Nasonia vitripennis females are typically monandrous and become sexually unreceptive after mating, but the molecular basis for this switch remains unclear. In this study, transcriptome analysis revealed a novel role for dopamine (DA) in modulating this switch. Courtship significantly upregulated DA-associated gene expression and increased DA levels in virgin females, leading to a loss of preference for male sex pheromones. More importantly, guarding (postcopulatory courtship) further elevated DA levels in females, resulting in sexual unreceptivity in mated females. Virgin females lost their preference for male sex pheromones after their DA levels were artificially elevated, and females were willing to mate multiple times when their DA receptors were antagonized. The switch in female pheromone preference caused by male oral pheromones (three fatty acid ethyl esters) was also mediated by elevated DA levels in females. In conclusion, our results indicate that DA plays a key role in the sexual receptivity switch of female N. vitripennis after mating. (c) 2026 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Copper homeostasis is critical for cellular function, yet its dysregulation triggers cytotoxicity through poorly defined mechanisms. Recent studies have identified cuproptosis, a novel copper-driven cell death pathway linked to mitochondrial metabolism, in mammalian systems. However, its conservation in eukaryotes remains unexplored. Here, we establish Tetrahymena thermophila as a model for studying copper toxicity and demonstrate that the copper ionophore elesclomol (ES) synergizes with copper to induce cuproptosis-like cell death. ES significantly enhances the toxicity of Cu (IC50 decreased from 1.126 mM to 0.451 μM) and this effect is strongly correlated with increased intracellular copper levels. Transcriptomic, metabolic and ultrastructural analyses show that ES-Cu disrupts mitochondrial dynamics, induces oxidative stress and DNA damage, and reprograms energy metabolism, culminating in non-apoptotic cell death. Dynamin-related protein 1 (DRP1)-mediated mitochondrial fission emerges as a key regulatory hub in T. thermophila in response to ES-Cu exposure. These findings underscore cuproptosis as an evolutionarily conserved pathway while highlighting species-specific adaptations, thereby advancing our understanding of copper toxicity and its therapeutic potential.

Nanoplastics (NPs) are prevalent throughout the environment and have raised growing environmental concerns. Although numerous studies have examined the toxicological aspects of NPs, few have investigated their environmental fate and behavior when affected by organisms other than bacteria or fungi. Planktonic ciliates are essential components of aquatic ecosystems and play important roles in decomposing organic matter and transferring energy from the microbial food web to higher trophic levels. To investigate the interplay between NPs and the ciliate Tetrahymena thermophila, we executed a sequence of feeding experiments utilizing 50 nm polystyrene nanoplastics (PS-NPs). In the presence of sufficient nutrition, exposure to PS-NPs (even at concentrations up to 500 mg/L) did not significantly inhibit growth in Tetrahymena thermophila, indicating only a mild toxic effect of PS-NPs. When ingested by T. thermophila, the PS-NPs are repackaged into aggregates with lysosomal components in the food vacuole and finally expelled as compacted "fecal pellets". This process modifies the physical attributes of PS-NPs, including their hydrophilicity, aggregability, and buoyancy, influencing their transportation, retention, deposition dynamics, and ultimately their bioavailability within the environment. A total of 73 proteins were identified from the fecal pellets, containing various hydrolases. Gel permeation chromatography (GPC), Fourier transform infrared (FTIR), and thermogravimetric analysis (TGA) were used to identify changes in molecular weights, functional groups, and thermal stabilities of PS-NP residues in fecal pellets. The results verified the degradation of PS-NPs during the passage through the T. thermophila cell.

Although fosthiazate (FOS) is a novel chiral organophosphorus nematicide, its enantioselective toxicity to Bombyx mori remains inadequately characterized. This study systematically evaluated the acute and developmental toxicity of FOS stereoisomers by determining LC50 values and assessing enzyme activity, oxidative stress biomarkers, growth metrics, and silk gland histopathology. Acute toxicity assays revealed profound stereoselectivity; specifically, (1S,3R)-fosthiazate (SR-FOS) and (1S,3S)-fosthiazate (SS-FOS) exhibited toxicity more than 10-fold higher than the other stereoisomers. Following sublethal exposure (8.54 mg/L), SR-FOS and SS-FOS significantly inhibited acetylcholinesterase (AChE) activity, upregulated the expression of ace1 and ace2 genes by 2.46- to 7.35-fold, and induced severe oxidative stress, marked by a 38.02-62.61 % increase in ROS, a 28.75-65.18 % increase in SOD activity, a 34.96-110.93 % increase in CAT activity, and a 1.91- to 3.88-fold elevation in MDA levels. In addition, developmental toxicity also varied significantly among stereoisomers. SR-FOS and SS-FOS reduced pupal weight by 16.82-17.76 % and cocoon shell weight by 21.88-28.13 %. These stereoisomers also caused severe damage to the silk glands, accompanied by a 1.21- to 6.45-fold downregulation of genes encoding fibroin (Fib-H, Fib-L), sericin (Ser1, Ser2, Ser3), and the silk gland factor 1 (SGF1), indicating that disrupted silk synthesis is a key mechanism of chronic toxicity. Furthermore, UPLC-MS/MS results showed that the residues of the four stereoisomers in silkworm excrement also showed significant differences (RS/RR-FOS > SR/SS-FOS). These findings establish critical structure-activity relationships for FOS, which are essential for the risk assessment of chiral pesticides.

The single von Willebrand factor C-domain proteins (SVWCs) are primarily found in arthropods and are involved in responding to environmental stress, nutritional availability, and pathogen infection. In this study, an SVWC family member from the Macrobrachium nipponense was identified that contains a conserved single von Willebrand factor C domain that is highly expressed in the nerve cord (designated MnSVWC2). The role of MnSVWC2 in resistance to bacteria and viruses was investigated. MnSVWC2 is upregulated in response to both bacterial challenge and viral infection. The recombinant MnSVWC2 (rMnSVWC2) exhibited binding activity to a range of pathogen-associated molecular patterns (PAMPs). Furthermore, it exhibited Ca2+-dependent binding and agglutination capabilities against Gram-negative, Gram-positive bacteria and yeast. The co-incubation of rMnSVWC2 with E. coli, followed by injection into prawns, resulted in an increase in the phagocytosis of E. coli by hemocytes in vivo. ELISA analysis indicated that rMnSVWC2 can bind to white spot syndrome virus. Knockdown of MnSVWC2 by RNA interference (RNAi) resulted in an increase in virus copies in prawns and a significant decrease in survival rate following viral infection. These findings provide important insights into the function of MnSVWC2 in the innate immunity of M. nipponense and the mechanisms of defense against pathogens.

Salinization of inland waters, driven by climate change and human activities, poses a major threat to aquatic ecosystems. While species can swiftly adapt to environmental stress, the molecular mechanisms underpinning this adaptation remain to be fully elucidated. This study seeks to clarify the complex adaptive strategies employed by the freshwater ciliate Tetrahymena thermophila in response to chronic salt stress through the methodologies of experimental evolution and multi-omics integration. The findings indicate that three lineages adapted to salt (ST-4, ST-8, and ST-12), which evolved under a regime of increasing NaCl concentration, demonstrated a trade-off between delayed growth and osmotic resilience. Transcriptomic and proteomic analyses revealed key evolutionary priorities, including (i) the co-upregulation of pathways related to DNA replication, glutathione metabolism, and endoplasmic reticulum (ER) protein processing, (ii) the suppression of lipid catabolism alongside the accumulation of lipid droplets mediated by START2, and (iii) mitochondrial remodeling through the expansion of ER contacts to sustain ATP production. Interestingly, the adaptation to salt appears to tolerate genome instability induced by replication stress through the dysregulation of replisome components, specifically the upregulation of Prim1 and downregulation of LIG, while also evading antioxidant defenses via the compartmentalization of oxidative damage. These results contribute to a framework in which protists effectively balance lipid-mediated osmoregulation, controlled mutagenesis, and organelle metabolism to navigate salinity challenges, thereby offering predictive insights into microbial adaptation thresholds within evolving ecosystems.IMPORTANCESalinization of inland waters is a growing concern due to climate change and human activities. Understanding how organisms adapt to saline environments is vital. Tetrahymena thermophila, a model organism, was studied to explore its adaptation mechanisms. The findings show that through gene regulation, it can acclimate to high salt conditions. The role of mitochondria in metabolic reprogramming during this process is significant. This research contributes to a more profound understanding of how organisms adapt to saline stress and the molecular mechanisms underlying such adaptations, which may aid in predicting and managing the impacts of salinization on aquatic ecosystems.

The activation of the immune system by pathogens imposes significant energetic costs on hosts, which may result in the diversion of resources away from other non-essential biological processes, such as growth and reproduction. The underlying mechanisms of trade-offs between immune responses and host fitness remain poorly understood. We used a Musca domestica mutant (pirk-KO) to evaluate the influence of non-infection-induced immune system activation on female reproduction and larval growth. Pirk, a negative feedback inhibitor of the immune deficiency (Imd) pathway expressed in intestine and fat body, was induced by bacteria. pirk loss enhanced the immune response of house flies, reflected in sustained upregulated antimicrobial peptide gene expression and improved resistance to bacterial infections. The phenotypic traits of pirk-KO house flies, including delayed larval growth, reduced the body weight, and impaired female fertility, were indicative of the adaptive costs associated with aberrant immune activation. The transcriptional heterogeneities between pirk-KO and wild-type (WT) male flies indicated the overactivation of the Imd signaling pathway, accompanied by significant metabolic adaptations to the loss of pirk. The upregulation of pivotal genes involved in glycolysis and the TCA cycle indicated an enhanced central carbon metabolism in pirk-KO. The downregulation of multiple key enzymes involved in the pentose phosphate pathway in pirk-KO flies suggests a reduction in metabolic flux through the pentose phosphate pathway, which in turn results in impaired anabolism. The collective findings indicate that the pirk-KO flies undergo metabolic reprogramming to increase ATP production as a response to excessive immune activation, rather than incorporating nutrients into cellular biomass for cell proliferation. The pirk-KO flies exhibited a significantly elevated food intake and elevated levels of free glucose, trehalose, and fructose in comparison to the WT flies. Nevertheless, the glycogen and triglyceride contents in the pirk-KO flies were observed to be slightly diminished in comparison to the WT group. When the immune defense is activated, the flies extract more free energy to fuel the immunological deployment by increasing nutrient intake, as well as reducing resource allocation to non-essential life-history traits, primarily reproduction and growth.

Antimicrobial peptides (AMPs) are increasingly being recognised as promising alternatives to conventional antibiotics due to their distinctive antimicrobial mechanisms and reduced likelihood of inducing drug resistance. Insects represent a significant source of AMPs. In this study, a potential AMP gene, MdAMP5, was identified based on its strong immunoinducibility and the presence of a signal peptide, and an amphipathic α-helix in the encoded protein. MdAMP5 encoded a 50-amino acid precursor protein with an N-terminal 22-amino acid signal peptide. The calculated molecular mass of the mature protein was 2.92 kDa, with an estimated isoelectric point of 6.23. Structural analyses revealed that the N-terminus of mature MdAMP5 contained an irregularly coiled segment, while the C-terminus featured an amphipathic α-helix with a glycine-lysine residue at the end. Furthermore, the MdAMP5 gene was successfully expressed in the yeast Pichia pastoris, and the recombinant MdAMP5 (rMdAMP5) protein exhibited effective and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria in vitro and in vivo. Treatment with rMdAMP5 resulted in significant changes in bacterial morphology, including cell lysis and deformation of bacteriophages. In conclusion, this study identified and successfully expressed a novel AMP that showed low cytotoxicity to mammalian cells and high selectivity towards bacterial cells. This research offers a new candidate for therapeutic drug development, and enhances the understanding of the mechanism and application of AMPs.

Oocyte aging is closely related to a decline in female fertility, accompanied by increased reactive oxygen species levels and changes in protein posttranslational modifications. However, the role of protein palmitoylation in oocyte aging has not been investigated. In the present study, a new association between redox and palmitoylation in aging oocytes was found. We found that the protein level of palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, was increased in maternally aged mice oocytes and follicular fluid of aged (age >35 years) patients with decreased ovarian reserve (DOR). Elevated PPT1 led to decreased S-palmitoylation levels in oocytes, which impaired oocyte maturation and spindle formation. Tubulin was identified as a critical palmitoylated protein regulated by PPT1, whose palmitoylation was also decreased by advanced age, accompanied by abnormalities in membrane localization and microtubule polymerization. Melatonin was found to down-regulate excessive PPT1 and rescue PPT1-induced damage in mouse oocytes, not only by regulating oxidative stress, but also by binding with PPT1 to regulate its lysosomal degradation. In summary, our data demonstrate that PPT1 participates in oocyte aging by regulating tubulin palmitoylation, providing evidence that oxidative stress regulates protein palmitoylation and revealing a novel mechanism of oocyte aging.

Metallothioneins (MTs) play pivotal roles in metal detoxification and homeostasis across diverse organisms. However, their functional diversity and evolutionary adaptations in non-model insects remain poorly characterized. Here, we systematically identified the MT gene family in the globally distributed sanitary pest Musca domestica and dissected its role in heavy metal stress responses. Genomic and transcriptomic analyses revealed ten MT genes (MdMTs), with MdMT1 exhibiting the highest basal expression in larvae and adults. Phylogenetically, MdMT1 clusters with dipteran orthologs and features a conserved β-domain architecture with cysteine-rich motifs. Exposure to Cd2+ or Cu2+ induced concentration-dependent MdMT1 upregulation, peaking at 3.0-fold (0.3 mM Cd2+) and 3.8-fold (3 mM Cu2+). Recombinant MdMT1 (rMdMT1) enhanced metal and oxidative tolerance in Escherichia coli, and isothermal titration calorimetry confirmed high-affinity Cd2+ binding but negligible Cu2+ binding. CRISPR/Cas9-mediated knockout of MdMT1 rendered M. domestica hypersensitive to Cd2+ and Cu2+, as evidenced by a significant reduction in survival compared to wild-type controls. Consistent with this observed phenotype, MdMT1 deficiency led to a significantly enhanced accumulation of Cd2+ and Cu2+ ions. Consequently, the loss of MdMT1 resulted in elevated levels of reactive oxygen species and malondialdehyde, indicating impaired antioxidant defense. Collectively, these findings establish MdMT1 as a key mediator of heavy metal detoxification in M. domestica, offering insights into pest adaptation to metal-contaminated environments.

Afforestation projects using species mixtures are expected to better support ecosystem services than monoculture plantations. While grassland studies have shown natural selection favoring high-performance genotypes in species-rich communities, this has not been explored in forests. We used seed-family identity (known maternity) to represent genetic identity and investigated how this affected the biomass accumulation (i.e. growth) of individual trees (n = 13 435) along a species richness gradient (1-16 species) and over stand age (9 yr) in a forest biodiversity experiment. We found that among the eight species tested, different seed families responded differently to species richness, some of them growing relatively better in low-diversity plots and others in high-diversity plots. Furthermore, within-species growth variation increased with species richness and stand age, while between-species variation decreased with stand age. These results indicate that seed families within species and their reaction norms along the species richness gradient vary considerably and thus can explain a substantial proportion of the overall variation in tree growth. Our findings suggest that the growth and associated ecosystem services of species-rich mixtures in afforestation projects can be optimized by artificially selecting seed families with high mixture performance in biodiversity experiments.

Hemocytes represent a pivotal element of insect defense against pathogens through the mechanisms of cellular and humoral immunity. However, hemocyte types and functions exhibit variation across different insect taxa, leading to the lack of a standardized classification system for insect hemocytes. To gain insight into the immune mechanisms of the house fly Musca domestica, we used a combination of morphological observations and single-cell RNA sequencing techniques (scRNA-seq) to taxonomically characterize house fly larval hemocytes and analyze their immune function. As a result, five different types of hemocytes were identified from house fly larvae, granulocytes (GR), spherulocytes (SP), plasmatocytes (PL), prohemocytes (PR), and oenocytoids (OE). On the basis of microscopic observations, flow cytometry analysis and gene expression profiles, the immune functions of house fly hemocytes were hypothesized to be as follows: the GRs are responsible for phagocytosis, the SPs are highly expressive of lectins, and the PLs are highly expressive of antimicrobial peptides (AMPs) and are involved in nodule formation, the PRs act as progenitor cells and retain the differentiation potential of stem cells, and the OEs are involved in melanization reactions mainly through the expression of phenoloxidase (PO). Based on the scRNA-seq data, the marker genes for each type of hemocyte were identified. The present study unveils the heterogeneity of house fly hemocytes in terms of morphology, gene expression characteristics, and function, thereby establishing the foundation for an in-depth understanding of the immune mechanisms in house fly.

Ofloxacin (OFL), a fluoroquinolone antibiotic, has attracted attention due to its extensive use and frequent environmental detection, posing ecological risks in aquatic systems. Herein, a multilevel ecotoxicity assessment of OFL (0.01, 0.1, 1, and 10 mg/L) was conducted on an ecotoxicology model, Daphnia magna, using a 21-day chronic test. The involved parameters included mitochondrial function, metabolites, digestive enzymes, oxidative stress, development, reproduction, locomotor behavior, and multi-omic analysis. Environmentally relevant concentration of OFL (0.01 mg/L) exposure elicited detrimental effects on redox homeostasis, intestinal flora, and lipid metabolism in D. magna. It is noteworthy that hormetic effects were observed in reproductive capacity in D. magna exposed to OFL. Furthermore, exposure to elevated contamination concentrations of OFL (0.1, 1, and 10 mg/L) led to pronounced oxidative stress in D. magna, resulting in gut damage and reduced digestive enzymes and metabolites. Notably, exposure to 10 mg/L OFL induced marked mitochondrial dysfunction and shortage of lipid metabolites in the D. magna, leading to the destruction of energy supply and impairment of locomotor, development, and reproduction. This study comprehensively assessed the aquatic ecological risks of OFL and provided insights into the mechanisms underlying the toxicity of OFL on the development, reproduction, and energy allocation of D. magna.

Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

Spermatogenesis is critical for insect reproduction and is regulated by many different genes. In this study, we found that Forkhead transcription factor Fd59a functions as a key factor in the spermatogenesis of Drosophila melanogaster. Fd59a contains a conversed Forkhead domain, and it is clustered to the FoxD subfamily with other FoxD members from some insect and vertebrate species. Mutations in Fd59a caused swelling in the apical region of the testis. More importantly, fewer mature sperm were present in the seminal vesicle of Fd59a mutant flies compared to the control flies, and the fertility of Fd59a2/2 mutant males was significantly lower than that of the control flies. Immunofluorescence staining showed that the homeostasis of the testis stem cell niche in Fd59a2/2 mutant and Fd59a RNAi flies was disrupted and the apoptosis of sperm bundles was increased. Furthermore, results from RNA sequencing and qRT-PCR suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Taken together, our results indicated that Fd59a plays a key role in the spermatogenesis of Drosophila.

Drosophila NF-κB transcription factor Dif has been well known for its function in innate immunity, and recent study also reveals its role in neuronal cells. However, the underlying mechanisms of Dif in the brain remain elusive. In this study, we aim to investigate the function of Dif in Drosophila brain development and how Dif regulates structure and plasticity of the brain to affect aging and behaviors. Based on the analysis of differentially expressed genes, we identified key genes associated with cell division, development and aging in the brain of Dif1 loss of function mutant. In Dif1 larvae, we found that the metamorphosis and brain development were delayed, and cell division was decreased. In Dif1 adults, the number of neuron cells was reduced in the brain, the lifespan and locomotor activity were decreased, protein markers associated with aging-related neurodegenerative diseases in the brain were altered in abundance or activity. Our results indicated that Dif plays a crucial role in brain plasticity and neurogenesis, dysfunction of Dif delays larval brain development and impacts proliferation of neuronal cells, resulting in aging adult brain by regulating expression of key genes in multiple signaling pathways involved in cell division, neurogenesis and aging.

The muscle LIM protein (MLP) is a member of the cysteine and glycine-rich protein (CSRP) family, composed of CSRP1, CSRP2 and CSRP3/MLP. MLP is involved in a multitude of functional roles, including cytoskeletal organization, transcriptional regulation, and signal transduction. However, the molecular mechanisms underlying its involvement in immune and stress responses remain to be elucidated. This study identified an MnMLP in the freshwater crustacean Macrobrachium nipponense. The isothermal titration calorimetry assay demonstrated that recombinant MnMLP was capable of coordinating with Zn2+. Upon challenge by Aeromonas veronii or WSSV, and exposure to CdCl2, up-regulation was recorded in the muscle and intestinal tissues, suggesting its involvement in immune and anti-stress responses. MnMLP protein was predominantly expressed in the cytoplasm of the transfected HEK-293T cells, but after treatment with LPS, Cd2+ or H2O2, the MnMLP was observed to be transferred into the nucleus. The comet assay demonstrated that the overexpression of MnMLP could mitigate the DNA damage induced by H2O2 in HEK-293T cells, suggesting the potential involvement of MnMLP in the DNA repair process. These findings suggest that DNA repair may represent a possible mechanism by which MnMLP may be involved in the host's defense against pathogens and stress.

Paris polyphylla var. yunnanensisis a perennial herb with significant medicinal properties in anticancer, anti-inflammatory, antibacterial immunomodulatory and antispasmodic activities (Duan et al. 2018). In April 2022, leaf blight disease emerged in Xiangtan City (Hunan), affecting P. polyphylla plantings over an area of 3×104 m2 (27.904°N, 112.918°E). The disease incidence reached an average of 22% of the plants in the field, with infected plants initially displaying water-soaked chlorosis, followed by dry yellow shrinkage that gradually spread from the leaf tips to the entire plant. To identify the causal agent, 20 leaf lesions (4 mm2) collected from 20 plants were surface sterilized with 75% ethanol for 10 s, 5% NaOCl for 30 s, rinsed in sterile distilled water three times, transferred to potato dextrose agar (PDA) plates with lactic acid (0.125%) , and incubated at 28 °C in the dark. Four isolates (PP21 to PP24) with similar morphologies were obtained and purified by the hyphal-tip method. Colonies on PDA initially appeared white with cottony mycelium, later turning light-yellow on the underside. Septate hyphae were branched at right angles with a small constriction at the departure of the branch point and measured 4.16 to 7.93 µm in diameter. Binucleate cells within the septate hyphae were visualized using Giemsa staining (Servicebio, China). For molecular identification, the rDNA internal transcribed spacer (ITS), the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2), and ATP synthase subunit 6 (ATP6) were amplified from genomic DNA of the isolates extracted by Fungus Genomic DNA Extraction Kit (Bioflux, China) using primers ITS1/ITS4 (White et al. 1990), bRPB2-6F/bRPB2-7.1R (Matheny 2005; Reeb et al. 2004) and ATP61/ATP62 (Kretzer and Bruns 1999), respectively. The ITS, RBP2 and ATP6 of the four isolates were sequenced and deposited in GenBank. BLASTn search of sequenced ITS (PQ187050, PQ187051, PP728052, PQ187052), RBP2 (PQ202833, PQ202834, PP735921, PQ202835) and ATP6 (PQ202837, PQ202838, PP735922, PQ202839) revealed a >99% identity with the type strain of Ceratobasidium ramicola CBS133.82 (NR138368, DQ301708, and DQ301577). For phylogenetic analysis, concatenated sequences of ITS, RBP2, and ATP6 were employed using the maximum-likelihood method in MEGA-X. Based on the morphological and molecular analyses, the isolates were classified into the C. ramicola clade (Bandoni 1979; Samuels et al. 2012; Jeong et al. 2023). To test the pathogenicity of the isolate PP23, mycelial plugs (5 mm in diameter) were placed directly on 15 healthy leaves from 15 three-year-old plants after puncturing with sterile needles. Sterile PDA plugs served as controls. All plants were kept in a greenhouse with conditions of 25°C, 80% relative humidity and a photoperiod of 12 h. After 5 days, all infected plants developed leaf blight symptoms similar to those described above, whereas the control plants remained asymptomatic. The pathogenicity test was conducted three times. C. ramicola was re-isolated from all symptomatic leaves and confirmed to be identical to the original isolate based on morphology and nucleotide sequences of ITS, RBP2, and ATP6. While C. ramicola is commonly reported to be isolated from diseased cacao, its pathogenicity to cacao remains unknown (Samuels et al. 2012). To our knowledge, this is the first report of C. ramicola causing leaf blight on P. polyphylla, a medicinal herb with significant economic importance in China.

Manganese (Mn) is an essential element for plants and plays a role in various metabolic processes. However, excess manganese can be toxic to plants. This study aimed to analyze the changes in various physiological activities and the transcriptome of Arabidopsis under different treatments: 1 mmol/L MnCl2 treatment for 1 day or 3 days, and 1 day of recovery on MS medium after 3 days of MnCl2 treatment. During the recovery phase, minor yellowing symptoms appeared on the leaves of Arabidopsis, and the content of chlorophyll and carotenoid decreased significantly, but the content of malondialdehyde and soluble sugar increased rapidly. Transcriptome sequencing data shows that the expression patterns of differentially expressed genes exhibit three major models: initial response model, later response model, recovery response model. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis identified several affected metabolic pathways, including plant hormone signal transduction mitosolysis activates protein kinase (MAPK) phytohormone signaling, phenylpropanoid biosynthesis, ATP binding cassette transporters (ABC transporter), and glycosphingolipid biosynthesis. Differential expressed genes (DEGs) involved in phenylpropanoid biosynthesis, ABC transporter, and glycosphingolipid biosynthesis, were identified. Sixteen randomly selected DEGs were validated through qRT-PCR and showed consistent results with RNA-seq data. Our findings suggest that the phenylpropanoid metabolic pathway is activated to scavenge reactive oxygen species, the regulation of ABC transporter improves Mn transport, and the adjustment of cell membrane lipid composition occurs through glycerophospholipid metabolism to adapt to Mn stress in plants. This study provides new insights into the molecular response of plants to Mn stress and recovery, as well as theoretical cues for cultivating Mn-resistant plant varieties.

Panax notoginseng is an important Chinese medicinal plant. Saponins are the major bioactive secondary metabolites with a wide range of medicinal and commercial value in P. notoginseng, so it is crucial to develop environmentally friendly methods to increase their production. The symbiotic relationship between endophytic bacteria and host plants offers a sustainable approach to enhance secondary metabolite biosynthesis. In this study, it was reported that the co-cultivation of an endophytic bacterium Enterobacter cloacae PN7, isolated from P. notoginseng and its host plant could greatly promote saponin accumulation in the root of seedlings. After six days of PN7 treatment, the total saponin concentration reached 21.64 mg/g, representing a 2.01-fold increase over the control. Transcriptome sequencing revealed that PN7 induction upregulated key genes in the saponin biosynthetic pathway (including DXS, HMGR, PMK, DS, CYP450, and GTs), modulated 253 plant hormone signaling genes (such as those related to JA, ETH, and ABA), and affected 284 transcription factor genes and 47 ABC transporter genes. Co-expression network analysis identified DEGs related to plant hormone signaling, transcription factors, and ABC transporters in saponin biosynthesis and distribution. The results suggested that JA signaling, mediated by transcription factors, such as bHLH and MYBs, and its interaction with ETH, played crucial roles in saponin biosynthesis. Additionally, potential ABC transporter candidates involved in saponin transport were identified. This study highlights the role of endophytic bacteria in enhancing saponin production in P. notoginseng and opens avenues for further research on microbial-plant interactions in secondary metabolite production.