High temperature induces oxidative stress in spotted seabass (Lateolabrax maculatus) and leads to inflammation and apoptosis.

Our study aims to examine the changes of long-term high temperature on the mortality and health status of spotted seabass (Lateolabrax maculatus), as well as to screen suitable biomarkers to determine whether the spotted seabass is under heat stress. In this study, 360 juvenile spotted seabass were evenly distributed into three temperature-controlled systems at 27°C (N, normal temperature), 31°C (M, moderate temperature), and 35°C (H, high temperature) for an 8-week aquaculture experiment. The results revealed that 35°C water temperature significantly increased the mortality and the MDA content in tissues (P < 0.05). Meanwhile, 35°C water temperature significantly increased the activity of SOD enzyme and T-AOC capacity in tissues, as well as the expression of hsp60, hsp70, and hsp90 (P < 0.05). Additionally, the expression of nrf2, il1ß, il8, caspase3, caspase9, and bax in the liver significantly increased (P < 0.05), while the expression of keap1, il10, tgfß, and bcl2 decreased significantly (P < 0.05). These results indicate that 35°C water temperature induces oxidative stress in spotted seabass, leading to tissue oxidative damage, promoting inflammation and apoptosis in liver, and increasing mortality. However, the organism compensates by heightening its antioxidant capacity via the Nrf2-Keap1 signaling pathway and inducing high expression of heat shock proteins for self-protection. Furthermore, the alterations in the mRNA level of hsp70 and MDA content in the liver, muscle, and kidney can serve as indicators for evaluating spotted seabass under prolonged heat stress.

Microplastic Contamination in Aquafeed Ingredients Used as Protein and Carbohydrate Sources.

The current study aimed to evaluate the occurrence of microplastics in feed ingredients commonly used as protein and carbohydrate (energy) sources to understand and mitigate microplastic contamination. Microplastics average was 1.27, 0.69, 2.85, 0.55, 0.07, and 0.17 particle g- 1 in fishmeal, soybean meal, poultry by-products, rice bran, wheat bran, and wheat flour, respectively. Notably, poultry by-products demonstrated significantly higher microplastic levels than other ingredients (p < 0.05). The dominant microplastic shape was microfibers, with prevalent sizes ranging from 500 to 1000 µm. We estimated that packaging materials are a significant source of pollution due to the high presence of polypropylene and other polymers. Top aquaculture species with the greatest microplastic exposure risk include the Catla catla, Hypophthalmichthys nobilis, and Oreochromis niloticus. This research extends our knowledge of microplastic pathways, contributes to improving aquafeed quality, and provides the basis for determining the risk of microplastic exposure in aquafeed.

Microplastic pollution in aquafeed of diverse aquaculture animals.

Microplastics have emerged as pervasive contaminants, and determining their occurrence in aquafeed is key for evaluating their risks to farmed animals and, by extension, humans. However, knowledge about microplastic in aquafeed is still limited. Herein, we determined microplastic characteristics in aquafeed for five important aquaculture animals with different feeding habits. Aquafeed samples were collected for spotted sea bass, shrimp, grass carp, Tilapia, and frogs from main companies in China. The samples were digested using chemical digestion, and the residuals were subjected to a density separation. Microplastics were identified under the microscope and characterized by their shape, color, size, and polymer type. The results showed that microplastics are highly abundant in the feed of frogs, followed by spotted sea bass, Tilapia, grass carp, and shrimp. We found that feed size contributes to the total microplastic abundance in the feed. Further, microplastics were mainly in microfiber form, and the dominant polymer type was propylene, suggesting that packaging and processing are the main sources of pollution. Additionally, the most abundant size of microplastics was 100-1000 µm. Calculating microplastic ingestion risk, the spotted sea bass had the greatest recorded risk of microplastic ingestion, followed by grass carp, frogs, Tilapia, and shrimp. This study lays a foundational step toward understanding microplastic effects on aquaculture animals and calls for further environmentally relevant laboratory experiments to assess the risk of microplastic ingestion on animals and potential transfer to humans.

Derivatives of postbiotics (cell wall constituents) from Bacillus subtilis (LCBS1) relieve soybean meal-induced enteritis in bullfrog (Aquarana catesbeianus).

Soybean meal (SM) serves as a primary alternative to fish meal in aquafeeds. However, a high-SM diet may result in intestinal injury. Our previous study demonstrated the probiotic effects of heat-inactivated Bacillus subtilis (LCBS1) on bullfrogs (Aquarana catesbeianus) fed a high-SM diet, probably attributed to the bioactive constituent of cell wall. Therefore, in this study, the main constituents of cell wall from LCBS1, including peptidoglycan (PGN), lipoteichoic acid (LTA), cell wall protein (CWP), and whole cell wall (WCW), were extracted and added to a high-SM (~55 %) diet to investigate their probiotic effects on bullfrogs and reveal the possible mechanisms. The results indicated that bullfrogs fed the LTA of LCBS1 showed the highest weight gain, feed efficiency, and protein efficiency ratio. Additionally, the LTA of LCBS1 could activate the humoral immunity and modulate intestinal microbiota. It might activate JAK2-STAT3 and MAPK-ERK pathways, as well as up-regulate tlr5 gene to promote intestinal cell proliferation, thereby alleviating jejunal injury. The WCW of LCBS1 effectively increased the growth performance of bullfrogs by improving the humoral immunity, enhancing intestinal barrier function, and alleviating intestinal inflammatory response. The PGN and CWP of LCBS1 could stimulate the humoral immunity and enhance intestinal barrier function, but had no significant effect on the growth performance of bullfrogs. In conclusion, the LTA might be the primary bioactive constituent of heat-inactivated LCBS1, with the beneficial effects of promoting intestinal cell proliferation and enhancing intestinal barrier function, therefore alleviating the intestinal injury induced by SM on bullfrogs. This study establishes a theoretical basis for the efficient utilization of plant proteins by the application of postbiotics additive in aquafeed, which further saves the feed costs and promotes development of economically sustainable aquaculture.

Combination of Transcriptomics and Metabolomics Analyses Provides Insights into the Mechanisms of Growth Differences in Spotted Seabass (Lateolabrax maculatus) Fed a Low-Phosphorus Diet.

To analyze the potential mechanisms of growth differences in spotted seabass (Lateolabrax maculatus) fed a low-phosphorus diet, a total of 150 spotted seabass with an initial body weight of 4.49 ± 0.01 g were used (50 fish per tank) and fed a low-phosphorus diet for eight weeks. At the end of the experiment, five of the heaviest and five of the lightest fish were selected from each tank as fast-growing spotted seabass (FG) and slow-growing spotted seabass (SG), respectively, and their livers were analyzed by metabolomics and transcriptomics. The hepatic antioxidant capacity of the FG fed a low-phosphorus diet was significantly higher than that of the SG. A total of 431 differentially expressed genes (DEGs) were determined in the two groups, and most of the DEGs were involved in metabolism-related pathways such as steroid biosynthesis, glycolysis/gluconeogenesis, and protein digestion and absorption. Substance transport-related regulators and transporters were predominantly up-regulated. Furthermore, a large number of metabolites in the liver of FG were significantly up-regulated, especially amino acids, decanoyl-L-carnitine and dehydroepiandrosterone. The integration analysis of differential metabolites and genes further revealed that the interaction between protein digestion and absorption, as well as phenylalanine metabolism pathways were significantly increased in the liver of FG compared to those of the SG. In general, FG fed a low-phosphorus diet induced an enhancement in hepatic immune response, substance transport, and amino acid metabolism. This study provides new information on genetic mechanisms and regulatory pathways underlying differential growth rate and provides a basis for the foundation of efficient utilization of low-phosphorus diets and selective breeding programs for spotted seabass.

Slow recovery in trophic structure of restored wetlands in Northeast China.

Restoration measures have been widely implemented in wetland ecosystems globally to bend the curve of biodiversity loss and restore associated ecological functions. However, assessments of the effectiveness of wetland restoration have predominantly focused on the recovery of taxonomic composition, while few studies have assessed the effectiveness of these efforts from a food web perspective. Here, we incorporated stable isotope approach to investigate trophic structure in natural and restored wetlands in Northeast China. The investigated consumers, including zooplankton, macroinvertebrates, and fish, exhibited lower δ15N and higher δ13C values in restored wetlands than in natural wetlands. Natural wetlands exhibited higher trophic positions and a wider range of trophic levels compared to restored wetlands. Primary consumers in natural wetlands relied more on particulate organic matter (POM, 42.9 % ± 24.1 %), while those in restored wetlands were more dependent on substrate organic matter (SOM, 42.3 % ± 23.9 %). Compared to natural wetlands, isotopic richness was significantly lower in restored wetlands, with smaller isotopic variation (SEAs) in basal resources, aquatic invertebrates, and fish. Our findings reveal that the recovery of trophic structures in restored wetlands lags behind that of taxonomic composition. Future restoration efforts should prioritize enhancing habitat heterogeneity and resource availability to support a diverse range of trophic levels. Monitoring trophic dynamics is essential for assessing the progress of wetland restoration and should be integrated into monitoring schemes.

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.

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.

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.

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.

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.

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.

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.

4-PBA Attenuates Fat Accumulation in Cultured Spotted Seabass Fed High-Fat-Diet via Regulating Endoplasmic Reticulum Stress.

Excessive fat accumulation is a common phenomenon in cultured fish, which can cause metabolic disease such as fatty liver. However, the relative regulatory approach remains to be explored. Based on this, two feeding trials were conducted. Firstly, fish were fed either a normal-fat diet (NFD) or a high-fat diet (HFD) for eight weeks and sampled at the 2nd, 4th, 6th, and 8th week after feeding (Experiment I). In the first four weeks, fish fed an HFD grew faster than those fed an NFD. Conversely, the body weight and weight gain were higher in the NFD group at the 6th and 8th weeks. Under light and transmission electron microscopes, fat accumulation of the liver was accompanied by an obvious endoplasmic reticulum (ER) swell. Accordingly, the expressions of atf-6, ire-1, perk, eif-2α, atf-4, grp78, and chop showed that ER stress was activated at the 6th and 8th weeks. In Experiment II, 50 mg/kg 4-PBA (an ERs inhibitor) was supplemented to an HFD; this was named the 4-PBA group. Then, fish was fed with an NFD, an HFD, and a 4-PBA diet for eight weeks. As the result, the excessive fat deposition caused by an HFD was reversed by 4-PBA. The expression of ER stress-related proteins CHOP and GRP78 was down-regulated by 4-PBA, and the transmission electron microscope images also showed that 4-PBA alleviated ER stress induced by the feeding of an HFD. Furthermore, 4-PBA administration down-regulated SREBP-1C/ACC/FAS, the critical pathways of fat synthesis. In conclusion, the results confirmed that ER stress plays a contributor role in the fat deposition by activating the SREBP-1C/ACC/FAS pathway. 4-PBA as an ER stress inhibitor could reduce fat deposition caused by an HFD via regulating ER stress.

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.

Effects of the Replacement of Dietary Fish Meal with Defatted Yellow Mealworm (Tenebrio molitor) on Juvenile Large Yellow Croakers (Larimichthys crocea) Growth and Gut Health.

This study was conducted to investigate the effects of Tenebrio molitor meal (TM) replacement for fish meal (FM) on growth performance, humoral immunity, and intestinal health of juvenile large yellow croakers (Larimichthys crocea). Four experimental diets were formulated by replacing FM with TM at different levels-0% (TM0), 15% (TM15), 30% (TM30), and 45% (TM45). Triplicate groups of juveniles (initial weight = 11.80 ± 0.02 g) were fed the test diets to apparent satiation two times daily for eight weeks. There was no significant difference in final body weight (FBW) and weight gain rate (WG) among TM0, TM15, and TM30, while TM45 feeding significantly reduced the FBW and WG. Compared with TM0, AKP activity in serum was significantly decreased in TM45, while the TM15 group remarkably increased LZM activity. TM30 showed significantly higher serum C3 levels compared to the TM0 group, while the TM addition groups decreased the C4 levels significantly in the serum. In terms of intestinal histology, the addition of TM increased the height and thickness of the intestinal villus and also increased the thickness of the intestinal muscles significantly. The addition of TM significantly reduced the serum DAO and D-lactate concentrations. The results of 16S rRNA gene sequencing showed that the addition of TM significantly enhanced the relative abundance of Bacilli and Lactobacillus and contributed to the decrease in the relative abundance of Plesiomonas. In addition, the TM30 and TM45 groups significantly reduced the abundance of Peptostreptococcaceae. Overall, our results indicated that TM could be a viable alternative protein source, 6.7% TM supplantation (replacing 15% FM) in large yellow croaker feed improved humoral immunity and intestinal health with no adverse effects on growth. Furthermore, the replacement of FM with 30% and 45% TM adversely affects growth and humoral immunity.

Oxidative Stress Can Be Attenuated by 4-PBA Caused by High-Fat or Ammonia Nitrogen in Cultured Spotted Seabass: The Mechanism Is Related to Endoplasmic Reticulum Stress.

Oxidative stress is a common phenomenon in aquaculture, which can be induced by nutritional or environmental factors. Generally, oxidative stress causes poor growth performance, metabolic dysregulation, and even the death of aquatic animals. To identify a nutritional intervention strategy, high-fat diet (HFD) feeding (Experiment I) and acute ammonia nitrogen challenge (Experiment II) tests were carried out. In Experiment I, HFD feeding significantly decreased the growth performance concomitantly with excessive fat deposition in the liver and abdomen. The addition of 4-PBA in the diet improved the excessive fat accumulation. The activities of antioxidative enzymes were suppressed, and the levels of lipid and protein peroxidation were increased, indicating that HFD feeding induced oxidative stress. The endoplasmic reticulum stress (ERs) related genes were downregulated in the HFD group. Under a transmission electron microscope (TEM), more swollen and dilated ER lumen could be observed. These results indicated that the HFD induced ERs activation. Although 4-PBA acted as a potent ERs inhibitor, as evidenced by the alleviated alterations of ERs molecules and the ER ultrastructure, the oxidative stress was also attenuated by 4-PBA. In Experiment II, dietary 4-PBA improved the tolerance to the acute ammonia nitrogen challenge, as lower mortality and serum aminotransferase activity was found. Further results showed that 4-PBA decreased the peroxidation content and attenuated ERs, thus confirming the correlation between oxidative stress and ERs. Our findings showed that dietary 4-PBA supplementation can attenuate oxidative stress induced by a HFD or acute ammonia challenge; the mechanism is related to its potent inhibition effect for ERs.

Hydroxytyrosol Promotes the Mitochondrial Function through Activating Mitophagy.

Emerging evidence suggests that mitochondrial dysfunction mediates the pathogenesis for non-alcoholic fatty liver disease (NAFLD). Hydroxytyrosol (HT) is a key component of extra virgin olive oil which can exert beneficial effects on NAFLD through modulating mitochondria. However, the mechanism of the impacts of HT still remains elusive. Thus, an in vivo and a series of in vitro experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in fish. For the in vivo experiment, two diets were produced to contain 10% and 16% fat as normal-fat and high-fat diets (NFD and HFD) and two additional diets were prepared by supplementing 200 mg/kg of HT to the NFD and HFD. The test diets were fed to triplicate groups of spotted seabass (Lateolabrax maculatus) juveniles for 8 weeks. The results showed that feeding HFD leads to increased fat deposition in the liver and induces oxidative stress, both of which were ameliorated by HT application. Furthermore, transmission electron microscopy revealed that HFD destroyed mitochondrial cristae and matrix and induced severe hydropic phenotype, while HT administration relieved these alterations. The results of in vitro studies using zebrafish liver cell line (ZFL) showed that HT promotes mitochondrial function and activates PINK1-mediated mitophagy. These beneficial effects of HT disappeared when the cells were treated with cyclosporin A (Csa) as a mitophagy inhibitor. Moreover, the PINK1-mediated mitophagy activation by HT was blocked when compound C (CC) was used as an AMPK inhibitor. In conclusion, our findings demonstrated that HT alleviates fat accumulation, oxidative stress and mitochondrial dysfunction, and its effects are deemed to be mediated via activating mitophagy through the AMPK/PINK1 pathway.

Effects of Supplementing Intestinal Autochthonous Bacteria in Plant-Based Diets on Growth, Nutrient Digestibility, and Gut Health of Bullfrogs (Lithobates catesbeianus).

Poor utilization efficiency of plant protein diets always leads to intestinal barrier dysfunction and growth inhibition in animals. Probiotics have shown promise in improving growth performance and gut health of the host. However, obtaining the host-beneficial probiotic from thousands of bacterial phylotypes is challenging. Here, four intestinal autochthonous bacteria were isolated from fast-growing bullfrog after a 60-day feeding on a soybean meal (SM)-based diet. Another feeding trial was conducted to evaluate the effects of supplementing these strains in an SM-based diet on growth, nutrient digestibility, immunity, and gut health of bullfrog. A high-SM basal diet was used as a non-supplemented control group (NC), and four other diets were prepared by supplementing the basal diet with 1 × 107 CFU/g of Bacillus siamensis, Bacillus tequilensis (BT), Bacillus velezensis, and Lactococcus lactis (LL). Results showed that weight gain, feed efficiency, nitrogen retention, and apparent digestibility coefficients of dry matter and protein were significantly higher in the LL group compared with the NC group (p < 0.05). Furthermore, compared with the NC group, both BT and LL groups showed markedly higher jejunal protease and amylase activities, serum complement 4 and immunoglobulin M levels, jejunal muscularis thickness (p < 0.05), and up-regulated expression of il-10 and zo-1 genes (p < 0.05). High-throughput sequencing revealed higher abundances of Bacillus and Cetobacterium in BT and LL groups, respectively, accompanied with decreased abundances of Enterobacter and Escherichia-Shigella. Besides, KEGG pathways related to metabolisms were significantly enhanced by the LL diet relative to the NC diet (p < 0.05). Overall, the beneficial effects of two frog-derived probiotics were determined: supplementation of L. lactis in SM-based diet promoted growth and nutrient digestibility; both B. tequilensis and L. lactis supplementation improved immune response and intestinal barrier function of bullfrogs.

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.