A Bacterial-Sourced Protein Diet Induces Beneficial Shifts in the Gut Microbiome of the Zebrafish, Danio rerio.

Background: Bacterial-sourced single-cell proteins (SCPs) offer an alternative protein source for diet formulation for Zebrafish (Danio rerio) and other aquaculture models. In addition, the use of a single-cell bacterial protein source derived from multiple species provides a unique insight into the interplay among nutrients in the diet, microbial populations in the diet, and the gut microbiome in D. rerio. Objective: Our objective in this study was to evaluate the impact of dietary replacement of fish protein hydrolysate in a standard reference (SR) with a single-cell bacterial protein source on D. rerio gut microbiome. Methods: We investigated gut microbial compositions of D. rerio fed an open-formulation standard reference (SR) diet or a bacterial-sourced protein (BP) diet, utilizing microbial taxonomic co-occurrence networks, and predicted functional profiles. Results: Microbial communities in the SR diet were primarily composed of Firmicutes. In contrast, the BP diet was mainly composed of Proteobacteria. Alpha diversity revealed significant differences in microbial communities between the 2 diets, and between the guts of D. rerio fed either of the 2 diets. D. rerio fed with the SR diet resulted in abundance of Aeromonas and Vibrio. In contrast, D. rerio fed with a BP diet displayed a large abundance of members from the Rhodobacteraceae family. Taxonomic co-occurrence networks display unique microbial interactions, and key taxons in D. rerio gut samples were dependent on diet and gender. Predicted functional profiling of the microbiome across D. rerio fed SR or BP diets revealed distinct metabolic pathway differences. Female D. rerio fed the BP diet displayed significant upregulation of pathways related to primary and secondary bile acid synthesis. Male D. rerio fed the BP diet revealed similar pathway shifts and, additionally, a significant upregulation of the polyketide sugar unit biosynthesis pathway. Conclusions: The use of a BP dramatically affects the composition and activity of the gut microbiome. Future investigations should further address the interplay among biological systems and diet and may offer insights into potential health benefits in preclinical and translational animal models.

Replacement of Dietary Fish Protein with Bacterial Protein Results in Decreased Adiposity Coupled with Liver Gene Expression Changes in Female Danio rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Objective: Our objective was to evaluate a bacterial-based single-cell protein (SCP) for the production of open-source standardized diets with defined health characteristics for the zebrafish research community. Methods: We completed a 16-wk feeding trial using juvenile D. rerio 31 d postfertilization (10 tanks per diet and 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient [standard reference (SR) diet] or a novel bacterial SCP source [bacterial protein (BP) diet]. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the BP diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments, suggesting normal physiological function. Genes differentially expressed in female D. rerio fed the BP diet compared with females fed the SR diet were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: Protein source substantially affected body growth metrics and composition as well as gene expression. These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Replacement of Dietary Fish Protein With Bacterial Single Cell Protein Results in Decreased Adiposity Coupled With Liver Expression Changes in Female Danio Rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Our objective was to evaluate single cell protein (SCP) for production of open-source standardized diets with defined heath characteristics for the zebrafish research community. We completed a 16-week feeding trial using juvenile D. rerio 31 days post-fertilization (dpf) (10 tanks per diet, 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient or a novel bacterial SCP source. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio only with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the SCP containing diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments. Genes differentially expressed in female D. rerio provided the bacterial SCP compared to females given fish protein were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Body Metrics and the Gut Microbiome in Response to Macronutrient Limitation in the Zebrafish Danio rerio.

Background: Healthy and predictable physiologic homeostasis is paramount in animal models for biomedical research. Proper macronutrient intake is an essential and controllable environmental factor for maintaining animal health and promoting experimental reproducibility. Objective and Methods: Evaluate reductions in dietary macronutrient composition on body weight metrics, composition, and gut microbiome in Danio rerio. Methods: D. rerio were fed reference diets deficient in either protein or lipid content for 14 weeks. Results: Diets of reduced-protein or reduced-fat resulted in lower weight gain than the standard reference diet in male and female D. rerio. Females fed the reduced-protein diet had increased total body lipid, suggesting increased adiposity compared with females fed the standard reference diet. In contrast, females fed the reduced-fat diet had decreased total body lipid compared with females fed the standard reference diet. The microbial community in male and female D. rerio fed the standard reference diet displayed high abundances of Aeromonas, Rhodobacteraceae, and Vibrio. In contrast, Vibrio spp. were dominant in male and female D. rerio fed a reduced-protein diet, whereas Pseudomonas displayed heightened abundance when fed the reduced-fat diet. Predicted functional metagenomics of microbial communities (PICRUSt2) revealed a 3- to 4-fold increase in the KEGG (Kyoto Encyclopedia of Genes and Genomes) functional category of steroid hormone biosynthesis in both male and female D. rerio fed a reduced-protein diet. In contrast, an upregulation of secondary bile acid biosynthesis and synthesis and degradation of ketone bodies was concomitant with a downregulation in steroid hormone biosynthesis in females fed a reduced-fat diet. Conclusions: These study outcomes provide insight into future investigations to understand nutrient requirements to optimize growth, reproductive, and health demographics to microbial populations and metabolism in the D. rerio gut ecosystem. These evaluations are critical in understanding the maintenance of steady-state physiologic and metabolic homeostasis in D. rerio. Curr Dev Nutr 20xx;x:xx.

Feed Intake as an Estimation of Attractability in Pacific White Shrimp Litopenaeus vannamei.

A simple bioassay that quantifies feed intake as an estimation of relative attractability of feeds containing different ingredients in the Pacific white shrimp Litopenaeus vannamei is described. Fish meal (FM), fish protein hydrolysate (FPH), squid meal (SqM) and casein (CN) were assessed at the same dietary level for their relative influence on feed intake rates of Litopenaeus vannamei. A bland diet containing 92% whole wheat grain meal, 6% diatomaceous earth and 2% alginate with a known low attractability was used as the standard control or base diet. Ingredients were added to the bland base control diet at a level of 3% as fed. Shrimp were stocked into 80 L glass tanks (n= 20 per tank) in a recirculating aquaculture system. Tanks were randomly assigned to one of five diet treatments (3tanks/treatment). Experiments measuring the attractability of each feed were conducted twice daily at 0900 hours and 1330 hours over a five day period. For each experiment, 40 feed pellets (ca. 1 g) corresponding to the assigned treatment were provided to each tank. To calculate the rate of feed intake, pellets remaining in each tank were counted at six minute intervals for a seventy-two minute period. Differences in rate of feed intake among diets were evaluated using Cox Regression Analysis. This attractability assay required only small amounts of ingredients and incorporated ingredients into a bland feed, which significantly reduces the influence from other ingredients or compound in the pellets. All of the test protein ingredients, especially SqM, in the feeds significantly increased the feed consumption rate. The diet containing SqM was consumed at a significantly higher rate than those containing casein and FM but not FPH. FPH and CN containing diets were not significantly different but consumed at a higher rate than the diet containing FM. Results of these trials indicate that the presence of certain ingredients can increase feed intake, thereby increasing nutrient availability of the diets. This reported method to determine consumption of diets containing certain ingredients can be considered as a valid method to estimate attractability.

Effects of fructooligosaccharides (FOS) on growth, survival, gut microflora, stress, and immune response in Pacific white shrimp, Litopenaeus vannamei, cultured in a recirculating system.

The present study investigated the effects of short-chain fructooligosaccharides (FOS) as dietary supplements on stress, immune response, gut microbiota, growth, and survivability of three different treatment groups of Pacific white shrimp, Litopaneous vennamei, cultured in a recirculating system. The experiment was conducted over a 35-day trial period. Shrimps were fed diets, 15 times a day using automated feeder, supplemented with GOS at 0%, 0.15%, and 0.30% by weight. Denaturing gradient gel electrophoresis analysis revealed that the enteric microbial community of shrimp fed the basal diet differed markedly (<80.0% similarity coefficient) from those fed FOS-supplemented diets. However, shrimp survival, weight gain, and immune responses among the treatment groups were good but not significantly different (P > 0.05), probably due to the limited length of the feeding trial.

Dietary supplementation of galactooligosaccharides (GOS) in Pacific white shrimp, Litopenaeus vannamei, cultured in a recirculating system and its effects on gut microflora, growth, stress, and immune response.

This study was designed to examine the effects of a prebiotic compound on the immune system, digestive tract histology, and stress physiology of shrimp. The specific effects of dietary supplementation of the prebiotic galactooligosaccharide (GOS or GTGOS) on shrimp health are scarce. This experiment, therefore attempted to evaluate the effects of GOS on growth, survival, intestinal microbiota, stress resistance and immune responses of Pacific white shrimp, Litopaneous vannamei. Over a 35-day trial, shrimps were fed diets, 15 times a day using automated feeder, supplemented with GOS at 0%, 0.25%, and 0.40% by weight. Shrimp survival and weight gain among the treatment groups were good but not significantly different (P > .05). Shrimp fed GOS-supplemented diets had reduced stress (glucose, P < .05) and increased immune responses (total hemocyte counts and phagocytic capacity, P < .05) compared to shrimps fed only basal diet with no supplementation. These results suggest that GOS not only changed the populations of gut microbiota but also reduced stress levels and enhanced immune response in shrimp.

Effects of Dietary Carbohydrate on Weight Gain and Gonad Production in Small Sea Urchins, Lytechinus variegatus.

In experiment 1, juvenile sea urchins (n = 80, 0.088 ± 0.001 g wet weight and 5.72 ± 0.04 mm diameter) were held individually and fed ad libitum one of three semi-purified formulated diets (n = 16 individuals treatment-1). In the diets, protein was held constant (310g kg-1 dry, as fed) and carbohydrate level varied (190, 260, or 380 g kg-1 dry, as fed). Wet weights were measured every 2 weeks. Total wet weight gain was inversely proportional to dietary carbohydrate level and energy content of the respective diet. In experiment 2, sea urchins (5.60 ± 0.48g wet weight, n= 40) fed 190 g kg-1 carbohydrate consumed significantly more dry feed than those fed 260 g kg-1, but not more than those fed 380 g kg-1 carbohydrate. Based on differential feed intake rates, sea urchins that consumed more feed also consumed higher levels of protein and had the highest weight gain. Consequently, protein content and/or protein: energy ratio may be important in determining feed utilization and growth among sea urchins in this study. The average digestible energy intake was approximately 70 kcal kg-1 body weight day-1, suggesting daily caloric intake of juvenile Lytechinus variegatus is lower than in shrimp and fish.

PRODUCTION AND ECONOMIC OPTIMIZATION OF DIETARY PROTEIN AND CARBOHYDRATE IN THE CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus.

Juvenile Lytechinus variegatus (ca. 3.95± 0.54 g) were fed one of 10 formulated diets with different protein (ranging from 11- 43%) and carbohydrate (12 or 18%; brackets determined from previous studies) levels. Urchins (n= 16 per treatment) were fed a daily sub-satiation ration equivalent to 2.0% of average body weight for 10 weeks. Our objective was (1) to create predictive models of growth, production and efficiency outcomes and (2) to generate economic analysis models in relation to these dietary outcomes for juvenile L. variegatus held in culture. At dietary protein levels below ca. 30%, models for most growth and production outcomes predicted increased rates of growth and production among urchins fed diets containing 18% dietary carbohydrate levels as compared to urchins fed diets containing 12% dietary carbohydrate. For most outcomes, growth and production was predicted to increase with increasing level of dietary protein up to ca. 30%, after which, no further increase in growth and production were predicted. Likewise, dry matter production efficiency was predicted to increase with increasing protein level up to ca. 30%, with urchins fed diets with 18% carbohydrate exhibiting greater efficiency than those fed diets with 12% carbohydrate. The energetic cost of dry matter production was optimal at protein levels less than those required for maximal weight gain and gonad production, suggesting an increased energetic cost (decreased energy efficiency) is required to increase gonad production relative to somatic growth. Economic analysis models predict when cost of feed ingredients are low, the lowest cost per gram of wet weight gain will occur at 18% dietary carbohydrate and ca. 25- 30% dietary protein. In contrast, lowest cost per gram of wet weight gain will occur at 12% dietary carbohydrate and ca. 35- 40% dietary protein when feed ingredient costs are high or average. For both 18 and 12% levels of dietary carbohydrate, cost per gram of wet weight gain is predicted to be maximized at low dietary protein levels, regardless of feed ingredient costs. These models will compare dietary requirements and growth outcomes in relation to economic costs and provide insight for future commercialization of sea urchin aquaculture.

Optimizing dietary levels of menhaden and soybean oils and soybean lecithin for pre-gonadal somatic growth in juveniles of the sea urchin Lytechinus variegatus.

Dietary lipids serve as important sources of energy and essential fatty acids for aquatic animals. Sources of animal and plant oils are increasingly limited as well as expensive, and dietary requirements associated with the inclusion of these oils must be carefully evaluated to facilitate sustainable and affordable formulations. In this study, we investigated quantities of menhaden oil (MO) with and without soybean lecithin or soybean oil (SO) to determine appropriate levels for optimal somatic growth for pre-gonadal juvenile Lytechinus variegatus. We prepared semi-purified diets that varied in neutral lipid content (0, 2, 4, or 8% dry matter) and soy lecithin (0 or 2%) and exchanged lipids reciprocally with purified starch while holding constant all other nutrients. We maintained laboratory-reared juvenile L. variegatus (average initial wet weight 82 ± 0.7 mg, mean ± SE , n = 9 treatment-1) in recirculating seawater systems and fed each daily a sub-satiation ration for five weeks. We assessed wet weights and test diameters every two weeks and at the end of the experiment (5 wk). Level of MO with or without soybean lecithin did not significantly affect wet weight gain; however, increasing levels of SO in the diet reduced wet weight gain and dry matter production efficiency and increased feed conversion ratio. Dry gut weight was positively correlated with level of MO. Lipid level in the gut increased with increasing dietary lipid level, regardless of source. These data suggest the composition of the SO is inhibitory for either nutrient absorption or metabolic processes associated with growth at this life stage. Diets containing total lipid levels of approximately 5 to 6% that include sources of n-3 fatty acids may support optimal growth for pre-gonadal juvenile L. variegatus.

EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE LEVELS ON WEIGHT GAIN AND GONAD PRODUCTION IN THE SEA URCHIN LYTECHINUS VARIEGATUS.

Adult Lytechinus variegatus were fed eight formulated diets with different protein (ranging from 12 to 36%) and carbohydrate (ranging from 21 to 39 %) levels. Each sea urchin (n = 8 per treatment) was fed a daily sub-satiation ration of 1.5% of average body weight for 9 weeks. Akaike information criterion analysis was used to compare six different hypothesized dietary composition models across eight growth measurements. Dietary protein level and protein: energy ratio were the best models for prediction of total weight gain. Diets with the highest (> 68.6 mg P kcal--1) protein: energy ratios produced the most wet weight gain after 9 weeks. Dietary carbohydrate level was a poor predictor for most growth parameters examined in this study. However, the model containing a protein × carbohydrate interaction effect was the best model for protein efficiency ratio (PER). PER decreased with increasing dietary protein level, more so at higher carbohydrate levels. Food conversion ratio (FCR) was best modeled by total dietary energy levels: Higher energy diets produced lower FCRs. Dietary protein level was the best model of gonad wet weight gain. These data suggest that variations in dietary nutrients and energy differentially affect organismal growth and growth of body components.

EFFECT OF DIET QUALITY ON NUTRIENT ALLOCATION TO THE TEST AND ARISTOTLE'S LANTERN IN THE SEA URCHIN LYTECHINUS VARIEGATUS (LAMARCK, 1816).

Small adult (19.50 ± 2.01g wet weight) Lytechinus variegatus were fed eight formulated diets with different protein (12 to 36% dry weight as fed) and carbohydrate (21 to 39 % dry weight) levels. Each sea urchin (n = 8 per treatment) was fed a daily ration of 1.5% of the average body weight of all individuals for 9 weeks. Akaike information criterion scores were used to compare six different dietary composition hypotheses for eight growth measurements. For each physical growth response, different mathematical models representing a priori hypotheses were compared using the Akaike Information Criterion (AIC) score. The AIC is one of many information-theoretic approaches that allows for direct comparison of non-nested models with varying number of parameters. Dietary protein level and protein: energy ratio were the best models for prediction of test diameter increase. Dietary protein level was the best model of test with spines wet weight gain and test with spines dry matter production. When the Aristotle's lantern was corrected for size of the test, there was an inverse relationship with dietary protein level. Log transformed lantern to test with spines index was also best associated with the dietary protein model. Dietary carbohydrate level was a poor predictor for growth parameters. However, the protein × carbohydrate interaction model was the best model of organic content (% dry weight) of the test without spines. These data suggest that there is a differential allocation of resources when dietary protein is limiting and the test with spines, but not the Aristotle's lantern, is affected by availability of dietary nutrients.

Acetylcholinesterase in the sea urchin Lytechinus variegatus: characterization and developmental expression in larvae.

Acetylcholinesterase (AChE) in the echinoid Lytechinus variegatus has been characterized. Kinetic parameters V(max), K(m), K(ss), and b were 2594+/-1048 nmol ATCh hydrolyzed/min/mg tissue wet weight, 185+/-11 microM, 308+/-100 mM, and 0.2, respectively for the substrate ATCh and 17.8+/-6.87 nmol BTCh hydrolyzed/min/mg tissue wet weight, 654+/-424 microM, 36+/-31 mM, and 0.6, respectively for BTCh. Pharmacologic analyses were performed with four inhibitors of cholinesterases, physostigmine, BW284c51, ethopropazine, and iso-OMPA, and yielded IC(50) values of 106+/-4 nM, 718+/-118 nM, 2.57+/-0.6 mM, and >0.0300 M, respectively. Both kinetic and pharmacologic results confirmed the existence of AChE in larval L. variegatus. Dimeric and tetrameric globular forms (determined by velocity sedimentation on sucrose gradients) were present in L. variegatus larvae. Activity of AChE increased significantly as larvae progressed in development from embryos to eight-arm larvae. Acetylcholinesterase activity of F1 larvae derived from sea urchins collected from wild populations and of F1 larvae derived from sea urchins cultured in the laboratory and fed two different diets suggest that the nutritional and/or environmental history of the adult sea urchin affect the developmental progression of AChE activity in the F1 offspring.

Dietary supplementation of short-chain fructooligosaccharides influences gastrointestinal microbiota composition and immunity characteristics of Pacific white shrimp, Litopenaeus vannamei, cultured in a recirculating system.

Supplementation of prebiotic compounds, including short-chain fructooligosaccharides (scFOS) has been shown to confer benefits on nutrient utilization, growth, and disease resistance of various animal species through improved gastrointestinal (GI) microbiota. However, potential uses of prebiotics for shrimp have not been defined. A 6-wk feeding trial was conducted in a recirculating system to determine the effects of scFOS supplementation on growth performance, immune functions, and GI microbiota composition of Pacific white shrimp (Litopenaeus vannamei). scFOS was supplemented in a nutritionally complete diet (35% crude protein) at 0.025, 0.0500, 0.075, 0.100, 0.200, 0.400, and 0.800% by weight. After 6 wk of feeding, shrimp fed 0, 0.1, and 0.8% scFOS were sampled for assays of immune function and GI microbiota. Dietary supplementation of scFOS did not improve weight gain, feed conversion ratio, or survival of shrimp. Denaturing gradient gel electrophoresis analysis suggested the intestinal tract microbial community from shrimp fed the basal diet was different from that of shrimp fed the scFOS diets [similarity coefficient (SC) = 74.9%)], although the intestinal tract microbial community from shrimp fed the scFOS-supplemented diets was very similar (SC = 92.3%). All the bacterial species contributing to the GI microbial differences were identified, although most of them are uncultured species. Both total hemocyte count and hemocyte respiratory burst increased (P < 0.05) by incremental dietary supplementation of scFOS (0-0.8%). This study is the first to our knowledge to show that dietary scFOS can selectively support growth of certain bacterial species in the GI tract of shrimp and enhance immunity, which may facilitate development of alternative strategies, including novel probiotics and synbiotics, for shrimp growth and health management.

The effect of feeding frequency on consumption of food, absorption efficiency, and gonad production in the sea urchin Lytechinus variegatus.

The frequency of feeding in the field is variable in sea urchins, ranging from nearly continuous to diel or intermittent. It is essential to know the effect of feeding interval on physiological and metabolic processes to understand the basis for production. Lytechinus variegatus (50 mm horizontal diameter) were collected in January 1999 and held in closed-circuit aquaria at 25 degrees C and 35 per thousand salinity. After 9 days without food, individuals were fed one of three treatments: food available ad libitum, food available for 1 day every 2 days or food available for 1 day every 4 days for 28 days. The rate of food consumption per day of all individuals was high the first week of feeding. It then decreased to a lower, constant rate in those fed ad libitum but remained high in those fed one day every 2 or 4 days. The total amount eaten was directly related to frequency of feeding. The apparent dry matter digestibility (absorption efficiency) did not vary with frequency of feeding. As the total amount of energy absorbed was directly related to the frequency of feeding, the increase in the rate of food consumption does not compensate for a decrease in frequency of feeding. Gonad production efficiency was directly related to frequency of feeding. Gonad gross production (assimilation) efficiencies were 8.4, 3.9 and 3.4% for individuals fed every day, or fed one day every 2 and 4 days, respectively. The corresponding gonad net assimilation efficiencies were 12.5, 5.5, and 4.8%. A decrease in frequency of food availability results in use of a greater proportion of the food consumed for maintenance and less for gonad production.

Fine structure of the midgut epithelium in the developing brown shrimp, Penaeus aztecus.

The midgut epithelium of larval and early postlarval brown shrimp has been studied with light and electron microscopy. Ultrastructurally the features of the midgut do not change during these stages of development. On the basis of electron density, two epithelial cell types can be distinguished, and these are referred to as light and dark cells. The dark cells contain more rough endoplasmic reticulum and more free ribosomes than the light cells. Mitochondria in the dark cells have a matrix which is less electron dense than the mitochondrial matrix of the light cells. Both cell types have a microvillous border with a surface coat. The microvilli lack microfilaments within their core, and a terminal web is not differentiated in the stages examined. Tubular smooth endoplasmic reticulum is abundant in the basal portions of the cells. Electron dense, membrane bound vesicles are consistently seen in association with the Golgi apparatus, apical cell surface, and gut lumen and therefore are believed to be secretory granules. Cells in the anterior portion of the midgut often contain very large lipid droplets in the cytoplasm.