Revealing the roles of egg darkness and nest similarity for a cryptic parasite egg versus host's cognition: an alternate coevolutionary trajectory.

As a text-book example of coevolution, the escalating interactions between egg mimicry by parasitic cuckoos and egg recognition by their hosts constitute a key battlefield for parasitism and anti-parasitism strategies. However, some parasite-host systems have deviated from this coevolutionary trajectory because some cuckoos do not lay mimetic eggs, while the hosts do not recognize them, even under the high costs of parasitism. The cryptic egg hypothesis was proposed to explain this puzzle, but the evidence to date is mixed and the relationship between the two components of egg crypticity, egg darkness (dim egg coloration) and nest similarity (similarity to host nest appearance), remains unknown. Here, we developed a 'field psychophysics' experimental design to dissect these components while controlling for undesired confounding factors. Our results clearly show that both egg darkness and nest similarity of cryptic eggs affect recognition by hosts, and egg darkness plays a more influential role than nest similarity. This study provides unambiguous evidence to resolve the puzzle of absent mimicry and recognition in cuckoo-host systems and explains why some cuckoo eggs were more likely to evolve dim coloration rather than similarity to host eggs or host nests.

Prediction Model for Severe Mycoplasma pneumoniae Pneumonia in Pediatric Patients by Admission Laboratory Indicators.

OBJECTIVE: The purpose of this study was to develop a model for predicting severe Mycoplasma pneumoniae pneumonia (SMPP) in pediatric patients with Mycoplasma pneumoniae pneumonia (MPP) on admission by laboratory indicators. METHODS: Pediatric patients with MPP from January 2019 to December 2020 in our hospital were enrolled in this study. SMPP was diagnosed according to guideline for diagnosis and treatment of community-acquired pneumonia in children (2019 version). Prediction model was developed according to the admission laboratory indicators. Receiver operating characteristic curve and Goodness-of-fit test were analyzed for the predictive value. RESULTS: A total of 233 MPP patients were included in the study, with 121 males and 112 females, aged 4.541 (1-14) years. Among them, 84 (36.1%, 95% CI 29.9-42.6%) pediatric patients were diagnosed as SMPP. Some admission laboratory indicators (immunoglobulins M (IgM), eosinophil proportion, eosinophil count, hemoglobin, erythrocyte sedimentation rate (ESR), total protein, albumin and prealbumin) were found statistically different (p < 0.05) between non-SMPP group and SMPP group. Logistic regress analysis showed IgM, eosinophil proportion, eosinophil count, ESR and prealbumin were independent risk factors for SMPP. According to these five admission laboratory indicators, the prediction model for SMPP in pediatric patients was developed. The area under curve of the prediction model was 0.777, and the goodness-of-fit test showed that the predicted SMPP incidence by the model was consistent with the actual incidence (χ2 = 244.51, p = 0.203). CONCLUSION: We developed a model for predicting SMPP in pediatric patients by admission laboratory indicators. This model has good discrimination and calibration, which provides a basis for the early identification SMPP on admission. However, this model should be validated by multicenter studies with large sample.

[Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress].

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Dietary iron deficiency impaired intestinal immune function of on-growing grass carp under the infection of Aeromonas hydrophila: Regulation of NF-κB and TOR signaling.

Iron is an important mineral element for fish. In this study, we investigated the influences of dietary iron deficiency on intestinal immune function as well as underlying signaling of on-growing grass carp (Ctenopharyngodon idella). Fish were fed with six graded level of dietary iron for sixty days, and a fourteen days' challenge test under infection of Aeromonas hydrophila thereafter. Results showed that compared with optimal iron level, iron deficiency increased enteritis morbidity, decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) concentrations and down-regulated mRNA levels of hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, Mucin2, ß-defensin-1, anti-inflammatory cytokines transforming growth factor ß1 (TGF-ß1), TGF-ß2, interleukin 4/13A (IL-4/13A), IL-4/13B, IL-10, IL-11 and IL-15, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated mRNA levels of pro-inflammatory cytokines IL-1ß, interferon γ2 (IFN-γ2), IL-8, IL-12p35, IL-12p40 and IL-17D, nuclear factor kappa B (NF-κB) p65, IκB kinases α (IKKα), IKKß and eIF4E-binding protein (4E-BP) in intestine of on-growing grass carp, indicating that iron deficiency impaired intestinal immune function of fish under infection of A. hydrophila. Besides, iron excess also increased enteritis morbidity and impaired immune function of fish under infection of A. hydrophila. In addition, the effect of ferrous fumarate on intestinal immune function of on-growing grass carp is more efficient than ferrous sulfate. Finally, based on ability against enteritis, LZ activities in mid intestine and distal intestine, we recommended adding 83.37, 86.71 and 85.39 mg iron/kg into diet, respectively.

Vitamin A deficiency impairs intestinal physical barrier function of fish.

The present study was the first to investigate the effects of dietary vitamin A (VA) on the intestinal physical barrier function associated with oxidation, antioxidant system, apoptosis and cell-cellular tight junction (TJ) in the proximal (PI), mid (MI) and distal (DI) intestines of young grass carp (Ctenopharyngodon idella). Fish were fed graded levels of dietary VA for 10 weeks, and then a challenge test using an injection of Aeromonas hydrophila was conducted for 14 days. Results indicated that dietary VA deficiency caused oxidative damage to fish intestine partly by the reduced non-enzymatic antioxidant components glutathione (GSH) and VA contents as well as reduced antioxidant enzyme activities [not including manganese superoxide dismutase (MnSOD)]. Further results observed that the decreased antioxidant enzyme activities by VA deficiency were partly related to the down-regulation of their corresponding mRNA levels which were regulated by the down-regulation of NF-E2-related factor 2 (Nrf2) mRNA levels and up-regulation of kelch-like-ECH-associated protein (Keap1a) (rather than Keap1b) mRNA levels in three intestinal segments of fish. Meanwhile, VA deficiency up-regulated the mRNA levels of the apoptosis signalling [caspase-3, caspase-8, caspase-9 (rather than caspase-7)] associated with the inhibition of the target of rapamycin (TOR) signalling pathway in three intestinal segments of fish. Additionally, VA deficiency decreased the mRNA levels of TJ complexes [claudin-b, claudin-c, claudin-3, claudin-12, claudin-15a, occludin and zonula occludens-1 (ZO-1) in the PI, MI and DI, as well as claudin-7 and claudin-11a in the MI and DI] linked to the up-regulation of myosin light chain kinase (MLCK) signalling. These results suggested that VA deficiency impaired structural integrity in three intestinal segments of fish. Meanwhile, excessive VA also showed similar negative effects on these indexes. Taken together, the current study firstly demonstrated that VA deficiency impaired physical barrier functions associated with impaired antioxidant capacity, aggravated cell apoptosis and disrupted TJ complexes in the PI, MI and DI, but different segments performed different actions in fish. Based on protecting fish against protein oxidation, the optimal VA levels for grass carp were estimated to be 2622 IU/kg diet.

Impairing of gill health through decreasing immune function and structural integrity of grass carp (Ctenopharyngodon idella) fed graded levels dietary lipids after challenged with Flavobacterium columnare.

The current study conducted to investigate the hypothesis that low or excess levels of lipids increased the gill rot morbidity through impairing the immune function and structural integrity in the gill of grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ±â€¯0.53 g were fed diets containing six graded levels of lipids at 0.59%, 2.14%, 3.60%, 5.02%, 6.66% and 8.01% diets for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that compared with optimal lipids supplementation (2.14%-8.01% lipids diets), low or excess levels of lipids impaired fish immune function through declining the activities of humoral compounds, down-regulated the mRNA levels of anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65) (not p52), IκB kinase α (IKKα) (not IKKß), IKKγ and eIF4E-binding protein (4EBP) in the gill of young grass carp. In addition, low or excess levels of lipids decreased young grass carp physical barrier function through down-regulating the mRNA levels of ZO-1 (rather than ZO-2b), Claudin b, c, 3, 12, 15a, 15b, 7b, 7a and Occludin through MAPKK 6/p38 MAPK/MLCK signaling molecules, decreasing antioxidant ability via Kelch-like ECH-associating protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2) signaling molecules, and down-regulating the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) in the gill of grass carp. Based on the quadratic regression analysis for the gill rot morbidity, C3 and MDA contents, the dietary lipids requirements for young grass carp have been estimated to be 5.60%, 6.01% and 4.58% diets.

Soybean glycinin decreased growth performance, impaired intestinal health, and amino acid absorption capacity of juvenile grass carp (Ctenopharyngodon idella).

The present study evaluated the influence of dietary soybean glycinin on growth performance, intestinal morphology, free intestinal amino acid (AA) content, and intestinal AA transporter (AAT) mRNA levels in juvenile grass carp (Ctenopharyngodon idella). Results were displayed as follows: (1) 8% dietary glycinin decreased growth performance, inhibited intestinal growth, and caused intestinal histology damage of grass carp; (2) dietary glycinin decreased the content of free neutral AAs including Val, Ser, Tyr, Ala, Pro, and Gln in all intestinal segments, and Thr, Ile, Leu, Phe, and Gly in the MI and DI while downregulated the mRNA levels of corresponding transporters including SLC38A2, SLC6A19b, and SLC6A14 in all intestinal segments, and SLC7A5, SLC7A8, and SLC1A5 in the MI and DI. Dietary glycinin decreased the content of free basic AAs including Arg in the MI and DI and His in all intestinal segments while downregulated cationic AAT SLC7A1 mRNA levels in the MI and DI. Dietary glycinin decreased the content of free acidic AAs including Glu in all intestinal segments and Asp in the MI and DI while decreased mRNA levels of corresponding transporters including SLC1A2a in all intestinal segments and SLC1A3 in the MI and DI; (3) the digestion trial showed that basic subunits of glycinin was hard to digest in the intestine of grass carp; (4) co-administration of glutamine with glycinin partially alleviated the negative effects. Overall, glycinin decreased intestinal AA absorption capacity partly contributed by decreased AATs' mRNA levels and the indigestibility of glycinin.

[Cloning and expression analysis of calmodulin gene from Pinellia ternate].

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.

Impaired intestinal immune barrier and physical barrier function by phosphorus deficiency: Regulation of TOR, NF-κB, MLCK, JNK and Nrf2 signalling in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

In aquaculture, the occurrence of enteritis has increased and dietary nutrition is considered as one of the major strategies to solve this problem. In the present study, we assume that dietary phosphorus might enhance intestinal immune barrier and physical barrier function to reduce the occurrence of enteritis in fish. To test this assumption, a total of 540 grass carp (Ctenopharyngodon idella) were investigated by feeding graded levels of available phosphorus (0.95-8.75 g/kg diet) and then infection with Aeromonas hydrophila. The results firstly showed that phosphorus deficiency decreased the ability to combat enteritis, which might be related to the impairment of intestinal immune barrier and physical barrier function. Compared with optimal phosphorus level, phosphorus deficiency decreased fish intestinal antimicrobial substances activities or contents and down-regulated antimicrobial peptides mRNA levels leading to the impairment of intestinal immune response. Phosphorus deficiency down-regulated fish intestinal anti-inflammatory cytokines mRNA levels and up-regulated the mRNA levels of pro-inflammatory cytokines [except IL-1ß and IL-12p35 in distal intestine (DI) and IL-12p40] causing aggravated of intestinal inflammatory responses, which might be related to the signalling molecules target of rapamycin and nuclear factor kappa B. In addition, phosphorus deficiency disturbed fish intestinal tight junction function and induced cell apoptosis as well as oxidative damage leading to impaired of fish intestinal physical barrier function, which might be partially associated with the signalling molecules myosin light chain kinase, c-Jun N-terminal protein kinase and NF-E2-related factor 2, respectively. Finally, based on the ability to combat enteritis, dietary available phosphorus requirement for grass carp (254.56-898.23 g) was estimated to be 4.68 g/kg diet.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling.

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ±â€¯0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKß, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.

Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study was conducted to investigate the effects of dietary threonine on the disease resistance, gill immune and physical barriers function of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juveniles were fed six iso-nitrogenous diets containing graded levels of threonine (3.99-21.66 g kg-1 diet) for 8 weeks, and then challenged with Flavobacterium columnare. Results showed that threonine deficiency (3.99 g kg-1 diet): (1) increased the gill rot morbidity after exposure to F. columnare; (2) attenuated the gill immune barrier function by decreasing antimicrobial substances production, up-regulating the mRNA levels of pro-inflammatory cytokines (except IL-12p40), and down-regulating the anti-inflammatory cytokines partly due to the modulation of NF-κB and TOR signaling. (3) disrupt the gill tight junction complexes by down-regulating TJs (claudin-3, -b, -c, 12, occludin, ZO-1 and ZO-2) and up-regulating TJs (claudin-7a, -7b) as well as related signaling molecule myosin light chain kinase mRNA levels (P < 0.05). (4) exacerbated the gill apoptosis by up-regulating cysteinyl aspartic acid-protease-3, 8, 9, c-Jun N-terminal kinases and mediating apoptosis related factors mRNA levels (P < 0.05); (5) exacerbated oxidative injury with increased reactive oxygen species, malondialdehyde and protein carbonyl contents (P < 0.05), decreased the antioxidant related enzymes activities and corresponding mRNA levels (except glutathione peroxidase-1b and glutathione-S-transferase-omega 2) as well as glutathione contents (P < 0.05) partly ascribe to the abridgement of NF-E2-related factor 2 signaling [Nrf2/Keap1a (not Keap1b)] in fish gill. Overall, threonine deficiency depressed the disease resistance, and impaired immune and physical barriers in fish gill. Finally, based on the gill rot morbidity and biochemical indices (immune indices LA activity and antioxidant indices MDA content), threonine requirements for juvenile grass carp (9.53-53.43 g) were estimated to be 15.32 g kg-1 diet (4.73 g 100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1 protein), 15.46 g kg-1 diet (4.77 g 100 g-1 protein), respectively.

Methionine hydroxy analogue supplementation modulates gill immunological and barrier health status of grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of methionine hydroxy analogue (MHA) on the physical barrier and immune defence in the gill of young grass carp (Ctenopharyngodon idella). A total 630 young grass carp with an average initial weight of 259.70 ±â€¯0.47 g were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. After feeding trial, 15 fish from each treatment were challenged with Flavobacterium columnare. Compared to the basal diet, optimal MHA improved cellular structure integrity of gill via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < .05). Simultaneously, optimal MHA supplementation improved cellular structure integrity of gill via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < .05). Besides, optimal MHA improved intercellular structure integrity of immune organs via up-regulating the mRNA levels of intercellular tight junctions-related genes, which might be owing to the down-regulation of myosin light chain kinase (MLCK) mRNA levels (P < .05). Summarily, MHA could improve the physical barrier of fish gill. In addition, optimal MHA supplementation increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin and ß-defensin, suggesting that MHA could enhance antimicrobial ability of fish gill. Meanwhile, optimal MHA supplementation enhanced the immune defence of gill via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase ß, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < .05). In conclusion, the positive effect of MHA on gill health is associated with the improvement of the defence against apoptosis, antioxidant status, tight junctions and immune defence of fish gill. Meanwhile, MHA was superior to DLM on improving the physical barrier of fish gill. For the direction to healthy breeding of young grass carp, the optimal MHA supplementation levels on the premise of 4.01 g/kg methionine basal were estimated by quadratic regression curve, such as 5.49, 6.17 and 6.02 g/kg diet bases on the defence against gill-rot, malondialdehyde content and LZ activity in the gill, respectively.

Low or excess levels of dietary cholesterol impaired immunity and aggravated inflammation response in young grass carp (Ctenopharyngodon idella).

The present study explored the effect of cholesterol on the immunity and inflammation response in the immune organs (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) fed graded levels of dietary cholesterol (0.041-1.526%) for 60 days and then infected with Aeromonas hydrophila for 14 days. The results showed that low levels of cholesterol (1) depressed the innate immune components [lysozyme (LZ), acid phosphatase (ACP), complements and antimicrobial peptides] and adaptive immune component [immunoglobulin M (IgM)], (2) up-regulated the mRNA levels of pro-inflammatory cytokines [interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p35, IL-12p40, IL-15, IL-17D, tumor necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2)], partly due to the activated nuclear factor kappa B (NF-κB) signalling, and (3) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-4/13B, IL-10, IL-11, transforming growth factor (TGF)-ß1 and TGF-ß2], partly due to the suppression of target of rapamycin (TOR) signalling in the immune organs of young grass carp. Interestingly, dietary cholesterol had no influences on the IκB kinase α (IKKα) and IL-4/13A mRNA levels in the head kidney, spleen and skin, the IL-1ß and IL-12p40 mRNA levels in the spleen and skin, or the ß-defensin-1 mRNA level in the skin of young grass carp. Additionally, low levels of cholesterol increased the skin haemorrhage and lesion morbidity. In summary, low levels of cholesterol impaired immunity by depressing the innate and adaptive immune components, and low levels of cholesterol aggravated the inflammation response via up-regulating the expression of pro-inflammatory cytokines as well as down-regulating the expression of anti-inflammatory cytokines partly through the modulation of NF-κB and TOR signalling in the immune organs of fish. Similar to the low level of cholesterol, the excess level of dietary cholesterol impaired immunity and aggravated inflammation response in the immune organs of fish. Finally, based on the percent weight gain (PWG), the ability against skin haemorrhage and lesions as well as the LZ activity in the head kidney and the ACP activity in the spleen, the optimal dietary cholesterol levels for young grass carp were estimated as 0.721, 0.826, 0.802 and 0.772% diet, respectively.

Dietary myo-inositol deficiency decreased intestinal immune function related to NF-κB and TOR signaling in the intestine of young grass carp (Ctenopharyngodon idella).

In this study, we investigated the effects of dietary myo-inositol on the intestinal immune barrier function and related signaling pathway in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (221.33 ±â€¯0.84 g) were fed six diets containing graded levels of myo-inositol (27.0, 137.9, 286.8, 438.6, 587.7 and 737.3 mg/kg) for 10 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila. The results indicated that compared with the optimal dietary myo-inositol level, myo-inositol deficiency (27.0 mg/kg diet): (1) decreased lysozyme (LZ) and acid phosphatase (ACP) activities, as well as complement 3 (C3), C4 and immunoglobulin M (IgM) contents in the proximal intestine (PI), middle intestine (MI) and distal intestine (DI) of young grass carp (P < 0.05). (2) down-regulated the mRNA levels of anti-microbial substance: liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, ß-defensin-1 and mucin2 in the PI, MI and DI of young grass carp (P < 0.05). (3) up-regulated pro-inflammatory cytokines [IL-1ß (not in DI), TNF-α and IL-8], nuclear factor kappa B P65 (not NF-κB P52), c-Rel, IκB kinaseα (IKKα), IKKß and IKKγ mRNA levels in the PI, MI and DI of young grass carp (P < 0.05); and down-regulated pro-inflammatory cytokines IL-15 (not in DI) and inhibitor of κBα (IκBα) mRNA levels (P < 0.05). (4) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-10 (not in DI), IL-11, IL-4/13B (not IL-4/13A), TGF-ß1 and TGF-ß2], target of rapamycin (TOR), eIF4E-binding proteins 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6k1) in the PI, MI and DI of young grass carp (P < 0.05). All data indicated that myo-inositol deficiency could decrease fish intestine immunity and cause inflammation under infection of A. hydrophila. Finally, the optimal dietary myo-inositol levels for the ACP and LZ activities in the DI were estimated to be 415.1 and 296.9 mg/kg diet, respectively.

Sodium butyrate enhanced physical barrier function referring to Nrf2, JNK and MLCK signaling pathways in the intestine of young grass carp (Ctenopharyngodon idella).

This study evaluated the effect of dietary sodium butyrate (SB) supplementation on the intestinal physical barrier function of young grass carp (Ctenopharyngodon idella). The fish were fed one powdery sodium butyrate (PSB) diet (1000.0 mg kg-1 diet) and five graded levels of microencapsulated sodium butyrate (MSB) diets: 0.0 (control), 500.0, 1000.0, 1500.0 and 2000.0 mg kg-1 diet for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila to explore the effect of SB supplementation on intestinal physical barrier function and the potential mechanisms in fish. The results showed that optimal SB supplementation: (1) down-regulated the cysteine-aspartic protease-2 (caspase-2), caspase-3 (rather than PI), caspase-7, caspase-8 (rather than PI), caspase-9, fatty acid synthetase ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell lymphoma 2 associated X protein (Bax) and c-Jun Nterminal protein kinase (JNK) mRNA levels, up-regulated the B-cell lymphoma protein-2 (Bcl-2) (rather than PI), inhibitor of apoptosis proteins (IAP) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in the intestine (P < 0.05), inhibited the intestinal cell apoptosis, maintained the intestine cell structure integrity; (2) increased NF-E2-related factor 2 (Nrf2) mRNA levels and nucleus protein levels, and down-regulated kelch-like-ECH-associated protein (Keap1b) (rather than Keap1a) mRNA levels in the intestine, up-regulated copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase 1a (GPx1a), GPx1b, GPx4a, GPx4b, glutathione S-transferases R (GSTR), GSTP1, GSTP2, GSTO1, GSTO2 and glutathione reductase (GR) mRNA levels in the intestine, increased the corresponding antioxidant enzymes activity (P < 0.05), thus enhancing the ability of scavenging free radicals and decreasing the reactive oxygen species (ROS) content, decreasing the lipid and protein peroxidation, as well as alleviating oxidative damage; (3) down-regulated the molecule myosin light-chain kinase (MLCK) mRNA levels in the intestine, and up-regulated the occludin, zonula occludens-1 (ZO-1), ZO-2, claudin-b, claudin-c, claudin-f, claudin-3c (rather than PI), claudin-7a, claudin-7b and claudin-11 mRNA levels, down-regulated claudin-12, claudin-15a and claudin-15b mRNA levels (P < 0.05), thus maintaining the structural integrity between cells. This study suggests that SB supplementation could improve fish intestinal physical barrier function. Furthermore, according to the positive effect, MSB was superior to PSB on improving intestinal physical barrier function of fish. Finally, based on protein carbonyl content in the PI, the optimal SB supplementation (MSB as SB source) for young grass carp was estimated to be 338.8 mg kg-1 diet.

[Mechanism exploration on nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract].

In order to study the mechanism of nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract, yeast extract（YE） was added to the culture medium. Then the changes of physiological and biochemical indexes of A. belladonna hairy roots after treatment with YE were detected. The results are as follows，the activity of key enzymes of nitrogen metabolism changed differently. Compared with the control group (CK), the activity of nitrate reductase (NR) and glutamine synthetase (GS) were significantly increased, while the activity of glutamate dehydrogenase (GDH) was not changed significantly. The content of nitrate nitrogen, ammonium nitrogen had a significant decrease,but the content of soluble protein, free amino acid, total nitrogen are significantly more than CK. Moreover, YE treatment led to the increase of the content precursor amino acids (ornithine and arginine) and precursor putrescine in secondary metabolic pathways of A. belladonna. The expression level of gene putrescine N-methyl transferase (pmt), tropinone reductase-I (trI) and hyoscyamine 6-ß-hydroxylase(h6h) all increased in a different rate caused by YE treatment, which eventually led to the increase of the yield of tropane alkaloids. The yield of hyoscyamine and scopolamine were 3.09 and 1.85 folds than that of CK after 16 days treatment time. The results indicated that YE can induce more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism, meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism.

[Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna].

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 µmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 µmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 µmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

[Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna].

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺4/NO⁻3) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺4/NO⁻3). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺4/NO⁻3) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.

Threonine deficiency decreased intestinal immunity and aggravated inflammation associated with NF-κB and target of rapamycin signalling pathways in juvenile grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

This study aimed to investigate the impacts of dietary threonine on intestinal immunity and inflammation in juvenile grass carp. Six iso-nitrogenous semi-purified diets containing graded levels of threonine (3·99-21·66 g threonine/kg) were formulated and fed to fishes for 8 weeks, and then challenged with Aeromonas hydrophila for 14 d. Results showed that, compared with optimum threonine supplementation, threonine deficiency (1) decreased the ability of fish against enteritis, intestinal lysozyme activities (except in the distal intestine), acid phosphatase activities, complement 3 (C3) and C4 contents and IgM contents (except in the proximal intestine (PI)), and it down-regulated the transcript abundances of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, IgZ, IgM and ß-defensin1 (except in the PI) (P<0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IL-17D mRNA levels partly related to NF-κB signalling; (3) could down-regulate intestinal anti-inflammatory cytokine transforming growth factor (TGF)-ß1, TGF-ß2, IL-4/13A (not IL-4/13B) and IL-10 mRNA levels partly by target of rapamycin signalling. Finally, on the basis of the specific growth rate, against the enteritis morbidity and IgM contents, the optimum threonine requirements were estimated to be 14·53 g threonine/kg diet (4·48 g threonine/100 g protein), 15.05 g threonine/kg diet (4·64 g threonine/100 g protein) and 15·17 g threonine/kg diet (4·68 g threonine/100 g protein), respectively.

Vitamin A deficiency suppresses fish immune function with differences in different intestinal segments: the role of transcriptional factor NF-κB and p38 mitogen-activated protein kinase signalling pathways.

The present study investigated the effects of dietary vitamin A on immune function in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp (Ctenopharyngodon idella). Fish were fed graded levels of dietary vitamin A for 10 weeks, and then a challenge test using an injection of Aeromonas hydrophila was conducted for 14 d. The results showed that, compared with the optimum vitamin A level, vitamin A deficiency significantly decreased fish growth performance, increased enteritis morbidity, decreased intestinal innate humoral immune response and aggravated intestinal inflammation. However, liver-expressed antimicrobial peptide 2A/B mRNA in the DI and IL-6, IL-17D, IL-10, transforming growth factor (TGF)-ß1 and TGF-ß2 mRNA in the PI were not affected by vitamin A levels. Meanwhile, vitamin A deficiency disturbed inflammatory cytokines in the PI, MI and DI, which might be partly linked to p38 mitogen-activated protein kinase (p38MAPK) signalling and NF-κB canonical signalling pathway (IκB kinase ß (IKKß), IKKγ, inhibitor of κBα, NF-κB p65 and c-Rel) rather than NF-κB non-canonical signalling pathway (NF-κB p52 and IKKα). However, the signalling molecules NF-κB p65 and p38MAPK did not participate in regulating cytokines in the PI. These results suggested that vitamin A deficiency decreased fish growth and impaired intestinal immune function, and that different immune responses in the PI, MI and DI were mediated partly by NF-κB canonical signalling and p38MAPK signalling pathways. On the basis of percentage of weight gain, to protect fish against enteritis morbidity and acid phosphatase activity, the optimum dietary vitamin A levels were estimated to be 0·664, 0·707 and 0·722 mg /kg, respectively.