High starch diets attenuate the immune function of Micropterus salmoides immune organs by modulating Keap1/Nrf2 and MAPK signaling pathways.

Based on their good physiological functions and physical properties, carbohydrates are widely used in fish feed. However, excessive use of carbohydrates such as starch in fish feed may reduce the immunity of the fish and cause a series of health problems. In order to more clearly clarify the effects of different starch levels in feed on the immune organs of Micropterus salmoides, this study took the immune organs as the entry point and explored it from several perspectives, including differences in enzyme activity in plasma, changes in gene expression in immune organs, and resistance to pathogenic bacteria. The results showed that (1) high starch feed activates inflammatory responses in the spleen and head kidney through the MAPK signaling pathway. This leads to a decrease in the number of lymphocytes and weakens the resistance to pathogens; (2) high starch diet affects the antioxidant capacity of the trunk kidney by regulating the Keap1/Nrf2 pathway; (3) There was a strong correlation between gene expression patterns in the head kidney and lysozyme content in plasma. This implies that the high starch diet may regulate lysozyme production by affecting gene expression in the head kidney and further affect immune function. This study helps to reveal the interaction between starch and the immune system and provide scientific basis for the development of reasonable dietary recommendations and disease prevention.

Transcriptome analysis of the immunomodulatory effects of Salvia miltiorrhiza polysaccharide on hemocyte immune response in Procambarus clarkii.

Natural plant polysaccharide as immune modulator is considered an effective strategy for healthy aquaculture to reduce medicine treatment. Salvia miltiorrhiza polysaccharides (SMP) had applications to regulate immune activity and enhance antioxidant in vertebrates, but the potential function has been rarely reported in crustaceans. In this study, the immunological effects of SMP on hemocytes of Procambarus clarkii were analyzed. Results showed that total superoxide dismutase (T-SOD), phenoloxidase (PO) activity and respiratory burst were up-regulated after SMP treatment. After high-throughput sequencing, 2170 differentially expressed genes (DEGs) including 1294 up-regulated and 876 down-regulated genes were identified. KEGG function enrichment analysis indicated that DEGs are involved in crustaceans cellular immune-related signaling pathways, including lysosome, phagosome and endocytosis. Transcriptome mining and qRT-PCR showed that SMP up-regulated humoral immunity factors gene expression. Diets supplemented with 0.8% SMP significantly up-regulated the total number of hemocytes (THC), T-SOD and PO activity, improved the survival of crayfish after Citrobacter freundii infection. This study suggested that SMP could improve the cellular and humoral immunity of P. clarkii. Furthermore, this finding supplied a molecular foundation for further comprehending the immunopotentiator effects of plant polysaccharides in crustaceans.

Identification, tissue distribution, and anorexigenic effect of amylin in Siberian sturgeon (Acipenser baeri).

Amylin is a 37-amino acid polypeptide that has been found to be involved in feeding regulation in some mammals, birds, and goldfish. We cloned amylin of Siberian sturgeon and detected its distribution pattern in 15 tissues. The expression levels in the periprandial period (pre-and post-feeding), the changes in the food intake, and the expression levels of related appetite factors after the intraperitoneal injection of amylin were detected. The expression of amylin was found to be the highest in the hypothalamus. Compared with 1 h pre-feeding, the expression levels of amylin in the hypothalamus and duodenum were increased significantly 1 h post-feeding. Compared with the control group (saline), intraperitoneal injection of 50 ng/g, 100 ng/g, and 200 ng/g of amylin significantly inhibited food intake at 1 h post injection, but not at 3 h and 6 h. The injection of 50 ng/g, 100 ng/g, and 200 ng/g amylin significantly inhibited the cumulative feed. After 1 h of 50 ng/g amylin injection, the levels of MC4R and somatostatin in the hypothalamus increased significantly, while the levels of amylin and NPY decreased significantly. The levels of CCK in the valvular intestine were increased significantly. Insulin in the duodenum was also increased significantly, but there was no significant change in ghrelin in the duodenum. These results show that amylin inhibits feeding in Siberian sturgeon by down-regulating the appetite-stimulating factor NPY and up-regulating the appetite-suppressing factors somatostatin, MC4R, CCK, and insulin. This study provides a theoretical basis for studying the feeding function and action mechanisms of amylin in Siberian sturgeon.

Transcriptome analysis and the effects of polyunsaturated fatty acids on the immune responses of the critically endangered angtze sturgeon (Acipenser dabryanus).

The poor understanding of nutrition needed has become a significant obstruction to artificial conservation of Yangtze sturgeon (Acipenser dabryanus) and the relationship between ployunsaturated fatty acid nutrition and the immune response of Yangtze sturgeon is remains unclear. To explore this relationship, the immune response was determined by the activities of serum immune-related enzymes and the transcriptome pattern in the spleen after feeding different fat source diets for 7 weeks. In addition, the gene expression pattern after a lipopolysaccharide (LPS) challenge was investigated in the presence of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Long-term feeding of the fish oil diets increased the serum immune-related enzyme activities, including lysozyme, acid phosphatase, and alkaline phosphatase of Yangtze sturgeon. More than 653,999 transcripts with an N50 length of 1047 bp were obtained and a final set of 280,408 unigenes was generated. After annotating the unigenes, 3549 genes were assigned to the immune system and 2839 were identified to participate in the response to the different fat sources. A transcriptome assay showed the fish oil diets moderately upregulated immune-related signaling pathways in the spleen of Yangtze sturgeon, including NLR signaling, platelet activation, Fc gamma R-mediated phagocytosis, Th17 cell differentiation, and Th1 and Th2 cell differentiation. The quantitative polymerase chain reaction (qPCR) results of candidate genes for these pathways showed similar results. The LPS challenge study revealed that DHA and EPA moderately upregulated the candidate immune-related genes and modulated excessive activation of the immune pathway by the pathogen. This study confirmed the immunomodulatory function of unsaturated fatty acids in Yangtze sturgeon. This research will provide a reference for the preparation of artificial diets for Yangtze sturgeon.

Hepatoprotective Activity of Vitamin E and Metallothionein in Cadmium-Induced Liver Injury in Ctenopharyngodon idellus.

As an environmental and industrial pollutant, cadmium (Cd) can cause a broad spectrum of toxicological effects. Multiple organs, especially the liver, are considerably affected by Cd in both humans and animals. We investigated the protective effects of metallothionein (MT) and vitamin E (VE) supplementation on Cd-induced apoptosis in the grass carp (Ctenopharyngodon idellus) liver. Grass carp were divided into four groups: the control group, Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and Cd + MT group. All fish were injected with CdCl2 on the first day and then VE, MT, and PBS were given 4 days postinjection, respectively. The results showed that Cd administration resulted in liver poisoning in grass carp, which was expressed as an increase in Cd contents, malondialdehyde (MDA) concentration, percentage of hepatocyte apoptosis, and apoptosis-related gene mRNA transcript expression. However, VE and MT treatments protected against Cd-induced hepatotoxicity in grass carp by decreasing Cd contents, lipid peroxidation, and histological damage and reducing the percentage of hepatocyte apoptosis by regulating related mRNA transcript expression. These data demonstrate that oxidative stress and activation of the caspase signaling cascade play a critical role in Cd-induced hepatotoxicity. However, VE and MT alleviate Cd-induced hepatotoxicity through their antioxidative and antiapoptotic effects, and MT has a more powerful effect than VE.

Effects of dietary vitamin E deficiency on systematic pathological changes and oxidative stress in fish.

The aim of this study was to investigate the effects of dietary vitamin E deficiency on systematic pathological changes and oxidative stress in fish. A total of 320 healthy common carp (Cyprinus carpio) were randomized into four groups; the control group was fed a basal diet supplemented with 100 IUkg-1 of vitamin E, while the three experimental groups were fed the same basal diet with reduced vitamin E content (0, 25, or 50 IUkg-1). Findings showed that fish in the experimental groups mainly presented with sekoke disease, exophthalmia, leprnorthsis, and ascites. Histopathological and ultrastructural changes comprised nutritional myopathy with muscle fiber denaturation and necrosis, and multi-tissue organ swelling, degeneration, and necrosis. Compared with the control group, RBC count, hemoglobin content, vitamin E concentration, and superoxide dismutase activity were significantly lower in all three experimental groups. However, malondialdehyde content was considerably higher in experimental groups than in the control group. However, there was no difference in glutathione peroxidase activity among groups. In conclusion, dietary vitamin E deficiency (<100 IUkg-1) can cause severe injury and, in particular, oxidative damage in common carp. The oxidative damage might be a main influence caused by vitamin E deficiency in fish. These findings reveal the complete systematic pathological effect of vitamin E deficiency in common carp, which may be applicable to other fish and animals.

Plant polysaccharides used as immunostimulants enhance innate immune response and disease resistance against Aeromonas hydrophila infection in fish.

Plant polysaccharides (PPS) are an important medicinal plant product, and play a major role in preventing and controlling infectious microbes in aquaculture. The present study investigated the effect of three PPS; Ficus carica polysaccharides (FCPS), Radix isatidis polysaccharides (RIPS), and Schisandra chinensis polysaccharides (SCPS), used as feed additives, on innate immune responses and disease resistance against Aeromonas hydrophila in crucian carp. Results show that crucian carp fed with these PPS showed significant (p < 0.05) enhancement of their innate immune response including leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, total protein level, complement C3, and superoxide dismutase activity compared with the control group. Their degree of influence on these immune parameters was in the order of FCPS > RIPS > SCPS, except for lysozyme activity (RIPS > FCPS > SCPS). In addition, fish cumulative mortalities in the three treatment groups were remarkably lower than in the control group (95%) when challenged with A. hydrophila, relative percent survivals were 57.9%, 47.4%, and 42.1% in FCPS, RIPS, and SCPS groups, respectively. These results suggest that FCPS, RIPS, and SCPS used as immunostimulants are capable of enhancing immune responses and disease resistance against A. hydrophila in crucian carp, and that FCPS was the most effective. The findings from this study will help accelerate research of this topic, and promote the application and development of immunostimulants, such as Chinese herbs, in aquaculture.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Molecular and physiological evidences for the role in appetite regulation of apelin and its receptor APJ in Ya-fish (Schizothorax prenanti).

Apelin is a recently discovered peptide produced by several tissues with diverse physiological actions mediated by its receptor APJ. In order to better understand the role of apelin in the regulation of appetite in fish, we cloned the cDNAs encoding apelin and APJ, and investigated their mRNA distributions in Ya-fish (Schizothorax prenanti) tissues. We also assessed the effects of different nutritional status on apelin and APJ mRNAs abundance. Apelin and APJ mRNAs were ubiquitously expressed in all tissues tested, relatively high expression levels were detected in the heart, spleen, hypothalamus and kidney. Short-term fasting significant increased APJ mRNA expression, but no significant difference between fasted fish and fed control on 5- and 7-day. Meanwhile, apelin mRNA expression consistently increased during the 7-day food deprivation. In order to further characterize apelin in fish, we performed intraperitoneal (i.p.) injection of apelin-13 and examined food intake of the injected fish. Apelin injected at a dose of 100 ng/g body weight induced a significant increase in food intake compared to saline injected fish. Our results suggest that apelin acts as an orexigenic factor in Ya-fish. Their widespread distributions also suggest that apelin and APJ might play multiple physiological regulating roles in fish.

Molecular characterization and tissue expression of peptide YY in Schizothorax prenanti: effects of periprandial changes and fasting on expression in the hypothalamus.

Peptide YY (PYY) is a potent anorectic neuropeptide implicated in feeding regulation in mammals. However, the involvement of PYY in the feeding behavior of teleosts has not been well understood. In this study, we employed molecular, real-time quantitative PCR and physiological studies to characterize the structure, distribution, and appetite regulatory effects of PYY in Schizothorax prenanti (S. prenanti). A very high conservation in PYY sequences was found in teleosts. PYY is widely expressed, with the highest levels of expression in telencephalon, medulla oblongata, pituitary and hypothalamus of S. prenanti. The PYY mRNA expression in the hypothalamus was highly elevated after a meal, suggesting a satiety signal role for PYY in S. prenanti. In addition, PYY gene expression in the hypothalamus was decreased after fasting and increased sharply after refeeding, which suggested that PYY might be involved in the central regulation of appetite in S. prenanti. Overall, our result provides basis for further investigation into the regulation of feeding in S. prenanti.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.

Schizothorax davidi ghrelin: cDNA cloning, tissue distribution and indication for its stimulatory character in food intake.

Ghrelin is a gut/brain hormone with a unique acyl modification and various biological functions in fish and mammals. The objectives of this project were to identify ghrelin gene organization, study tissue specific ghrelin mRNA expression and investigate the short- (0, 0.5, 1.5, 3, 6, 9, 12h post-fasting) and long- (1, 3, 5, 7 days) term fasting as well as refeeding after a 7 day fasting induced changes in the expression of ghrelin mRNA in Schizothorax davidi. Our reverse transcription polymerase chain reaction analysis confirmed the predicted ghrelin sequence available in the GenBank and identified ghrelin mRNA expression in several tissues including the gut, liver, brain, heart, spleen, head kidney, gill and muscle. Quantitative PCR studies indicated that the expression level of ghrelin mRNA presented ascendant trend in short-term fasting group compared to the fed group, but it did not reach the significant level on statistics, while there is a significant increase in ghrelin mRNA expression in the gut of Schizothorax davidi fasted for 3, 5 and 7 days when compared to the expression in ad libitum fed fish. Refeeding after a 7 day fasting caused a significant and dramatic decrease in ghrelin mRNA expression in the gut of Schizothorax davidi. An increase in the expression of ghrelin mRNA during fasting, and its decrease following refeeding suggests an orexigenic role for ghrelin in Schizothorax davidi. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin during evolution.