Alteration of eggs biochemical composition and progeny survival by maternal high carbohydrate nutrition in a teleost fish.

Reproductive performances, and the factors affecting them, are of major importance especially for farmed fish in the context of the development of a sustainable aquaculture. Dietary maternal lipids have been identified as a major factor affecting reproductive performances. Nevertheless, the consequences of carbohydrates have been little studied while plant-derived carbohydrates could be increasingly used in broodstock diets. To explore this issue, 2-year-old female trout were fed either a control diet that contains no carbohydrate and a high protein content (65.7%) or a diet formulated with plant-derived carbohydrates containing 32.5% carbohydrate and 42.9% protein ('HC diet') for an entire reproductive cycle. The reproductive performances, the quality of the unfertilized eggs and the development of the progeny were carefully monitored. Although the one year HC nutrition had not impaired female growth nor spawns quality, such nutrition had increased the variability of eggs size within spawns (+ 34.0%). Moreover, the eggs produced had a modified fatty acid profile, including a significant reduction in EPA content (- 22.9%) and a significant increase in the AA/EPA ratio (+ 33.3%). The progeny were impacted by such alterations as their survival rates were significantly reduced. A lower plant-derived carbohydrate inclusion (20%) should be considered in aquafeed for female broodstock in trout.

Tissue origin of circulating microRNAs and their response to nutritional and environmental stress in rainbow trout (Oncorhynchus mykiss).

Stresses associated with changes in diet or environmental disturbances are common situations that fish encounter during their lifetime. The stability and ease of measuring microRNAs (miRNAs) present in biological fluids make these molecules particularly interesting biomarkers for non-lethal assessment of stress in animals. Rainbow trout were exposed for four weeks to abiotic stress (moderate hypoxia) and/or nutritional stress (a high-carbohydrate/low-protein diet). Blood plasma and epidermal mucus were sampled at the end of the experiment, and miRNAs were assessed using small RNA sequencing. We identified four miRNAs (miR-122-5p, miR-184-3p, miR-192-5p and miR-194a-5p) and three miRNAs (miR-210-3p, miR-153a-3p and miR-218c-5p) that accumulated in response to stress in blood plasma and epidermal mucus, respectively. In particular, the abundance of miR-210-3p, a hypoxamiR in mammals, increased strongly in the epidermal mucus of rainbow trout subjected to moderate hypoxia, and can thus be considered a relevant biomarker of hypoxic stress in trout. We explored the contribution of 22 tissues/organs to the abundance of circulating miRNAs (c-miRNAs) in blood plasma and epidermal mucus influenced by the treatments. Some miRNAs were tissue-specific, while others were distributed among several tissues. Some c-miRNAs (e.g., miR-210-3p, miR184-3p) showed similar variations in both tissues and fluids, while others showed an inverse trend (e.g., miR-122-5p) or no apparent relationship (e.g. miR-192-5p, miR-194a-5p. Overall, these results demonstrate that c-miRNAs can be used as non-lethal biomarkers to study stress in fish. In particular, the upregulation of miR-210-3p in epidermal mucus induced by hypoxia demonstrates the potential of using epidermal mucus as a matrix for identifying non-invasive biomarkers of stress. This study provides information about the tissue sources of c-miRNAs and highlights the potential difficulty in relating variations in miRNA abundance in biological fluids to that in tissues.

Physical Enrichment Triggers Brain Plasticity and Influences Blood Plasma Circulating miRNA in Rainbow Trout (Oncorhynchus mykiss).

Physical enrichment is known to improve living conditions of fish held in farming systems and has been shown to promote behavioral plasticity in captive fish. However, the brain's regulatory-mechanism systems underlying its behavioral effects remain poorly studied. The present study investigated the impact of a three-month exposure to an enriched environment (EE vs. barren environment, BE) on the modulation of brain function in rainbow trout (Oncorhynchus mykiss) juveniles. Using high-throughput RT-qPCR, we assessed mRNA genes related to brain function in several areas of the trout brain. These included markers of cerebral activity and plasticity, neurogenesis, synaptogenesis, or selected neurotransmitters pathways (dopamine, glutamate, GABA, and serotonin). Overall, the fish from EE displayed a series of differentially expressed genes (neurotrophic, neurogenesis, and synaptogenesis markers) essentially localized in the telencephalon, which could underpin the beneficial effects of complexifying the environment on fish brain plasticity. In addition, EE significantly affected blood plasma c-miRNA signatures, as revealed by the upregulation of four c-miRNAs (miR-200b/c-3p, miR-203a-3p, miR-205-1a-5p, miR-218a-5p) in fish blood plasma after 185 days of EE exposure. Overall, we concluded that complexifying the environment through the addition of physical structures that stimulate and encourage fish to explore promotes the trout's brain function in farming conditions.

Effect of micro-algae Schizochytrium sp. supplementation in plant diet on reproduction of female rainbow trout (Oncorhynchus mykiss): maternal programming impact of progeny.

BACKGROUND: The broodstock diet, and in particular the lipid and fatty acid composition of the diet, is known to play a key role in reproductive efficiency and survival of the progeny in fish. A major problem when replacing both fish meal and fish oil by plant sources is the lack of n-3 long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To address this problem, we studied the effect of the plant-based diet supplemented with Schizochytrium sp. microalgae, source of DHA, compared to a conventional commercial diet rich in fish meal and fish oil on reproductive performance and egg quality and the consequences on progeny, in female rainbow trout broodstock. RESULTS: The results demonstrated that DHA-rich microalgae supplementation in a plant-based diet allowed for the maintenance of reproductive performance and egg quality comparable to a conventional commercial feed rich in fish meal and fish oil and led to an increased significant fry survival after resorption. Moreover, when females were fed a plant-based diet supplemented with micro-algae, the 4-month-old progenies showed a significant higher growth when they were challenged with a similar diet as broodstock during 1 month. We provide evidence for metabolic programming in which the maternal dietary induced significant protracted effects on lipid metabolism of progeny. CONCLUSIONS: The present study demonstrates that supplementation of a plant-based diet with DHA-rich microalgae can be an effective alternative to fish meal and fish oil in rainbow trout broodstock aquafeed.

Circulating miRNA repertoire as a biomarker of metabolic and reproductive states in rainbow trout.

BACKGROUND: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. RESULTS: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. CONCLUSIONS: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

VisEgg: a robust phenotyping tool to assess rainbow trout egg features and viability.

Assessing female fish reproductive success requires a thorough evaluation of egg characteristics, including egg number, size, and variability as well as egg developmental potential through the monitoring of embryo survival after fertilization. While embryonic success relies, at least in part, on paternal contribution, some parameters are strictly related to egg characteristics, one of the main ones being the viability of the egg when released into the water at spawning. It is however not necessarily possible, at least in salmonid fish that lay nontransparent eggs, to separate the different causes of egg/embryo failure. In this context, our aim was (i) to develop a simple and rapid system to capture images of rainbow trout eggs combined with computerized processing of these images to perform a fully automatic individual characterization of egg features including number and size (ii) to estimate unfertilized egg viability through the monitoring of the percentage of eggs that will not survive to water hydration. To evaluate the VisEgg system, unfertilized eggs (approximatively 400 eggs per batch) originating from 105 different females were hydrated in water. After 24 h, a picture of the eggs was obtained using a dedicated shooting system consisting of a light source and a digital single-lens reflex (SLR) camera. An image processing algorithm was developed to allow the automatic detection and separation of the eggs and to perform automatic measurements of egg number and individual egg size. The presence of white egg was used as an indirect measure of egg integrity, the "whitening" being the result of water entry into the egg through the vitelline membrane. These white eggs were therefore considered nonviable, as a result of their lack of physical integrity. Fertilization assays were performed in parallel using a subsample of the same egg batch. Embryonic development was monitored and hatching rate was calculated. A significant correlation between white egg percentage after hydration and hatching rate was observed (Spearman coefficient = -0.557, p < 0.001), in consistency with the fact that nonviable egg will not allow successful embryonic development. In contrast, the percentage of eggs that do not successfully hatch includes egg/embryo failures of different nature including reduced egg viability. Using the VisEgg, we were able to quantify the lack of viability of the eggs separately from the different other events that may occur during fertilization and incubation. the VisEgg is a convenient and reliable tool to obtain individual measures on trout eggs. It can be used to assess not only egg size and egg number but also unfertilized egg viability before fertilization.

C-ECi: a CUBIC-ECi combined clearing method for three-dimensional follicular content analysis in the fish ovary†.

Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (3D) imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.

Genes Involved in Drosophila melanogaster Ovarian Function Are Highly Conserved Throughout Evolution.

This work presents a systematic approach to study the conservation of genes between fruit flies and mammals. We have listed 971 Drosophila genes involved in female reproduction at the ovarian level and systematically looked for orthologs in the Ciona, zebrafish, coelacanth, lizard, chicken, and mouse. Depending on the species, the percentage of these Drosophila genes with at least one ortholog varies between 69% and 78%. In comparison, only 42% of all the Drosophila genes have an ortholog in the mouse genome (P < 0.0001), suggesting a dramatically higher evolutionary conservation of ovarian genes. The 177 Drosophila genes that have no ortholog in mice and other vertebrates correspond to genes that are involved in mechanisms of oogenesis that are specific to the fruit fly or the insects. Among 759 genes with at least one ortholog in the zebrafish, 73 have an expression enriched in the ovary in this species (RNA-seq data). Among 760 genes that have at least one ortholog in the mouse; 76 and 11 orthologs are reported to be preferentially and exclusively expressed in the mouse ovary, respectively (based on the UniGene expressed sequence tag database). Several of them are already known to play a key role in murine oogenesis and/or to be enriched in the mouse/zebrafish oocyte, whereas others have remained unreported. We have investigated, by RNA-seq and real-time quantitative PCR, the exclusive ovarian expression of 10 genes in fish and mammals. Overall, we have found several novel candidates potentially involved in mammalian oogenesis by an evolutionary approach and using the fruit fly as an animal model.

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system.

BACKGROUND: Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. RESULTS: Analyses of the bacterial community identified in water from BFT and "clear seawater" (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. CONCLUSION: This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Rearing effect of biofloc on antioxidant and antimicrobial transcriptional response in Litopenaeus stylirostris shrimp facing an experimental sub-lethal hydrogen peroxide stress.

This study compares the antioxidant and antimicrobial transcriptional expression of blue shrimps reared according to two different systems, BioFloc Technology (BFT) and Clear sea Water (CW) and their differential responses when facing an experimental sublethal hydrogen peroxide stress. After 30 days of rearing, juvenile shrimps were exposed to H2O2 stress at a concentration of 30 ppm during 6 h. The oxidative stress caused by H2O2 was examined in the digestive glands of the shrimp, in which antioxidant enzyme (AOE) and antimicrobial peptide (AMP) gene expression were analysed by quantitative real-time PCR. Results showed that rearing conditions did not affect the expression of genes encoding AOEs or AMPs. However, H2O2 stress induced a differential response in expression between shrimps from the two rearing treatments (BFT and CW). Comparative analysis of the expression profiles indicates that catalase transcripts were significantly upregulated by H2O2 stress for BFT shrimps while no change was observed for CW shrimps. In contrast, H2O2 caused down-regulation of superoxide dismutase and glutathione transferase transcripts and of the three AMP transcripts studied (penaeidin 2 and 3, and crustin) for CW shrimps, while no effect was observed on BFT shrimp transcript levels. These results suggested that BFT shrimps maintained antioxidant and AMP responses after stress and therefore can effectively protect their cells against oxidative stress, while CW shrimp immune competence seems to decrease after stress.