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Sesarmops sinensis is a dominant omnivorous crab species, which plays an important ecological function in salt marsh ecosystems. To better understand its immune system and immune related genes under pathogen infection, the transcriptome was analyzed by comparing the data of S. sinensis hepatopancreas stimulated by PBS and PGN. A set of assembly and annotation identified 39,039 unigenes with an average length of 1105 bp, obtaining 1300 differentially expressed genes (DEGs) in all, which included 466 remarkably up-regulated unigenes and 834 remarkably down-regulated unigenes. In addition, based on mensurable real time-polymerase chain reaction and high-throughput sequencing, several immune responsive genes were found to be markedly up-regulated under PGN stimulation. In conclusion, in addition to enriching the existing transcriptome data of S. sinensis, this study also clarified the immune response of S. sinensis to PGN stimulation, which will help us to further understand the crustacean's immune system.

Poor utilisation efficiency of carbohydrate always leads to metabolic phenotypes in fish. The intestinal microbiota plays an important role in carbohydrate degradation. Whether the intestinal bacteria could alleviate high-carbohydrate diet (HCD)-induced metabolic phenotypes in fish remains unknown. Here, a strain affiliated to Bacillus amyloliquefaciens was isolated from the intestine of Nile tilapia. A basal diet (CON), HCD or HCD supplemented with B. amy SS1 (HCB) was used to feed fish for 10 weeks. The beneficial effects of B. amy SS1 on weight gain and protein accumulation were observed. Fasting glucose and lipid deposition were decreased in the HCB group compared with the HCD group. High-throughput sequencing showed that the abundance of acetate-producing bacteria was increased in the HCB group relative to the HCD group. Gas chromatographic analysis indicated that the concentration of intestinal acetate was increased dramatically in the HCB group compared with that in the HCD group. Glucagon-like peptide-1 was also increased in the intestine and serum of the HCB group. Thus, fish were fed with HCD, HCD supplemented with sodium acetate at 900 mg/kg (HLA), 1800 mg/kg (HMA) or 3600 mg/kg (HHA) diet for 8 weeks, and the HMA and HHA groups mirrored the effects of B. amy SS1. This study revealed that B. amy SS1 could alleviate the metabolic phenotypes caused by HCD by enriching acetate-producing bacteria in fish intestines. Regulating the intestinal microbiota and their metabolites might represent a powerful strategy for fish nutrition modulation and health maintenance in future.

The National Natural Science Fund of China (31830102) and the Program of Shanghai Academic Research Leader (19XD1421200)

A high-carbohydrate diet could achieve a protein-sparing effect, but it may cause negative impacts on the growth condition of fish due to their poor utilisation ability of carbohydrate. How to reduce the adverse effects caused by a high-carbohydrate diet is important for the development of aquaculture. In the present study, we aimed to identify whether inulin could attenuate the metabolic syndrome caused by a high-carbohydrate diet in fish. Nile tilapia (Oreochromis niloticus) (1·19 (sd0·01) g) were supplied with 35 % carbohydrate (CON), 45 % carbohydrate (HC) and 45 % carbohydrate + 5 g/kg inulin (HCI) diets for 10 weeks. The results showed that addition of inulin improved the survival rate when fish were challenged withAeromonas hydrophila, indicating that inulin had an immunostimulatory effect. Compared with the HC group, the HCI group had lower lipid accumulation in liver and the gene expression analyses indicated that addition of inulin down-regulated genes related to lipogenesis and up-regulated genes relevant toβ-oxidation significantly (P< 0·05). Higher liver glycogen and glucose tolerance were found in the HCI group compared with the HC group (P< 0·05). These results indicated that inulin could alleviate the metabolic syndrome induced by a high-carbohydrate diet. Furthermore, addition of inulin to a high-carbohydrate diet changed the intestinal bacterial composition and significantly increased the concentration of acetic acid and propionic acid in fish gut which have the potential to increase pathogen resistance and regulate metabolic characteristics in fish. Collectively, our results demonstrated a possible causal role for the gut microbiome in metabolic improvements induced by inulin in fish.

Key points The pparab subtype in zebrafish is much more highly expressed in tissues with high oxidative activity than pparaa. The pparab deficiency in zebrafish reduces fatty acid β-oxidation both in liver and muscle, illustrating its functional homology as a mammalian peroxisome proliferator-activated receptor α (PPARα). pparab deficiency promotes metabolic reprogramming by increasing glucose utilization and inhibiting amino acid breakdown. The present study brings new insights into the comprehensive regulatory roles of PPARα in the cellular fuel selection and provides a valuable animal model for PPARα studies from a viewpoint of comparative physiology. Abstract Dysfunction of lipid metabolism is involved in the pathogenesis of several chronic metabolic diseases. Peroxisome proliferator-activated receptor α (PPARα) is essential for normal metabolic homeostasis and, in particular, for the regulation of fatty acid β-oxidation (FAO). However, little is known about its regulation roles in systemic nutrient metabolism. To explore the underlying modulation role of PPARα in metabolic homeostasis, we generated a pparab-knockout zebrafish (Danio rerio) model. The pparab mutants demonstrated lower expression of key enzymes involved in FAO, as well as lower mitochondrial and peroxisomal FAO in tissues, which was associated with lipid accumulation in liver and visceral mass. Conversely, glucose utilization was higher because they demonstrated lower blood glucose and tissue glycogen concentrations, as well as activation of the phosphoinositide 3-kinase/AKT pathway. In addition, pparab-deficient zebrafish demonstrated activation of AKT/mammalian target of rapamycin signalling and higher protein content, implying greater protein synthesis and/or lower amino acid breakdown. These data clearly revealed that pparab deletion reduces FAO but increases glucose utilization and protein deposition to maintain energy homeostasis. The present study provides new insights into the comprehensive regulatory role of PPARα in systemic energy metabolism in fish, and this pparab-deficient zebrafish also constitutes a valuable model for investigating the functions of PPARα in mammals from comparative physiology aspects.

Many metabolic diseases in fish are often associated with lowered peroxisomal fatty acid (FA) β-oxidation. However, the physiological role of peroxisomal FA oxidation in lipid metabolism in fish still remains unclear. In the present study, a specific peroxisomal FA β-oxidation inhibitor, 10,12-tricosadiynoic acid (TDYA), was used to investigate the effects of impaired peroxisomal β-oxidation on growth performance, health status, and lipid metabolism in Nile tilapia. The results showed that the dietary TDYA treatment did not affect weight gain, but significantly decreased peroxisomal β-oxidation in the liver, and increased body fat accumulation. The fish with impaired peroxisomal β-oxidation exhibited higher contents of serum lipid and peroxidation products, and alanine aminotransferase activity, and significantly lowered hepatic activities of superoxide dismutase and catalase. The inhibited peroxisomal β-oxidation did not enhance mitochondrial β-oxidation activity, but compensatorily upregulated FA β-oxidation-related gene expression, and downregulated the gene expressions in lipolysis and lipogenesis. Taken together, TDYA treatment markedly induced lipid accumulation and hepatic oxidative damage via systemically depressing lipid catabolism and antioxidant capacity. Our findings reveal the pivotal roles of peroxisomal β-oxidation in maintaining health and lipid homeostasis in fish, and could be helpful in understanding metabolic diseases in fish.

To limit the spread of severe acute respiratory syndrome coronavirus 2, the government of China has been monitoring infected travelers and minimizing cold-chain contamination. However, other factors might contribute to recurring outbreaks. We analyze the role of undocumented migrants as potential transmitters of severe acute respiratory syndrome coronavirus 2 in China.

Oxygen deprivation (hypoxia) is a common challenge in water environment, which causes lack of energy and oxidative damage in organisms. Many studies have indicated a number of physiological and metabolic changes under hypoxia, but the effects of dietary nutrients on hypoxia tolerance have not been well evaluated. In the present 7-week feeding trial, we fed zebrafish with low-protein diet (LP), high-protein diet (HP), low-fat diet (LF), high-fat diet (HF), low-carbohydrate diet (LC), and high-carbohydrate diet (HC), respectively. Afterward, the resistance to acute hypoxia challenge, growth, body composition, activities of metabolic enzymes, and expressions of energy homeostasis-related genes and six hifαs genes were measured. The results indicated that only the HC diet could significantly improve the resistance to hypoxia challenge. Moreover, the HC diet feeding caused higher glycogen deposition in the liver and muscle, and these glycogens were significantly reduced after 6-h acute hypoxia challenge. Meanwhile, the lactate content in the liver and blood was increased in the HC groups. At hypoxia status, the relative mRNA expressions of the genes related to glycolysis, ATP production, insulin signaling pathway, and hif-3a (hif1al) were all significantly increased in the muscle of the HC diet-fed fish. This study revealed that high-carbohydrate diet could improve the resistance to hypoxia by activating glycolysis and hif/insulin signaling pathway in zebrafish, mainly in the muscle, to efficiently supply energy. Therefore, our results highlight the importance of dietary carbohydrate in resisting hypoxia in fish.

Elucidating the closest living relatives of extant primates is essential for fully understanding important biological processes related to the genomic and phenotypic evolution of primates, especially of humans. However, the phylogenetic placement of these primate relatives remains controversial, with three primary hypotheses currently espoused based on morphological and molecular evidence. In the present study, we used two algorithms to analyze differently partitioned genomic datasets consisting of 45.4 Mb of conserved non-coding elements and 393 kb of concatenated coding sequences to test these hypotheses. We assessed different genomic histories and compared with other molecular studies found solid support for colugos being the closest living relatives of primates. Our phylogeny showed Cercopithecinae to have low levels of nucleotide divergence, especially for Papionini, and gibbons to have a high rate of divergence. The MCMCtree comprehensively updated divergence dates of early evolution of Primatomorpha and Primates.

Peroxisome proliferator-activated receptor α (PPARα) plays critical physiological roles in energy metabolism, antioxidation and immunity of mammals, however, these functions have not been fully understood in fish. In the present study, Nile tilapia (Oreochromis niloticus) were fed with fenofibrate, an agonist of PPARα, for six weeks, and subsequently challenged with Aeromonas hydrophila. The results showed that PPARα was efficiently activated by fenofibrate through increasing mRNA and protein expressions and protein dephosphorylation. PPARα activation increased significantly mitochondrial fatty acid β-oxidation efficiency, the copy number of mitochondrial DNA and expression of monoamine oxidase (MAO), a marker gene of mitochondria. Meanwhile, PPARα activation also increased significantly the expression of NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 9 (NDUFA9, complex I) and mitochondrial cytochrome c oxidase 1 (MTCO1, complex IV). The fenofibrate-fed fish had higher survival rate when exposed to A. hydrophila. Moreover, the fenofibrate-fed fish also had higher activities of immune and antioxidative enzymes, and gene expressions of anti-inflammatory cytokines, while had lower expressions of pro-inflammatory cytokine genes. Taken together, PPARα activation improved the ability of Nile tilapia to resist A. hydrophila, mainly through enhancing mitochondrial fatty acids β-oxidation, immune and antioxidant capacities, as well as inhibiting inflammation. This is the first study showing the regulatory effects of PPARα activation on immune functions through increasing mitochondria-mediated energy supply in fish.

Cold stress is a major threat to fish in both nature and aquaculture, and can induce oxidative stress in various fish. While the exact role of oxidative stress in cold-caused mortality is still unknown. The purpose of the present study was to evaluate the effects of oxidative stress on cold tolerance in fish and verify whether changing oxidative status could affect cold tolerance. We firstly demonstrated that acute cold exposure induced high oxidative stress in zebrafish liver, which may lead to mortality. Then we performed in vivo and in vitro experiments to determine the effects of the altered oxidative status on cold tolerance in zebrafish and zebrafish liver cell line (ZFL), respectively. In the in vivo study, the zebrafish which were fed with α-lipoic acid or reduced glutathione had lower cold-caused oxidative stress and tissues damage, and showed higher cold tolerance. In the experiment using zebrafish cells, increasing oxidative stress by H2O2 decreased the cellular cold tolerance, and the cold tolerance was partly recovered when oxidative stress was reduced by the addition of Vitamin C (VC). Taken together, we conclude that the reduction of oxidative stress increases cold tolerance in fish.

The dominant fatty acids (FAs) in oils are often used to explain different nutritional effects of dietary oils in fish. However, the amounts of dominant FAs among oils are different, and the nutritional roles of these important FAs in fish have not been precisely compared at similar levels in feeding trials. In the present study, different amounts of palmitic acid were added to safflower oil (SO), olive oil (OO) and fish oil (FO) to obtain comparable amounts (about 550 g/kg of total FAs) of 18:2n‐6, 18:1n‐9 and 20:5n‐3 + 22:6n‐3 and subsequently fed to Nile tilapia (11.1 ± 0.01 g) for 8 weeks. The results showed similar growth among groups but FO group obtained lower fat deposition, serum ALT and AST activities, compared to OO. Lipogenesis‐related gene expressions were higher in OO group than FO group in liver, muscle and adipose tissue, but there were only few differences in these genes between SO and FO groups. Lipid catabolism genes in FO group were higher than OO and SO groups in adipose tissue, but not in muscle, and the significantly higher expressions of CPT1b and PPARα were only observed in liver. Overall, dietary 18:2n‐6, 20:5n‐3 and 22:6n‐3 were beneficial to normal growth and lipid metabolism, whereas high amount of 18:1n‐9 induced lipid deposition and liver damage in Nile tilapia.

Key points In a cold environment, mammals increase their food intake while fish decrease or stop feeding. However, the physiological value of fasting during cold resistance in fish is currently unknown. Fasting for more than 48 h enhanced acute cold resistance in zebrafish, which correlated with lipid catabolism and cell damage attenuation. Lipid catabolism and autophagy were necessary for cold resistance in fish and the inhibition of mitochondrial fatty acid β-oxidation or autophagy weakened the fasting-induced cold resistance. Repression of mechanistic target of rapamycin (mTOR) signalling pathway by rapamycin largely mimicked the beneficial effects of fasting in promoting cold resistance, suggesting mTOR signalling may be involved in the fasting-induced cold resistance in fish. Our study demonstrates that fasting may be a protective strategy for fish to survive under cold stress. Abstract In cold environments, most homeothermic animals increase their food intake to supply more energy to maintain body temperature, whereas most poikilothermic animals such as fishes decrease or even stop feeding under cold stress. However, the physiological value of fasting during cold resistance in poikilotherms has not been explained. Here, we show that moderate fasting largely enhanced cold resistance in fish. By using pharmacological (fenofibrate, mildronate, chloroquine and rapamycin) and nutritional approaches (fatty acids diets and amino acids diets) in wild-type or specific gene knock-out zebrafish models (carnitine palmitoyltransferase-1b-deficient strain, CPT1b-/- , or autophagy-related protein 12-deficient strain, ATG12-/- ), we verified that fasting-stimulated lipid catabolism and autophagy played essential roles in the improved cold resistance. Moreover, suppression of the mechanistic target of rapamycin (mTOR) pathway by using rapamycin mostly mimicked the beneficial effects of fasting in promoting cold resistance as either the physiological phenotype or transcriptomic pattern. However, these beneficial effects were largely reduced when the mTOR pathway was activated through high dietary leucine supplementation. We conclude that fasting helps fish to resist cold stress by modulating lipid catabolism and autophagy, which correlates with the mTOR signalling pathway. Therefore, fasting can act as a protective strategy of fish in resisting coldness.

Background Due to the special anatomy morphology and physiological function of the mandible, it has always become a challenge to accurately reconstruct the mandibular defect in maxillofacial surgery. Digital three dimensions (3D) printing surgical guide, as the effective method for individual and accurate surgery, is a hotspot of clinical research at present. In this study, 3D printing PLA surgical guide plate was applied to reconstruct the mandibular defect with fibula flap, its clinical effect and accuracy were evaluated, which aimed to improve the accurate reconstruction of mandibular defects. Methods After sterilization, the dimension deformation of the PLA standard specimen were measured. Eighteen patients diagnose with mandibular tumor were collected as observation objects. Then partial mandible resection and simultaneous mandible reconstruction with fibula graft were implemented according to the computer-aided design plan. The clinical effects of 3D printing PLA guide plates application were evaluated by facial contours, occlusal stability and chewing function. Through registering the postoperative computed images reconstruction with preoperative designed shape, the reconstruction accuracy was evaluated by detecting the maximum difference including the distance between lateral convex point of the condyles, the distance between medial convex point of the condyles and the horizontal contained angle between long axis of the condyles. Results After high temperature steam sterilization, the curvature of the PLA specimen with 100% filling rate and 4.8 mm thickness were the smallest and their dimension deformation had no statistical significance (P>0.05). The minimally deformed 3D printing PLA guide plate were smoothly placed in the right place during the operation. After surgery, the face was symmetrical, the occlusal relationship was restored well and no deviation of the mandibular movement were found. The spiral computed tomography (SCT) scanning showed that the distance between lateral/medial convex points of the condyle and the horizontal contained angle were 128.34±8.68 mm, 88.69±6.75 mm and 145.87°±12.01°. Compared with preoperative design, the maximum deviation of the actual postoperative registration was 1.67±0.63, and the difference was not statistically significant (P>0.05). Conclusions The application of 3D printing PLA guide plate in the segmental section and reconstruction of the mandible can effectively simplify the operation, and better reconstruct the continuity of the mandible. The surgical accuracy can fully meet clinical needs with relatively low prices.

Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl-/-; AKO fish) or lysosomal acid lipase (lal-/-; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.

Background Diabetic cardiomyopathy (DCM) is a serious complication of diabetes that can lead to significant mortality. Cyclocarya paliurus is a tree, the leaves of which are often utilized to prevent and treat diabetes mellitus. Whether C. paliurus leaves can prevent or treat DCM, however, it remains to be formally assessed. The present study was therefore designed to assess the ability of C. paliurus to protect against DCM in db/db mice. Methods Male wild-type (WT) and db/db mice were administered C. paliurus ethanol leaf extracts (ECL) or appropriate vehicle controls daily via gavage, and levels of blood glucose in treated animals were assessed on a weekly basis. After a 10-week treatment, the levels of cardiac troponin I (cTn-I), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), aspartate transaminase (AST), total triglycerides (TG), and total cholesterol (TC) in serum were measured. Activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in heart tissues were detected. Hematoxylin-eosin (HE) and Masson staining were conducted. The protein expression that related with oxidative stress and inflammatory reaction was evaluated by Western blotting. Results Compared with WT mice, the TG, TC, and blood glucose levels in db/db mice increased significantly, which were reduced by ECL treatment. Compared with WT mice, the levels of LDH, CK-MB, AST, and cTn-I in serum and MDA in heart tissues of db/db mice increased significantly. Activities of SOD, GSH-Px, and CAT in heart tissues of db/db mice decreased significantly. The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in heart tissues of db/db mice increased remarkably. However, ECL treatment improved the above pathological changes significantly. ECL alleviated pathological injury and fibrosis in heart tissues of mice. Western blotting showed that ECL increased Bcl-2 level and decreased Bax, cle-caspase-3, and cle-caspase-9 expression. Furthermore, ECL inhibited NF-κB nuclear translocation and increased PI3K and p-Akt expressions. Conclusion Our results indicate that ECL treatment can markedly reduce pathological cardiac damage in db/db mice through antiapoptotic, antifibrotic, and anti-inflammatory mechanisms. Specifically, this extract was able to suppress NF-κB activation via the PI3K/Akt signaling pathway. Given its diverse activities and lack of significant side effects, ECL may thus have therapeutic value for the treatment of DCM.

L-carnitine is a functional aquafeed additive for enhancing lipid catabolism by elevating mitochondrial fatty acid β-oxidation and modulating energy metabolism to provide a “protein sparing effect”. However, results on the effects of dietary l -carnitine on nutrient metabolism in fish are still conflicting. We explored comprehensively the effects of dietary l -carnitine on energy metabolism in Nile tilapia. We fed Nile tilapia for eight weeks with diets supplemented with l -carnitine or not. We conducted metabolic tracking tests by intraperitoneally injecting individual fish with 14C-labeled palmitic acid (PA), glucose (Glu) and an amino acid mixture (AAs). After the feeding trial, insignificant growth-promoting effect of l -carnitine was obtained in treated fish. However, l -carnitine significantly reduced the lipid content in whole body and muscle accompanied by increasing the free carnitine concentration and fatty acid β-oxidation efficiency. Moreover, l -carnitine elevated concentrations of serum glucose, pyruvate and lactate, and increased glycogen and protein deposition in muscle. These results suggest that ingested glucose and protein prefer to be reserved in carnitine-fed fish with sufficient fatty acids oxidation for energy. Nevertheless, after a 14C-labeled single nutrient injection, carnitine-fed fish showed a higher oxidation rate of [1-14C]-PA, d -[1-14C]-Glu and l -[14C (U)]-AAs. Our study indicates that, the effects of l -carnitine on nutrient metabolism are correlated with the abundance of individual macronutrients such that an inadequate lipid supply would cause dietary l -carnitine supplementation to elevate higher breakdown of glucose and protein for energy generation. The present study provides new insights on the regulation mechanism of l -carnitine on nutrient metabolism in fish.

This study shows that the ability of gut microbiota members to enhance host energy harvest from a high-fat diet is a conserved feature of host-microbe interactions in fish, as in mammals. It also underscores that gut microbiota members are able to significantly impact host biology even when at low abundance., An efficient energy harvesting mechanism is likely critical for animals in their natural environment. Intestinal microbiota enriched by a high-fat diet aid in lipid accumulation, a strategy likely evolved for energy harvest in mammals. However, whether this strategy is conserved among vertebrate organisms remains unclear. A bacterial strain (S1), enriched on soybean oil rich medium, was isolated from the gut of Nile tilapia and demonstrated to be a member of the Citrobacter genus. Although a high-fat diet increased the number of Citrobacter spp., these bacteria were not abundant in the intestine by high-throughput sequencing. Addition of bacterium S1 to a high-fat diet modulated intestinal microbial composition and increased high-fat diet-induced lipid accumulation in mesenteric adipose tissue, accompanied by (i) increased triglyceride absorption efficiency and triglyceride reesterification and (ii) increased intestinal permeability. Collectively, our results provide evidence that specific intestinal bacteria aid the host in harvesting more energy from a high-fat diet in fish. Furthermore, the results from the present study also suggest that nondominant bacteria in the gut may play an important role in regulating host metabolism. IMPORTANCE This study shows that the ability of gut microbiota members to enhance host energy harvest from a high-fat diet is a conserved feature of host-microbe interactions in fish, as in mammals. It also underscores that gut microbiota members are able to significantly impact host biology even when at low abundance.

Potassium (K) is essential for plant growth and development. Here, we show that the KUP/HAK/KT K+ transporter KUP9 controls primary root growth in Arabidopsis thaliana. Under low-K+ conditions, kup9 mutants displayed a short-root phenotype that resulted from reduced numbers of root cells. KUP9 was highly expressed in roots and specifically expressed in quiescent center (QC) cells in root tips. The QC acts to maintain root meristem activity, and low-K+ conditions induced QC cell division in kup9 mutants, resulting in impaired root meristem activity. The short-root phenotype and enhanced QC cell division in kup9 mutants could be rescued by exogenous auxin treatment or by specifically increasing auxin levels in QC cells, suggesting that KUP9 affects auxin homeostasis in QC cells. Further studies showed that KUP9 mainly localized to the endoplasmic reticulum (ER), where it mediated K+ and auxin efflux from the ER lumen to the cytoplasm in QC cells under low-K+ conditions. These results demonstrate that KUP9 maintains Arabidopsis root meristem activity and root growth by regulating K+ and auxin homeostasis in response to low-K+ stress.

l-Carnitine is essential for mitochondrialβ-oxidation and has been used as a lipid-lowering feed additive in humans and farmed animals.d-Carnitine is an optical isomer ofl-carnitine anddl-carnitine has been widely used in animal feeds. However, the functional differences betweenl- andd-carnitine are difficult to study because of the endogenousl-carnitine background. In the present study, we developed a low-carnitine Nile tilapia model by treating fish with a carnitine synthesis inhibitor, and used this model to investigate the functional differences betweenl- andd-carnitine in nutrient metabolism in fish.l- ord-carnitine (0·4 g/kg diet) was fed to the low-carnitine tilapia for 6 weeks.l-Carnitine feeding increased the acyl-carnitine concentration from 3522 to 10 822 ng/g and alleviated the lipid deposition from 15·89 to 11·97 % in the liver of low-carnitine tilapia. However, as compared withl-carnitine group,d-carnitine feeding reduced the acyl-carnitine concentration from 10 822 to 5482 ng/g, and increased lipid deposition from 11·97 to 20·21 % and the mRNA expression of the genes involved inβ-oxidation and detoxification in the liver.d-Carnitine feeding also induced hepatic inflammation, oxidative stress and apoptosis. A metabolomic investigation further showed thatd-carnitine feeding increased glycolysis, protein metabolism and activity of the tricarboxylic acid cycle and oxidative phosphorylation. Thus,l-carnitine can be physiologically utilised in fish, whereasd-carnitine is metabolised as a xenobiotic and induces lipotoxicity.d-Carnitine-fed fish demonstrates increases in peroxisomalβ-oxidation, glycolysis and amino acid degradation to maintain energy homeostasis. Therefore,d-carnitine is not recommended for use in farmed animals.

Dietary usage of microencapsulated oils has been proved to promote growth and gut health in land animals, however, these beneficial effects have not been intensively investigated in aquatic animals. The present study hypothesized that dietary microencapsulated oils could alleviate negative effects caused by high-fat diet (HFD) in fish, thus we fed juvenile Nile tilapia with HFD (15% fat) by using normal or microencapsulated oils as lipid sources for 8 weeks. To compare the effects between different microencapsulated oils with different fatty acid composition, two oil mixtures (PL: 60% palm oil +40% linseed oil; FL: 60% fish oil +40% linseed oil) were used. After feeding trial, the growth, body composition, fatty acid composition, resistance to pathogenic bacteria challenge, activities of immune enzymes, expressions of inflammation genes, intestinal structure and microbiota were measured, respectively. The results indicated that microencapsulated oils didn’t affect fish growth, body composition, and muscle fatty acid composition, but improved the intestinal activities of lysozyme (LZM) and resistance against Aeromonas hydrophila. Further tests showed that microencapsulated oils not only improved the structure of intestine, but also increased the number of beneficial bacteria (Bacillus and Paenibacillus) and decreased the number of harmful bacteria (Pseudoalteromonas and Roseovarius) in the intestine. Those beneficial effects are more significant between normal PL oil and microencapsulated PL oil, but no significant difference in FL oil groups. Taken together, these results suggested that microencapsulated oil might enhance immune activities in the Nile tilapia fed with HFD by improving intestinal structure and microflora. Moreover, the beneficial effects of the microencapsulated oil are correlated to the oil composition. To the best of our knowledge, this is the first study illustrating the beneficial effects of dietary microencapsulated oil in the fish fed with HFD.

Purpose: Compared relative translucency of IPS e.max CAD,Vita Suprinity,Lava Zirconia,Cercon Zirconia,Zenostar and upcera Zirconia, to provide a reference for the clinical aesthetic choice. Method: Relative translucency were calculated from the luminous reflectance (Y) of the specimens with a black (Yb) and a white backing (Yw) to give Yb/Yw with Minolta CR-321.One-way analysis of variance and Tukey’s multiple comparison test were used to analyze the data (PVita Suprinity >Lava Zirconia >Cercon Zirconia >Zenostar >upcera Zirconia. Conclusion: There were significantly different between the six groups, for patients with anterior tooth areas restoration and high aesthetic requirements, IPS e.max CAD and Vita Suprinity are recommended; For patients with poor transmittance of adjacent teeth, and the abutments are dark which need to be covered can choose Lava Zirconia and Cercon Zirconia; For low transparency like posterior areas and severe tetracycline teeth, upcera Zirconia and Zenostar ceramic blocks may be considered.

Background: Antibiotics used globally to treat human and animal diseases exist ubiquitously in the environment at low doses because of misuse, overdose and poor absorption after ingestion, coupled with their high-water solubility and degradation resistance. However, the systemic chronic effects of exposure to low environmental concentrations of antibiotics (LECAs) and legal aquaculture doses of antibiotics (LADAs) in fish and their human health risk are currently unknown. Objective: To investigate the in vivo chronic effects of exposure to LECAs and LADAs using oxytetracycline (OTC) and sulfamethoxazole (SMZ) in Nile tilapia (Oreochromis niloticus) and their human health risk. Methods: Twenty O. niloticus weighing 27.73 ± 0.81 g were exposed to water containing LECAs (OTC at 420 ng/L and SMZ at 260 ng/L) and diets supplemented with LADAs (OTC 80 mg/kg/day and SMZ 100 mg/kg/day) for twelve weeks. General physiological functions, metabolic activities, intestinal and hepatic health were systemically evaluated. The possible human health risks of the consumption of the experimental Nile tilapia fillets in adults and children were assessed by using risk quotient. Results: After exposure, we observed retarded growth performance accompanied by reduced nutrients digestibility, feed efficiency, organ indices, and lipid body composition in treated fish. Antibiotics distorted intestinal morphological features subsequently induced microbiota dysbiosis and suppressed intestinal tight junction proteins. Exposure of fish to LECAs and LADAs induced oxidative stress, suppressed innate immunity, stimulated inflammatory and detoxification responses, concomitantly inhibited antioxidant capacity and caused lipid peroxidation in intestine and liver organs. Both LECAs and LADAs enhanced gluconeogenesis, inhibited lipogenesis and fatty acid beta oxidation in intestine and liver organs. The exposure of fish to LECAs and LADAs induced anaerobic glycolytic pathway and affected intestinal fat catabolism in intestine while halted aerobic glycolysis, increased hepatic fat catabolism, and induced DNA damage in liver. The hazard risk quotient in children for fish treated with OTCD was >1 indicating human health risk. Conclusion: Overall, both LECAs and LADAs impair general physiological functions, nutritional metabolism, and compromise fish immune system. Consumption of fish fed with legal OTC provokes health risk in children. Global stringent prohibition policy for use of antibiotics in aquaculture production and strategies to limit their release into the environment are urgently required to protect human health. Keywords: Low environmental antibiotics, Legal aquaculture antibiotics, Toxicological effects, Human health risk

Antibiotics have been widely used in human and veterinary medicine to both treat and prevent disease. Due to their high water solubility and low bioavailability, many antibiotic residues have been found in aquatic environments. Fish are an indispensable link between the environmental pollution and human health. However, the chronic effects of environmental concentrations of antibiotics in fish have not been thoroughly investigated. Sulfamethoxazole (SMX) and oxytetracycline (OTC) are frequently detected in aquatic environments. In this study, zebrafish were exposed to SMX (260 ng/L) and OTC (420 ng/L) for a six-week period. Results indicated that exposure to antibiotics did not influence weight gain of fish but increased the metabolic rate and caused higher mortality when treated fish were challenged with Aeromonas hydrophila. Furthermore, exposure to antibiotics in water resulted in a significant decrease in intestinal goblet cell numbers, alkaline phosphatase (AKP), acid phosphatase (ACP) activities, and the anti-oxidant response while there was a significant increase in expression of inflammatory factors. Antibiotic exposure also disturbed the intestinal microbiota in the OTC-exposed group. Our results indicated that environmental antibiotic concentrations can impair the gut health of zebrafish. The potential health risk of antibiotic residues in water should be evaluated in the future.

The Pacific white shrimp Litopenaeus vannamei is the most important crustacean species in aquaculture, accounting for 70% of the total shrimp production in the world. In the past decade, development of the shrimp industry has been greatly impeded by disease prevalence, poor growth in low salinity inland water, dietary use of plant ingredients, and antibiotic overuse. The animal gut is a vital organ, and most of the gut functions, such as the functions of immunity, health regulation and nutrient absorption, are achieved by bacterial metabolism in the gut. Therefore, understanding the role of the gut microbiota of L. vannamei is important for improving the performance and production of L. vannamei in aquaculture. This paper summarizes the recent advancements in research on the composition of the gut microbiota of L. vannamei, the factors that influence the microbiota, and nutritional manipulation of the gut microbiota. In addition, based on the review, current problems are raised and future studies ...

Long-term feeding of fish with a high-fat diet (HFD) causes excess fat deposition and an impairment of immune function. In the present study, we aimed to determine whether dietary tea polyphenols (TPs) would ameliorate the adverse effects of HFD-feeding in GIFT tilapia. Juvenile GIFT tilapias (5.4 ± 0.9 g) were raised in twelve 200-L tanks (three tanks per diet, 20 fish per tank) and fed a control diet (6% fat, 36% protein), an HFD (12% fat, 36% protein), or an HFD supplemented with 50 mg/kg or 200 mg/kg TP for 8 weeks. The fish were hand-fed 5% of their body weight per day in three feeds, and maintained at 28 ± 1 °C under a 14-h light/10-h dark cycle. The fish in each tank were bulk weighed and counted fortnightly, and the daily feed amount was adjusted accordingly. At the end of the trial, the cumulative survival rate was calculated, and the weight gain and feed conversion ratio were calculated according to the bulk weight of fish in each tank. Tissues were collected from nine fish per diet, their organs were weighed, and biochemical and molecular indices were subsequently measured. HFD-feeding significantly increased lipid deposition, reduced cumulative survival from 96% to 75%, reduced hepatic alkaline phosphate activity (AKP) and serum total antioxidant capacity (T-AOC); and reduced the hepatic expression of immunoglobulin M (IgM), transforming growth factor-beta (TGF-β) and glutathione-S-transferase (GST) genes versus the control diet. The addition of TPs at 50 or 200 mg/kg both ameliorated the HFD-induced increase in lipid droplets in the liver (50 mg/kg TP from 40.83% to 17.27%; 200 mg/kg TP to 25.33%), and increased the cumulative survival rate of the tilapia. The addition of 50 mg/kg TP had a marked effect increasing cumulative survival to 90%, and increasing the activities of serum acid phosphatase (ACP), T-AOC; and IgM, TGF-β, nuclear factor-κB (NF-κB), superoxide dismutase (SOD), and GST gene expression to the highest level of the HFD-fed groups. The 50 mg/kg TP-containing diet also significantly increased the hepatic expression of carnitine palmitoyltransferase 1 alpha (CPT1α) versus the control diet. In contrast, the tilapia fed an HFD supplemented with 200 mg/kg TPs had the lowest expression of adipose triglyceride lipase, hormone-sensitive lipase, CPT1α, fatty acid synthase and acetyl-CoA carboxylase alpha genes of any of the groups, which implies that the lower and higher levels of TP supplementation have differing effects on lipid metabolism. The 200 mg/kg supplement had lower cumulative survival rate (82%), and smaller effects on serum ACP and hepatic AKP activities than the 50 mg/kg dose, and had no significant effect on serum T-AOC or the expression of IgM, TGF-β, GST, or NF-κB genes in the tilapia. These results indicate that the beneficial effects of TPs on the lipid metabolism and health of fish fed an HFD are dose-related. Moreover, they are likely to be largely mediated through lipid catabolism.

Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SCAT) have different structures and metabolic functions and play different roles in the regulation of the mammal endocrine system. However, little is known about morphology and physiological and metabolic functions between VAT and SCAT in fish. We compared the morphological, physiological, and biochemical characteristics of VAT and SCAT in Nile tilapia and measured their functions in energy intake flux, lipolytic ability, and gene expression patterns. SCAT contained more large adipocytes and nonadipocytes than VAT in Nile tilapia. VAT had higher lipid content and was the primary site for lipid deposition. Conversely, SCAT had higher hormone-induced lipolytic activity. Furthermore, SCAT had a higher percentage of monounsaturated and lower polyunsaturated fatty acids than VAT. SCAT had higher mitochondrial DNA, gene expression for fatty acid β-oxidation, adipogenesis, and brown adipose tissue characteristics, but it also had a lower gene expression for inflammation and adipocyte differentiation than VAT. SCAT and VAT have different morphological structures, as well as physiological and metabolic functions in fish. VAT is the preferable lipid deposition tissue, whereas SCAT exhibits higher lipid catabolic activity than VAT. The physiological functions of SCAT in fish are commonly overlooked. The present study indicates that SCAT has specific metabolic characteristics that differ from VAT. The differences between VAT and SCAT should be considered in future metabolism studies using fish as models, either in biomedical or aquaculture studies.

Colonizing germ-free (GF) zebrafish with specific bacterial species provides the possibility of understanding the influence on host biological processes including gene expression, development, immunity, and behavioral responses. It also enlightens our understanding on the host-microbe interactions within the physiological context of a living host. However, the responses of GF zebrafish to various colonization conditions such as bacterial concentrations, colonization time points, and exposure duration remain unclear. To address this issue, we explored the responses of GF zebrafish by using two bacterial species at varying concentrations, colonization time points and exposure duration. Therefore, we mono-associated GF zebrafish with Escherichia coli DH5α or Bacillus subtilis WB800N at concentrations ranging from 102 to 107 CFU/ml either at 3 day post fertilization (dpf) or 5 dpf for 24 or 48 h. We evaluated the responses of GF zebrafish by analyzing the survival rate, colonization efficiency, nutrients metabolism, intestinal cell proliferation, innate immunity, stress, and behavior responses by comparing it to conventionally raised zebrafish (CONR) and GF zebrafish. The results indicated that the final bacteria concentrations ranging from 102 to 104 CFU/ml did not cause any mortality when GF mono-associated larvae were exposed to either E. coli DH5α or B. subtilis WB800N at 3 or 5 dpf, while concentrations ranging from 106 to 107 CFU/ml increased the mortality, particularly for 5 dpf owing to the decrease in dissolved oxygen level. The E. coli DH5α mainly induced the expression of genes related to nutrients metabolism, cell proliferation and immunity, while B. subtilis WB800N mainly upregulated the expression of genes related to immunity and stress responses. Moreover, our data revealed that GF zebrafish showed higher levels of physical activity than CONR and the microbial colonization reduced the hyperactivity of GF zebrafish, suggesting colonization of bacteria affected behavior characteristics. This study provides useful information on bacterial colonization of GF zebrafish and the interaction between the host and microbiota.

With the development of aquaculture industry, high-carbohydrate diet is used to stimulate protein-sparing effect and reduce feed cost. However, fish utilize carbohydrates poorly in general, and instead, high level of carbohydrates in the diet influence the growth condition of fish. How to alleviate the side effects of high carbohydrate diet on fish health has attracted more and more attentions. In the present study, Nile tilapia (Oreochromis niloticus) were fed with 25% and 45% of carbohydrate diet for eight weeks. Higher body weight but lower resistance to pathogen was found in 45% carbohydrate diet group. Higher expression level of inflammation cytokines, increased expression of total NF-κB protein and phosphorylated NF-κB protein (p-NF-κB) were detected in higher carbohydrate group. Concentration of short-chain fatty acids (SCFAs) was measured and the results indicated that high-carbohydrate diet decreased acetate content in the intestine. In order to detect the relationship between the decreased concentration of acetate and lower resistance to pathogen in high-carbohydrate group, 45% of carbohydrate diets (HC) supplemented with different concentrations of sodium acetate (HC + LA, 100 mmol/L; HC + MA, 200 mmol/L; HC + HA, 400 mmol/L) were used to raise Nile Tilapia for eight weeks. The results indicated that addition of 200 mmol/L sodium acetate (HC + MA) reduced the mortality when fish were challenged with Aeromonas hydrophila. Furthermore, we also found that addition of 200 mmol/L sodium acetate mainly inhibited p38 mitogen-activated protein kinase (p38MAPK) and NF-κB phosphorylation to decrease the expression level of inflammation cytokines (IL-8, IL-12, TNF-α and IL-1β) in the intestine. The present study indicated that certain concentration of sodium acetate could alleviate high-carbohydrate induced intestinal inflammation mainly by suppressing MAPK activation and NF-κB phosphorylation.

Autophagy is a conserved cellular degradation process through which intracellular components are degraded by the lysosome, but its roles in fish metabolism have not been studied in depth. Therefore, the present study aimed to investigate whether autophagy plays a key role in maintaining metabolic homeostasis in fish. In an 8-week feeding trial, Nile tilapia were fed either a control diet with medium fat and medium carbohydrate (Control), or a control diet supplemented with a classic autophagy inhibitor (chloroquine, CQ). CQ supplementation significantly inhibited autophagy and impaired fish growth and protein synthesis, and the glycolysis was stimulated, accompanied by fat accumulation, high oxidative stress and inflammation. Physiological status and gene expressions suggested that impaired autophagy might be at least one cause of the metabolic diseases which has been commonly seen in aquaculture. These results indicate that inhibition of autophagy could significantly affect the metabolism of lipid, carbohydrate and protein in fish; hence, autophagy could play important roles in maintaining homeostasis of nutrient metabolism in cultured fish.

Dietary contamination of aflatoxin is commonly detected in aquafeeds, and its adverse effects on growth and health of farmed fish have been frequently reported. However, the effects of dietary aflatoxin on flesh quality in fish has not been well demonstrated. To evaluate these effects, in the present study, 360 yellow catfish (25 g ± 0.05 g) were randomly divided into three groups and fed with the diets containing different concentration of aflatoxin B1(AFB1) (Control diet; AFB44 diet: 44 μg/kg; AFB234 diet: 234 μg/kg) for 8 weeks. The results showed that the dietary AFB1 exposure didn’t significantly change fish growth, organ weights, fatty acid and amino acid composition, and AFB1 residues in muscle. However, the dietary AFB1 exposure with 234 μg/kg significantly increased the total lipid and triglycerides (TG) contents in muscle, while decreased flesh contents of moisture, total protein and phospholipids (PL), and reduced myofiber diameter. Moreover, the reduced myofiber number and increased connective tissue ratio were observed in the both AFB groups. The expressions of the genes related to myofibers structural proteins were reduced with the increase of dietary AFB1 concentration. Taken together, the present results demonstrated that the 8-week dietary exposure of AFB1 with 44 or 234 μg/kg in yellow catfish would not inhibit growth or cause high AFB1 residues in flesh, but could reduce the protein content and PL/TG ratio in flesh, and significantly change the characteristics of myofibers, thereby impaired the nutritional value, flavor and sensory quality of fish flesh.

This study investigates the effect of arginine (Arg) on the growth, antioxidant capacity, inflammation and immunity of juvenile Chinese mitten crab Eriocheir sinensis under high pH stress. Three diets containing 23.7 g/kg (control) 31.4 g/kg and 40.1 g/kg Arg were formulated and fed to crabs (3.37 ± 0.01 g) at high pH (pH = 9.5) for eight weeks. The weight gain and specific growth rate significantly increased in crabs fed the 31.4 g/kg and 40.1 g/kg Arg diets (P < 0.05). Furthermore, Arg improved the antioxidant capacity by increasing the activities of superoxide dismutase, catalase and glutathione production in the hepatopancreas (P < 0.05). In addition, Arg significantly downregulated the inflammatory and apoptotic genes including LPS-induced TNF-α factor, p38 mitogen-activated protein kinase, a disintegrin and metalloprotease 17, Bcl-2-associated X and cysteine-aspartic acid protease 3 (P < 0.05). Arg significantly upregulated the immune genes including crustin, relish and peroxinectin (P < 0.05). The respiratory burst of crabs fed the control diet was significantly lower than those fed the 31.4 g/kg Arg and 40.1 g/kg Arg diets after LPS injection of 0 h, 6 h and 12 h (P < 0.05). This study indicates that dietary Arg can alleviate oxidative stress and inflammation and improve the immunity of juvenile Chinese mitten crab under high pH stress.

This study was to evaluate the relationship between myo-inositol synthesis and carbohydrate metabolism in Mozambique tilapia (Oreochromis mossambicus). Fish were immediately transferred from 0 psu (practical salinity units, the control) to 20 psu for 96 h with 60 fish in each group. Compared with the control group, the volume of the mitochondria-rich cell in the gills of O. mossambicus was significantly increased by acute hypersaline stress. The osmotic pressure and the contents of Na+, K+ and Cl− in the serum were significantly increased in the 20 psu group. Moreover, fish in the 20 psu group had significantly higher activities of glutathione peroxidase and superoxide dismutase and a higher malondialdehyde content than the control in the liver. Salinity stress significantly increased the serum glucose but decreased the liver glycogen. The activities of glucokinase and phosphofructokinase in the liver and gills were significantly higher in the 20 psu group, indicating that glycolysis could be significantly enhanced by salinity stress. The activities of glucose-6-phosphate synthase and phosphoenolpyruvate carboxykinase in the liver were significantly decreased by salinity stress. The expressions of myo-inositol-1-phosphate synthase and myo-inositol monophosphatase were significantly up-regulated in the gills and kidney. However, the expressions of these two genes were significantly down-regulated in the liver. The myo-inositol content in the gills, kidney and liver in the 20 psu group were significantly increased by salinity stress. This study indicates that salinity stress can impair the gill structure and reduce the antioxidant capacity of O. mossambicus. The changes in MI biosynthesis and carbohydrate metabolism could be an effective strategy to alleviate the adverse effect of salinity stress.

Because of the increasing market potential of Pacific white shrimp (Litopenaeus vannamei) and limited availability of coastal areas for production, culture of L. vannamei at low salinity is a growing trend throughout the world. Dietary manipulation could improve the growth performance of L. vannamei at low salinity. This study reared L. vannamei with glucose, sucrose and corn starch as dietary carbohydrate sources, respectively, at a low salinity. The results indicated that the sucrose and glucose feeding groups showed better growth performance. An Illumina-based sequencing method was used to examine the intestinal bacterial composition and the results indicated that Proteobacteria were the most prevalent members, but abundance of Actinobacteria decreased while Firmicutes increased in the corn starch-fed group. Furthermore, bacteria related to complex carbohydrate degradation were in lower abundance, whereas the abundance of opportunistic pathogenic bacteria increased in corn starch-fed group than the other two groups, suggesting that the diet imposes selective pressure on the intestinal microbiota. Complex carbohydrates were not the ideal energy sources for L. vannamei at low salinity because the host has higher energy demand in the stressful conditions while the complex carbohydrate degradation efficiency of the gut microbiota in L. vannamei is limited.

While a digestive tract is known to harbor complex assemblages of microbiota, maintaining a functional, stable microbial community is important to the host's health but can be constantly influenced by various environmental factors. Salinity can lead to stress, including hyposaline or hypersaline stress to aquatic animals, which influence the growth condition of the animals. However, the relationship among the salinity, intestinal microbiota and growth condition of the aquatic animals remains unclear. In this study, two euryhaline species, Oreochromis niloticus (Nile tilapia) and Litopenaeus vannamei (Pacific white shrimp) were reared in water with different salinity and the growth condition verified that Nile tilapia and Pacific white shrimp had reverse salinity preference. The gut microbiota of Nile tilapia and Pacific white shrimp were compared by using 16S rRNA gene Illumina sequencing. Analysis of more than 280,000 quality-filtered sequences obtained from guts of Nile tilapia and Pacific white shrimp reared in different salinities showed that both host phylogeny and water salinity influenced the intestinal microbiota composition. Redundancy analysis combined with the heat map analysis indicated that among the shared operational taxonomy units (OTUs) in both Nile tilapia and Pacific white shrimp, 14 OTUs increased while 10 OTUs decreased in abundance toward to the increase of salt concentration. The proportion of bacteria regarded as opportunists increased while those regarded as commensal or beneficial bacteria decreased when the host facing hyposaline or hypersaline stress. The alteration of intestinal microbiota was likely attributed to the environmental selection for microbes that could grow better under high or low salinity, the host response to the salinity stress and subsequent stress exerting on the gut microbiota, or both. Statement of relevance This manuscript studied the correlation between the intestinal microbiota and two euryhaline aquatic animals, Oreochromis niloticus (Nile tilapia) and Litopenaeus vannamei (Pacific white shrimp).

Suppressor of cytokine signaling (SOCS) proteins are inverse feedback regulators of cytokine and hormone signaling mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway that are involved in immunity, growth and development of organisms. In the present study, three SOCS genes, SOCS-1, SOCS-2 and SOCS-3, were identified in an economically important fish, Nile tilapia (Oreochromis niloticus) referred to as NtSOCS-1, NtSOCS-2 and NtSOCS-3. Multiple alignments showed that, the three SOCS molecules share highly conserved functional domains, including the SRC homology 2 (SH2) domain, the extended SH2 subdomain (ESS) and the SOCS box with others vertebrate counterparts. Phylogenetic analysis indicated that NtSOCS-1, 2 and 3 belong to the SOCS type II subfamily. Whereas NtSOCS-1 and 3 showed close evolutionary relationship with Perciformes, NtSOCS-2 was more related to Salmoniformes. Tissue specific expression results showed that, NtSOCS-1, 2 and 3 were constitutively expressed in all nine tissues examined. NtSOCS-1 and 3 were highly expressed in immune-related tissues, such as gills, foregut and head kidney. However, NtSOCS-2 was superlatively expressed in liver, brain and heart. In vivo, NtSOCS-1 and 3 mRNA levels were up-regulated after lipopolysaccharide (LPS) challenge while NtSOCS-2 was down-regulated. In vitro, LPS stimulation increased NtSOCS-3 mRNA expression, however it inhibited the transcription of NtSOCS-1 and 2. Collectively, our findings suggest that, the NtSOCS-1 and 3 might play significant role(s) in innate immune response, while NtSOCS-2 may be more involved in metabolic regulation.

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.

High energy diets (HED) have been widely used in farmed fish, but HED also caused some metabolic diseases. The rapamycin-induced inhibition of mechanistic target of rapamycin (mTOR) signalling has been verified to alleviate metabolic syndromes in mammals, however, the functions of rapamycin in regulating metabolism in the fish fed with HED has not been well investigated. In the present 8-week feeding trial, Nile tilapia were fed with a normal diet with medium fat and medium carbohydrate (MFMC), a high fat and high carbohydrate (HFHC) diet, or the HFHC diet supplemented with rapamycin (Rap). Compared to MFMC diet, the HFHC diet significantly increased phosphorylation of mTOR protein, lipid deposition in tissues, glycogen degradation, oxidative stress and inflammation, but decreased lipid catabolism capability. The supplementation of Rap in the HFHC diet inhibited phosphorylation of mTOR protein and protein synthesis, but elevated protein catabolism. Rap addition also increased catabolism of lipid and carbohydrate, reduced oxidative stress and inflammation, and promoted autophagic activities, meaning Rap addition could alleviate the most of the adverse effects caused by the HFHC diet. All these indicate that the elevated mTOR activity was highly correlated to the adverse effects of HFHC diet on physiological functions in fish, and the inhibition of mTOR activity could largely help to maintain normal physiological status in the HED-fed fish. Therefore, mTOR could be a promising regulatory target to alleviate the metabolic dysfunctions caused by HED in fish, however, the possible negative effects of TOR pathway regulation on protein retention and growth should also be carefully evaluated in practices.

Intestinal microbiota plays profound roles in host nutrition, physiology, and evolution. The development of DNA sequencing technologies has increased dramatically research on fish intestinal microbiota. However, most studies conducted so far have focused on the microbial structure and diversity. Studies targeting the exact function of commensal microbes in aquatic animals are still scarce, which limits the application of microbiota related knowledge in aquaculture. Gnotobiotic models (animals cultured in axenic conditions or with defined microbial lineages) are excellent tools for identifying the molecular interactions between intestinal microbiota and host, which drive studies of microbiota from correlation to causality in mammals. In recent decades, gnotobiotic fish models have been established and applied in aquaculture research. This review summarizes the colonization conditions in gnotobiotic zebrafish model and its application in understanding intestinal microbiota-host interactions in aquaculture. Furthermore, methods and research progress on other gnotobiotic models including freshwater and marine fishes, molluscs and crustaceans are also discussed. Application of gnotobiotic models in aquaculture has deepened our understanding of the relationship between the host and intestinal microbiota, which will facilitate the modulation of intestinal microbiota for production of healthy animals and sustainable development of aquaculture.

The requirements of essential fatty acids (EFAs) varies among fish species, however, the possible different cellular metabolism patterns of EFAs among fish species have not been well addressed. In the present study, we compared activities of cellular FA uptake, β-oxidation and esterification towards [1–14C]-labeled linoleic acid (LA, 18:2n-6) and linolenic acid (LNA, 18:3n-3) in the primary hepatocytes between grass carp (GCH) and Nile tilapia (NTH), which are two freshwater fish but have specific requirement of LNA or LA, respectively. The results showed that LNA had a higher FA-serum binding efficiency than LA in both GCH and NTH. GCH preferentially took up LNA, while NTH primarily took up LA. Both GCH and NTH had higher β-oxidation activity to LA than to LNA. LNA caused higher esterification than LA in GCH, but LA and LNA caused similar esterification effects in NTH. In GCH, LNA tended to induce higher expressions of the genes related to lipid transportation and lipogenesis, but lowered expressions of the lipid catabolism-related genes. However, the regulation of LNA or LA on gene expressions in NTH was marginal. Taken together, GCH had specific preference to take up and esterify LNA, while NTH preferentially took up LA. These differences are in accordance with the specific EFA requirements of grass carp and Nile tilapia. Our study provides new information for understanding the relationships between the specific EFA requirements and cellular metabolism of EFAs in fish.

Dietary α-lipoic acid (LA), β-glucan (Gluc) and l-carnitine (L-Ca) are commonly used additives to promote fish growth and stress resistance in aquaculture production. However their mechanisms and efficiencies in helping fish to resist diseases have not been compared before. In this study, we fed Nile tilapia (Oreochromis niloticus) with diets containing appropriate doses of LA, Gluc and L-Ca for five weeks and further intraperitoneally injected the fish with Aeromonas hydrophila. After dietary treatment, none of the additives affected the fish growth, but dietary Gluc and L-Ca reduced protein and lipid body contents in fish, respectively. After A. hydrophila challenge, all fish treated with the three dietary additives showed higher survival rate, but those fed on dietary L-Ca had lower survival than those fed on LA and Gluc diets, indicating high protection efficiency of LA and Gluc. The protective mechanisms of the three feed additives were quite different under A. hydrophila infection. Dietary LA induced higher total antioxidant capacity and higher mRNA expression of anti-oxidative genes than other additives in liver and also activated partly the immune function in serum and spleen. Gluc largely increased the immune function by activating the immunity enzymes in serum, inducing inflammation in liver and increasing the expression of immune genes in spleen and head kidney. Gluc also increased partly the antioxidant capacity in serum and liver and lipid catabolism in liver. L-Ca largely increased lipid catabolism in liver while it increased partly the antioxidant capacities in serum and liver. Taken together, these results indicate that, dietary LA, Gluc and L-Ca have various protective mechanisms and differ in their efficiencies on resisting A. hydrophila infection in Nile tilapia.

Leptin is known to inhibit appetite and promote energy metabolism in vertebrates. Leptin resistance (LR) commonly occurs in diet-induced obesity (DIO) in mammals. However, the roles of leptin in the energy homeostasis in DIO animals with LR remain unclear. Here we first verified the high expression of leptin in subcutaneous adipose tissue (SCAT) as in liver in Nile tilapia. Furthermore, we produced two types of DIO Nile tilapia by using a high-carbohydrate diet (HCD) or a high-fat diet (HFD), and confirmed the existence of LR in both models. Notably, we found that HCD-DIO fish retained leptin action in the activation of lipid metabolism and showed LR in glucose metabolism regulation, while this selective leptin action between lipid and glucose metabolism was reversed in HFD-DIO fish. Fasting the fish for 1 week completely recovered leptin actions in the regulation of lipid and glucose metabolism. Therefore, leptin may retain more of its activities in animals with LR than previously believed. Evolutionally, this selective regulation of leptin in nutrients metabolism could be an adaptive mechanism in animals to store surplus calories when different types of food are abundant.

Understanding how intestinal microbiota alters energy homeostasis and lipid metabolism is a critical process in energy balance and health. However, the exact role of intestinal microbiota in the regulation of lipid metabolism in fish remains unclear. Here, we used two zebrafish models (germ-free and antibiotics-treated zebrafish) to identify the role of intestinal microbiota in lipid metabolism. Conventional and germ-free zebrafish larvae were fed with egg yolk. Transmission electron microscopy was used to detect the presence of lipid droplets in the intestinal epithelium. The results showed that, microbiota increased lipid accumulation in the intestinal epithelium. The mRNA sequencing technology was used to assess genes expression level. We found majority of the differentially expressed genes were related to lipid metabolism. Due to the limitation of germ-free zebrafish larvae, antibiotics-treated zebrafish were also used to identify the relationship between the gut microbiota and the host lipid metabolism. Oil-red staining showed antibiotics-treated zebrafish had less intestinal lipid accumulation than control group. The mRNA expression of genes related to lipid metabolism in liver and intestine was also quantified by using real-time PCR. The results indicated that apoa4, hsl, cox15, slc2a1a, and lss were more related to intestinal bacteria in fish, while the influence of intestinal microbiota on the activity of fabp6, acsl5, cd36, and gpat2 was different between the liver and intestine. This study identified several genes regulated by intestinal microbiota. Furthermore, the advantages and disadvantages of each model have been discussed. This study provides valuable information for exploring host-microbiota interactions in zebrafish in future.

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor (0.05% body weight (BW) /d) for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations but not in muscle carnitine concentrations. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies.

Lipophagy degrades lipid droplets (LDs) through the lysosomal degradative pathway, thus plays important roles in regulating lipid metabolism in mammals. However, information on the existence and functions of lipophagy in fish lipid metabolism is still limited. In the present study, we confirmed the existence of lipophagy by observing the structures of LDs sequestered in autophagic vacuoles in the zebrafish liver cell line (ZFL) via electronic microscopy. Moreover, starved cells increased the mRNA expression of the microtubule-associated protein 1A/1B light chain 3 beta (LC3), which is a marker protein for autophagy and protein conversion from LC3-I to LC3-II. Inhibiting autophagy with chloroquine increased significantly the LDs content and decreased fatty acid β-oxidation and esterification activities in the ZFL cells cultured in the fed state. Furthermore, inhibiting autophagy function downregulated the mRNA expression of the genes and their proteins related to lipid metabolism. Altogether, the present study verified the existence of lipophagy and its essential regulatory roles in lipid metabolism in fish cells.

The use of antibiotics for anti-infection and growth promotion has caused the overuse of antibiotics in aquaculture. However, the benefit or risk of the long-term use of antibiotics on fish growth or health has not been fully addressed. In the present study, zebrafish were fed with sulfamethoxazole (SMX) or oxytetracycline (OTC) at the therapeutic concentrations (100 and 80 mg/kg body weight per day, respectively) for 6 weeks to mimic the long-term use of antibiotics. The digestive enzyme activities were higher in both antibiotic treatments, and higher oxygen consumption rate was found in OTC treated group. As a result, SMX increased the weight gain of zebrafish, and OTC treatment did not show significant prompting effect on growth. The mortality was higher in SMX or OTC treated group on 2nd-4th day after exposure to Aeromonas hydrophila. Lower alkaline phosphatase (AKP) and acid phosphatase (ACP) activities were found in OTC treated group, while higher malondialdehyde (MDA) content was found in the intestine of both SMX and OTC treated zebrafish. Furthermore, feeding OTC decreased the intestinal microbial richness. This study revealed that long-term use of legal aquaculture concentrations of antibiotics caused systemic adverse effects on fish gut health; stringent policy for use of antibiotics in fish is urgent.

Osmotic stress is a major form of abiotic stress that adversely affects growth and development of plants and subsequently reduces yield and quality of crops. In this study, the effect of nitric oxide (NO) and calcium (Ca2+) on the process of adventitious rooting in cucumber (Cucumis sativus L.) under simulated osmotic stress was investigated. The results revealed that the effect of exogenous NO and Ca2+ in promoting the development of adventitious roots in cucumber seedlings under simulated osmotic stress was dose-dependent, with a maximal biological response at 10 μM NO donor nitroprusside (SNP) or 200 μM Ca2+. The application of Ca2+ chelators or channel inhibitors and calmodulin (CaM) antagonists significantly reversed NO-induced adventitious rooting, implying that endogenous Ca2+/CaM might be involved in NO-induced adventitious rooting under osmotic stress. Moreover, intracellular Ca amount was also increased by NO in cucumber hypocotyls during the development of adventitious roots under osmotic stress. This increase of endogenous Ca2+ was inhibited by NO specific scavenger 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), nitrate reductase inhibitors tungstate (Na2WO4) and sodium azide (NaN3). This gives an indication that Ca2+ might be a downstream signaling molecule in the adventitious root development by NO under osmotic condition. The results also show that NO or Ca2+ play a positive role in improving plant water status and photosynthetic system by increasing chlorophyll content and photochemical activity in leaves. Furthermore, NO and Ca2+ treatment might alleviate the negative effects of osmotic stress by decreasing membrane damage and reactive oxygen species (ROS) production by enhancing the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Therefore, Ca2+/CaM may act as a downstream signaling molecule in NO-induced development of adventitious root under simulated osmotic stress through improving the photosynthetic performance of leaves and activating antioxidative system in plants.

Peroxisome proliferation activated receptor α (PPARα) is an important transcriptional regulator of lipid metabolism and is activated by high-fat diet (HFD) and fibrates in mammals. However, whether nutritional background affects PPARα activation and the hypolipidemic effects of PPARα ligands have not been investigated in fish. In the present two-phase study of Nile tilapia (Oreochromis niloticus), fish were first fed a HFD (13% fat) or low-fat diet (LFD; 1% fat) diet for 10 weeks, and then fish from the first phase were fed the HFD or LFD supplemented with 200 mg/kg body weight fenofibrate for 4 weeks. The results indicated that the HFD did not activate PPARα or other lipid catabolism-related genes. Hepatic fatty acid β-oxidation increased significantly in the HFD and LFD groups after the fenofibrate treatment, when exogenous substrates were sufficiently provided. Only in the HFD group, fenofibrate significantly increased hepatic PPARα mRNA and protein expression, and decreased liver and plasma triglyceride concentrations. This is the first study to show that body fat deposition and dietary lipid content affects PPARα activation and the hypolipidemic effects of fenofibrate in fish, and this could be due to differences in substrate availability for lipid catabolism in fish fed with different diets.

Excess fat accumulation has been observed widely in farmed fish; therefore, efficient lipid-lowering factors have obtained high attention in the current fish nutrition studies. Dietary L-carnitine can increase fatty acid β-oxidation in mammals, but has produced contradictory results in different fish species. To date, the mechanisms of metabolic regulation of L-carnitine in fish have not been fully determined. The present study used zebrafish to investigate the systemic regulation of nutrient metabolism by dietary L-carnitine supplementation. L-carnitine significantly decreased the lipid content in liver and muscle, accompanied by increased concentrations of total and free carnitine in tissues. Meanwhile, L-carnitine enhanced mitochondrial β-oxidation activities and the expression of carnitine palmitoyltransferase 1 mRNA significantly, whereas it depressed the mRNA expression of adipogenesis-related genes. In addition, L-carnitine caused higher glycogen deposition in the fasting state, and increased and decreased the mRNA expressions of gluconeogenesis-related and glycolysis-related genes, respectively. L-carnitine also increased the hepatic expression of mTOR in the feeding state. Taken together, dietary L-carnitine supplementation decreased lipid deposition by increasing mitochondrial fatty acid β-oxidation, and is likely to promote protein synthesis. However, the L-carnitine-enhanced lipid catabolism would cause a decrease in glucose utilization. Therefore, L-carnitine has comprehensive effects on nutrient metabolism in fish.