Low phosphorus increases hepatic lipid deposition, oxidative stress and inflammatory response via Acetyl-CoA carboxylase-dependent manner in zebrafish liver cells.

Acetyl-CoA carboxylase (ACC) plays a regulatory role in both fatty acid synthesis and oxidation, controlling the process of lipid deposition in the liver. Given that existing studies have shown a close relationship between low phosphorus (P) and hepatic lipid deposition, this study was conducted to investigate whether ACC plays a crucial role in this relationship. Zebrafish liver cell line (ZFL) was incubated under low P medium (LP, P concentration: 0.77 mg/L) or adequate P medium (AP, P concentration: 35 mg/L) for 240 h. The results showed that, compared with AP-treated cells, LP-treated cells displayed elevated lipid accumulation, and reduced fatty acid ß-oxidation, ATP content, and mitochondrial mass. Furthermore, transcriptomics analysis revealed that LP-treated cells significantly increased lipid synthesis (Acetyl-CoA carboxylases (acc), Stearyl coenzyme A dehydrogenase (scd)) but decreased fatty acid ß-oxidation (Carnitine palmitoyltransferase I (cptI)) and (AMP-activated protein kinase (ampk)) mRNA levels compared to AP-treated cells. The phosphorylation of AMPK and ACC, and the protein expression of CPTI were significantly decreased in LP-treated cells compared with those in AP-treated cells. After 240 h of LP treatment, PF-05175157 (an ACC inhibitor) was supplemented in the LP treatment for an additional 12 h. PF-05175157-treated cells showed higher phosphorylation of ACC, higher protein expression of CPTI, and lower protein expression of FASN, lower TG content, enhanced fatty acid ß-oxidation, increased ATP content, and mitochondrial mass compared with LP-treated cells. PF-05175157 also relieved the LP-induced oxidative stress and inflammatory response. Overall, these findings suggest that ACC is a promising target for treating LP-induced elevation of lipid deposition in ZFL, and can alleviate oxidative stress and inflammatory response.

Combination of mineral protection and molecular characteristics rather than alone to govern soil organic carbon stability in Qinghai-Tibetan plateau wetlands.

Wetlands in the Yarlung Tsangpo River Basin (YTR) on the Qinghai-Tibet Plateau provide immense soil organic carbon (SOC) storage, which is highly susceptible to climate warming and requires urgent deciphering SOC stabilization mechanisms of long-term protection of SOC against decomposition. Conflicting views exist regarding whether persistent SOC is controlled by molecular features or by mineral protection. As such, this study quantified SOC stability using two thermal indices (TG-T50, and DSC), described molecular features of SOC using pyrolysis-gas chromatography-mass spectrometry, and measured SOC protection by minerals using a chemical extraction method. Results indicated SOC of topsoils had higher thermal stability, with TG-T50 and DSC-T50 of 337.61 °C and 384.58 °C, than that of subsoils with TG-T50 and DSC-T50 of 337.32 and 382.67 °C, respectively. We found subsoils had significantly higher proportions of aliphatic and aromatic compounds, while existed higher SOC associated with minerals. It seemed SOC stabilization differed with soil depths, in which mineral protection dictated SOC thermal stability in topsoils while molecular features posed a more important constraint on SOC stabilization in subsoils. Overall, our findings support the hypothesis of physical and chemical protection but emphasized that SOC thermal stability largely depended on to extent of the combination between molecular features and mineral protection, which explained 55% in topsoils and 73% in subsoils, respectively.

High water temperature raised the requirements of methionine for spotted seabass (Lateolabrax maculatus).

This study evaluated the effects of dietary methionine level and rearing water temperature on growth, antioxidant capacity, methionine metabolism, and hepatocyte autophagy in spotted seabass (Lateolabrax maculatus). A factorial design was used with six methionine levels [0.64, 0.85, 1.11, 1.33, 1.58, and 1.76%] and two temperatures [moderate temperature (MT): 27 ℃, and high temperature (HT): 33 ℃]. The results revealed the significant effects of both dietary methionine level and water temperature on weight gain (WG) and feed efficiency (FE), and their interaction effect was found on WG (P < 0.05). In both water temperatures tested, fish WG increased with increasing methionine level up to 1.11% and decreased thereafter. The groups of fish reared at MT exhibited dramatically higher WG and FE than those kept at HT while an opposite trend was observed for feed intake. Liver antioxidant indices including reduced glutathione and malondialdehyde (MDA) concentrations, and catalase and superoxide dismutase (SOD) activities remarkably increased in the HT group compared to the MT group. Moreover, the lowest MDA concentration and the highest SOD activity were recorded at methionine levels between 1.11% and 0.85%, respectively, regardless of water temperatures. Expression of methionine metabolism-related key enzyme genes (mat2b, cbs, ms, and bhmt) in the liver was increased at moderate methionine levels, and higher expression levels were detected at MT compared to HT with the exception of ms gene relative expression. Relative expression of hepatocyte autophagy-related genes (pink1, atg5, mul1, foxo3) and hsp70 was upregulated by increasing methionine level up to a certain level and decreased thereafter and increasing water temperature led to significantly enhanced expression of hsp70. In summary, HT induced heat stress and reduced fish growth, and an appropriate dietary methionine level improved the antioxidant capacity and stress resistance of fish. A second-order polynomial regression analysis based on the WG suggested that the optimal dietary methionine level for maximum growth of spotted seabass is 1.22% of the diet at 27 ℃ and 1.26% of the diet at 33 ℃, then 1.37 g and 1.68 g dietary methionine intake is required for 100 g weight gain at 27 ℃ or 33 ℃, respectively.

Molecular Characterization of PGC-1ß (PPAR Gamma Coactivator 1ß) and its Roles in Mitochondrial Biogenesis in Blunt Snout Bream (Megalobrama amblycephala).

This study aimed at achieving the molecular characterization of peroxisome proliferator-activated receptor-gamma coactivator 1ß (PGC-1ß) and exploring its modulatory roles in mitochondria biogenesis in blunt snout bream (Megalobrama amblycephala). A full-length cDNA of PGC-1ß was cloned from liver which covered 3110 bp encoding 859 amino acids. The conserved motifs of PGC-1ß family proteins were gained by MEME software, and the phylogenetic analyses showed motif loss and rearrangement of PGC-1ß in fish. The function of PGC-1ß was evaluated through overexpression and knockdown of PGC-1ß in primary hepatocytes of blunt snout bream. We observed overexpression of PGC-1ß along with enhanced mitochondrial transcription factor A (TFAM) expression and mtDNA copies in hepatocytes, and its knockdown led to slightly reduced NRF1 expression. However, knockdown of PGC-1ß did not significantly influence TFAM expression or mtDNA copies. The alterations in mitochondria biogenesis were assessed following high-fat intake, and the results showed that it induces downregulation of PGC-1ß. Furthermore, significant decreases in mitochondrial respiratory chain activities and mitochondria biogenesis were observed by high-fat intake. Our findings demonstrated that overexpression of PGC-1ß induces the enhancement of TFAM expression and mtDNA amount but not NRF-1. Therefore, it could be concluded that PGC-1ß is involved in mitochondrial biogenesis in blunt snout bream but not through PGC-1ß/NRF-1 pathway.

Molecular cloning, expression HSP70 and its response to bacterial challenge and heat stress in Microptenus salmoides.

The gene encoding HSP70 was isolated from Microptenus salmoides by homologous cloning and rapid amplification of cDNA ends (RACE). The HSP70 transcripts were 2116 bp long and contained 1953 open reading frames encoding proteins of 650 amino acids with a molecular mass of 71.2 kDa and theoretical isoelectric point of 5.22. The qRT-PCR analysis showed that the HSP70 gene was differentially expressed in various tissues under normal conditions, and the highest HSP70 level was observed in the spleen and the lowest levels in the muscle and heart. The clear time-dependent expression level of HSP70 was observed after bacterial challenge and heat stress. A significant increase in HSP70 expression level was detected and reached a maximum at 3 h and 6 h in liver, spleens and gill tissues after Aeromonas hydrophila infection and heat stress, respectively (P < 0.05). As time progressed, the expression of HSP70 transcript was downregulated and mostly dropped back to the original level at 48 h. The concentration of cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased as the time of stress progressed, with the highest level found on 3 h and later declined rapidly and reached to the control levels at the 48 h. Those results suggested that HSP70 was involved in the immune response to bacterial challenge and heat stress. The cloning and expression analysis of the HSP70 provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides.

Molecular characterization of p38 MAPK from blunt snout bream (Megalobrama amblycephala) and its expression after ammonia stress, and lipopolysaccharide and bacterial challenge.

p38 mitogen-activated protein kinase (MAPK) is an important protein which plays a key role in regulating the innate immunity, so exploring its molecular characterization is helpful in understanding the resistance against microbial infections in cultured fish. Here, a full-length cDNA of p38 MAPK was cloned from liver of blunt snout bream (Megalobrama amblycephala) which covered 2419 bp with an open reading frame of 1086 bp encoding 361 amino acids. p38 MAPK contained the characteristic structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW), which are conserved in MAPK family. To investigate p38 MAPK functions, two in vivo experiments were carried out to examine its expression following ammonia exposure and bacterial challenge. Also, an in vitro experiment was conducted to assess the role of p38 MAPK in inflammation of primary hepatocytes induced by lipopolysaccharide (LPS). The results showed the ubiquitous expression of p38 MAPK in all the tested tissues with varying levels. p38 MAPK mRNA expression was significantly up-regulated by ammonia stress and Aeromonas hydrophila challenge, and altered in a time-dependent manner. Moreover, the results indicated that the inflammatory response induced by LPS in hepatocytes is p38 MAPK dependent as knockdown of p38 MAPK using siRNA technology depressed the expression of IL-1ß and IL-6. The findings in this study showed that p38 MAPK has anti-stress property, and plays key role in protection against bacterial infection and inflammation in blunt snout bream.

Replacement of fish meal with Bacillus pumillus SE5 and Pseudozyma aphidis ZR1 fermented soybean meal in diets for Japanese seabass (Lateolabrax japonicus).

This study examined the effects of replacing fish meal (FM) with three different types of soybean meal (SM) including untreated SM, Bacillus pumillus SE5 (BP) fermented SM (BPFSM) and Pseudozyma aphidis ZR1 (PA) fermented SM (PAFSM) in diets for Japanese seabass (Lateolabrax japonicus). A basal diet was formulated using FM (FM diet), and six other diets were produced by substituting 40 or 80% of FM with SM, BPFSM or PAFSM (SM40, SM80, BPFSM40, BPFSM80, PAFSM40 and PAFSM80 diets). Each diet was fed to triplicate groups of fish (7.14 ±â€¯0.05 g) twice daily for eight weeks. Replacing 40% of FM with SM sources did not significantly influence growth (P > 0.05), while increasing the substitution level to 80% led to reduced growth rates (P < 0.05). The groups received SM80 and PAFSM80 diets showed significantly higher feed conversion ratio and lower protein digestibility than FM group. Furthermore, notably lower dry matter digestibility was detected in SM80 group. Remarkably lower serum total antioxidant capacity was found in the SM80 group, and catalase activity did not significantly differ between FM and BPFSM40 groups. Serum malondialdehyde concentration was enhanced by increasing FM replacement level and the highest value was observed in the SM80 fed fish. FM and PAFSM40 groups showed significantly higher lysozyme activity than the SM80 group. Fish fed the BPFSM40 diet exhibited the highest complement C3 activity and the lowest value was observed in the SM80 group. Expression of lysozyme gene in spleen was down-regulated in the SM80 group, and no significant difference in expression of C3 gene was found among FM, BPFSM40 and PAFSM40 groups. Digestive enzymes activity and gut morphology were significantly influenced by FM replacement. Expression of HSP70 and pro-inflammatory genes including TNF-α and IL-1ß were up-regulated by FM replacement and relatively lower expression levels were found by using fermented SM. An opposite trend was observed for the anti-inflammatory TGF-ß gene expression. Serum d-lactate concentration was significantly increased by replacing 80% of FM with any of the SM sources. These findings indicated that using fermented SM, particularly BPFSM, beneficially influences feed utilization, antioxidant capacity, innate immunity and gut health in juvenile Japanese seabass.

Bacillus pumilus SE5 originated PG and LTA tuned the intestinal TLRs/MyD88 signaling and microbiota in grouper (Epinephelus coioides).

The normal microbiota plays a key role in the health of host, but little is known of how the fish immune system recognizes and responds to indigenous bacteria/probiotics. Our previous studies have showed that heat-inactivated indigenous Bacillus pumilus SE5 activate the TLR2 signaling pathways and modulate the intestinal microbiota in grouper (Epinephelus coioides), suggesting microbial-associated molecular patterns (MAMPs) involved. In this study, whole cell wall (CW) and two possible MAMPs, peptidoglycan (PG) and lipoteichoic acid (LTA) have been extracted from B. pumilus SE5 and their effects on intestinal immune related genes expression and microbiota were evaluated in a 60 days feeding trial. Significantly elevated expression of TLR1, TLR2, TLR5 and MyD88 was observed in fish fed the CW, PG and LTA containing diets, and the highest expression was observed in groups PG and LTA. At the same time, significantly upregulated expression of antimicrobial effectors, such as antimicrobial peptides (epinecidin-1, hepcidin-1 and ß-defensin), C-type Lectin and IgM was observed in fish fed PG and LTA containing diets. This induced activation of intestinal immunity was consistent with the microbiota data showing that CW, PG and LTA originated from SE5 modulated the overall structure of intestinal microbiota, and the relative abundance of potentially pathogenic Vibrio decreased significantly while beneficial Lactobacillus increased significantly in fish fed PG and LTA. In conclusion, both the PG and LTA originated from B. pumilus SE5 could activate TLRs/MyD88 signaling and expression of wide-ranging antibacterial effectors, and therefore shape the intestinal microbiota in grouper.

Comparative analysis of vertebral transcriptome in Japanese seabass (Lateolabrax japonicus) fed diets with varying phosphorus/calcium levels.

Aquaculture jeopardizes the aquatic environment by discharge of the most dietary phosphorus (P) into the water. Reducing the dietary P level is a common approach for decreasing the P discharge but it may result in increased risk of P deficiency leading to vertebral deformities. However, the molecular mechanism of vertebral deformities is poorly understood. We assessed vertebral transcriptome and compared the genes associated with bone metabolism in Japanese seabass (Lateolabrax japonicus) fed three diets containing different P and Ca levels including: diet I (0.4% P, 0.3% Ca), diet II (0.8% P, 0.3% Ca) and diet III (0.8% P, 3% Ca). The results showed that P deficiency reduces the ossification of vertebrae and induces visible vertebral deformities. Moreover, 256 gens were up-regulated and 125 genes were down-regulated in fish fed P deficient diets. Furthermore, administration of the diet with adequate P and Ca excess (diet III) resulted in the significant enhancement in expression of 19 genes and reduced expression of 93 genes. Comparing group II with group III, expression of 109 genes was up-regulated and expression of 1369 genes was down-regulated. Gene ontology enrichment analysis revealed significant alterations in biological functions by P deficiency. In summary, these findings indicated that both dietary P shortage and Ca excess lead to reduced differentiation and proliferation of osteoblast and induce a higher activity of osteoclastogenesis, which could subsequently impair vertebral mineralization and cause skeletal deformities.

Effects of berberine on growth, liver histology, and expression of lipid-related genes in blunt snout bream (Megalobrama amblycephala) fed high-fat diets.

Fatty liver of cultured fish often correlates closely with poor growth and low harvest yield. Some Chinese herbs can reduce hepatic fat storage. This study aimed to examine lipid-lowering effect of berberine (BBR) in blunt snout bream (Megalobrama amblycephala). Triplicate groups of fish were fed four experimental diets: low-fat diet (LFD, 5% fat), high-fat diet (HFD, 15% fat), and HFD supplemented with 50 or 100 mg BBR/kg diet (BBR50, BBR100). After 8-week feeding, growth performance, liver histology and fat deposition, and hepatic genes expression were examined. The results showed significant reduction of growth performance and feed intake in fish fed HFD compared to those fed the LFD and BBR50 diets. Supplementing 50 mg BBR/kg to the HFD significantly improved weight gain and feed intake. Higher hepatic fat content and histological abnormalities were found in the liver of fish receiving HFD, and BBR50 and BBR100 could attenuate these abnormalities of liver. Expression of CPT I, AOX, ApoB100, ApoE, and PGC-1α genes was significantly decreased in fish fed HFD, and 50 and 100 mg/kg BBR supplementation could revert the downregulation of these genes. Also, the expression of FATP, LPL, and LDLR genes was upregulated in HFD-fed fish, and their expression was significantly decreased by 50 and 100 mg/kg BBR supplementation. In conclusion, supplementing BBR to HFD could attenuate liver fat deposition and disorders. The fat-lowering effects of BBR appear to be mediated by activating genes related with fatty acid oxidation and decreasing genes for fatty acid uptake.

Chitooligosaccharide supplementation in low-fish meal diets for Pacific white shrimp (Litopenaeus vannamei): Effects on growth, innate immunity, gut histology, and immune-related genes expression.

This study evaluated the effects of supplementing chitooligosaccharide (COS) in low fish meal (FM) diets on growth, immune response, intestine and hepatopancrease histology, and expression of inflammatory and immune-related genes in Pacific white shrimp (Litopenaeus vannamei). A basal diet was formulated using FM and soybean meal (SM) as primary protein sources and considered as a high FM (HFM) diet, then a low FM (LFM) diet was prepared by substituting 50% of FM with SM and supplemented with 0, 0.3, 0.6, 0.9, 1.2 or 1.5 g COS kg-1 diet (LFM, COS3, COS6, COS9, COS12 and COS15 diets). Each diet was fed to quadruplicate groups of shrimp (0.9 g) to apparent satiation three times daily for eight weeks. At the end of the experiment no significant changes in growth and survival rate were observed among treatments (P > 0.05). FM replacement led to significant (P < 0.05) reduction of serum lysozyme activity and significant improvements were obtained by adding 0.3 or 0.6 g kg-1 COS to the LFM diet. A significant decrease in nitric oxide synthase activity was found in LFM group and no beneficial effects could be achieved by COS application. LFM group showed higher hepatopancrease superoxide dismutase and glutathione peroxidase activities than HFM group and further enhancements were obtained by COS application. Hepatopancrease total antioxidant capacity and alkaline phosphatase activity decreased in LFM group and COS supplementation improved their values. Expression of lysozyme, crustin, Pen3 and proPo genes were significantly up-regulated in hepatopancrease of groups received 0.3-0.9 g COS kg-1 diet. FM substitution enhanced the expression of HSP70 and inflammatory genes such as AIF and TNF in hepatopancrease and intestine, and COS administration at a moderate level down-regulated their expression level. Remarkable enhancement in intestinal fold height was obtained by inclusion of 0.3 or 0.6 g COS kg-1 diet compared to the group received LFM diet. Shrimps fed HFM and COS containing diets exhibited higher number of E-cells within their hepatopancrease tubules than the LFM group. The findings in this study clearly demonstrated that COS could enhance non-specific immune response and antioxidant activity, and ameliorate the negative impacts of high SM diets on gut and hepatopancrease health in pacific white shrimp. The optimum inclusion level of COS seems to be 0.3-0.6 g kg-1 of diet.

Effects of organic acids and essential oils blend on growth, gut microbiota, immune response and disease resistance of Pacific white shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus.

An 8-week feeding trial was undertaken to evaluate supplemental effects of AviPlus® (AP), a blend of organic acids [citric acid, 25%; sorbic acid, 16.7%] and essential oils [thymol, 1.7%; vanillin, 1.0%], on growth, gut microbiota, innate immunity and disease resistance of Pacific white shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus. A basal experimental diet was formulated and supplemented with 0, 0.3, 0.6, 0.9 and 1.2 g kg-1 AP to produce five test diets (Con, AP0.3, AP0.6, AP0.9 and AP1.2). Each diet was fed to triplicate groups of shrimp (0.2 ± 0.01 g, mean ± SE) to apparent satiation three times daily. Growth performance and survival rate were not significantly influenced by AP supplementation (P > 0.05). Significantly (P < 0.05) higher serum total protein was found in groups fed ≥ 0.6 g kg-1 AP compared to control. Serum alkaline phosphatase and phenoloxidase activities were significantly increased in AP0.9 and AP1.2 groups. Also, the group received AP0.6 diet showed significantly higher glutathione peroxidase activity than control. Expression of gut pro-inflammatory genes including TNF-α, LITAF and RAB6A were down-regulated by AP administration. Gut microbiota analysis showed the significant enhancement of the operational taxonomic unit (OTU) diversity and richness indices by AP application. AP supplementation led to increased abundance of Firmicutes and a reduction in abundance of Proteobacteria. Also, dietary inclusion of 1.2 g kg-1 AP led to a significant increase in the abundance of Lactobacillus in shrimp gut. The group offered AP0.3 diet showed significantly higher disease resistance than control group. Furthermore, AP application significantly enhanced relative expression of immune related genes including lysozyme, penaeidin and catalase at 48 h post challenge. In conclusion, these findings show that the tested organic acids and essential oils mixture beneficially affects intestinal microflora and improves immune response and disease resistance of L. vannamei.

Berberine attenuates oxidative stress and hepatocytes apoptosis via protecting mitochondria in blunt snout bream Megalobrama amblycephala fed high-fat diets.

High-fat diets may have favorable effects on growth and cost, but high-fat diets often induce excessive fat deposition, resulting in liver damage. This study aimed to identify the hepatoprotective of a Chinese herb (berberine) for blunt snout bream (Megalobrama amblycephala). Fish were fed with a normal diet (LFD, 5 % fat), high-fat diet (HFD, 15 % fat) or berberine-supplemented diets (BSD, 15 % fat with berberine 50 or 100 mg/kg level) for 8 weeks. After the feeding, histology, oxidative status and mitochondrial function of liver were assessed. The results showed that HFD caused fat accumulation, oxidative stress and apoptosis in hepatocytes of fish. Hepatocytes in HFD group appeared to be hypertrophied, with larger liver cells diameter than these of LFD group. Berberine-supplemented diets could attenuate oxidative stress and hepatocytes apoptosis. HFD induced the decreasing mitochondrial complexes activities and bulk density and surface area density. Berberine improved function of mitochondrial respiratory chain via increasing the complex activities. Moreover, the histological results showed that berberine has the potential to repair mitochondrial ultrastructural damage and elevate the density in cells. In conclusion, our study demonstrated that berberine has attenuated liver damage induced by the high fat mainly via the protection for mitochondria.

Effects of fructooligosaccharide on immune response, antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream (Megalobrama amblycephala) under high ammonia stress.

This study aimed to determine the effects of fructooligosaccharide (FOS) on immune response, antioxidant capability and HSP70 and HSP90 mRNA expressions of blunt snout bream (Megalobrama amblycephala) under high ammonia stress. A total of 360 fish were randomly distributed into three groups (each with four replicates) and were fed three levels of FOS (0, 0.4 and 0.8 %) for 8 weeks. After the feeding trial, 24 fish per tank were exposed to ammonia at 10 mg L(-1). After stress, plasma cortisol and glucose levels of fish fed 0.4 % FOS were all significantly lower than that of the control group at 6 and 3 h, respectively. Plasma lysozyme and alternative complement pathway (ACH50) activities as well as nitrogen monoxide (NO) levels all increased significantly with the maximum levels being attained at 6, 6 and 3 h, respectively. Thereafter, these parameters all decreased significantly. In addition, fish fed 0.4 % FOS showed higher immune parameters under stress compared with that of control group. In addition, liver superoxide dismutase and catalase activities of fish fed 0.4 % FOS were both significantly higher than that of the control group before and after stress, while the opposite was true for malondialdehyde content. After stress, the expression of HSP70 and HSP90 of fish fed FOS was significantly higher than that of the control group at 6 and 12 h, respectively. After 12 h stress, the cumulative mortality of fish fed FOS was significantly lower than that of the control. The results indicated that the supplementation of 0.4 % FOS could increase the nonspecific immunity, antioxidant capacity and HSP70 and HSP90 expression of blunt snout bream and enhance its resistance to high ammonia stress.

The impacts of reduced connectivity on multiple facets of aquatic insect diversity in floodplain wetlands, Northeast China.

River-floodplain ecosystems are highly complex and dynamic systems that are subjected to human disturbance, such as the construction of levees. Aquatic insects are among the most widely used indicators to assess human impacts on floodplain wetlands. Most studies are still based on taxonomic biodiversity. However, trait-based approaches remain limited, which could impede the development of effective management strategies. Here, we investigated aquatic insect assemblages in eleven pairs of wetlands along the Wusuli River in two seasons (Spring and Autumn) and assessed their responses to the impact of levee construction, considering taxonomic and functional diversity. We found that pooled species richness (73 taxa) in river-connected wetlands was almost twice of that in levee-blocked wetlands (37 taxa). Six and one indicator taxa for river-connected wetlands were identified in May and October, respectively, while no indicator taxon for the levee-blocked wetlands was identified. Moreover, taxonomic and functional alpha diversity in river-connected wetlands was much higher than in levee-blocked wetlands, but beta diversity showed a contrasting pattern. Additionally, multivariate dispersion analysis indicated a more evident difference in beta diversity between river-connected and levee-blocked wetlands in May than in October, likely due to the temporary lateral connection in summer (i.e., water flowed over levees during flood events). Our results revealed that anthropogenic impacts (e.g., levee construction and agricultural activity) weakened the connectivity of floodplain wetland ecosystems, leading to decreased taxonomic and functional diversity of aquatic insects in isolated wetlands. Our study highlights the importance of combining taxonomic and trait-based approaches in biomonitoring programs of floodplain wetland ecosystems. It also underscores the necessity of restoring habitat connectivity of wetland ecosystems (e.g., river-floodplain connectivity and connections between different wetlands) to facilitate biodiversity recovery and enhance ecological functions and services supported by these valuable ecosystems.

Effects of Dietary Supplementation of Bile Acids on Growth, Glucose Metabolism, and Intestinal Health of Spotted Seabass (Lateolabrax maculatus).

An 8-week feeding trial was performed to investigate the effects of dietary bile acids on growth, glucose metabolism, and intestinal health in spotted seabass (Lateolabrax maculatus) reared at high temperatures (33 °C). The fish (20.09 ± 1.12 g) were fed diets supplemented with bile acids: 0 (Con), 400 (BA400), 800 (BA800), and 1200 (BA1200) mg/kg, respectively. The results showed that the growth was promoted in fish at the BA800 treatment compared with the control (p < 0.05). Increased enzyme activities and transcripts of gluconeogenesis in the liver were observed, whereas decreased enzyme activities and transcripts of glycolysis, as well as glycogen content, were shown in the BA800 treatment (p < 0.05). The transcripts of bile acid receptors fxr in the liver were up-regulated in the BA800 treatment (p < 0.05). A bile acid supplementation of 800 mg/kg improved the morphological structure in the intestine. Meanwhile, intestinal antioxidant physiology and activities of lipase and trypsin were enhanced in the BA800 treatment. The transcripts of genes and immunofluorescence intensity related to pro-inflammation cytokines (il-1ß, il-8, and tnf-α) were inhibited, while those of genes related to anti-inflammation (il-10 and tgf-ß) were induced in the BA800 treatment. Furthermore, transcripts of genes related to the NF-κB pathway in the intestine (nfκb, ikkα, ikkß, and ikbα1) were down-regulated in the BA800 treatment. This study demonstrates that a dietary bile acid supplementation of 800 mg/kg could promote growth, improve glucose metabolism in the liver, and enhance intestinal health by increasing digestive enzyme activity and antioxidant capacity and inhibiting inflammatory response in L. maculatus.

Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate immunity, antioxidant capability and disease resistance of triangular bream (Megalobrama terminalis).

This study was conducted to investigate the effects of fructooligosaccharide (FOS) and Bacillus licheniformis (B. licheniformis) and their interaction on innate immunity, antioxidant capability and disease resistance of triangular bream Megalobrama terminalis (average initial weight 30.5 ± 0.5 g). Nine experimental diets were formulated to contain three FOS levels (0, 0.3% and 0.6%) and three B. licheniformis levels (0, 1 × 10(7), 5 × 10(7) CFU g(-1)) according to a 3 × 3 factorial design. At the end of the 8-week feeding trial, fish were challenged by Aeromonas hydrophila (A. hydrophila) and survival rate was recorded for the next 7 days. The results showed that leucocyte counts, alternative complement activity as well as total serum protein and globulin contents all increased significantly (P < 0.05) as dietary B. licheniformis levels increased from 0 to 1 × 10(7) CFU g(-1), while little difference (P > 0.05) was observed in these parameters in terms of dietary FOS levels. Both plasma alkaline phosphatase and phenoloxidase activities were significantly (P < 0.05) affected only by dietary FOS levels with the highest values observed in fish fed 0.6 and 0.3% FOS, respectively. Both immunoglobulin M content and liver superoxide dismutase (SOD) activity were significantly affected (P > 0.05) by both FOS and B. licheniformis. Liver catalase, glutathione peroxidase as well as plasma SOD activities of fish fed 1 × 10(7) CFU g(-1)B. licheniformis were all significantly (P < 0.05) higher than that of the other groups, whereas the opposite was true for malondialdehyde content. After A. hydrophila challenge, survival rate was not affected (P > 0.05) by either FOS levels or B. licheniformis contents, whereas a significant (P < 0.05) interaction between these two substances was observed with the highest value observed in fish fed 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis. The results of this study indicated that dietary FOS and B. licheniformis could significantly enhance the innate immunity and antioxidant capability of triangular bream, as well as improve its disease resistance. The best combination of these two prebiotics and/or probiotics was 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis.

Physiological Response of Spotted Seabass (Lateolabrax maculatus) to Different Dietary Available Phosphorus Levels and Water Temperature: Changes in Growth, Lipid Metabolism, Antioxidant Status and Intestinal Microbiota.

A 10-week growth experiment was conducted to assess the physiological response of spotted seabass (Lateolabrax maculatus) raised at moderate (27 °C) and high temperatures (33 °C) to different dietary available phosphorus (P) levels. Five diets with available P levels of 0.35, 0.55, 0.71, 0.82 and 0.92% were formulated, respectively. A water temperature of 33 °C significantly decreased growth performance and feed utilization, and increased oxidative stress and lipid deposition of spotted seabass compared with 27 °C. A second-order polynomial regression analysis based on weight gain (WG) showed that the available P requirement of spotted seabass raised at 27 °C and 33 °C was 0.72% and 0.78%, respectively. The addition of 0.71-0.82% P to the diet improved the growth performance, feed utilization, and antioxidant capacity of spotted seabass and alleviated the excessive lipid deposition compared with the low-P diet (0.35% P). Moreover, the addition of 0.71-0.92% P to diets increased the diversity of intestinal microbiota and the relative abundance of Lactococcus lactis and decreased the relative abundance of Plesiomonas compared with the low-P diet. Thus, dietary supplementation with 0.71-0.82% P improved the growth performance, antioxidant capacity and microbial composition of spotted seabass, and alleviated the disturbance of lipid metabolism caused by high temperature or low-P diet.

Dietary n-3/n-6 polyunsaturated fatty acid ratio modulates growth performance in spotted seabass (Lateolabrax maculatus) through regulating lipid metabolism, hepatic antioxidant capacity and intestinal health.

An 8-week feeding experiment was carried out to explore the effects of dietary n-3/n-6 polyunsaturated fatty acid (PUFA) ratio on growth performance, lipid metabolism, hepatic antioxidant status, and gut flora of spotted seabass (Lateolabrax maculatus). Six experimental diets were formulated to contain different levels of two purified oil sources including docosahexaenoic and eicosapentaenoic acids enriched oil (n-3) and linoleic acid-enriched oil (n-6) leading to n-3/n-6 PUFA ratios of 0.04, 0.35, 0.66, 1.35, 2.45 and 16.17. Each diet was fed to triplicate groups of juvenile L. maculatus (11.06 ± 0.20 g, 30 fish/tank). Final body weight (FBW), weight gain (WG), specific growth rates (SGR), protein efficiency ratio (PER) and feed utilization efficiency increased as n-3/n-6 PUFA ratio increased up to a certain level, and then decreased thereafter. Fish fed the diet with n-3/n-6 PUFA ratio of 0.66 exhibited the highest FBW, WG, SGR and PER and the lowest feed conversion ratio. Lower n-3/n-6 PUFA ratios induced up-regulated expression of lipid synthesis-related genes (fas, acc2 and srebp-1c) and down-regulated expression of lipolysis related genes (atgl, pparα, cpt-1 and aox). Higher expression of lipolysis-related genes (atgl, pparα and cpt-1) was recorded at moderate n-3/n-6 PUFA ratios (0.66 to 1.35). Moreover, inappropriate n-3/n-6 PUFA ratios triggered up-regulation of pro-inflammatory genes (il-6 and tnf-α) and down-regulation of anti-inflammatory genes (il-4 and il-10) in the intestine. The diet with n-3/n-6 PUFA ratio of 0.66 inhibited intestine inflammation, improved intestinal flora richness, increased the abundance of beneficial bacteria such as Lactobacillus, Alloprevotella and Ruminococcus, and reduced the abundance of harmful bacteria including Escherichia-Shigella and Enterococcus. In summary, it could be suggested that a dietary n-3/n-6 PUFA ratio of 0.66 can improve growth performance and feed utilization in L. maculatus, as is deemed to be mediated through regulation of lipid metabolism and intestinal flora.

Differential distribution patterns and assembly processes of soil microbial communities under contrasting vegetation types at distinctive altitudes in the Changbai Mountain.

Diversity patterns and community assembly of soil microorganisms are essential for understanding soil biodiversity and ecosystem processes. Investigating the impacts of environmental factors on microbial community assembly is crucial for comprehending the functions of microbial biodiversity and ecosystem processes. However, these issues remain insufficiently investigated in related studies despite their fundamental significance. The present study aimed to assess the diversity and assembly of soil bacterial and fungal communities to altitude and soil depth variations in mountain ecosystems by using 16S and ITS rRNA gene sequence analyses. In addition, the major roles of environmental factors in determining soil microbial communities and assembly processes were further investigated. The results showed a U-shaped pattern of the soil bacterial diversity at 0-10 cm soil depth along altitudes, reaching a minimum value at 1800 m, while the fungal diversity exhibited a monotonically decreasing trend with increasing altitude. At 10-20 cm soil depth, the soil bacterial diversity showed no apparent changes along altitudinal gradients, while the fungal Chao1 and phylogenetic diversity (PD) indices exhibited hump-shaped patterns with increasing altitude, reaching a maximum value at 1200 m. Soil bacterial and fungal communities were distinctively distributed with altitude at the same depth of soil, and the spatial turnover rates in fungi was greater than in bacteria. Mantel tests suggested soil physiochemical and climate variables significantly correlated with the ß diversity of microbial community at two soil depths, suggesting both soil and climate heterogeneity contributed to the variation of bacterial and fungal community. Correspondingly, a novel phylogenetic null model analysis demonstrated that the community assembly of soil bacterial and fungal communities were dominated by deterministic and stochastic processes, respectively. The assembly processes of bacterial community were significantly related to the soil DOC and C:N ratio, while the fungal community assembly processes were significantly related to the soil C:N ratio. Our results provide a new perspective to assess the responses of soil microbial communities to variations with altitude and soil depth.