Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Nonmammalian jawed vertebrates lack lymph nodes but maintain diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing nonhematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen-presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ. This lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system.

Epigenomic analysis reveals prevalent contribution of transposable elements to cis-regulatory elements, tissue-specific expression, and alternative promoters in zebrafish.

Transposable elements (TEs) encode regulatory elements that impact gene expression in multiple species, yet a comprehensive analysis of zebrafish TEs in the context of gene regulation is lacking. Here, we systematically investigate the epigenomic and transcriptomic landscape of TEs across 11 adult zebrafish tissues using multidimensional sequencing data. We find that TEs contribute substantially to a diverse array of regulatory elements in the zebrafish genome and that 37% of TEs are positioned in active regulatory states in adult zebrafish tissues. We identify TE subfamilies enriched in highly specific regulatory elements among different tissues. We use transcript assembly to discover TE-derived transcriptional units expressed across tissues. Finally, we show that novel TE-derived promoters can initiate tissue-specific transcription of alternate gene isoforms. This work provides a comprehensive profile of TE activity across normal zebrafish tissues, shedding light on mechanisms underlying the regulation of gene expression in this widely used model organism.

Effects of polyethylene, polylactic acid, and tire particles on the sediment microbiome and metabolome at high and low temperatures.

Global warming has led to a high incidence of extreme heat events, and the frequent occurrence of extreme heat events has had extensive and far-reaching impacts on wetland ecosystems. The widespread distribution of plastics in the environment, including polyethylene (PE), polylactic acid (PLA), and tire particles (TPs), has caused various environmental problems. Here, high-throughput sequencing techniques and metabolomics were used for the first time to investigate the effects of three popular microplastic types: PE, PLA, and TP, on the sediment microbiome and the metabolome at both temperatures. The microplastics were incorporated into the sediment at a concentration of 3% by weight of the dry sediment (wt/wt), to reflect environmentally relevant conditions. Sediment enzymatic activity and physicochemical properties were co-regulated by both temperatures and microplastics producing significant differences compared to controls. PE and PLA particles inhibited bacterial diversity at low temperatures and promoted bacterial diversity at high temperatures, and TP particles promoted both at both temperatures. For bacterial richness, only PLA showed inhibition at low temperature; all other treatments showed promotion. PE, PLA, and TP microplastics changed the community structure of sediment bacteria, forming two clusters at low and high temperatures. Furthermore, PE, PLA, and TP changed the sediment metabolic profiles, producing differential metabolites such as lipids and molecules, organic heterocyclic compounds, and organic acids and their derivatives, especially TP had the most significant effect. These findings contribute to a more comprehensive understanding of the potential impact of microplastic contamination.IMPORTANCEIn this study, we added 3% (wt/wt) microplastic particles, including polyethylene, polylactic acid, and tire particles, to natural sediments under simulated laboratory conditions. Subsequently, we simulated the sediment microbial and ecosystem responses under different temperature conditions by incubating them for 60 days at 15°C and 35°C, respectively. After synthesizing these results, our study strongly suggests that the presence of microplastics in sediment ecosystems and exposure under different temperature conditions may have profound effects on soil microbial communities, enzyme activities, and metabolite profiles. This is important for understanding the potential hazards of microplastic contamination on terrestrial ecosystems and for developing relevant environmental management strategies.

Variations in antibiotic resistomes associated with archaeal, bacterial, and viral communities affected by integrated rice-fish farming in the paddy field ecosystem.

Antibiotic resistance genes (ARGs) serving as a newly recognized pollutant that poses potential risks to global human health, which in the paddy soil can be potentially altered by different agricultural production patterns. To elucidate the impacts and mechanisms of the widely used and sustainable agricultural production pattern, namely integrated rice-fish farming, on the antibiotic resistomes, we applied metagenomic sequencing to assess ARGs, mobile genetic elements (MGEs), bacteria, archaea, and viruses in paddy soil. There were 20 types and 359 subtypes of ARGs identified in paddy soil. The integrated rice-fish farming reduced the ARG and MGE diversities and the abundances of dominant ARGs and MGEs. Significantly decreased ARGs were mainly antibiotic deactivation and regulator types and primarily ranked level IV based on their potential threat to human health. The integrated rice-fish farming decreased the alpha diversities and altered microbial community compositions. MGEs, bacteria, archaea, and virus exhibited significant correlations with ARGs, while integrated rice-fish farming effectively changed their interrelationships. Viruses, bacteria, and MGEs played crucial roles in affecting the ARGs by the integrated rice-fish farming. The most crucial pathway by which integrated rice-fish farming affected ARGs was through the modulation of viral communities, thereby directly or indirectly influencing ARG abundance. Our research contributed to the control and restoration of ARGs pollution from a new perspective and providing theoretical support for the development of clean and sustainable agricultural production.

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats.

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.

Influences of the Integrated Rice-Crayfish Farming System with Different Stocking Densities on the Paddy Soil Microbiomes.

Integrated rice-fish farming has emerged as a novel agricultural production pattern to address global food security challenges. Aiming to determine the optimal, scientifically sound, and sustainable stocking density of red claw crayfish (Cherax quadricarinatus) in an integrated rice-crayfish farming system, we employed Illumina high-throughput 16S rRNA gene sequencing to evaluate the impact of different stocking densities of red claw crayfish on the composition, diversity, function, and co-occurrence network patterns of soil bacterial communities. The high stocking density of red claw crayfish reduced the diversity and evenness of the soil bacterial community during the mid-culture stage. Proteobacteria, Actinobacteria, and Chloroflexi emerged as the most prevalent phyla throughout the experimental period. Low stocking densities initially boosted the relative abundance of Actinobacteria in the paddy soil, while high densities did so during the middle and final stages. There were 90 distinct functional groups identified across all the paddy soil samples, with chemoheterotrophy and aerobic chemoheterotrophy being the most abundant. Low stocking densities initially favored these functional groups, whereas high densities enhanced their relative abundances in the later stages of cultivation. Medium stocking density of red claw crayfish led to a more complex bacterial community during the mid- and final culture stages. The experimental period showed significant correlations with soil bacterial communities, with total nitrogen (TN) and total phosphorus (TP) concentrations emerging as primary factors contributing to the alterations in soil bacterial communities. In summary, our findings demonstrated that integrated rice-crayfish farming significantly impacted the soil microbiomes and environmental factors at varying stocking densities. Our study contributed to theoretical insights into the profound impact of integrated rice-crayfish farming with various stocking densities on bacterial communities in paddy soils.

The Association between Discrepancies in Parental Emotional Expressivity, Adolescent Loneliness and Depression: A Multi-Informant Study Using Response Surface Analysis.

Incongruent perceptions of parental emotional expressivity between parents and adolescents may signify relational challenges, potentially impacting adolescents' socioemotional adjustment. Direct evidence is still lacking and father-adolescent discrepancies are overlooked. This study employed a multi-informant design to investigate whether both mother-adolescent and father-adolescent discrepancies in perceptions of parental expressivity are related to adolescents' mental well-being, specifically focusing on loneliness and depression. Analyzing data from 681 families (mean age of adolescents = 15.5 years old, 51.2% girls, 40% only-children) in China revealed that adolescents tended to perceive paternal and maternal emotional expressivity more negatively than their parents, particularly fathers. Polynomial regression and response surface analysis showed significant links between parent-adolescent congruence and incongruence and adolescent loneliness. (In)Congruence between adolescents and mothers or fathers predicted later adolescent depression, mediated by adolescent loneliness and varied by the dimension of emotional expressivity. These findings provide insights into the roles of mothers' and fathers' emotional expressivity in shaping children's mental well-being during adolescence.

Transcriptome Analysis Reveals the Effect of Stocking Density on Energy Metabolism in the Gills of Cherax quadricarinatus under Rice-Crayfish Co-Culture.

Stocking density is a crucial factor affecting productivity in aquaculture, and high stocking density is a stressor for aquatic animals. In this study, we aimed to investigate the effects of stocking densities on oxidative stress and energy metabolism in the gills of Cherax quadricarinatus under rice-crayfish farming. The C. quadricarinatus were reared at low density (LD), medium density (MD), and high density (HD) for 90 days. The results showed that the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) levels were higher in the HD group than those in the LD group. Transcriptomic analysis revealed 1944 upregulated and 1157 downregulated genes in the gills of the HD group compared to the LD group. Gene ontology (GO) enrichment analysis indicated that these differentially expressed genes (DEGs) were significantly associated with ATP metabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis also showed that high stocking density resulted in the dysregulation of oxidative phosphorylation. Furthermore, high stocking density upregulated six lipid metabolism-related pathways. Overall, our findings, despite the limited number of samples, suggested that high stocking density led to oxidative stress and dysregulation of energy metabolism in the gills of C. quadricarinatus under rice-crayfish co-culture. Alteration in energy metabolism may be an adaptive response to adverse farming conditions.

Dynamic changes in microbial community structure in farming pond water and their effect on the intestinal microbial community profile in juvenile common carp (Cyprinus carpio L.).

Water quality parameter dynamics, gut, sediment and water bacteria communities were studied to understand the environmental influence on the gut microbial community of a new strain of Huanghe common carp. A total of 3,384,078 raw tags and 5105 OTUs were obtained for the gut, water and sediment bacteria. The water quality had a stronger influence on the water bacteria community than gut and sediment bacteria communities. The ambient water quality parameters also significantly influenced the water and sediment bacteria communities. Comparing the gut, sediment, and water microbial communities, a relationship was found among them. However, gut bacteria were more closely related to sediment bacterial communities than to water bacteria communities. The results showed that the top three bacterial taxa were identical in gut and sediment samples in the early days of rearing. Interestingly, bacterial communities in the carp gut, water, and sediment had different adaptabilities to variations in environmental factors.

Apex Predators Enhance Environmental Adaptation but Reduce Community Stability of Bacterioplankton in Crustacean Aquaculture Ponds.

Aquaculture environments harbor complex bacterial communities that are critical for the growth and health of culture species. Apex predators are frequently added to aquaculture ponds to improve ecosystem stability. However, limited research has explored the effects of apex predators on the composition and function of bacterioplankton communities, as well as the underlying mechanisms of community assembly. Using 16S ribosomal RNA (rRNA) high-throughput sequencing, we investigated bacterioplankton communities of crustacean aquaculture ponds with and without apex predators (mandarin fish, Siniperca chuatsi) throughout the culture process. In addition to investigating differences in bacterioplankton communities, we also explored variations in environmental adaptation, functional redundancy, and community stability. Significant differences were observed in bacterioplankton composition among different cultural stages; there was an increase in Bacteriobota and fermentation-related bacteria, but a decrease in Firmicutes and pathogens in the middle stages of aquaculture. Apex predators increased the abundance of organic matter degradation bacteria and decreased pathogens. Bacterioplankton communities under apex predator disturbances had a wider environmental breadth, indicating broader environmental adaptation. Moreover, functional prediction and network analyses revealed that communities under apex predator disturbances were less functionally redundant and unstable. Based on the null model, stochastic processes drove community assembly during aquaculture, whereas apex predators elevated the contribution of deterministic processes. Greater changes in nitrate in culture ponds caused by apex predator disturbances were decisive in controlling the balance between stochasticity and determinism in community assembly. Our study provided insight into the mechanisms underlying bacterioplankton community assembly in aquaculture systems in response to apex predator disturbances.

Advancing chemokine research: the molecular function of CXCL8.

CXCL8 and other chemokines have been implicated in tissue inflammation and are attractive candidates for therapeutic targeting to treat human disease.

Effects of Berberine on Lipid Metabolism, Antioxidant Status, and Immune Response in Liver of Tilapia (Oreochromis niloticus) under a High-Fat Diet Feeding.

Berberine, a natural alkaloid found abundantly in various medicinal plants, exhibits antioxidative, anti-inflammatory, and lipid metabolism-regulatory properties. Nonetheless, its protective effects and the molecular mechanisms underlying liver injury in fish have not been fully elucidated. The aims of this study were to investigate the antioxidative, anti-inflammatory, and lipid metabolism-regulating effects of berberine against high-fat diet (HFD)-induced liver damage and to clarify the underlying molecular mechanisms. Tilapia were fed diets containing two doses of berberine (50 and 100 mg/kg diet) alongside high fat for 60 days. The results showed that berberine treatments (50 and/or 100 mg/kg) significantly reduced elevated aminotransferases, triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) in the plasma. In the liver, berberine treatments significantly increased the expression of peroxisome proliferator-activated receptor α (pparα) and carnitine palmitoyltransferase 1 (cpt-1) genes, leading to a reduction in lipid accumulation. Meanwhile, berberine treatment suppressed lipid peroxidation formation and enhanced antioxidant capacity. Berberine upregulated the mRNA levels of erythroid 2-related factor 2 (nrf2) and its downstream genes including heme oxygenase 1 (ho-1) and glutathione-S-transferase (gstα). Additionally, berberine attenuated the inflammation by inhibiting the expression of toll-like receptor 2 (tlr2), myeloid differential protein-88 (myd88), relb, and inflammatory cytokines such as interleukin-1ß (il-1ß), tumor necrosis factor-α (tnf-α), and il-8. In summary, this study suggested that berberine offers protection against HFD-induced liver damage in tilapia via regulating lipid metabolism, antioxidant status, and immune response. This protective effect may be attributed to the modulation of the Nrf2, TLR2/MyD88/NF-κB, and PPARα signaling pathways.

Influences of Stocking Density on Antioxidant Status, Nutrients Composition, and Lipid Metabolism in the Muscles of Cyprinus carpio under Rice-Fish Co-Culture.

Cyprinus carpio is a significant freshwater species with substantial nutritional and economic value. Rice-carp co-culture represents one of its principal cultivation methods. However, in the system, the optimal farming density for carp and the impact of high stocking density on their muscle nutritional composition have yet to be explored. Thus, the objective of the current study was to investigate the influences of stocking density on the muscle nutrient profiles and metabolism of C. carpio in rice-fish co-culture systems. Common carp were cultured at three stocking densities, low density (LD), medium density (MD), and high density (HD), over a period of 60 days. Following this, comprehensive analyses incorporating physiological, biochemical, and multi-omics sequencing were conducted on the muscle tissue of C. carpio. The results demonstrated that HD treatment led to a reduction in the antioxidant capacity of C. carpio, while resulting in elevated levels of various fatty acids in muscle tissue, including saturated fatty acids (SFAs), omega-3 polyunsaturated fatty acids (n-3 PUFAs), and omega-6 polyunsaturated fatty acids (n-6 PUFAs). The metabolome analysis showed that HD treatment caused a marked reduction in 43 metabolites and a significant elevation in 30 metabolites, primarily linked to lipid and amino acid metabolism. Additionally, transcriptomic analysis revealed that the abnormalities in lipid metabolism induced by high-stocking-density treatment may be associated with significant alterations in the PPAR signaling pathway and adipokine signaling pathway. Overall, our findings indicate that in rice-fish co-culture systems, high stocking density disrupted the balance of antioxidant status and lipid metabolism in the muscles of C. carpio.

Integrated rice-yellow catfish farming resulting in variations in the agricultural environment, rice growth performance, and soil bacterial communities.

Different rice production patterns exert varying comprehensive impacts on the agricultural environment. Integrated rice-fish farming, an advanced and rapidly developing agricultural production pattern, aims to improve resource utilization efficiency and enhance food productivity. To unravel the responses and internal interactions of the agricultural ecological environment to integrated rice-fish farming, we assessed and compared environmental factor, rice growth performance, and soil microbiome in both integrated rice-yellow catfish farming (IRYF) and rice monoculture (RM) systems. Our results revealed significant increases in the total nitrogen and ammonia concentrations in the paddy water and soil induced by the IRYF. Rice growth performance in the IRYF group surpassed that in the RM group. IRYF obviously impacted almost all dominant bacterial phyla, genera, and functional groups (top ten most abundant), enhancing the ability of bacteria to degrade and utilize organic matter. Additionally, IRYF led to noticeable reductions in the Shannon, Simpson, Chao 1, and Pielou_J indices. IRYF strengthened the interconnections between various taxonomic units in bacterial co-occurrence network, resulting in increased complexity, stability, and disturbance resistance in the soil bacterial community. IRYF notably facilitated the transition from a community assembly dominated by stochastic processes to one dominated by deterministic processes for the soil bacterial community. The deterministic process driving this transition was variable selection. All the environmental factors, except for soil nitrate, demonstrated relatively high contributions to alterations in soil bacterial communities, with environmental variables significantly positively correlated with the soil bacterial community in the IRYF group. Alterations in functionality, composition, and diversity of the soil bacterial community were clearly associated with most environmental variables and rice growth performance indices. Our research contributed to understanding the comprehensive impacts of integrated rice-fish farming on agricultural ecosystems and provide theoretical support for achieving the sustainable agricultural production and optimizing the rice production patterns.

Comparison of Intestinal Bacteria of Procambarus clarkii Farmed in Various Rice Paddy Regions.

The aim of this study was to assess the regional differences of Procambarus clarkii through analyzing gut microbiota in specimens from different areas in China. The P. clarkii were collected from ten integrated rice-crayfish farming systems locating across ten major producing areas as follows: Feixi (FX), Suqian (SQ), Yangzhou (YZ), Xuyi (XY), Qianjiang (QJ), Jianli (JL), Honghu (HH), Yueyang (YY), Changsha (CS), and Nanxian (NX). The composition of gut microbiota was assessed by analyzing 16S rRNA sequences. The PCoA results indicated significant differences in microbial community composition among the ten areas (R = 0.999, p = 0.001). The intestinal microbial diversity in P. clarkii cultured in rice fields from YY and CS exceeded that of other regions, with NX displaying the least diversity. At the phylum level, Proteobacteria were most abundant in HH, while Firmicutes showed increased relative abundances in FX and SQ, contrasted by lower relative abundances of Bacteroidetes in these areas. At the genus level, Ralstonia, Amedibacillus, Bacteroides, Anaerorhabdus, and Dysgonomonas were the dominant bacteria. The bacterial co-occurrence networks analysis revealed that the community structures in locations FX, SQ, XY, HH, and NX were comparatively simplistic, whereas those in the YZ, QJ, JL, YY, and CS regions displayed as more complex. In summary, the diversity and relative abundance of intestinal bacteria exhibits regional variability. These findings can offer theoretical data for evaluating the quality of P. clarkii aquaculture.

Temperature-dependent effects of microplastics on sediment bacteriome and metabolome.

The increasing prevalence of microplastics in the environment has become a concern for various ecosystems, including wetland ecosystems. Here, we investigated the effects of three popular microplastic types: polyethylene, polylactic acid, and tire particles at 5 °C and 25 °C on the sediment microbiome and metabolome at the 3% (w/w) level. Results indicated that temperature greatly influenced catalase and neutral phosphatase activities, whereas the type of microplastic had a more significant impact on urease and dehydrogenase activities. The addition of microplastic, especially tire particles, increased microbial diversity and significantly altered the microbial community structure and metabolic profile, leading to the formation of different clusters of microbial communities depending on the temperature. Nonetheless, the effect of temperature on the metabolite composition was less significant. Functional prediction showed that the abundance of functional genes related to metabolism and biogeochemical cycling increased with increasing temperature, especially the tire particles treatment group affected the nitrogen cycling by inhibiting ureolysis and nitrogen fixation. These observations emphasize the need to consider microplastic type and ambient temperature to fully understand the ecological impact of microplastics on microbial ecosystems.

Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. Here, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-day time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds the molecular foundation and a comparative single-cell genomic framework to identify neutrophil markers of tissue damage using model organisms.

The axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish.

Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the ALO is readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.

Effects of Stocking Density on the Growth Performance, Physiological Parameters, Antioxidant Status and Lipid Metabolism of Pelteobagrus fulvidraco in the Integrated Rice-Fish Farming System.

Pelteobagrus fulvidraco is a freshwater fish commonly raised in rice fields, yet the optimal stocking density for this species remains unknown. Therefore, this study aimed to investigate the appropriate stocking density of P. fulvidraco in integrated rice-fish farming systems. Three different stocking densities--low density (LD, 125 g/m2), middle density (MD, 187.5 g/m2), and high density (HD, 250 g/m2)--were set up to evaluate P. fulvidraco's growth performance, stress indices, immune function, antioxidant status, and lipid metabolism after 90 days of farming. The results indicated that HD treatment had a detrimental effect on P. fulvidraco's growth parameters. HD treatment led to an increase in cortisol (Cor) and lactate (La) levels, but a decrease in glucose (Glu) content in serum. After 90 days of farming, an immune response accompanied by the increase of complement 3 (C3), C4, and immunoglobulin M (IgM) was observed in the HD group. Meanwhile, HD treatment induced oxidative stress and altered antioxidative status evidenced by the levels of catalase (CAT), glutathione peroxidase (Gpx), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) in serum or liver. Additionally, the lipid metabolism-related genes including lipoprotein lipase (lpl), peroxisome proliferators-activated receptor (pparα), carnitine palmitoyltransferase-1 (cpt-1), and sterol regulatory element binding protein-1 (srebp-1) were markedly downregulated in the HD and/or MD group after 90 days of farming. In conclusion, this study contributes to a better understanding of P. fulvidraco's response to different stocking densities in integrated rice-fish farming systems. We suggest that the appropriate stocking density for P. fulvidraco in these farming systems should be below 250 g/m2, considering both fish growth and physiological responses.