Unraveling the ecological mechanisms of Aluminum on microbial community succession in epiphytic biofilms on Vallisneria natans leaves: Novel insights from microbial interactions.

The extensive use of aluminum (Al) poses an escalating ecological risk to aquatic ecosystems. The epiphytic biofilm on submerged plant leaves plays a crucial role in the regulation nutrient cycling and energy flow within aquatic environments. Here, we conducted a mesocosm experiment aimed at elucidating the impact of different Al concentrations (0, 0.6, 1.2, 2.0 mg/L) on microbial communities in epiphytic biofilms on Vallisneria natans. At 1.2 mg/L, the highest biofilms thickness (101.94 µm) was observed. Al treatment at 2.0 mg/L significantly reduced bacterial diversity, while micro-eukaryotic diversity increased. Pseudomonadota and Bacteroidota decreased, whereas Cyanobacteriota increased at 1.2 mg/L and 2.0 mg/L. At 1.2 and 2.0 mg/L. Furthermore, Al at concentrations of 1.2 and 2.0 mg/L enhanced the bacterial network complexity, while micro-eukaryotic networks showed reduced complexity. An increase in positive correlations among microbial co-occurrence patterns from 49.51% (CK) to 57.05% (2.0 mg/L) was indicative of augmented microbial cooperation under Al stress. The shift in keystone taxa with increasing Al concentration pointed to alterations in the functional dynamics of microbial communities. Additionally, Al treatments induced antioxidant responses in V. natans, elevating leaf reactive oxygen species (ROS) content. This study highlights the critical need to control appropriate concentration Al concentrations to preserve microbial diversity, sustain ecological functions, and enhance lake remediation in aquatic ecosystems.

Gastrodin protects porcine sertoli cells from zearalenone-induced abnormal secretion of glial cell line-derived neurotrophic factor through the NOTCH signaling pathway.

Zearalenone (ZEA) is a prevalent mycotoxin found in moldy diets and is associated with reproductive dysfunction. However, the molecular underpinning of ZEA in impairment of spermatogenesis remains largely unknown. To unveil the toxic mechanism of ZEA, we established a co-culture model using porcine Sertoli cells and porcine spermatogonial stem cells (pSSCs) to investigate the impact of ZEA on these cell types and their associated signaling pathways. Our findings showed that low concentration of ZEA inhibited cell apoptosis, while high concentration induced cell apoptosis. Furthermore, the expression levels of Wilms' tumor 1 (WT1), proliferating cell nuclear antigen (PCNA) and glial cell line-derived neurotrophic factor (GDNF) were significantly decreased in ZEA treatment group, while concurrently upregulating the transcriptional levels of the NOTCH signaling pathway target genes HES1 and HEY1. The addition of the NOTCH signaling pathway inhibitor DAPT (GSI-IX) alleviated the damage to porcine Sertoli cells caused by ZEA. Gastrodin (GAS) significantly increased the expression levels of WT1, PCNA and GDNF, and inhibited the transcription of HES1 and HEY1. GAS also efficiently restored the decreased expression levels of DDX4, PCNA and PGP9.5 in co-cultured pSSCs suggesting its potential in ameliorating the damage caused by ZEA to Sertoli cells and pSSCs. In conclusion, the present study demonstrates that ZEA disrupts pSSCs self-renewal by affecting the function of porcine Sertoli cell, and highlights the protective mechanism of GAS through the regulation of the NOTCH signaling pathway. These findings may offer a novel strategy for alleviating ZEA-induced male reproductive dysfunction in animal production.

Aluminum phytotoxicity induced structural and ultrastructural changes in submerged plant Vallisneria natans.

Aluminum (Al) is a concentration-dependent toxic metal found in the crust of earth that has no recognized biological use. Nonetheless, the mechanism of Al toxicity to submerged plants remains obscure, especially from a cell/subcellular structure and functional group perspective. Therefore, multiple dosages of Al3+ (0, 0.3, 0.6, 1.2, and 1.5 mg/L) were applied hydroponically to the submerged plant Vallisneria natans in order to determine the accumulation potential of Al at the subcellular level and their ultrastructural toxicity. More severe structural and ultrastructural damage was determined when V. natans exposed to ≥ 0.6 mg/L Al3+. In 1.2 and 1.5 mg/L Al3+ treatment groups, the total chlorophyll content of leaves significantly reduced 3.342, 3.838 mg/g FW, some leaves even exhibited chlorosis and fragility. Under 0.3 mg/L Al3+ exposure, the middle-age and young leaves were potent phytoexcluders, whereas at 1.5 mg/L Al3+, a large amount of Al could be transferred from the roots to other parts, among which the aged leaves were the most receptive tissues (7.306 mg/g). Scanning/Transmission electron microscopy analysis displayed the Al-mediated disruption of vascular bundle structure in leaf cells, intercellular space and several vegetative tissues, and demonstrated that Al in vacuole and chloroplast subcellular segregation into electron dense deposition. Al and P accumulation in the roots, stolons and leaves varied significantly among treatments and different tissues (P < 0.05). Fourier transform infrared spectroscopy of plant biomass also indicated possible metabolites (amine, unsaturated hydrocarbon, etc.) of V. natans that may bind Al3+. Conclusively, results revealed that Al3+ disrupts the cellular structure of leaves and roots or binds to functional groups of biological tissues, thereby affecting plant nutrient uptake and photosynthesis. Findings might have scientific and practical significance for the restoration of submerged vegetation in Al-contaminated lakes.

Combined Effects of Allelopathic Polyphenols on Microcystis aeruginosa and Response of Different Chlorophyll Fluorescence Parameters.

Polyphenols are allelochemicals secreted by aquatic plants that effectively control cyanobacteria blooms. In this study, sensitive response parameters (including CFPs) of Microcystis aeruginosa were explored under the stress of different polyphenols individually and their combination. The combined effects on M. aeruginosa were investigated based on the most sensitive parameter and cell densities. For pyrogallic acid (PA) and gallic acid (GA), the sensitivity order of parameters based on the EC50 values (from 0.73 to 3.40 mg L-1 for PA and from 1.05 to 2.68 mg L-1 for GA) and the results of the hierarchical cluster analysis showed that non-photochemical quenching parameters [NPQ, q N, q N(rel) and q CN] > photochemical quenching parameters [YII, q P, q P(rel) and q L] or others [F v/F m, F' v /F' m, q TQ and UQF(rel)] > cell densities. CFPs were not sensitive to ellagic acid (EA) and (+)-catechin (CA). The sensitivity order of parameters for M. aeruginosa with PA-GA mixture was similar to that under PA and GA stress. The quantitative (Toxicity Index, TI) and qualitative (Isobologram representation) methods were employed to evaluate the combined effects of PA, GA, and CA on M. aeruginosa based on cell densities and NPQ. TI values based on the EC50 cells suggested the additive effects of binary and multiple polyphenols, but synergistic and additive effects according to the EC50 NPQ (varied from 0.16 to 1.94). In terms of NPQ of M. aeruginosa, the binary polyphenols exhibited synergistic effects when the proportion of high toxic polyphenols PA or GA was lower than 40%, and the three polyphenols showed a synergistic effect only at the ratio of 1:1:1. Similar results were also found by isobologram representation. The results showed that increasing the ratio of high toxic polyphenols would not enhance the allelopathic effects, and the property, proportion and concentrations of polyphenols played an important role in the combined effects. Compared with cell densities, NPQ was a more suitable parameter as evaluating indicators in the combined effects of polyphenols on M. aeruginosa. These results could provide a method to screen the allelochemicals of polyphenols inhibiting cyanobacteria and improve the inhibitory effects by different polyphenols combined modes.

Response of Vallisneria natans to aluminum phytotoxicity and their synergistic effect on nitrogen, phosphorus change in sediments.

Increasing aluminum (Al) use and its effects on aquatic systems have been a global issue, however the Al impacts on submerged plants and their ecological functions were poorly understood. Aquatic simulation experiments were performed to study Al-toxicity on the germination and seedling morphological and physiological characteristics of Vallisneria natans, and investigate their synergistic effect on nitrogen (N), phosphorus (P) change and microbial community in sediment. The seeds germination characteristics, growth and physiological parameters of seedlings, including root activity, were significantly affected by alum treatments and the inhibition levels increased with Al3+ concentration. The Al accumulation in roots and leaves were significantly different. Al3+ concentration above 0.3 mg/L showed toxic to V. natans. TN, TP, IP, Fe/Al-P contents in sediments varied markedly under co-existence of Al and V. natans. Additionally, the relative abundance of sediment microbial community related to N, P cycle was effected. Results concluded that the increasing aquatic Al-concentration inhibits growth and propagation of submerged plants and the ecological restoration effect, and exerts synergistic effect with submerged plants on N, P components in sediments. Such findings were helpful for Al ecological evaluation, and were instructive for the submerged plants restoration in shallow eutrophic lakes with Al input.

Aluminum distribution heterogeneity and relationship with nitrogen, phosphorus and humic acid content in the eutrophic lake sediment.

Increasing amount of aluminum (Al) gets into aquatic ecosystem through anthropogenic activity, but the knowledge about Al migration and relationships with sediments possessing different physico-chemical properties in eutrophic lakes is limited. Here, the Al migration rule and relationships with sediment nutritions in the Hangzhou West Lake, China was investigated, where a certain amount of residual Al-salts can enter because of the pre-treatment of the Qiantang River diversion project every day. Results revealed the obvious spatial distribution heterogeneity of Al in sediment vertical direction and horizontal direction following water flow. The Al content in sediment ranged 0.463-1.154 g kg-1 in Maojiabu Lake, and ranged 9.862-40.442 g kg-1 in Xiaonanhu Lake. Higher Al content distributed in upper layer sediment in lake with more disturbance. Total nitrogen (TN) contents were higher 0.917-3.387 mg g-1 and 0.627-0.786 mg g-1 in upper layer sediment than that in lower layer in Maojiabu Lake and Xiaonanhu Lake, respectively. Total phosphorus (TP) content ranged 0.779-2.580 mg g-1, in which IP and Fe/Al-P contributed 24.9-80.8% and 17.0-51.6%, respectively. Correlations between Al content with nutrition, humic acid (HA) etc. of sediment regionally varied in Maojiabu and Xiaonanhu Lake. Spatial distribution of Al-salt in eutrophic lakes closely related with the physico-chemical characteristics of nutrients, humus, human disturbance and water division parameters. Results provides new insight into Al-salts migration and references for Al-risk evaluating in eutrophic lakes.

Impacts of residual aluminum from aluminate flocculant on the morphological and physiological characteristics of Vallisneria natans and Hydrilla verticillata.

Aluminate is generally used as a flocculant in water and wastewater treatment processes, but the residual aluminum (Al) may have toxic effects on aquatic organisms when the concentration accumulates beyond a threshold level. The in situ and laboratory tests were conducted to evaluate the impact of residual Al on submerged macrophytes in West Lake, Hangzhou, China, which receives Al flocculant-purified water diverted from the Qiantang River. The responses of Vallisneria natans and Hydrilla verticillata were investigated based on their morphological and physiological parameters in pot culture and aquarium simulation experiments. In the pot culture experiments, the biomass, seedling number, plant height, stolon number, stolon length, and root weight were significantly higher at a site located 150m from the inlet compared with those at a site located 15m from the inlet (P < 0.05), thereby indicating that the residual Al significantly inhibited the morphological development of V. natans and H. verticillata. The variations in the chlorophyll-a, protein, and malondialdehyde contents of the two species in both the pot culture and aquarium simulation experiments also demonstrated that the two submerged macrophytes were stressed by residual Al. V. natans and H. verticillata accumulated 0.052-0.227mg of Al per gram of plant biomass (fresh weight, mg/g FW) and 0.045-0.205mg Al/g FW in the in situ experiments, respectively, where the amounts of Al were significantly higher in the plants in the treatment aquaria during the laboratory experiments than those in the controls. These results may have important implications for the restoration of submerged macrophytes and ecological risk assessments in Al-exposed lakes. It is recommended that the Al salt concentration used for the control of lake eutrophication should be reduced to an appropriate level.

Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa.

N-Phenyl-1-naphthylamine (P1NA) and N-Phenyl-2-naphthylamine (P2NA) are both widely used as antioxidant and plant secondary metabolites. In this study, growth, esterase, photosynthetic activity and cell membrane integrity were used as biomarkers to compare biotoxicity of P1NA and P2NA on Microcystis aeruginosa. According to the results, a dose-response relationship was observed only between P1NA concentrations and growth inhibition. The EC50 (48 h) of P1NA calculated from growth inhibition was 16.62 µM, while that of P2NA was not detected. When the esterase and photosynthetic activity were applied to evaluate the biotoxicity, it was found that a concentration of 20 µM P1NA, P2NA caused reduction of esterase activity and Fv/Fm of M. aeruginosa to 22.2 and 3.3%, 97.5 and 92.1%, respectively, after 48 h exposure. The percentage of membrane-damaged cells was increased as P1NA exposure concentration increased, but that was not detected when exposure to P2NA. The difference substituted position in the molecular structure of P1NA and P2NA leads to different toxicological properties and only P1NA was found highly toxic to M. aeruginosa. The toxicity is due to that only P1NA can be biotransformed to 1,4-naphthoquinone, which could induce overproduction of intracellular ROS as well as result in oxidative damage and growth inhibition of test organism.

Effects of pyrogallic acid on Microcystis aeruginosa: oxidative stress related toxicity.

Pyrogallic acid (PA) is used in various industrial and consumer products. The molecular mechanisms underlying PA's toxicity was not fully understood. In this study, toxicity of PA on Microcystis aeruginosa with reactive oxygen species (ROS) generation as an end point was investigated. The results showed an increase in the percentage of cells with loss of membrane integrity and enhanced intracellular ROS production. Exposure to 50mgL(-1) PA for 48h caused the highest percentage of loss of membrane integrity (56.7%), and a 2.54-fold higher intracellular ROS level compared to control. Further investigation revealed that PA caused a dose-dependent increase in DNA strand breaks (DSB) of M. aeruginosa at exposure concentration from 2 to 50mgL(-1). The incubation of cells with ROS scavengers ascorbic acid, N-acetyl-l-cysteine (NAC) and tocopherol markedly alleviated the level of PA-induced DSB. Analysis of PA autoxidized products in culture solution showed that PA was quickly converted to purpurogallin (PG), and PG was further autoxidized to other polyphenolic compounds. PA and PG might participate a futile redox cycle, which mediated ROS production in M. aeruginosa. These results suggested DNA strands and cell membrane were two targets of ROS induced by PA, and oxidative damage was an important mechanism for the toxicity of PA against M. aeruginosa.

Chloro-benzoquinones cause oxidative DNA damage through iron-mediated ROS production in Escherichia coli.

Chloro-benzoquinones (CBQs) are a group of disinfection byproducts that are suspected to be potentially carcinogenic. Here, the mechanism of DNA damage caused by CBQs in the presence of ferrous ions was investigated in an Escherichia coli wild type M5 strain and a mutant L5 (ahpCF katEG mutant) strain that carried an enhanced green fluorescent protein reporter under the control of a SOS response gene (recA) promoter. All tested CBQs (including para-benzoquinone, 2-chloro-para-benzoquinone, and dichloro-para-benzoquinones with different substitutes) caused substantial oxidative DNA damage with EC50 values in the micromolar range. Moreover, 2,5-dichloro-para-benzoquinone (2,5-DCBQ), a typical CBQ, caused substantial ROS production in E. coli mutant cells. And ROS scavengers provided partial protective effects on genotoxicity of 2,5-DCBQ to E. coli mutant cells. The addition of Fe(2+) to the 2,5-DCBQ exposure system caused an increase in DNA oxidative damage; iron-chelating agents could partially prevent these cells from DNA damage. Finally, intracellular AhpCF, catalase E, and catalase G were all found to play an important role in the survival of E. coli cells exposed to CBQs, as indicated by an increased sensitivity of the ahpCF katEG mutant L5 strain to treatment compared with wild type M5 cells. Taken together, these results suggest that CBQs cause oxidative DNA damage in E. coli cells through the participation of iron-mediated ROS production.

Generation of reactive oxygen species in cyanobacteria and green algae induced by allelochemicals of submerged macrophytes.

Inhibition of phytoplankton by allelochemicals released by submerged macrophytes is reported to be one of the mechanisms that maintain a clear-water state in shallow lakes. In order to elucidate this mechanism, the ability of six polyphenols and two long-chain fatty acids to induce the generation of reactive oxygen species (ROS) in phytoplankton was studied using the ROS sensitive probe 2',7'- dichlorodihydrofluorescein diacetate (DCFH-DA). The results showed that only (+)-catechin (CA) and pyrogallic acid (PA) could induce ROS formation in Microcystis aeruginosa and Pseudokirchneriella subcapitata. 25 mg L⁻¹ CA caused 1.2, 1.4 and 1.8 times increase of ROS levels in M. aeruginosa at 1, 2 and 4h exposure, respectively, and, correspondingly in P. subcapitata cells, these values were 3.7, 6.2 and 7.7, respectively. PA also significantly increased the levels of intracellular ROS in P. subcapitata (P < 0.01); however, significant ROS generation in M. aeruginosa was observed at only 4h exposure (P < 0.01). Light enhanced ROS generation in CA treated cells, but not in the cells treated with PA. CA and PA may act as redox cyclers after uptake by test organisms and produce ROS successively. These results suggest that the oxidative stress induced by the redox cycling property of allelochemicals may be one of the important causes for the inhibitory effect of some submerged macrophytes towards undesired phytoplankton in natural aquatic ecosystems.

Study on the mechanism of allelopathic influence on cyanobacteria and chlorophytes by submerged macrophyte (Myriophyllum spicatum) and its secretion.

For revealing the mechanism of allelopathic influence on phytoplankton by aquatic macrophytes, the growth and photosynthetic activities of cyanobacteria Microcystis aeruginosa and the chlorophyte Selenastrum capricornutum were investigated when they coexisted with submerged macrophyte Myriophyllum spicatum and were exposed to allelopathic polyphenols: pyrogallic acid (PA), gallic acid (GA), ellagic acid (EA) and (+)-catechin (CA). According to the results of coexistence assays, the non-photochemical quenching (NPQ) and effective quantum efficiency (YII) of M. aeruginosa were affected earlier and more rapidly than the cell density. However, the influence of M. spicatum on S. capricornutum was not found. When the Toxicity Index (TI) was applied to evaluate the combined effects of binary and multiple mixtures of polyphenols, it was found that the four tested polyphenols with the proportion identified in the M. spicatum-cultured solution were observed to present synergistic effect (0.36-0.49) according to the cell density, NPQ and YII of M. aeruginosa. With the combined effects of polyphenols on S. capricornutum, only additive action (0.52-1.62) was found. On the other hand, PA (2.97mgL(-1)), GA (2.65mgL(-1)) caused significant reductions of photosystem II (PSII) and whole electron transport chain activities of M. aeruginosa by 71.43 and 18.37%, 70.95 and 40.77% (P<0.05), respectively, after 24-h exposure, but no inhibition effect was found in S. capricornutum. The dark respiration and photosystem I (PSI) activities of M. aeruginosa were significantly increased by exposure to PA and GA (P<0.05). Nevertheless, EA and CA had no influence on the electron transport activities of the tested organisms. These results indicate that the reduction in photosynthetic activity of M. aeruginosa and the synergistic effect of allelochemicals may be two important causes for the inhibition of undesired phytoplankton by submersed macrophytes in natural aquatic ecosystems, and PSII in cyanobacteria is considered to be one of the target sites attacked by allelopathic polyphenols.