortality. Methyl tert-butyl ether exposure had no significant impact on egg viability, whereas TBA induced a decline of hatch rate. The MTBE can be regarded as a pollutant with toxicological effects on catfish larvae at concentrations above 50 mg/L. Although such concentrations greatly surpass present-day concentrations found in surface water (0.088 mg/L), concentrations up to 200 mg/L have been detected in groundwater.

In vitro adenine nucleotide catabolism in African catfish spermatozoa.

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 degrees C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 degrees C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP-->ADP-->AMP (adenosine/IMP)-->inosine-->hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.

Comparative study of the reproductive characteristics of XY male and hormonally sex-reversed XX male Eurasian perch, Perca fluviatilis.

In order to compare the reproductive capacity of XY male versus XX male (neomales) Eurasian perch (Perca fluviatilis), we determined the sperm quality (sperm concentration and motility) and reproductive characteristics such as gonadosomatic index (GSI), fertilization rate and sex steroid levels (testosterone, T; 17beta-estradiol, E2 and 11-ketotestosterone, 11KT) during the reproductive season. Median GSI was not significantly different between XY males (7.9%) and XX males (7.5%). Fertilization rates ranged between 30.0 and 98.0%. Sperm concentration ranged between 27.9 x 10(9) and 42.0 x 10(9) spermatozoa ml(-1). Median level of T, 11KT and E2 levels increased in the middle of the reproductive season (2136.0, 2409.0 and 3252.0 pg ml(-1), respectively) and decreased at the end (1657.0, 2006.6 and 431.0 pg ml(-1), respectively). Sperm motility was assessed by CASA and expressed by the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), percentage of motile sperm (% MOT) and motile concentration (MOC). Overall, there were not any significant differences between XY and XX males. In conclusion, no differences of reproductive capacities were observed between XY males and XX males suggesting that the last can be crossed with females to improve the productivity of Eurasian perch by producing all-female stock.