This study aimed to examine the impact of varying concentrations of bile acids (BA) added to the feed on several aspects of Penaeus vannamei. The purity of BA was 25.29%, and its main components were 5.74% chenodeoxycholic acid, 6.27% allocholic acid, 3.20% cholic acid, 5.79% hyodeoxycholic acid, and 2.31% hyocholic acid. The experiment was designed with four groups: CT, BA1, BA2, and BA3, where BA were added to the shrimp basal diet at concentrations of 0.0 mg/kg, 0.1 mg/kg, 1.0 mg/kg, and 10.0 mg/kg, respectively. After 60 days of farming P. vannamei (initial body weight: 1.21 ± 0.05 g), the results showed that BA supplementation significantly improved growth performance, and BA2 group was the most significant, which increased the final weight (FBW) by 18.6%, weight gain rate (WGR) by 19.5%, and survival rate (SR) by 5.8% compared with the CT group (p < 0.05). Additionally, the activities of trypsin and lipase in gut tissue were significantly increased (p < 0.05). Furthermore, BA supplementation increased the activity of antioxidant-related enzymes in the hepatopancreas and enhanced the mRNA expression levels of gut-associated immune genes. In addition, the supplementation of 0.1 mg/kg BA significantly altered the gut microbial composition, reducing the proportion of harmful Proteobacteria while enhancing the relative abundance of beneficial microorganisms such as Firmicutes and Bacteroides. In conclusion, 1.0 mg/kg and 10.0 mg/kg BA supplementation significantly improved the growth performance, digestive capacity, and antioxidant capacity of shrimp, among which 1.0 mg/kg supplementation had the most significant effect and improved the intestinal microbial composition of shrimp.

The past decades have marked the rise of metabolomics and lipidomics as the -omics sciences which reflect the most phenotypes in living systems. Mass spectrometry-based approaches are acknowledged for both quantification and identification of molecular signatures, the latter relying primarily on fragmentation spectra interpretation. However, the high structural diversity of biological small molecules poses a considerable challenge in compound annotation. Feature-based molecular networking (FBMN) combined with database searches currently sets the gold standard for annotation of large datasets. Nevertheless, FBMN is usually based on collision-induced dissociation (CID) data, which may lead to unsatisfying information. The use of alternative fragmentation methods, such as electron-activated dissociation (EAD), is undergoing a re-evaluation for the annotation of small molecules, as it gives access to additional fragmentation routes. In this study, we apply the performances of data-dependent acquisition mass spectrometry (DDA-MS) under CID and EAD fragmentation along with FBMN construction, to perform extensive compound annotation in the crude extracts of the freshwater sentinel organism Gammarus fossarum. We discuss the analytical aspects of the use of the two fragmentation modes, perform a general comparison of the information delivered, and compare the CID and EAD fragmentation pathways for specific classes of compounds, including previously unstudied species. In addition, we discuss the potential use of FBMN constructed with EAD fragmentation spectra to improve lipid annotation, compared to the classic CID-based networks. Our approach has enabled higher confidence annotations and finer structure characterization of 823 features, including both metabolites and lipids detected in G. fossarum extracts.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins prevalent in the environment and food chain, posing severe health risks to humans and animals. Bile acids are natural detergents synthesized from cholesterol and play a key role in the excretion of toxins in vertebrates. Here, pacific white shrimp (Litopenaeus vannamei) served as an animal model to examine the toxicity mechanisms of AFB1 and assess the potential alleviating effects of bile acids against AFB1. Our results revealed that AFB1 exposure significantly inhibited the growth performance and immune response of shrimp, accompanied by AFB1 accumulation and histological damage. Mechanistically, AFB1-induced DNA damage activated DNA repair mechanisms and induced the arrest of cell cycle via the ATR-cyclin B/cdc2 pathway. Additionally, AFB1 directly suppressed the immune response and growth performance of shrimp by inhibiting Toll and IMD pathways and the secretion of digestive enzymes. Notably, dietary bile acids significantly reduced AFB1 accumulation and alleviated AFB1-induced growth retardation and immunotoxicity in shrimp, and CCKAR, ATR, and Relish may be key mediators of the alleviating effects of bile acids. Our study provided new insights into the toxicity mechanisms of AFB1 in invertebrates and highlighted the potential of bile acids to alleviate AFB1 toxicity.

An 8-week feeding trial was conducted to evaluate the effects of chenodeoxycholic acid (CDCA) on growth performance, body composition, lipid metabolism, and intestinal health of juvenile white shrimp, Litopenaeus vannamei fed a low fishmeal diet. Four practical diets were formulated: HFM (25% fishmeal), LFM (15% fishmeal), LB1 (LFM + 0.04% CDCA), LB2 (LFM + 0.08% CDCA). Each diet was assigned to four tanks with forty shrimp (initial weight 0.33 ± 0.03 g) per tank. The results indicated that the growth performance of shrimp were similar between the four groups; the crude lipid content of shrimp fed the LB2 diet was significantly lower than those fed the HFM diet (P < 0.05). The lipase activity content in hepatopancreatic were significantly higher in the two CDCA supplemented groups than that in LFM group; the contents of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol in hemolymph were significantly lower in LFM group, LB1 group and LB2 group than that in HFM group (P < 0.05). The shrimp fed LB1 diet was significantly decreased the intestinal expression levels of tube than those fed in HFM diet; the intestinal gene expression of imd and toll were significantly lower in LB2 group than those in HFM group (P < 0.05). The results of hepatopancreas gene expression suggest that shrimp fed the LFM diet showed significantly upregulated expression levels of sterol regulatory element-binding protein (srebp), acetyl-CoA carboxylase (acc), and carnitine palmitoyltransferase 1 (cpt-1) than those fed the HFM diet; shrimp fed the LB1 diet showed significantly upregulated expression levels of srebp, acc, and AMP-activated protein kinase (ampk) than those fed the HFM diet; shrimp fed the LB2 diet had higher expression levels of srebp, acc, and cpt-1 than those fed the HFM diet (P < 0.05). In the hepatopancreas, the shrimp fed the LFM diet shown significantly up-regulated the expression levels of beclin1 compared to those fed HFM diet; the expression levels of autophagy-related protein13 (atg3), autophagy-related protein 12 (atg12) of in shrimp fed the LB1 diet were significantly higher than those fed the HFM diet; and the expression levels of autophagy-related protein13 (atg13), beclin1, atg3, atg12, autophagy-related protein 9 (atg9) of shrimp fed LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). The atg3 in intestine of shrimp fed the LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). Intestinal mucous fold were damaged, hepatic tubules were disorganized and B cells appeared to be swollen in LFM group. The fold height and width of shrimp fed the diets supplemented with CDCA increased significantly than those fed the LFM diet (P < 0.05), the hepatic tubules were neatly arranged, and R cells increased. In conclusion, supplementary CDCA in a low fishmeal diet promoted lipid metabolism, enhanced autophagy of shrimp, also improved the health of the intestine and hepatopancreas.

The potential of bile acids (BAs) to reduce aflatoxin B₁ (AFB₁) residues and toxicity in Litopenaeus vannamei was evaluated. Both juveniles and subadults were treated with 0, 0.05, 0.15 and 0.25 g/kg BAs for 60 days followed by 10-d AFB₁ exposure (2000 μg/kg), and fifteen shrimp (five shrimp were pooled into one sample, n = 3) from each treatment were collected at five time points (30, 60, 63, 66 and 70 d). All parameters were determined using accepted and standard methods with acceptable accuracy (recovery) of 90-110%. Results demonstrated that BAs reduced the AFB₁ residues in shrimp (limit of detection: 0.01 μg/L, relative standard deviation < 10% and recovery: 92.1-96.8%). BAs increased the detoxification of AFB₁ and decreased the levels of oxidative stress products by increasing Phase II and antioxidant systems, avoiding AFB₁-induced deterioration of shrimp meat and health risks to human. The confidence level was 95%.

The ∼15.000 decapod crustaceans that are mostly omnivorous have evolved a structurally and functionally complex digestive system. They have highly effective cuticular chewing and filtering structures in the stomach, which are regularly renewed by moulting. Decapods produce a broad range of digestive enzymes including chitinases, cellulases, and collagenases with unique properties. These enzymes are synthesized in the F-cells of the hepatopancreas and are encoded in the genome as pre-pro-proteins. In contrast to mammals, they are stored in a mature form in the lumen of the stomach to await the next meal, and therefore, the enzymes are particularly stable. The fat emulsifiers are fatty acyl-dipeptides rather than bile salts. After mechanical and chemical processing of the food in the cardiac stomach, the chyme is filtered by two unique filter systems of different mesh-size. The filtrate is then transferred to the hepatopancreas where the nutrients are absorbed by the R-cells, mostly via carriers, resembling nutrient absorption in the small intestine of mammals. The absorbed nutrients are used to fuel the metabolism of the hepatopancreas, are supplied to other organs, and are stored in the R-cells as glycogen and lipid reserves. Export lipids are secreted from the R-cells into the haemolymph as high density lipoproteins that mainly consist of phospholipids. In contrast to mammals, the midgut tube and hindgut contribute only little to food processing and nutrient absorption. The oesophagus, stomach and hindgut are well innervated but the hepatopancreas lacks nerves. Hormone cells are abundant in the midgut and hepatopancreas epithelia. Microorganisms are often present in the intestine of decapods, but they are apparently not essential for digestion and nutrition.

Chemical risk assessment remains entrenched in chronic toxicity tests that set safety thresholds based on animal pathology or fitness. Chronic tests are resource expensive and lack mechanistic insight. Discovering a chemical's mode-of-action can in principle provide predictive molecular biomarkers for a toxicity endpoint. Furthermore, since molecular perturbations precede pathology, early-response molecular biomarkers may enable shorter, more resource efficient testing that can predict chronic animal fitness. This study applied untargeted metabolomics to attempt to discover early-response metabolic biomarkers that can predict reproductive fitness of Daphnia magna, an internationally-recognized test species. First, we measured the reproductive toxicities of cadmium, 2,4-dinitrophenol and propranolol to individual Daphnia in 21-day OECD toxicity tests, then measured the metabolic profiles of these animals using mass spectrometry. Multivariate regression successfully discovered putative metabolic biomarkers that strongly predict reproductive impairment by each chemical, and for all chemicals combined. The non-chemical-specific metabolic biomarkers were then applied to metabolite data from Daphnia 24-h acute toxicity tests and correctly predicted that significant decreases in reproductive fitness would occur if these animals were exposed to cadmium, 2,4-dinitrophenol or propranolol for 21 days. While the applicability of these findings is limited to three chemicals, they provide proof-of-principle that early-response metabolic biomarkers of chronic animal fitness can be discovered for regulatory toxicity testing.

To develop a simple and cost-effective bioavailability test for sediment-bound contaminants, the solubilization strengths of mixtures of four commercially available surfactants and four proteins were compared to that of digestive fluids from a deposit-feeding benthic polychaete Arenicola marina. Initial tests indicated that sodium taurocholate, a vertebrate bile salt, was the most accurate mimic of A. marina gut fluids' solubilization of individual polycyclic aromatic hydrocarbons (PAH). Further testing with nutritional lipids and other hydrophobic contaminants confirmed the similarities of these fluids. Bovine serum albumin (BSA) solubilization of PAH was the most efficient of all the proteins tested. A cocktail of sodium taurocholate and BSA was compared to A. marina's solubilization of 12 PAH from four different contaminated sediments (from Boston, Charleston, Jacksonville, and San Diego harbors). The two solutions released most PAH to similar extents; 40 of 48 PAH-sediment combinations were released at amounts within a factor of 2 in cocktail and gut fluid solutions. Therefore, the cocktail may serve as a surrogate for real gut fluids and allow easier adoption of the in vitro incubation approach to bioavailability testing.

Coastal sediments contain complex mixtures of hydrophobic compounds including organic contaminants such as polycyclic aromatic hydrocarbons and biogenic compounds such as cholesterol and phospholipids. Within the guts of benthic invertebrates, these mixtures are subjected to digestive, chemical conditions that can be rich in surfactants and proteinaceous material. Using in vitro incubations as proxy for digestive exposure, we studied the solubilization of binary mixtures of nutritional and contaminant lipids into artificial seawater and six marine invertebrate gut fluids (Molpadia intermedia, Cucumaria frondosa, Arenicola marina, Arenicola brasiliensis, Parastichopus californicus, and Nereis virens). For animals with surfactant micelles or high protein concentrations, solubilization interactions were frequent. For example, in Arenicola marina gut fluid, benzo(a)pyrene enhanced the solubilization of hexadecane (491% of the compound alone) and palmitic acid (130%) but hindered cholesterol (83%). Benzo(a)pyrene concentrations increased in gut fluids in the presence of cholesterol (137% of BaP alone), phenanthrene (154%), lecithin (140%), and hexadecanol (232%). In A. marina gut fluid, dilution with seawater indicated that these enhancements occur only when micelles are present. Sediment-water partitioning models, used to predict the bioavailability of hydrophobic organic chemicals, do not account for such interactions between solubilizates (compounds solubilized in micelles). However, for animals exposed via a digestive tract containing micelles or high protein concentrations, digestive bioavailability and perhaps bioaccumulation are likely influenced by these interactions.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

The inclusion of phosphatidylcholine (PC) in a purified diet is essential for the survival of juvenile lobsters. Attempts to substitute this substance with its alkaline hydrolysis products, fatty acids, an emulsifier or other phospholipids (cephalin, phosphatidylinositol) were not successful. The effectiveness of the PC ingredient in reducing mortality related to a molt death syndrome is dependent upon the source of the compound. Those PC molecules comprised of polyunsaturated fatty acids, were most effective. The lobster's nutritional requirement for PC is probably the result of a low rate of biosynthesis and the physiological role of PC is more probably associated with hemolymph lipoproteins and the transport of lipids, possible cholesterol.